TGFβ and BMP Dependent Cell Fate Changes Due to Loss of Filamin B Produces Disc Degeneration and Progressive Vertebral Fusions

PubMed Central

Zieba, Jennifer; Forlenza, Kimberly Nicole; Khatra, Jagteshwar Singh; Sarukhanov, Anna; Duran, Ivan; Rigueur, Diana; Lyons, Karen M.; Cohn, Daniel H.; Merrill, Amy E.; Krakow, Deborah

2016-01-01

Spondylocarpotarsal synostosis (SCT) is an autosomal recessive disorder characterized by progressive vertebral fusions and caused by loss of function mutations in Filamin B (FLNB). FLNB acts as a signaling scaffold by linking the actin cytoskleteon to signal transduction systems, yet the disease mechanisms for SCT remain unclear. Employing a Flnb knockout mouse, we found morphologic and molecular evidence that the intervertebral discs (IVDs) of Flnb–/–mice undergo rapid and progressive degeneration during postnatal development as a result of abnormal cell fate changes in the IVD, particularly the annulus fibrosus (AF). In Flnb–/–mice, the AF cells lose their typical fibroblast-like characteristics and acquire the molecular and phenotypic signature of hypertrophic chondrocytes. This change is characterized by hallmarks of endochondral-like ossification including alterations in collagen matrix, expression of Collagen X, increased apoptosis, and inappropriate ossification of the disc tissue. We show that conversion of the AF cells into chondrocytes is coincident with upregulated TGFβ signaling via Smad2/3 and BMP induced p38 signaling as well as sustained activation of canonical and noncanonical target genes p21 and Ctgf. These findings indicate that FLNB is involved in attenuation of TGFβ/BMP signaling and influences AF cell fate. Furthermore, we demonstrate that the IVD disruptions in Flnb–/–mice resemble aging degenerative discs and reveal new insights into the molecular causes of vertebral fusions and disc degeneration. PMID:27019229

Computation models simulating notochordal cell extinction during early ageing of an intervertebral disc.

PubMed

Louman-Gardiner, K M; Coombe, D; Hunter, C J

2011-12-01

Lower back pain due to intervertebral disc (IVD) degeneration is a prevalent problem which drastically affects the quality of life of millions of sufferers. Healthy IVDs begin with high populations of notochordal cells in the nucleus pulposus, while by the second stage of degeneration, these cells will be replaced by chondrocyte-like cells. Because the IVD is avascular, these cells rely on passive diffusion of nutrients to survive. It is thought that this transition in cell phenotype causes the shift of the IVD's physical properties, which impede the flow of nutrients. Our computational model of the IVD illustrates its ability to simulate the evolving chemical and mechanical environments occurring during the early ageing process. We demonstrate that, due to the insufficient nutrient supply and accompanying changes in physical properties of the IVD, there was a resultant exponential decay in the number of notochordal cells over time.

Human umbilical cord mesenchymal stromal cells exhibit immature nucleus pulposus cell phenotype in a laminin-rich pseudo-three-dimensional culture system.

PubMed

Chon, Brian H; Lee, Esther J; Jing, Liufang; Setton, Lori A; Chen, Jun

2013-10-02

Cell supplementation to the herniated or degenerated intervertebral disc (IVD) is a potential strategy to promote tissue regeneration and slow disc pathology. Human umbilical cord mesenchymal stromal cells (HUCMSCs) - originating from the Wharton's jelly - remain an attractive candidate for such endeavors with their ability to differentiate into multiple lineages. Previously, mesenchymal stem cells (MSCs) have been studied as a potential source for disc tissue regeneration. However, no studies have demonstrated that MSCs can regenerate matrix with unique characteristics matching that of immature nucleus pulposus (NP) tissues of the IVD. In our prior work, immature NP cells were found to express specific laminin isoforms and laminin-binding receptors that may serve as phenotypic markers for evaluating MSC differentiation to NP-like cells. The goal of this study is to evaluate these markers and matrix synthesis for HUCMSCs cultured in a laminin-rich pseudo-three-dimensional culture system. HUCMSCs were seeded on top of Transwell inserts pre-coated with Matrigel™, which contained mainly laminin-111. Cells were cultured under hypoxia environment with three differentiation conditions: NP differentiation media (containing 2.5% Matrigel™ solution to provide for a pseudo-three-dimensional laminin culture system) with no serum, or the same media supplemented with either insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1). Cell clustering behavior, matrix production and the expression of NP-specific laminin and laminin-receptors were evaluated at days 1, 7, 13 and 21 of culture. Data show that a pseudo-three-dimensional culture condition (laminin-1 rich) promoted HUCMSC differentiation under no serum conditions. Starting at day 1, HUCMSCs demonstrated a cell clustering morphology similar to that of immature NP cells in situ and that observed for primary immature NP cells within the similar laminin-rich culture system (prior study). Differentiated HUCMSCs under all conditions were found to contain glycosaminoglycan, expressed extracellular matrix proteins of collagen II and laminin α5, and laminin receptors (integrin α3 and β4 subunits). However, neither growth factor treatment generated distinct differences in NP-like phenotype for HUCMSC as compared with no-serum conditions. HUCMSCs have the potential to differentiate into cells sharing features with immature NP cells in a laminin-rich culture environment and may be useful for IVD cellular therapy.

Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

PubMed

Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

2015-12-01

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. Copyright © 2015 by the Genetics Society of America.

Use of Adipose Derived Stem Cells to Treat Large Bone Defects. Addendum

DTIC Science & Technology

2009-07-01

optimal delivery . We have also completed characterization of our segmental defect model, including analysis of vascular ingrowth during defect healing...cells seeded in 1.2% Keltone alginate at a density of 12-15x106cells/ml were loaded on 24-well transwell insert membranes [6]. Once hydrogel discs...process from tissue culture plates and hydrogels does not alter the surface phenotype. Gene expression of surface markers and proteins associated with

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

PubMed Central

van den Akker, Guus G. H.; Surtel, Don A. M.; Cremers, Andy; Richardson, Stephen M.; Hoyland, Judith A.; van Rhijn, Lodewijk W.

2016-01-01

Introduction Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. Methods Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). Results The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. Conclusion We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair. PMID:26794306

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus.

PubMed

van den Akker, Guus G H; Surtel, Don A M; Cremers, Andy; Richardson, Stephen M; Hoyland, Judith A; van Rhijn, Lodewijk W; Voncken, Jan Willem; Welting, Tim J M

2016-01-01

Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

Hypoxic Regulation of Functional Extracellular Matrix Elaboration by Nucleus Pulposus Cells in Long-Term Agarose Culture

PubMed Central

Gorth, Deborah J; Lothstein, Katherine E; Chiaro, Joseph A; Farrell, Megan J; Dodge, George R; Elliott, Dawn M; Malhotra, Neil R; Mauck, Robert L; Smith, Lachlan J

2015-01-01

Degeneration of the intervertebral discs is strongly implicated as a cause of low back pain. Since current treatments for discogenic low back pain show poor long-term efficacy, a number of new, biological strategies are being pursued. For such therapies to succeed, it is critical that they be validated in conditions that mimic the unique biochemical microenvironment of the nucleus pulposus (NP), which include low oxygen tension. Therefore, the objective of this study was to investigate the effects of oxygen tension on NP cell functional extracellular matrix elaboration in 3D culture. Bovine NP cells were encapsulated in agarose constructs and cultured for 14 or 42 days in either 20% or 2% oxygen in defined media containing transforming growth factor beta-3. At each time point, extracellular matrix composition, biomechanics and mRNA expression of key phenotypic markers were evaluated. Results showed that while bulk mechanics and composition were largely independent of oxygen level, low oxygen promoted improved restoration of the NP phenotype, higher mRNA expression of extracellular matrix and NP specific markers, and more uniform matrix elaboration. These findings indicate that culture under physiological oxygen levels is an important consideration for successful development of cell and growth factor-based regenerative strategies for the disc. PMID:25640328

Phenotype variations affect genetic association studies of degenerative disc disease: conclusions of analysis of genetic association of 58 single nucleotide polymorphisms with highly specific phenotypes for disc degeneration in 332 subjects.

PubMed

Rajasekaran, S; Kanna, Rishi Mugesh; Senthil, Natesan; Raveendran, Muthuraja; Cheung, Kenneth M C; Chan, Danny; Subramaniam, Sakthikanal; Shetty, Ajoy Prasad

2013-10-01

Although the influence of genetics on the process of disc degeneration is well recognized, in recently published studies, there is a wide variation in the race and selection criteria for such study populations. More importantly, the radiographic features of disc degeneration that are selected to represent the disc degeneration phenotype are variable in these studies. The study presented here evaluates the association between single nucleotide polymorphisms (SNPs) of candidate genes and three distinct radiographic features that can be defined as the degenerative disc disease (DDD) phenotype. The study objectives were to examine the allelic diversity of 58 SNPs related to 35 candidate genes related to lumbar DDD, to evaluate the association in a hitherto unevaluated ethnic Indian population that represents more than one-sixth of the world population, and to analyze how genetic associations can vary in the same study subjects with the choice of phenotype. A cross-sectional, case-control study of an ethnic Indian population was carried out. Fifty-eight SNPs in 35 potential candidate genes were evaluated in 342 subjects and the associations were analyzed against three highly specific markers for DDD, namely disc degeneration by Pfirrmann grading, end-plate damage evaluated by total end-plate damage score, and annular tears evaluated by disc herniations and hyperintense zones. Genotyping of cases and controls was performed on a genome-wide SNP array to identify potential associated disease loci. The results from the genome-wide SNP array were then used to facilitate SNP selection and genotype validation was conducted using Sequenom-based genotyping. Eleven of the 58 SNPs provided evidence of association with one of the phenotypes. For annular tears, rs1042631 SNP of AGC1 and rs467691 SNP of ADAMTS5 were highly significantly associated (p<.01) and SNPs in NGFB, IL1B, IL18RAP, and MMP10 were also significantly associated (p<.05). The rs4076018 SNP of NGFB was highly significant (p<.01) and rs2292657 SNP of GLI1 was significantly (p<.05) correlated to disc degeneration. For end-plate damage, the rs2252070 SNP of MMP 13 showed a significant association (p<.05). Previously associated genes such as COL 9, SKT, CHST 3, CILP, IGFR, SOXp, BMP, MMP 2-12, ADH2, IL1RN, and COX2 were not significantly associated and new associations (NGFB and GLI1) were identified. The validity of all the associations was found to be phenotype dependent. For the first time, genetic associations with DDD have been performed in an Indian population. Apart from identifying new associations, the highlight of the study was that in the same study population with DDD, SNP associations completely changed when different radiographic features were used to define the DDD phenotype. Our study results therefore indicate that standardization of the phenotypes chosen to study the genetics of disc degeneration is essential and should be strongly considered before planning genetic association studies. Copyright © 2013 Elsevier Inc. All rights reserved.

How Reliable Are the Reported Genetic Associations in Disc Degeneration?: The Influence of Phenotypes, Age, Population Size, and Inclusion Sequence in 809 Patients.

PubMed

Rajasekaran, S; Kanna, Rishi Mugesh; Reddy, Ranjani Raja; Natesan, Senthil; Raveendran, Muthuraja; Cheung, Kenneth M C; Chan, Danny; Kao, Patrick Y P; Yee, Anita; Shetty, Ajoy Prasad

2016-11-01

Prospective genetic association study. The aim of this study was to document the variations in the genetic associations, when different magnetic resonance imaging (MRI) phenotypes, age stratification, cohort size, and sequence of cohort inclusion are varied in the same study population. Genetic associations with disc degeneration have shown high inconsistency, generally attributed to hereditary factors and ethnic variations. However, the effect of different phenotypes, size of the study population, age of the cohort, etc have not been documented clearly. Seventy-one single-nucleotide polymorphisms (SNPs) of 41 candidate genes were correlated to six MRI markers of disc degeneration (annular tears, Pfirmann grading, Schmorl nodes, Modic changes, Total Endplate Damage score, and disc bulge) in 809 patients with back pain and/or sciatica. In the same study group, the correlations were then retested for different age groups, different sample, size and sequence of subject inclusion (first 404 and the second 405) and the differences documented. The mean age of population (M: 455, F: 354) was 36.7 ± 10.8 years. Different genetic associations were found with different phenotypes: disc bulge with three SNPs of CILP; annular tears with rs2249350 of ADAMTS5 and rs11247361 IGF1R; modic changes with VDR and MMP20; Pfirmann grading with three SNPs of MMP20 and Schmorl node with SNPs of CALM1 and FN1 and none with Total End Plate Score.Subgroup analysis based on three age groups and dividing the total population into two groups also completely changed the associations for all the six radiographic parameters. In the same study population, SNP associations completely change with different phenotypes. Variations in age, inclusion sequence, and sample size resulted in change of genetic associations. Our study questions the validity of previous studies and necessitates the need for standardizing the description of disc degeneration, phenotype selection, study sample size, age, and other variables in future studies. 4.

Sonic hedgehog in the notochord is sufficient for patterning of the intervertebral discs

PubMed Central

Choi, Kyung-Suk; Lee, Chanmi; Harfe, Brian D.

2012-01-01

The intervertebral discs, located between adjacent vertebrae, are required for stability of the spine and distributing mechanical load throughout the vertebral column. All cell types located in thes middle regions of the discs, called nuclei pulposi, are derived from the embryonic notochord. Recently, it was shown that the hedgehog signaling pathway plays an essential role during formation of nuclei pulposi. However, during the time that nuclei pulposi are forming, Shh is expressed in both the notochord and the nearby floor plate. To determine the source of SHH protein sufficient for formation of nuclei pulposi we removed Shh from either the floor plate or the notochord using tamoxifen-inducible Cre alleles. Removal of Shh from the floor plate resulted in phenotypically normal intervertebral discs, indicating that Shh expression in this tissue is not required for disc patterning. In addition, embryos that lacked Shh in the floor plate had normal vertebral columns, demonstrating that Shh expression in the notochord is sufficient for pattering the entire vertebral column. Removal of Shh from the notochord resulted in the absence of Shh in the floor plate, loss of intervertebral discs and vertebral structures. These data indicate that Shh expression in the notochord is sufficient for patterning of the intervertebral discs and the vertebral column. PMID:22841806

Sonic hedgehog in the notochord is sufficient for patterning of the intervertebral discs.

PubMed

Choi, Kyung-Suk; Lee, Chanmi; Harfe, Brian D

2012-01-01

The intervertebral discs, located between adjacent vertebrae, are required for stability of the spine and distributing mechanical load throughout the vertebral column. All cell types located in the middle regions of the discs, called nuclei pulposi, are derived from the embryonic notochord. Recently, it was shown that the hedgehog signaling pathway plays an essential role during formation of nuclei pulposi. However, during the time that nuclei pulposi are forming, Shh is expressed in both the notochord and the nearby floor plate. To determine the source of SHH protein sufficient for formation of nuclei pulposi we removed Shh from either the floor plate or the notochord using tamoxifen-inducible Cre alleles. Removal of Shh from the floor plate resulted in phenotypically normal intervertebral discs, indicating that Shh expression in this tissue is not required for disc patterning. In addition, embryos that lacked Shh in the floor plate had normal vertebral columns, demonstrating that Shh expression in the notochord is sufficient for pattering the entire vertebral column. Removal of Shh from the notochord resulted in the absence of Shh in the floor plate, loss of intervertebral discs and vertebral structures. These data indicate that Shh expression in the notochord is sufficient for patterning of the intervertebral discs and the vertebral column. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Classification of High Intensity Zones of the Lumbar Spine and Their Association with Other Spinal MRI Phenotypes: The Wakayama Spine Study.

PubMed

Teraguchi, Masatoshi; Samartzis, Dino; Hashizume, Hiroshi; Yamada, Hiroshi; Muraki, Shigeyuki; Oka, Hiroyuki; Cheung, Jason Pui Yin; Kagotani, Ryohei; Iwahashi, Hiroki; Tanaka, Sakae; Kawaguchi, Hiroshi; Nakamura, Kozo; Akune, Toru; Cheung, Kenneth Man-Chee; Yoshimura, Noriko; Yoshida, Munehito

2016-01-01

High intensity zones (HIZ) of the lumbar spine are a phenotype of the intervertebral disc noted on MRI whose clinical relevance has been debated. Traditionally, T2-weighted (T2W) magnetic resonance imaging (MRI) has been utilized to identify HIZ of lumbar discs. However, controversy exists with regards to HIZ morphology, topography, and association with other MRI spinal phenotypes. Moreover, classification of HIZ has not been thoroughly defined in the past and the use of additional imaging parameters (e.g. T1W MRI) to assist in defining this phenotype has not been addressed. A cross-sectional study of 814 (69.8% females) subjects with mean age of 63.6 years from a homogenous Japanese population was performed. T2W and T1W sagittal 1.5T MRI was obtained on all subjects to assess HIZ from L1-S1. We created a morphological and topographical HIZ classification based on disc level, shape type (round, fissure, vertical, rim, and enlarged), location within the disc (posterior, anterior), and signal type on T1W MRI (low, high and iso intensity) in comparison to the typical high intensity on T2W MRI. HIZ was noted in 38.0% of subjects. Of these, the prevalence of posterior, anterior, and both posterior/anterior HIZ in the overall lumbar spine were 47.3%, 42.4%, and 10.4%, respectively. Posterior HIZ was most common, occurring at L4/5 (32.5%) and L5/S1 (47.0%), whereas anterior HIZ was most common at L3/4 (41.8%). T1W iso-intensity type of HIZ was most prevalent (71.8%), followed by T1W high-intensity (21.4%) and T1W low-intensity (6.8%). Of all discs, round types were most prevalent (anterior: 3.6%, posterior: 3.7%) followed by vertical type (posterior: 1.6%). At all affected levels, there was a significant association between HIZ and disc degeneration, disc bulge/protrusion and Modic type II (p<0.01). Posterior HIZ and T1W high-intensity type of HIZ were significantly associated with disc bulge/protrusion and disc degeneration (p<0.01). In addition, posterior HIZ was significantly associated with Modic type II and III. T1W low-intensity type of HIZ was significantly associated with Modic type II. This is the first large-scale study reporting a novel classification scheme of HIZ of the lumbar spine. This study is the first that has utilized T2W and T1W MRIs in differentiating HIZ sub-phenotypes. Specific HIZ sub-phenotypes were found to be more associated with specific MRI degenerative changes. With a more detailed description of the HIZ phenotype, this scheme can be standardized for future clinical and research initiatives.

Circadian factors BMAL1 and RORα control HIF-1α transcriptional activity in nucleus pulposus cells: implications in maintenance of intervertebral disc health

PubMed Central

Suyama, Kaori; Silagi, Elizabeth S.; Choi, Hyowon; Sakabe, Kou; Mochida, Joji; Shapiro, Irving M.; Risbud, Makarand V.

2016-01-01

BMAL1 and RORα are major regulators of the circadian molecular oscillator. Since previous work in other cell types has shown cross talk between circadian rhythm genes and hypoxic signaling, we investigated the role of BMAL1 and RORα in controlling HIF-1-dependent transcriptional responses in NP cells that exist in the physiologically hypoxic intervertebral disc. HIF-1-dependent HRE reporter activity was further promoted by co-transfection with either BMAL1 or RORα. In addition, stable silencing of BMAL1 or inhibition of RORα activity resulted in decreased HRE activation. Inhibition of RORα also modulated HIF1α-TAD activity. Interestingly, immunoprecipitation studies showed no evidence of BMAL1, CLOCK or RORα binding to HIF-1α in NP cells. Noteworthy, stable silencing of BMAL1 as well as inhibition of RORα decreased expression of select HIF-1 target genes including VEGF, PFKFB3 and Eno1. To delineate if BMAL1 plays a role in maintenance of disc health, we studied the spinal phenotype of BMAL1-null mice. The lumbar discs of null mice evidenced decreased height, and several parameters associated with vertebral trabecular bone quality were also affected in nulls. In addition, null animals showed a higher ratio of cells to matrix in NP tissue and hyperplasia of the annulus fibrosus. Taken together, our results indicate that BMAL1 and RORα form a regulatory loop in the NP and control HIF-1 activity without direct interaction. Importantly, activities of these circadian rhythm molecules may play a role in the adaptation of NP cells to their unique niche. PMID:27049729

[Research progress of intervertebral disc endogenous stem cells for intervertebral disc regeneration].

PubMed

Liang, Hang; Deng, Xiangyu; Shao, Zengwu

2017-10-01

To summarize the research progress of intervertebral disc endogenous stem cells for intervertebral disc regeneration and deduce the therapeutic potential of endogenous repair for intervertebral disc degeneration. The original articles about intervertebral disc endogenous stem cells for intervertebral disc regeneration were extensively reviewed; the reparative potential in vivo and the extraction and identification in vitro of intervertebral disc endogenous stem cells were analyzed; the prospect of endogenous stem cells for intervertebral disc regeneration was predicted. Stem cell niche present in the intervertebral discs, from which stem cells migrate to injured tissues and contribute to tissues regeneration under certain specific microenvironment. Moreover, the migration of stem cells is regulated by chemokines system. Tissue specific progenitor cells have been identified and successfully extracted and isolated. The findings provide the basis for biological therapy of intervertebral disc endogenous stem cells. Intervertebral disc endogenous stem cells play a crucial role in intervertebral disc regeneration. Therapeutic strategy of intervertebral disc endogenous stem cells is proven to be a promising biological approach for intervertebral disc regeneration.

Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.

PubMed

Saggese, Taryn; Thambyah, Ashvin; Wade, Kelly; McGlashan, Susan Read

2018-05-01

Objective The nucleus pulposus of the human intervertebral disc contains 2 cell types: notochordal (NC) and mature nucleus pulposus (MNP) cells. NC cell loss is associated with disc degeneration and this process may be initiated by mechanical stress and/or nutrient deprivation. This study aimed to investigate the functional responses of NC and MNP cells to hydrostatic pressures and glucose restriction. Design Bovine MNP and NC cells were cultured in 3-dimensional alginate beads under low (0.4-0.8 MPa) and high (1.6-2.4 MPa) dynamic pressure for 24 hours. Cells were cultured in either physiological (5.5 mM) glucose media or glucose-restriction (0.55 mM) media. Finally, the combined effect of glucose restriction and high pressure was examined. Results Cell viability and notochordal phenotypic markers were not significantly altered in response to pressure or glucose restriction. MNP cells responded to low pressure with an increase in glycosaminoglycan (GAG) production while high pressure significantly decreased ACAN gene expression compared with atmospheric controls. NC cells showed no response in matrix gene expression or GAG production with either loading regime. Glucose restriction decreased NC cell TIMP-1 expression but had no effect on MNP cells. The combination of glucose restriction and high pressure only affected MNP cell gene expression, with decreased ACAN, Col2α1, and ADAMTS-5 expression. Conclusion This study shows that NC cells are more resistant to acute mechanical stresses than MNP cells and provides a strong rationale for future studies to further our understanding the role of NC cells within the disc, and the effects of long-term exposure to physical stresses.

N-Cadherin Maintains the Healthy Biology of Nucleus Pulposus Cells under High-Magnitude Compression.

PubMed

Wang, Zhenyu; Leng, Jiali; Zhao, Yuguang; Yu, Dehai; Xu, Feng; Song, Qingxu; Qu, Zhigang; Zhuang, Xinming; Liu, Yi

2017-01-01

Mechanical load can regulate disc nucleus pulposus (NP) biology in terms of cell viability, matrix homeostasis and cell phenotype. N-cadherin (N-CDH) is a molecular marker of NP cells. This study investigated the role of N-CDH in maintaining NP cell phenotype, NP matrix synthesis and NP cell viability under high-magnitude compression. Rat NP cells seeded on scaffolds were perfusion-cultured using a self-developed perfusion bioreactor for 5 days. NP cell biology in terms of cell apoptosis, matrix biosynthesis and cell phenotype was studied after the cells were subjected to different compressive magnitudes (low- and high-magnitudes: 2% and 20% compressive deformation, respectively). Non-loaded NP cells were used as controls. Lentivirus-mediated N-CDH overexpression was used to further investigate the role of N-CDH under high-magnitude compression. The 20% deformation compression condition significantly decreased N-CDH expression compared with the 2% deformation compression and control conditions. Meanwhile, 20% deformation compression increased the number of apoptotic NP cells, up-regulated the expression of Bax and cleaved-caspase-3 and down-regulated the expression of Bcl-2, matrix macromolecules (aggrecan and collagen II) and NP cell markers (glypican-3, CAXII and keratin-19) compared with 2% deformation compression. Additionally, N-CDH overexpression attenuated the effects of 20% deformation compression on NP cell biology in relation to the designated parameters. N-CDH helps to restore the cell viability, matrix biosynthesis and cellular phenotype of NP cells under high-magnitude compression. © 2017 The Author(s). Published by S. Karger AG, Basel.

Lubricin is Required for the Structural Integrity and Post-natal Maintenance of TMJ

PubMed Central

Koyama, E.; Saunders, C.; Salhab, I.; Decker, R.S.; Chen, I.; Um, H.; Pacifici, M.; Nah, H.D.

2014-01-01

The Proteoglycan 4 (Prg4) product lubricin plays essential roles in boundary lubrication and movement in limb synovial joints, but its roles in temporomandibular joint (TMJ) are unclear. Thus, we characterized the TMJ phenotype in wild-type and Prg4 –/– mouse littermates over age. As early as 2 weeks of age, mutant mice exhibited hyperplasia in the glenoid fossa articular cartilage, articular disc, and synovial membrane. By 1 month of age, there were fewer condylar superficial tenascin-C/Col1-positive cells and more numerous apoptotic condylar apical cells, while chondroprogenitors displayed higher mitotic activity, and Sox9-, Col2-, and ColX-expressing chondrocyte zones were significantly expanded. Mutant subchondral bone contained numerous Catepsin K- expressing osteoclasts at the chondro-osseous junction, increased invasive marrow cavities, and suboptimal subchondral bone. Mutant glenoid fossa, disc, synovial cells, and condyles displayed higher Hyaluronan synthase 2 expression. Mutant discs also lost their characteristic concave shape, exhibited ectopic chondrocyte differentiation, and occasionally adhered to condylar surfaces. A fibrinoid substance of unclear origin often covered the condylar surface. By 6 months of age, mutant condyles displayed osteoarthritic degradation with apical/mid-zone separation. In sum, lubricin exerts multiple essential direct and indirect roles to preserve TMJ structural and cellular integrity over post-natal life. PMID:24834922

Primary human osteoblasts grow into porous tantalum and maintain an osteoblastic phenotype.

PubMed

Welldon, Katie J; Atkins, Gerald J; Howie, Donald W; Findlay, David M

2008-03-01

Porous tantalum (Ta) has found application in orthopedics, although the interaction of human osteoblasts (HOB) with this material has not been reported. The aim of this study was to investigate the interaction of primary HOB with porous tantalum, using 5-mm thick discs of porous tantalum. Comparison was made with discs of solid tantalum and tissue culture plastic. Confocal microscopy was used to investigate the attachment and growth of cells on porous Ta, and showed that HOB attached successfully to the metal "trabeculae," underwent extensive cell division, and penetrated into the Ta pores. The maturation of HOB on porous Ta was determined in terms of cell expression of the osteoblast phenotypic markers, STRO-1, and alkaline phosphatase. Despite some donor-dependent variation in STRO-1/AlkPhos expression, growth of cells grown on porous Ta either promoted, or did not impede, the maturation of HOB. In addition, the expression of key osteoblastic genes was investigated after 14 days of culture. The relative levels of mRNA encoding osteocalcin, osteopontin and receptor activator of NFkappaB ligand (RANKL) was not different between porous or solid Ta or plastic, although these genes were expressed differently by cells of different donors. However, bone sialoprotein and type I collagen mRNA species showed a decreased expression on porous Ta compared with expression on plastic. No substrate-dependent differences were seen in the extent of in vitro mineralization by HOB. These results indicate that porous Ta is a good substrate for the attachment, growth, and differentiated function of HOB. (c) 2007 Wiley Periodicals, Inc.

Three-dimensional development of tensile pre-strained annulus fibrosus cells for tissue regeneration: An in-vitro study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuah, Yon Jin; Lee, Wu Chean; Wong, Hee Kit

Prior research has investigated the immediate response after application of tensile strain on annulus fibrosus (AF) cells for the past decade. Although mechanical strain can produce either catabolic or anabolic consequences to the cell monolayer, little is known on how to translate these findings into further tissue engineering applications. Till to date, the application and effect of tensile pre-strained cells to construct a three-dimensional (3D) AF tissue remains unknown. This study aims to investigate the effect of tensile pre-strained exposure of 1 to 24 h on the development of AF pellet culture for 3 weeks. Equibiaxial cyclic tensile strain wasmore » applied on AF monolayer cells over a period of 24 h, which was subsequently developed into a cell pellet. Investigation on cellular proliferation, phenotypic gene expression, and histological changes revealed that tensile pre-strain for 24 h had significant and lasting effect on the AF tissue development, with enhanced cell proliferation, and up-regulation of collagen type I, II, and aggrecan expression. Our results demonstrated the regenerative ability of AF cell pellets subjected to 24 h tensile pre-straining. Knowledge on the effects of tensile pre-strain exposure is necessary to optimize AF development for tissue reconstruction. Moreover, the tensile pre-strained cells may further be utilized in either cell therapy to treat mild disc degeneration disease, or the development of a disc construct for total disc replacement. - Highlights: • Establishment of tensile pre-strained cell line population for annulus development. • Tensile strain limits collagen gene expression declination in monolayer culture. • Tensile pre-strained cells up-regulate their matrix protein in 3D pellet culture.« less

Implantation of hyaluronic acid hydrogel prevents the pain phenotype in a rat model of intervertebral disc injury

PubMed Central

Sakai, Daisuke; Dockery, Peter

2018-01-01

Painful intervertebral disc degeneration is mediated by inflammation that modulates glycosylation and induces hyperinnervation and sensory sensitization, which result in discogenic pain. Hyaluronic acid (HA) used as a therapeutic biomaterial can reduce inflammation and pain, but the effects of HA therapy on glycosylation and pain associated with disc degeneration have not been previously determined. We describe a novel rat model of pain induced by intervertebral disc injury, with validation of the pain phenotype by morphine treatment. Using this model, we assessed the efficacy of HA hydrogel for the alleviation of pain, demonstrating that it reduced nociceptive behavior, an effect associated with down-regulation of nociception markers and inhibition of hyperinnervation. Furthermore, HA hydrogel altered glycosylation and modulated key inflammatory and regulatory signaling pathways, resulting in attenuation of inflammation and regulation of matrix components. Our results suggest that HA hydrogel is a promising clinical candidate for the treatment of back pain caused by degenerated discs. PMID:29632893

Drosophila Big bang regulates the apical cytocortex and wing growth through junctional tension.

PubMed

Tsoumpekos, Giorgos; Nemetschke, Linda; Knust, Elisabeth

2018-03-05

Growth of epithelial tissues is regulated by a plethora of components, including signaling and scaffolding proteins, but also by junctional tension, mediated by the actomyosin cytoskeleton. However, how these players are spatially organized and functionally coordinated is not well understood. Here, we identify the Drosophila melanogaster scaffolding protein Big bang as a novel regulator of growth in epithelial cells of the wing disc by ensuring proper junctional tension. Loss of big bang results in the reduction of the regulatory light chain of nonmuscle myosin, Spaghetti squash. This is associated with an increased apical cell surface, decreased junctional tension, and smaller wings. Strikingly, these phenotypic traits of big bang mutant discs can be rescued by expressing constitutively active Spaghetti squash. Big bang colocalizes with Spaghetti squash in the apical cytocortex and is found in the same protein complex. These results suggest that in epithelial cells of developing wings, the scaffolding protein Big bang controls apical cytocortex organization, which is important for regulating cell shape and tissue growth. © 2018 Tsoumpekos et al.

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

PubMed Central

2014-01-01

Introduction Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. Methods Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. Results A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)–negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. Conclusions Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease. PMID:24972717

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus.

PubMed

van den Akker, Guus G H; Surtel, Don A M; Cremers, Andy; Rodrigues-Pinto, Ricardo; Richardson, Stephen M; Hoyland, Judith A; van Rhijn, Lodewijk W; Welting, Tim J M; Voncken, Jan Willem

2014-06-27

Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)-negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease.

Expression of DISC1-interactome members correlates with cognitive phenotypes related to schizophrenia.

PubMed

Rampino, Antonio; Walker, Rosie May; Torrance, Helen Scott; Anderson, Susan Maguire; Fazio, Leonardo; Di Giorgio, Annabella; Taurisano, Paolo; Gelao, Barbara; Romano, Raffaella; Masellis, Rita; Ursini, Gianluca; Caforio, Grazia; Blasi, Giuseppe; Millar, J Kirsty; Porteous, David John; Thomson, Pippa Ann; Bertolino, Alessandro; Evans, Kathryn Louise

2014-01-01

Cognitive dysfunction is central to the schizophrenia phenotype. Genetic and functional studies have implicated Disrupted-in-Schizophrenia 1 (DISC1), a leading candidate gene for schizophrenia and related psychiatric conditions, in cognitive function. Altered expression of DISC1 and DISC1-interactors has been identified in schizophrenia. Dysregulated expression of DISC1-interactome genes might, therefore, contribute to schizophrenia susceptibility via disruption of molecular systems required for normal cognitive function. Here, the blood RNA expression levels of DISC1 and DISC1-interacting proteins were measured in 63 control subjects. Cognitive function was assessed using neuropsychiatric tests and functional magnetic resonance imaging was used to assess the activity of prefrontal cortical regions during the N-back working memory task, which is abnormal in schizophrenia. Pairwise correlations between gene expression levels and the relationship between gene expression levels and cognitive function and N-back-elicited brain activity were assessed. Finally, the expression levels of DISC1, AKAP9, FEZ1, NDEL1 and PCM1 were compared between 63 controls and 69 schizophrenic subjects. We found that DISC1-interactome genes showed correlated expression in the blood of healthy individuals. The expression levels of several interactome members were correlated with cognitive performance and N-back-elicited activity in the prefrontal cortex. In addition, DISC1 and NDEL1 showed decreased expression in schizophrenic subjects compared to healthy controls. Our findings highlight the importance of the coordinated expression of DISC1-interactome genes for normal cognitive function and suggest that dysregulated DISC1 and NDEL1 expression might, in part, contribute to susceptibility for schizophrenia via disruption of prefrontal cortex-dependent cognitive functions.

Expression of DISC1-Interactome Members Correlates with Cognitive Phenotypes Related to Schizophrenia

PubMed Central

Rampino, Antonio; Walker, Rosie May; Torrance, Helen Scott; Anderson, Susan Maguire; Fazio, Leonardo; Di Giorgio, Annabella; Taurisano, Paolo; Gelao, Barbara; Romano, Raffaella; Masellis, Rita; Ursini, Gianluca; Caforio, Grazia; Blasi, Giuseppe; Millar, J. Kirsty; Porteous, David John; Thomson, Pippa Ann; Bertolino, Alessandro; Evans, Kathryn Louise

2014-01-01

Cognitive dysfunction is central to the schizophrenia phenotype. Genetic and functional studies have implicated Disrupted-in-Schizophrenia 1 (DISC1), a leading candidate gene for schizophrenia and related psychiatric conditions, in cognitive function. Altered expression of DISC1 and DISC1-interactors has been identified in schizophrenia. Dysregulated expression of DISC1-interactome genes might, therefore, contribute to schizophrenia susceptibility via disruption of molecular systems required for normal cognitive function. Here, the blood RNA expression levels of DISC1 and DISC1-interacting proteins were measured in 63 control subjects. Cognitive function was assessed using neuropsychiatric tests and functional magnetic resonance imaging was used to assess the activity of prefrontal cortical regions during the N-back working memory task, which is abnormal in schizophrenia. Pairwise correlations between gene expression levels and the relationship between gene expression levels and cognitive function and N-back-elicited brain activity were assessed. Finally, the expression levels of DISC1, AKAP9, FEZ1, NDEL1 and PCM1 were compared between 63 controls and 69 schizophrenic subjects. We found that DISC1-interactome genes showed correlated expression in the blood of healthy individuals. The expression levels of several interactome members were correlated with cognitive performance and N-back-elicited activity in the prefrontal cortex. In addition, DISC1 and NDEL1 showed decreased expression in schizophrenic subjects compared to healthy controls. Our findings highlight the importance of the coordinated expression of DISC1-interactome genes for normal cognitive function and suggest that dysregulated DISC1 and NDEL1 expression might, in part, contribute to susceptibility for schizophrenia via disruption of prefrontal cortex-dependent cognitive functions. PMID:24940743

Imaginal Disc Abnormalities in Lethal Mutants of Drosophila

PubMed Central

Shearn, Allen; Rice, Thomas; Garen, Alan; Gehring, Walter

1971-01-01

Late lethal mutants of Drosophila melanogaster, dying after the larval stage of development, were isolated. The homozygous mutant larvae were examined for abnormal imaginal disc morphology, and the discs were injected into normal larval hosts to test their capacities to differentiate into adult structures. In about half of the mutants analyzed, disc abnormalities were found. Included among the abnormalities were missing discs, small discs incapable of differentiating, morphologically normal discs with limited capacities for differentiation, and discs with homeotic transformations. In some mutants all discs were affected, and in others only certain discs. The most extreme abnormal phenotype is a class of “discless” mutants. The viability of these mutant larvae indicates that the discs are essential only for the development of an adult and not of a larva. The late lethals are therefore a major source of mutants for studying the genetic control of disc formation. Images PMID:5002822

Rare disruptive variants in the DISC1 Interactome and Regulome: association with cognitive ability and schizophrenia.

PubMed

Teng, S; Thomson, P A; McCarthy, S; Kramer, M; Muller, S; Lihm, J; Morris, S; Soares, D C; Hennah, W; Harris, S; Camargo, L M; Malkov, V; McIntosh, A M; Millar, J K; Blackwood, D H; Evans, K L; Deary, I J; Porteous, D J; McCombie, W R

2018-05-01

Schizophrenia (SCZ), bipolar disorder (BD) and recurrent major depressive disorder (rMDD) are common psychiatric illnesses. All have been associated with lower cognitive ability, and show evidence of genetic overlap and substantial evidence of pleiotropy with cognitive function and neuroticism. Disrupted in schizophrenia 1 (DISC1) protein directly interacts with a large set of proteins (DISC1 Interactome) that are involved in brain development and signaling. Modulation of DISC1 expression alters the expression of a circumscribed set of genes (DISC1 Regulome) that are also implicated in brain biology and disorder. Here we report targeted sequencing of 59 DISC1 Interactome genes and 154 Regulome genes in 654 psychiatric patients and 889 cognitively-phenotyped control subjects, on whom we previously reported evidence for trait association from complete sequencing of the DISC1 locus. Burden analyses of rare and singleton variants predicted to be damaging were performed for psychiatric disorders, cognitive variables and personality traits. The DISC1 Interactome and Regulome showed differential association across the phenotypes tested. After family-wise error correction across all traits (FWER across ), an increased burden of singleton disruptive variants in the Regulome was associated with SCZ (FWER across P=0.0339). The burden of singleton disruptive variants in the DISC1 Interactome was associated with low cognitive ability at age 11 (FWER across P=0.0043). These results identify altered regulation of schizophrenia candidate genes by DISC1 and its core Interactome as an alternate pathway for schizophrenia risk, consistent with the emerging effects of rare copy number variants associated with intellectual disability.

Nemo regulates cell dynamics and represses the expression of miple, a midkine/pleiotrophin cytokine, during ommatidial rotation

PubMed Central

Muñoz-Soriano, Verónica; Ruiz, Carlos; Pérez-Alonso, Manuel; Mlodzik, Marek; Paricio, Nuria

2013-01-01

Ommatidial rotation is one of the most important events for correct patterning of the Drosophila eye. Although several signaling pathways are involved in this process, few genes have been shown to specifically affect it. One of them is nemo (nmo), which encodes a MAP-like protein kinase that regulates the rate of rotation throughout the entire process, and serves as a link between core planar cell polarity (PCP) factors and the E-cadherin–β-catenin complex. To determine more precisely the role of nmo in ommatidial rotation, live-imaging analyses in nmo mutant and wild-type early pupal eye discs were performed. We demonstrate that ommatidial rotation is not a continuous process, and that rotating and non-rotating interommatidial cells are very dynamic. Our in vivo analyses also show that nmo regulates the speed of rotation and is required in cone cells for correct ommatidial rotation, and that these cells as well as interommatidial cells are less dynamic in nmo mutants. Furthermore, microarray analyses of nmo and wild-type larval eye discs led us to identify new genes and signaling pathways related to nmo function during this process. One of them, miple, encodes the Drosophila ortholog of the midkine/pleiotrophin secreted cytokines that are involved in cell migration processes. miple is highly up-regulated in nmo mutant discs. Indeed, phenotypic analyses reveal that miple overexpression leads to ommatidial rotation defects. Genetic interaction assays suggest that miple is signaling through Ptp99A, the Drosophila ortholog of the vertebrate midkine/pleiotrophin PTPζ receptor. Accordingly, we propose that one of the roles of Nmo during ommatial rotation is to repress miple expression, which may in turn affect the dynamics in E-cadherin–β-catenin complexes. PMID:23428616

Isolation and expression of scabrous, a gene regulating neurogenesis in Drosophila.

PubMed

Mlodzik, M; Baker, N E; Rubin, G M

1990-11-01

Mutations in the Drosophila scabrous (sca) gene affect eye and bristle development, leading to irregular spacing of ommatidia and bristle duplications in the adult fly. We have cloned the sca gene by P-element tagging. The sca transcription unit is 12 kb and consists of four exons that are joined in a 3.2-kb mRNA. In an enhancer trap screen we have isolated several P[lacZ] insertions close to the sca transcription start site. We have examined the expression pattern of sca by in situ hybridization to sca transcripts, by beta-galactosidase localization in the P[lacZ] lines, and by immunocytochemistry with an anti-sca antiserum. During embryogenesis, sca is expressed in a dynamic pattern associated with neural development. During imaginal development, sca is mainly expressed in the R8 photoreceptor precursor cells in the eye imaginal disc and in sensory organ precursor cells in other discs. In the wing disc, sca expression is coextensive with the anlagen for bristles and is controlled by genes of the achaete-scute complex. Based on its loss-of-function phenotype, expression pattern, and the predicted structure of its product, a secreted peptide with homology to the fibrinogen gene family, we propose that sca encodes a signal involved in lateral inhibition within individual domains of the developing nervous system.

Dominant Drop mutants are gain-of-function alleles of the muscle segment homeobox gene (msh) whose overexpression leads to the arrest of eye development.

PubMed

Mozer, B A

2001-05-15

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the Dr(Mio) allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the Dr(Mio) mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins. Copyright 2001 Academic Press.

Development of a two-step protocol for culture expansion of human annulus fibrosus cells with TGF-β1 and FGF-2.

PubMed

Chou, Po-Hsin; Wang, Shih-Tien; Ma, Hsiao-Li; Liu, Chien-Lin; Chang, Ming-Chau; Lee, Oscar Kuang-Sheng

2016-07-12

Different biologic approaches to treat disc regeneration, including growth factors (GFs) application, are currently under investigation. Human annulus fibrosus (hAF) repair or regeneration is one of the key elements for maintenance and restoration of nucleus pulposus function. However, so far there is no effective treatment for this purpose. The aim of the present study was to investigate the response of hAF cells to different combinations of GFs, and develop a protocol for efficient culture expansion. hAF cells were harvested from degenerated disc tissues during surgical intervertebral disc removal, and hAF cells were expanded in a monolayer. The experiments were categorized based on different protocols with transforming growth factor (TGF-β1) and fibroblast growth factor (FGF-2) culture for 14 days: group 1 had no GFs (control group); group 2 received TGF-β1; group 3 received FGF-2; group 4 received both GFs; and group 5 (two-step) received both GFs for the first 10 days and TGF-β1 only for the next 4 days. Cell proliferation, collagen, and noncollagen extracellular matrix (ECM) production and genes expression were compared among these groups. At days 3, 7 and 10 of cultivation, groups 4 and 5 had significantly more cell numbers and faster cell proliferation rates than groups 1, 2, and 3. At 14 days of cultivation, significantly more cell numbers were observed in groups 3 and 4 than in group 5. The group 4 had the most cell numbers and the fastest proliferation rate at 14 days of cultivation. After normalization for cell numbers, group 5 (two-step) produced the most collagen and noncollagen ECM at 10 and 14 days of cultivation among the five groups. In group 5, ECM gene expression was significantly upregulated. High expression of matrix metalloproteinase-1 was upregulated with FGF-2 on the different days as compared to the other groups. Annulus fibrosus cell phenotypes were only marginally retained under the different protocols based on quantitative polymerase chain reaction results. Taken together, the two-step protocol was the most efficient among these different protocols with the most abundant ECM production after normalization for cell numbers for culture expansion of hAF cells. The protocol may be useful in further cell therapy and tissue engineering approaches for disc regeneration.

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

PubMed

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2016-01-01

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres

PubMed Central

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2016-01-01

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering. PMID:26760956

Which iodinated contrast media is the least cytotoxic to human disc cells?

PubMed

Kim, Kyung-Hyun; Park, Jeong-Yoon; Park, Hyo-Suk; Kuh, Sung-Uk; Chin, Dong-Kyu; Kim, Keun-Su; Cho, Yong-Eun

2015-05-01

Iodinated contrast media (CM) is commonly used for various intradiscal injections such as in discography and endoscopic spinal surgery. However, CM has been shown to be toxic to renal tissue due to its ionic strength and osmolarity and as a result of iodine-induced cytotoxicity, which has raised concern over whether there are similar negative effects on disc cells. This in vitro study was designed to identify the least cytotoxic iodinated CM to the human disc cell among four different physiochemical iodinated contrast dyes. In vitro laboratory study. Intervertebral disc tissue was obtained by discectomy from a total of 10 lumbar disc patients undergoing surgery and disc cells were isolated. The human disc cells were grown in 3D alginate bead culture with 0, 0.1, 10, and 100 mg/mL CM solutions (ionic dimer, ionic monomer, non-ionic dimer, and non-ionic monomer) and mannitol as a control for 2 days. The living cells were analyzed with trypan blue staining. Fluorescence-activated cell sorting analysis was performed using Annexin V and propidium iodide (PI) and 3D alginate bead immunostaining to identify live, apoptotic, and necrotic cells. Human disc cell death was time- and dose-dependent in response to CM and more necrosis was observed than apoptosis. In addition, non-ionic dimeric CM (iodixanol) showed the least toxic effect on human disc cells, followed by non-ionic monomeric (iopromide), ionic dimeric (ioxaglate), and ionic monomeric CM (ioxithalamate). Contrast media is cytotoxic to human disc cells in a dose- and time-dependent manner. This in vitro study revealed that, among four different CM preparations, non-ionic dimeric CM is the least detrimental to human disc cell viability. Careful attention should be paid to the type of CM chosen for discography and endoscopic spinal surgery. It is also necessary to investigate the detrimental effects of CM on disc cells and disc degeneration in further in vivo studies. Copyright © 2015 Elsevier Inc. All rights reserved.

A combined disc method with resazurin agar plate assay for early phenotypic screening of KPC, MBL and OXA-48 carbapenemases among Enterobacteriaceae.

PubMed

Teethaisong, Y; Eumkeb, G; Nakouti, I; Evans, K; Hobbs, G

2016-08-01

To validate a combined disc method along with resazurin chromogenic agar for early screening and differentiation of Klebsiella pneumoniae carbapenemase, metallo-β-lactamase and OXA-48 carbapenemase-producing Enterobacteriaceae. The combined disc test comprising of meropenem alone and with EDTA, phenylboronic acid or both EDTA and phenylboronic acid, and temocillin alone were evaluated with the resazurin chromogenic agar plate assay against a total of 86 molecularly confirmed Enterobacteriaceae clinical isolates (11 metallo-β-lactamases, eight Kl. pneumoniae carbapenemases, 11 OXA-48, 32 AmpC and 15 extended-spectrum-β-lactamase producers and nine co-producers of extended-spectrum-β-lactamase and AmpC). The inhibition zone diameters were measured and interpreted at 7 h for the presence of carbapenemase. All carbapenemase producers were phenotypically distinguished by this assay with 100% sensitivity and specificity. This early phenotypic method is very simple, inexpensive, and reliable in the detection and differentiation of carbapenemase-producing Enterobacteriaceae. It could be exploited in any microbiological laboratory for diagnosis of these recalcitrant bacteria. This assay poses excellent performance in discrimination of Kl. pneumoniae carbapenemase, metallo-β-lactamase and OXA-48 carbapenemases within 7 h, which is much faster than conventional disc diffusion methods. The rapid detection could help clinicians screen patients, control infection and provide epidemiological surveillance. © 2016 The Society for Applied Microbiology.

Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway

PubMed Central

Yanai, H; Yoshioka, Y; Yoshida, H; Nakao, Y; Plessis, A; Yamaguchi, M

2014-01-01

Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis. PMID:24752236

Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway.

PubMed

Yanai, H; Yoshioka, Y; Yoshida, H; Nakao, Y; Plessis, A; Yamaguchi, M

2014-04-21

Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis.

A large-scale, in vivo transcription factor screen defines bivalent chromatin as a key property of regulatory factors mediating Drosophila wing development

PubMed Central

Schertel, Claus; Albarca, Monica; Rockel-Bauer, Claudia; Kelley, Nicholas W.; Bischof, Johannes; Hens, Korneel

2015-01-01

Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise ∼5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such “bivalent” chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue. PMID:25568052

Disc cell clusters in pathological human intervertebral discs are associated with increased stress protein immunostaining.

PubMed

Sharp, Christopher A; Roberts, Sally; Evans, Helena; Brown, Sharon J

2009-11-01

Intervertebral disc (IVD) cells within the annulus fibrosus (AF) and nucleus pulposus (NP) maintain distinct functional extracellular matrices and operate within a potentially noxious and stressful environment. How disc cells respond to stress and whether stress is responsible for triggering degeneration is unknown. Disc cell proliferation and cluster formation are most marked in degenerate IVDs, possibly indicating attempts at matrix repair. In other tissues, stress proteins increase rapidly after stress protecting cell function and, although implicated in degeneration of articular cartilage, have received little attention in degenerative IVD pathologies. We have compared the distribution of stress protein immunolocalization in pathological and control IVDs. Disc tissues were obtained at surgery from 43 patients with degenerative disc disease (DDD) and herniation, and 12 controls at postmortem. Tissues were immunostained with a polyclonal antibody for heat shock factor 1 (HSF-1) and monoclonal antibodies for the heat shock proteins, Hsp27 and Hsp72, using an indirect immunoperoxidase method. Positively stained cells were expressed as a percentage of the total. Cell cluster formation was also assessed. The proportion of cells in clusters was similar in the AF (both 2%) and NP (8 and 9%) of control and DDD samples, whereas in herniated tissues this was increased (AF 12%, NP 14%). Stress antigen staining tended to be more frequent in clustered rather than in single/doublet cells, and this was significant (P < 0.005) in both the AF and NP of herniated discs. Clustered cells, which are most common in herniated discs, may be mounting a protective response to abnormal environmental factors associated with disc degeneration. A better understanding of the stress response in IVD cells may allow its utilization in disc cell therapies.

Drosophila Lyra mutations are gain-of-function mutations of senseless

NASA Technical Reports Server (NTRS)

Nolo, R.; Abbott, L. A.; Bellen, H. J.

2001-01-01

The Lyra mutation was first described by Jerry Coyne in 1935. Lyra causes recessive pupal lethality and adult heterozygous Lyra mutants exhibit a dominant loss of the anterior and posterior wing margins. Unlike many mutations that cause loss of wing tissue (e.g., scalloped, Beadex, cut, and apterous-Xasta), Lyra wing discs do not exhibit increased necrotic or apoptotic cell death, nor do they show altered BrdU incorporation. However, during wing disc eversion, loss of the anterior and posterior wing margins is apparent. We have previously shown that senseless, a gene that is necessary and sufficient for peripheral nervous system (PNS) development, is allelic to Lyra. Here we show by several genetic criteria that Lyra alleles are neomorphic alleles of senseless that cause ectopic expression of SENSELESS in the wing pouch. Similarly, overexpression of SENSELESS in the wing disc causes loss of wing margin tissue, thereby mimicking the Lyra phenotype. Lyra mutants display aberrant expression of DELTA, VESTIGIAL, WINGLESS, and CUT. As in Lyra mutants, overexpression of SENSELESS in some areas of the wing pouch also leads to loss of WINGLESS and CUT. In summary, our data indicate that overexpression of SENSELESS causes a severe reduction in NOTCH signaling that in turn may lead to decreased transcription of several key genes required for wing development, leading to a failure in cell proliferation and loss of wing margin tissue.

Function of Lipid Storage Droplet 1 (Lsd1) in Wing Development of Drosophila melanogaster.

PubMed

Men, Tran Thanh; Binh, Tran Duy; Yamaguchi, Masamitsu; Huy, Nguyen Tien; Kamei, Kaeko

2016-04-29

Perilipins are evolutionarily conserved from Drosophila to humans, the lipid storage droplet 1 (Lsd1) is a Drosophila homolog of human perilipin 1. The function of Lsd1 as a regulator of lipolysis in Drosophila has been demonstrated, as the Lsd1 mutant causes an increase of lipid droplet size. However, the functions of this gene during development are still under investigation. In order to determine the function of Lsd1 during development, Lsd1 was knocked down in Drosophila using the GAL4-UAS system. Selective knockdown of Lsd1 in the dorsal wing disc caused an atrophied wing phenotype. The generation of reactive oxygen species in the wing pouch compartment of the Lsd1-knockdown flies was significantly higher than in the control. Immunostaining with caspase-3 antibody revealed a greater number of apoptotic cells in Lsd1-knockdown wing discs than in the control. Cell death by autophagy was also induced in the knockdown flies. Moreover, cells deprived of Lsd1 showed mitochondrial expansion and decreased ATP levels. These results strongly suggest that knockdown of Lsd1 induces mitochondrial stress and the production of reactive oxygen species that result in cell death, via apoptosis and the autophagy pathway. These results highlight the roles of Drosophila Lsd1 during wing development.

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimamura, Mai; Kyotani, Akane; Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585

2014-08-01

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdownmore » of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila. - Highlights: • Knockdown of Cabeza induced rough eye phenotype. • Knockdown of Cabeza induced fusion of cone cells in pupal retinae. • Knockdown of Cabeza induced apoptosis in pupal retinae. • Mutation in EGFR pathway-related genes suppressed the rough eye phenotype. • Cabeza may negatively regulate the EGFR pathway.« less

[Expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro].

PubMed

Yu, Sheng-ji; Qiu, Gui-xing; Burton, Yang; Sandra, Roth; Cari, Whyne; Albert, Yee

2005-12-15

To investigate the expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro. The porcine lumbar intervertebral disc cells were isolated and cultured in vitro, and the cells underwent cyclic hydrostatic loading. After that, the expression of integrin alpha5 and actin in intervertebral disc cells were studied by means of morphology observing, Western blot and immunohistochemistry staining. The morphology of intervertebral disc cells were changed into smaller and flatten shape, and the expression of integrin alpha5 and actin were decreased after loading. The expression of integrin alpha5 decreases under cyclic hydrostatic pressure, and the actin is affected at the same time when signals are transferred into the cells by integrin alpha5. That may be one of the important mechanisms of the mechanotransduction in the cells of intervertebral disc.

The generation and functional characterization of induced pluripotent stem cells from human intervertebral disc nucleus pulposus cells.

PubMed

Zhu, Yanxia; Liang, Yuhong; Zhu, Hongxia; Lian, Cuihong; Wang, Liang; Wang, Yiwei; Gu, Hongsheng; Zhou, Guangqian; Yu, Xiaoping

2017-06-27

Disc degenerative disease (DDD) is believed to originate in the nucleus pulposus (NP) region therefore, it is important to obtain a greater number of active NP cells for the study and therapy of DDD. Human induced pluripotent stem cells (iPSCs) are a powerful tool for modeling the development of DDD in humans, and have the potential to be applied in regenerative medicine. NP cells were isolated from DDD patients following our improved method, and then the primary NP cells were reprogramed into iPSCs with Sendai virus vectors encoding 4 factors. Successful reprogramming of iPSCs was verified by the expression of surface markers and presence of teratoma. Differentiation of iPSCs into NP-like cells was performed in a culture plate or in hydrogel, whereby skin fibroblast derived-iPSCs were used as a control. Results demonstrated that iPSCs derived from NP cells displayed a normal karyotype, expressed pluripotency markers, and formed teratoma in nude mice. NP induction of iPSCs resulted in the expression of NP cell specific matrix proteins and related genes. Non-induced NP derived-iPSCs also showed some NP-like phenotype. Furthermore, NP-derived iPSCs differentiate much better in hydrogel than that in a culture plate. This is a novel method for the generation of iPSCs from NP cells of DDD patients, and we have successfully differentiated these iPSCs into NP-like cells in hydrogel. This method provides a novel treatment of DDD by using patient-specific NP cells in a relatively simple and straightforward manner.

In vivo biofunctional evaluation of hydrogels for disc regeneration.

PubMed

Reitmaier, Sandra; Kreja, Ludwika; Gruchenberg, Katharina; Kanter, Britta; Silva-Correia, Joana; Oliveira, Joaquim Miguel; Reis, Rui Luís; Perugini, Valeria; Santin, Matteo; Ignatius, Anita; Wilke, Hans-Joachim

2014-01-01

Regenerative strategies aim to restore the original biofunctionality of the intervertebral disc. Different biomaterials are available, which might support disc regeneration. In the present study, the prospects of success of two hydrogels functionalized with anti-angiogenic peptides and seeded with bone marrow derived mononuclear cells (BMC), respectively, were investigated in an ovine nucleotomy model. In a one-step procedure iliac crest aspirates were harvested and, subsequently, separated BMC were seeded on hydrogels and implanted into the ovine disc. For the cell-seeded approach a hyaluronic acid-based hydrogel was used. The anti-angiogenic potential of newly developed VEGF-blockers was investigated on ionically crosslinked metacrylated gellan gum hydrogels. Untreated discs served as nucleotomy controls. 24 adult merino sheep were used. After 6 weeks histological, after 12 weeks histological and biomechanical analyses were conducted. Biomechanical tests revealed no differences between any of the implanted and nucleotomized discs. All implanted discs significantly degenerated compared to intact discs. In contrast, there was no marked difference between implanted and nucleotomized discs. In tendency, albeit not significant, degeneration score and disc height index deteriorated for all but not for the cell-seeded hydrogels from 6 to 12 weeks. Cell-seeded hydrogels slightly decelerated degeneration. None of the hydrogel configurations was able to regenerate biofunctionality of the intervertebral disc. This might presumably be caused by hydrogel extrusion. Great importance should be given to the development of annulus sealants, which effectively exploit the potential of (cell-seeded) hydrogels for biological disc regeneration and restoration of intervertebral disc functioning.

Tracing notochord-derived cells using a Noto-cre mouse: implications for intervertebral disc development.

PubMed

McCann, Matthew R; Tamplin, Owen J; Rossant, Janet; Séguin, Cheryle A

2012-01-01

Back pain related to intervertebral disc degeneration is the most common musculoskeletal problem, with a lifetime prevalence of 82%. The lack of effective treatment for this widespread problem is directly related to our limited understanding of disc development, maintenance and degeneration. The aim of this study was to determine the developmental origins of nucleus pulposus cells within the intervertebral disc using a novel notochord-specific Cre mouse. To trace the fate of notochordal cells within the intervertebral disc, we derived a notochord-specific Cre mouse line by targeting the homeobox gene Noto. Expression of this gene is restricted to the node and the posterior notochord during gastrulation [embryonic day 7.5 (E7.5)-E12.5]. The Noto-cre mice were crossed with a conditional lacZ reporter for visualization of notochord fate in whole-mount embryos. We performed lineage-tracing experiments to examine the contribution of the notochord to spinal development from E12.5 through to skeletally mature mice (9 months). Fate mapping studies demonstrated that, following elongation and formation of the primitive axial skeleton, the notochord gives rise to the nucleus pulposus in fully formed intervertebral discs. Cellular localization of β-galactosidase (encoded by lacZ) and cytokeratin-8 demonstrated that both notochordal cells and chondrocyte-like nucleus pulposus cells are derived from the embryonic notochord. These studies establish conclusively that notochordal cells act as embryonic precursors to all cells found within the nucleus pulposus of the mature intervertebral disc. This suggests that notochordal cells might serve as tissue-specific progenitor cells within the disc and establishes the Noto-cre mouse as a unique tool to interrogate the contribution of notochordal cells to both intervertebral disc development and disc degeneration.

Tracing notochord-derived cells using a Noto-cre mouse: implications for intervertebral disc development

PubMed Central

McCann, Matthew R.; Tamplin, Owen J.; Rossant, Janet; Séguin, Cheryle A.

2012-01-01

SUMMARY Back pain related to intervertebral disc degeneration is the most common musculoskeletal problem, with a lifetime prevalence of 82%. The lack of effective treatment for this widespread problem is directly related to our limited understanding of disc development, maintenance and degeneration. The aim of this study was to determine the developmental origins of nucleus pulposus cells within the intervertebral disc using a novel notochord-specific Cre mouse. To trace the fate of notochordal cells within the intervertebral disc, we derived a notochord-specific Cre mouse line by targeting the homeobox gene Noto. Expression of this gene is restricted to the node and the posterior notochord during gastrulation [embryonic day 7.5 (E7.5)-E12.5]. The Noto-cre mice were crossed with a conditional lacZ reporter for visualization of notochord fate in whole-mount embryos. We performed lineage-tracing experiments to examine the contribution of the notochord to spinal development from E12.5 through to skeletally mature mice (9 months). Fate mapping studies demonstrated that, following elongation and formation of the primitive axial skeleton, the notochord gives rise to the nucleus pulposus in fully formed intervertebral discs. Cellular localization of β-galactosidase (encoded by lacZ) and cytokeratin-8 demonstrated that both notochordal cells and chondrocyte-like nucleus pulposus cells are derived from the embryonic notochord. These studies establish conclusively that notochordal cells act as embryonic precursors to all cells found within the nucleus pulposus of the mature intervertebral disc. This suggests that notochordal cells might serve as tissue-specific progenitor cells within the disc and establishes the Noto-cre mouse as a unique tool to interrogate the contribution of notochordal cells to both intervertebral disc development and disc degeneration. PMID:22028328

Wing Defects in Drosophila xenicid Mutant Clones Are Caused by C-Terminal Deletion of Additional Sex Combs (Asx)

PubMed Central

Bischoff, Kara; Ballew, Anna C.; Simon, Michael A.; O'Reilly, Alana M.

2009-01-01

Background The coordinated action of genes that control patterning, cell fate determination, cell size, and cell adhesion is required for proper wing formation in Drosophila. Defects in any of these basic processes can lead to wing aberrations, including blisters. The xenicid mutation was originally identified in a screen designed to uncover regulators of adhesion between wing surfaces [1]. Principal Findings Here, we demonstrate that expression of the βPS integrin or the patterning protein Engrailed are not affected in developing wing imaginal discs in xenicid mutants. Instead, expression of the homeotic protein Ultrabithorax (Ubx) is strongly increased in xenicid mutant cells. Conclusion Our results suggest that upregulation of Ubx transforms cells from a wing blade fate to a haltere fate, and that the presence of haltere cells within the wing blade is the primary defect leading to the adult wing phenotypes observed. PMID:19956620

Epistatic and Independent Effects on Schizophrenia-Related Phenotypes Following Co-disruption of the Risk Factors Neuregulin-1 × DISC1.

PubMed

O'Tuathaigh, Colm M P; Fumagalli, Fabio; Desbonnet, Lieve; Perez-Branguli, Francesc; Moloney, Gerard; Loftus, Samim; O'Leary, Claire; Petit, Emilie; Cox, Rachel; Tighe, Orna; Clarke, Gerard; Lai, Donna; Harvey, Richard P; Cryan, John F; Mitchell, Kevin J; Dinan, Timothy G; Riva, Marco A; Waddington, John L

2017-01-01

Few studies have addressed likely gene × gene (ie, epistatic) interactions in mediating risk for schizophrenia. Using a preclinical genetic approach, we investigated whether simultaneous disruption of the risk factors Neuregulin-1 (NRG1) and Disrupted-in-schizophrenia 1 (DISC1) would produce a disease-relevant phenotypic profile different from that observed following disruption to either gene alone. NRG1 heterozygotes exhibited hyperactivity and disruption to prepulse inhibition, both reversed by antipsychotic treatment, and accompanied by reduced striatal dopamine D2 receptor protein expression, impaired social cognition, and altered glutamatergic synaptic protein expression in selected brain areas. Single gene DISC1 mutants demonstrated a disruption in social cognition and nest-building, altered brain 5-hydroxytryptamine levels and hippocampal ErbB4 expression, and decreased cortical expression of the schizophrenia-associated microRNA miR-29b. Co-disruption of DISC1 and NRG1, indicative of epistasis, evoked an impairment in sociability and enhanced self-grooming, accompanied by changes in hypothalamic oxytocin/vasopressin gene expression. The findings indicate specific behavioral correlates and underlying cellular pathways downstream of main effects of DNA variation in the schizophrenia-associated genes NRG1 and DISC1. © The Author 2016. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Disabled infectious single cycle herpes simplex virus (DISC-HSV) is a candidate vector system for gene delivery/expression of GM-CSF in human prostate cancer therapy.

PubMed

Parkinson, Richard J; Mian, Shahid; Bishop, Michael C; Gray, Trevor; Li, Geng; McArdle, Stephanie E B; Ali, Selman; Rees, Robert C

2003-06-15

DISC-HSV is a replication incompetent herpes simplex virus that is a highly efficient vector for the transduction of genes in vivo and in vitro. We examine the ability of DISC-HSV to infect human prostate cancer cell-lines and xenograft tumor models, and induce expression of reporter and therapeutic cytokine genes. Infection was confirmed by cellular staining for the beta-galactosidase reporter gene product, and by EM. Human GM-CSF production following DISC-hGMCSF infection was measured using ELISA. The metabolic activity of infected cells was determined by NADP/NADPH assay. Cell death was estimated by cell-cycle analysis using flow cytometry with propidium iodide staining. Infection of DU145, PC3 and LNCaP cells with DISC-HSV was dose dependent. Cells infected with DISC-hGM-CSF released significant levels of hGM-CSF for 3 days. NADP/NADPH assay suggested that infected cells continued to be metabolically active for 3 days post-infection, which was consistent with flow cytometry findings that cell death did not occur within 7 days of infection. Tumor xenografts injected with DISC-HSV expressed beta-galactosidase, and intracellular viral particles were demonstrated using EM. We have previously reported the rejection of established tumors following intra-tumoral injection of DISC-GMCSF. This study demonstrates the ability of DISC-HSV to infect prostate cancer and express GMCSF at significant levels. We suggest that prostate cancer is a potential target for therapy using DISC-HSV containing GM-CSF. Copyright 2003 Wiley-Liss, Inc.

The presence of pleiotrophin in the human intervertebral disc is associated with increased vascularization: an immunohistologic study.

PubMed

Johnson, William E B; Patterson, Angela M; Eisenstein, Stephen M; Roberts, Sally

2007-05-20

An immunohistological study of surgical specimens of human intervertebral disc. To examine the presence of pleiotrophin in diseased or damaged intervertebral disc tissue and the association between its presence and the extent of tissue vascularization and innervation. Increased levels of pleiotrophin, a growth and differentiation factor that is active in various pathophysiologic processes, including angiogenesis, has been associated with osteoarthritic changes of human articular cartilage. The association between pleiotrophin expression and pathologic conditions of the human intervertebral disc is unknown. Specimens of human lumbar intervertebral discs, obtained following surgical discectomy, were divided into 3 groups: non-degenerated discs (n = 7), degenerated discs (n = 6), and prolapsed discs (n = 11). Serial tissue sections of each specimen were immunostained to determine the presence of pleiotrophin, blood vessels (CD34-positive endothelial cells), and nerves (neurofilament 200 kDa [NF200]-positive nerve fibers). Pleiotrophin immunoreactivity was seen in disc cells, endothelial cells, and in the extracellular matrix in most specimens of intervertebral disc but was most prevalent in vascularized tissue in prolapsed discs. There was a significant correlation between the presence of pleiotrophin-positive disc cells and that of CD34-positive blood vessels. NF200-positive nerves were seen in vascularized areas of more degenerated discs, but nerves did not appear to codistribute with blood vessels or pleiotrophin positivity in prolapsed discs. Pleiotrophin is present in pathologic human intervertebral discs, and its prevalence and distribution suggest that it may play a role in neovascularization of diseased or damaged disc tissue.

Serial sectioning for examination of photoreceptor cell architecture by focused ion beam technology

PubMed Central

Mustafi, Debarshi; Avishai, Amir; Avishai, Nanthawan; Engel, Andreas; Heuer, Arthur; Palczewski, Krzysztof

2011-01-01

Structurally deciphering complex neural networks requires technology with sufficient resolution to allow visualization of single cells and their intimate surrounding connections. Scanning electron microscopy (SEM), coupled with serial ion ablation (SIA) technology, presents a new avenue to study these networks. SIA allows ion ablation to remove nanometer sections of tissue for SEM imaging, resulting in serial section data collection for three-dimensional reconstruction. Here we highlight a method for preparing retinal tissues for imaging of photoreceptors by SIA-SEM technology. We show that this technique can be used to visualize whole rod photoreceptors and the internal disc elements from wild-type (wt) mice. The distance parameters of the discs and photoreceptors are in good agreement with previous work with other methods. Moreover, we show that large planes of retinal tissue can be imaged at high resolution to display the packing of normal rods. Finally, SIA-SEM imaging of retinal tissue from a mouse model (Nrl−/−) with phenotypic changes akin to the human disease enhanced S-cone syndrome (ESCS) revealed a structural profile of overall photoreceptor ultrastructure and internal elements that accompany this disease. Overall, this work presents a new method to study photoreceptor cells at high structural resolution that has a broad applicability to the visual neuroscience field. PMID:21439323

Microarray analysis of laser capture microdissected-anulus cells from the human intervertebral disc.

PubMed

Gruber, Helen E; Mougeot, Jean-Luc; Hoelscher, Gretchen; Ingram, Jane A; Hanley, Edward N

2007-05-15

Five Thompson Grade I/II discs (Group 1), 7 Grade III discs (Group 2), and 3 Grade IV discs (Group IV) were studied here in a project approved by the authors' Human Subjects Institutional Review Board. Our objective was to use laser capture microdissection (LCM) to harvest cells from the human anulus and to derive gene expression profiles using microarray analysis. Appropriate gene expression is essential in the intervertebral disc for maintenance of extracellular matrix (ECM), ECM remodeling, and maintenance of a viable disc cell population. During disc degeneration, cell numbers drop, making gene expression studies challenging. LCM was used to harvest cells from paraffin-embedded sections of human anulus tissue. Gene profiling used Affymetrix GeneChip Human X3P arrays. ANOVA and SAM permutation analysis were applied to dCHIP normalized, filtered, and log-transformed gene expression data ( approximately 33,500 probes), and data analyzed to identify genes that were significantly differentially expressed between the 3 groups. We identified 47 genes that were significantly differentially expressed between the 3 groups (P < 0.001 and lowest q values). Compared with the healthiest discs (Grade I/II), 13 genes were up-regulated and 19 down-regulated in both the Grade III and the Grade IV discs. Genes with biologic significance regulated during degeneration involved cell senescence, low cell division rates, hypoxia-related genes, heat-shock protein 70 interacting protein, neuropilin 2, and interleukin-23p19 (interleukin-12 family). Results expand our understanding of disc aging and degeneration and show that LCM is a valuable technique that can be used to collect mRNA amounts adequate for microarray analysis from the sparse cell population of the human anulus.

Fine structures of embryonic discs of in vivo post-hatching porcine blastocysts at the pre-primitive streak stage.

PubMed

Xia, P; Liu, Z; Qin, P

2011-04-01

To date, reports about the ultrastructure of porcine embryonic discs have not shown details of the primitive streak. The main objective of this study was to examine the ultrastructure of interior and exterior embryonic discs in porcine in vivo blastocysts with diameters of 1, 3 and 9 mm using scanning electron microscopy and transmission electron microscopy. For the first time, we revealed the ultrastructure of the unusual group of cells in the pre-primitive streak area of embryonic discs. The cells were 1-2 μm in diameter, had high electron density and contained abundant, free ribosomes and endoplasmic reticulum. These primitive streak cells could represent original embryonic stem cells or represent a stem cell niche. The results also showed three types of cells on the exterior surface of the embryonic discs. Moreover, our results provided morphological evidence of condensed nuclei in the smooth cells on the surface of the embryonic disc. © 2010 Blackwell Verlag GmbH.

Human Disc Nucleus Properties and Vertebral Endplate Permeability

PubMed Central

Rodriguez, Azucena G.; Slichter, Chloe K.; Acosta, Frank L.; Rodriguez-Soto, Ana E.; Burghardt, Andrew J.; Majumdar, Sharmila; Lotz, Jeffrey C.

2010-01-01

Study of human cadaveric discs quantifying endplate permeability and porosity and correlating these with measures of disc quality: cell density, proteoglycan content, and overall degeneration. Permeability and porosity increased with age and were not correlated with cell density or overall degeneration, suggesting that endplate calcification may not accelerate disc degeneration. Study Design Experimental quantification of relationships between vertebral endplate morphology, permeability, disc cell density, glycosaminoglycan content and degeneration in samples harvested from human cadaveric spines. Objective To test the hypothesis that variation in endplate permeability and porosity contribute to changes in intervertebral disc cell density and overall degeneration. Summary of Background Data Cells within the intervertebral disc are dependent on diffusive exchange with capillaries in the adjacent vertebral bone. Previous findings suggest that blocked routes of transport negatively affect disc quality, yet there are no quantitative relationships between human vertebral endplate permeability, porosity, cell density and disc degeneration. Such relationships would be valuable for clarifying degeneration risk factors, and patient features that may impede efforts at disc tissue engineering. Methods Fifty-one motion segments were harvested from 13 frozen cadaveric human lumbar spines (32 to 85 years) and classified for degeneration using the MRI-based Pfirrmann scale. A cylindrical core was harvested from the center of each motion segment that included vertebral bony and cartilage endplates along with adjacent nucleus tissue. The endplate mobility, a type of permeability, was measured directly using a custom-made permeameter before and after the cartilage endplate was removed. Cell density within the nucleus tissue was estimated using the picogreen method while the nuclear GAG content was quantified using the DMMB technique. Specimens were imaged at 8 μm resolution using microCT, bony porosity was calculated. Analysis of variance, linear regression, and multiple comparison tests were used to analyze the data. Results Nucleus cell density increased as the disc height decreased (R2=0.13; p=0.01) but was not related to subchondral bone porosity (p>0.5), total mobility (p>0.4) or age (p>0.2). When controlling for disc height however, a significant, negative effect of age on cell density was observed (p=0.03). In addition to this, GAG content decreased with age non-linearly (R2=0.83, p<0.0001) and a cell function measurement, GAGs/cell decreased with degeneration (R2=0.24; p<0.0001). Total mobility (R2=0.14; p<0.01) and porosity (R2=0.1, p<0.01) had a positive correlation with age. Conclusion Although cell density increased with degeneration, cell function indicated that GAGs/cell decreased. Since permeability and porosity increase with age and degeneration, this implies that cell dysfunction, rather than physical barriers to transport, accelerate disc disease. PMID:21240044

In Vitro Maturation and In Vivo Integration and Function of an Engineered Cell-Seeded Disc-like Angle Ply Structure (DAPS) for Total Disc Arthroplasty.

PubMed

Martin, J T; Gullbrand, S E; Kim, D H; Ikuta, K; Pfeifer, C G; Ashinsky, B G; Smith, L J; Elliott, D M; Smith, H E; Mauck, R L

2017-11-17

Total disc replacement with an engineered substitute is a promising avenue for treating advanced intervertebral disc disease. Toward this goal, we developed cell-seeded disc-like angle ply structures (DAPS) and showed through in vitro studies that these constructs mature to match native disc composition, structure, and function with long-term culture. We then evaluated DAPS performance in an in vivo rat model of total disc replacement; over 5 weeks in vivo, DAPS maintained their structure, prevented intervertebral bony fusion, and matched native disc mechanical function at physiologic loads in situ. However, DAPS rapidly lost proteoglycan post-implantation and did not integrate into adjacent vertebrae. To address this, we modified the design to include polymer endplates to interface the DAPS with adjacent vertebrae, and showed that this modification mitigated in vivo proteoglycan loss while maintaining mechanical function and promoting integration. Together, these data demonstrate that cell-seeded engineered discs can replicate many characteristics of the native disc and are a viable option for total disc arthroplasty.

Postnatal extra-embryonic tissues as a source of multiple cell types for regenerative medicine applications.

PubMed

Gubar, O S; Rodnichenko, A E; Vasyliev, R G; Zlatska, A V; Zubov, D O

2017-09-01

We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73 + CD90 + CD105 + CD146 + CD166+CD34 - CD45 - HLA-DR - . HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use.

Analysis of the Function of Apoptosis during Imaginal Wing Disc Regeneration in Drosophila melanogaster.

PubMed

Diaz-Garcia, Sandra; Ahmed, Sara; Baonza, Antonio

2016-01-01

Regeneration is the ability that allows organisms to replace missing organs or lost tissue after injuries. This ability requires the coordinated activity of different cellular processes, including programmed cell death. Apoptosis plays a key role as a source of signals necessary for regeneration in different organisms. The imaginal discs of Drosophila melanogaster provide a particularly well-characterised model system for studying the cellular and molecular mechanisms underlying regeneration. Although it has been shown that signals produced by apoptotic cells are needed for homeostasis and regeneration of some tissues of this organism, such as the adult midgut, the contribution of apoptosis to disc regeneration remains unclear. Using a new method for studying disc regeneration in physiological conditions, we have defined the pattern of cell death in regenerating discs. Our data indicate that during disc regeneration, cell death increases first at the wound edge, but as regeneration progresses dead cells can be observed in regions far away from the site of damage. This result indicates that apoptotic signals initiated in the wound spread throughout the disc. We also present results which suggest that the partial inhibition of apoptosis does not have a major effect on disc regeneration. Finally, our results suggest that during disc regeneration distinct apoptotic signals might be acting simultaneously.

Formation of the sacrum requires down-regulation of sonic hedgehog signaling in the sacral intervertebral discs.

PubMed

Bonavita, Raffaella; Vincent, Kathleen; Pinelli, Robert; Dahia, Chitra Lekha

2018-05-21

In humans, the sacrum forms an important component of the pelvic arch, and it transfers the weight of the body to the lower limbs. The sacrum is formed by collapse of the intervertebral discs (IVDs) between the five sacral vertebrae during childhood, and their fusion to form a single bone. We show that collapse of the sacral discs in the mouse is associated with the down-regulation of sonic hedgehog (SHH) signaling in the nucleus pulposus (NP) of the disc, and many aspects of this phenotype can be reversed by experimental postnatal activation of HH signaling. We have previously shown that SHH signaling is essential for the normal postnatal growth and differentiation of intervertebral discs elsewhere in the spine, and that loss of SHH signaling leads to pathological disc degeneration, a very common disorder of aging. Thus, loss of SHH is pathological in one region of the spine but part of normal development in another. © 2018. Published by The Company of Biologists Ltd.

Disc size regulation in the brood cell building behavior of leaf-cutter bee, Megachile tsurugensis.

PubMed

Kim, Jong-yoon

2007-12-01

The leaf-cutter bee, Megachile tsurugensis, builds a brood cell in a preexisting tunnel with leaf discs that she cuts in decreasing sizes and assembles them like a Russian matryoshka doll. By experimentally manipulating the brood cell, it was investigated how she regulates the size of leaf discs that fit in the brood cell's internal volume. When the internal volume was artificially increased by removing a bulk of leaf discs, she decreased the leaf disc size, although increasing it would have made the leaf disc more fitting in the increased internal volume. As a reverse manipulation, when the internal volume was decreased by inserting a group of inner layers of preassembled leaf discs to a brood cell, she decreased the leaf disc size, so that the leaf disc could fit in the decreased internal volume. These results suggest that she uses at least two different mechanisms to regulate the disc size: the use of some internal memory about the degree of building work accomplished in the first and of sensory feedback of dimensional information at the construction site in the second manipulation, respectively. It was concluded that a stigmergic mechanism, an immediate sensory feedback from the brood cell changed by the building work, alone cannot explain the details of the bee's behavior particularly with respect to her initial response to the first manipulation. For a more complete explanation of the behavior exhibited by the solitary bee, two additional behavioral elements, reinforcement of building activity and processing of dimensional information, were discussed along with stigmergy.

Condensing Raman spectrum for single-cell phenotype analysis.

PubMed

Sun, Shiwei; Wang, Xuetao; Gao, Xin; Ren, Lihui; Su, Xiaoquan; Bu, Dongbo; Ning, Kang

2015-01-01

In recent years, high throughput and non-invasive Raman spectrometry technique has matured as an effective approach to identification of individual cells by species, even in complex, mixed populations. Raman profiling is an appealing optical microscopic method to achieve this. To fully utilize Raman proling for single-cell analysis, an extensive understanding of Raman spectra is necessary to answer questions such as which filtering methodologies are effective for pre-processing of Raman spectra, what strains can be distinguished by Raman spectra, and what features serve best as Raman-based biomarkers for single-cells, etc. In this work, we have proposed an approach called rDisc to discretize the original Raman spectrum into only a few (usually less than 20) representative peaks (Raman shifts). The approach has advantages in removing noises, and condensing the original spectrum. In particular, effective signal processing procedures were designed to eliminate noise, utilising wavelet transform denoising, baseline correction, and signal normalization. In the discretizing process, representative peaks were selected to signicantly decrease the Raman data size. More importantly, the selected peaks are chosen as suitable to serve as key biological markers to differentiate species and other cellular features. Additionally, the classication performance of discretized spectra was found to be comparable to full spectrum having more than 1000 Raman shifts. Overall, the discretized spectrum needs about 5storage space of a full spectrum and the processing speed is considerably faster. This makes rDisc clearly superior to other methods for single-cell classication.

Differentiation of Mouse Induced Pluripotent Stem Cells (iPSCs) into Nucleus Pulposus-Like Cells In Vitro

PubMed Central

Chen, Jun; Lee, Esther J.; Jing, Liufang; Christoforou, Nicolas; Leong, Kam W.; Setton, Lori A.

2013-01-01

A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration – a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient’s own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24+ iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24+ cell fraction via real time RT-PCR. CD24+ iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24+ fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD. PMID:24086564

Floral development and evolution of capitulum structure in Anacyclus (Anthemideae, Asteraceae).

PubMed

Bello, M Angélica; Álvarez, Inés; Torices, Rubén; Fuertes-Aguilar, Javier

2013-11-01

Most of the diversity in the pseudanthia of Asteraceae is based on the differential symmetry and sexuality of its flowers. In Anacyclus, where there are (1) homogamous capitula, with bisexual, mainly actinomorphic and pentamerous flowers; and (2) heterogamous capitula, with peripheral zygomorphic, trimerous and long-/short-rayed female flowers, the floral ontogeny was investigated to infer their origin. Floral morphology and ontogeny were studied using scanning electron microscope and light microscope techniques. Disc flowers, subtended by paleae, initiate acropetally. Perianth and androecium initiation is unidirectional/simultaneous. Late zygomorphy occurs by enlargement of the adaxial perianth lobes. In contrast, ray flowers, subtended by involucral bracts, initiate after the proximal disc buds, breaking the inflorescence acropetal pattern. Early zygomorphy is manifested through the fusion of the lateral and abaxial perianth lobes and the arrest of the adaxials. We report atypical phenotypes with peripheral 'trumpet' flowers from natural populations. The peripheral 'trumpet' buds initiate after disc flowers, but maintain an actinomorphic perianth. All phenotypes are compared and interpreted in the context of alternative scenarios for the origin of the capitulum and the perianth identity. Homogamous inflorescences display a uniform floral morphology and development, whereas the peripheral buds in heterogamous capitula display remarkable plasticity. Disc and ray flowers follow different floral developmental pathways. Peripheral zygomorphic flowers initiate after the proximal actinomorphic disc flowers, behaving as lateral independent units of the pseudanthial disc from inception. The perianth and the androecium are the most variable whorls across the different types of flowers, but their changes are not correlated. Lack of homology between hypanthial appendages and a calyx, and the perianth double-sided structure are discussed for Anacyclus together with potential causes of its ray flower plasticity.

Floral development and evolution of capitulum structure in Anacyclus (Anthemideae, Asteraceae)

PubMed Central

Bello, M. Angélica; Álvarez, Inés; Torices, Rubén; Fuertes-Aguilar, Javier

2013-01-01

Background and Aims Most of the diversity in the pseudanthia of Asteraceae is based on the differential symmetry and sexuality of its flowers. In Anacyclus, where there are (1) homogamous capitula, with bisexual, mainly actinomorphic and pentamerous flowers; and (2) heterogamous capitula, with peripheral zygomorphic, trimerous and long-/short-rayed female flowers, the floral ontogeny was investigated to infer their origin. Methods Floral morphology and ontogeny were studied using scanning electron microscope and light microscope techniques Key Results Disc flowers, subtended by paleae, initiate acropetally. Perianth and androecium initiation is unidirectional/simultaneous. Late zygomorphy occurs by enlargement of the adaxial perianth lobes. In contrast, ray flowers, subtended by involucral bracts, initiate after the proximal disc buds, breaking the inflorescence acropetal pattern. Early zygomorphy is manifested through the fusion of the lateral and abaxial perianth lobes and the arrest of the adaxials. We report atypical phenotypes with peripheral ‘trumpet’ flowers from natural populations. The peripheral ‘trumpet’ buds initiate after disc flowers, but maintain an actinomorphic perianth. All phenotypes are compared and interpreted in the context of alternative scenarios for the origin of the capitulum and the perianth identity. Conclusions Homogamous inflorescences display a uniform floral morphology and development, whereas the peripheral buds in heterogamous capitula display remarkable plasticity. Disc and ray flowers follow different floral developmental pathways. Peripheral zygomorphic flowers initiate after the proximal actinomorphic disc flowers, behaving as lateral independent units of the pseudanthial disc from inception. The perianth and the androecium are the most variable whorls across the different types of flowers, but their changes are not correlated. Lack of homology between hypanthial appendages and a calyx, and the perianth double-sided structure are discussed for Anacyclus together with potential causes of its ray flower plasticity. PMID:23287557

Lumbar spine intervertebral disc gene delivery: a pilot study in lewis rats.

PubMed

Damle, Sheela R; Rawlins, Bernard A; Boachie-Adjei, Oheneba; Crystal, Ronald G; Hidaka, Chisa; Cunningham, Matthew E

2013-02-01

Basic research toward understanding and treating disc pathology in the spine has utilized numerous animal models, with delivery of small molecules, purified factors, and genes of interest. To date, gene delivery to the rat lumbar spine has only been described utilizing genetically programmed cells in a matrix which has required partial disc excision, and expected limitation of treatment diffusion into the disc. This study was designed to develop and describe a surgical technique for lumbar spine exposure and disc space preparation, and use of a matrix-free method for gene delivery. Naïve or genetically programmed isogeneic bone marrow stromal cells were surgically delivered to adolescent male Lewis rat lumbar discs, and utilizing quantitative biochemical and qualitative immunohistological assessments, the implanted cells were detected 3 days post-procedure. Statistically significant differences were noted for recovery of the β-galactosidase marker gene comparing delivery of naïve or labeled cells (10(5) cells per disc) from the site of implantation, and between delivery of 10(5) or 10(6) labeled cells per disc at the site of implantation and the adjacent vertebral body. Immunohistology confirmed that the β-galactosidase marker was detected in the adjacent vertebra bone in the zone of surgical implantation. The model requires further testing in larger cohorts and with biologically active genes of interest, but the observations from the pilot experiments are very encouraging that this will be a useful comparative model for basic spine research involving gene or cell delivery, or other locally delivered therapies to the intervertebral disc or adjacent vertebral bodies in rats.

The potential of chondrogenic pre-differentiation of adipose-derived mesenchymal stem cells for regeneration in harsh nucleus pulposus microenvironment.

PubMed

Wang, Jingkai; Tao, Yiqing; Zhou, Xiaopeng; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qi-Xin

2016-12-01

Recent studies indicated that cell-based therapy could be a promising approach to treat intervertebral disc degeneration. Though the harsh microenvironment in disc is still challenging to implanted cells, it could be overcome by pre-conditioning graft cells before transplantation, suggested by previous literatures. Therefore, we designed this study to identify the potential effect of chondrogenic pre-differentiation on adipose-derived mesenchymal stem cells in intervertebral disc-like microenvironment, characterized by limited nutrition, acidic, and high osmosis in vitro. Adipose-derived mesenchymal stem cells of rat were divided into five groups, embedded in type II collagen scaffold, and cultured in chondrogenic differentiation medium for 0, 3, 7, 10, and 14 days. Then, the adipose-derived mesenchymal stem cells were implanted and cultured in intervertebral disc-like condition. The proliferation and differentiation of adipose-derived mesenchymal stem cells were evaluated by cell counting kit-8 test, real-time quantitative polymerase chain reaction, and Western blotting and immunofluorescence analysis. Analyzed by the first week in intervertebral disc-like condition, the results showed relatively greater proliferative capability and extracellular matrix synthesis ability of the adipose-derived mesenchymal stem cells pre-differentiated for 7 and 10 days than the control. We concluded that pre-differentiation of rat adipose-derived mesenchymal stem cells in chondrogenic culture medium for 7 to 10 days could promote the regeneration effect of adipose-derived mesenchymal stem cells in intervertebral disc-like condition, and the pre-differentiated cells could be a promising cell source for disc regeneration medicine.

The Boston Keratoprosthesis: Comparing Corneal Epithelial Cell Compatibility with Titanium and PMMA

PubMed Central

Ament, Jared D.; Spurr-Michaud, Sandra J.; Dohlman, Claes H.; Gipson, Ilene K.

2014-01-01

Purpose To determine in vitro whether titanium is superior in corneal cell compatibility to standard polymethyl-methacrylate (PMMA) for the Boston Keratoprosthesis (KPro). Methods Human corneal-limbal epithelial (HCLE) cells were cultured 24, 48, 72, 96, 120, 144, or 168 hours in culture plates alone (controls) or with PMMA or titanium discs. Experiments were performed in triplicate and repeated (final n = 6). To determine if a soluble, toxic factor is emitted from materials, concurrent experiments at 48 and 144 hours were performed with discs placed in Transwell Supports, with HCLE cells plated beneath. As an additional test for soluble factors, cells were incubated 24 hours with disc-conditioned media, and number of viable cells per well was quantified at each timepoint by proliferation assay. To determine if delayed cell proliferation was attributable to cell death, HCLE cell death was measured under all conditions and quantified at each timepoint by cytotoxicity assay. The effects of material on HCLE cell proliferation over time was determined by repeated measures ANOVA. P < 0.05 was statistically significant. Results HCLE cell proliferation was greater in wells with titanium discs compared to PMMA. Differences between the test discs and control non-disc cocultures were statistically significant over time for both cell proliferation (P = 0.001) and death (P = 0.0025). No significant difference was found using Transwells (P = 0.9836) or disc-conditioned media (P = 0.36). Conclusion This in vitro HCLE cell model demonstrates significantly increased cell proliferation and decreased cell death with cell/titanium contact compared to cell/PMMA contact. Moreover, differences are unlikely attributable to a soluble factor. Prospective in vivo analysis of the two KPro biomaterials is indicated. PMID:19574903

Substance P stimulates production of inflammatory cytokines in human disc cells.

PubMed

Kepler, Christopher K; Markova, Dessislava Z; Hilibrand, Alan S; Vaccaro, Alexander R; Risbud, Makarand V; Albert, Todd J; Anderson, D Greg

2013-10-01

Laboratory study. The aims of this study were as follows: (1) to confirm that Substance P (SP) is expressed by nucleus pulposus (NP) and annulus fibrosus (AF) cells; (2) to determine the effect of SP on expression of inflammatory mediators in human disc cells and the effect of inflammatory mediators on the expression of SP; and (3) to characterize the relative expression of SP receptor isoforms in disc tissue and describe whether exposure to SP changes receptor expression. SP, classically described as a neurotransmitter, acts as an inflammatory regulator in other tissue types, but its role within the intervertebral disc has not been characterized. Human AF and NP cells from 7 individuals were expanded in monolayer and maintained in alginate bead culture. Cells were treated with SP or interleukin (IL)-1β/tumor necrosis factor-α (TNF-α). After treatment, the cells were recovered and then RNA was isolated and transcribed into cDNA. Quantitative reverse-transcriptase polymerase chain reaction was performed to evaluate expression of inflammatory mediators and SP and its receptors. Disc cells treated with SP demonstrated significant upregulation of IL-1β, IL-6, and IL-8 in NP and AF cells whereas significant upregulation of RANTES and TNF occurred only in the AF cells. AF and NP cells expressed SP at low levels; expression did not change significantly with SP treatment but was significantly upregulated after treatment with IL-1β/TNF-α. Both SP receptor isoforms were expressed by NP and AF cells. SP upregulates inflammatory mediators in disc cells. SP and its receptors were expressed in both NP and AF cells, and expression did not change after treatment with SP but increased after treatment with IL-1β/TNF-α. SP likely acts in an autocrine or paracrine manner in intervertebral disc cells and may be involved in "crosstalk" between disc cells and neurons, providing a potential mechanism for transmission of painful discogenic stimuli.

Human disc degeneration is associated with increased MMP 7 expression.

PubMed

Le Maitre, C L; Freemont, A J; Hoyland, J A

2006-01-01

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.

DNA sequence-level analyses reveal potential phenotypic modifiers in a large family with psychiatric disorders.

PubMed

Ryan, Niamh M; Lihm, Jayon; Kramer, Melissa; McCarthy, Shane; Morris, Stewart W; Arnau-Soler, Aleix; Davies, Gail; Duff, Barbara; Ghiban, Elena; Hayward, Caroline; Deary, Ian J; Blackwood, Douglas H R; Lawrie, Stephen M; McIntosh, Andrew M; Evans, Kathryn L; Porteous, David J; McCombie, W Richard; Thomson, Pippa A

2018-06-07

Psychiatric disorders are a group of genetically related diseases with highly polygenic architectures. Genome-wide association analyses have made substantial progress towards understanding the genetic architecture of these disorders. More recently, exome- and whole-genome sequencing of cases and families have identified rare, high penetrant variants that provide direct functional insight. There remains, however, a gap in the heritability explained by these complementary approaches. To understand how multiple genetic variants combine to modify both severity and penetrance of a highly penetrant variant, we sequenced 48 whole genomes from a family with a high loading of psychiatric disorder linked to a balanced chromosomal translocation. The (1;11)(q42;q14.3) translocation directly disrupts three genes: DISC1, DISC2, DISC1FP and has been linked to multiple brain imaging and neurocognitive outcomes in the family. Using DNA sequence-level linkage analysis, functional annotation and population-based association, we identified common and rare variants in GRM5 (minor allele frequency (MAF) > 0.05), PDE4D (MAF > 0.2) and CNTN5 (MAF < 0.01) that may help explain the individual differences in phenotypic expression in the family. We suggest that whole-genome sequencing in large families will improve the understanding of the combined effects of the rare and common sequence variation underlying psychiatric phenotypes.

[Self-assembly tissue engineering fibrocartilage model of goat temporomandibular joint disc].

PubMed

Kang, Hong; Li, Zhen-Qiang; Bi, Yan-Da

2011-06-01

To construct self-assembly fibrocartilage model of goat temporomandibular joint disc and observe the biological characteristics of the self-assembled fibrocartilage constructs, further to provide a basis for tissue engineering of the temporomandibular joint disc and other fibrocartilage. Cells from temporomandibular joint discs of goats were harvested and cultured. 5.5 x 10(6) cells were seeded in each agarose well with diameter 5 mm x depth 10 mm, daily replace of medium, cultured for 2 weeks. One day after seeding, goat temporomandibular joint disc cells in agarose wells were gathered and began to self-assemble into a disc-shaped base, then gradually turned into a round shape. When cultured for 2 weeks, hematoxylin-eosin staining was conducted and observed that cells were round and wrapped around by the matrix. Positive Safranin-O/fast green staining for glycosaminoglycans was observed throughout the entire constructs, and picro-sirius red staining was examined and distribution of numerous type I collagen was found. Immunohistochemistry staining demonstrated brown yellow particles in cytoplasm and around extracellular matrix, which showed self-assembly construct can produce type I collagen as native temporomandibular joint disc tissue. Production of extracellular matrix in self-assembly construct as native temporomandibular joint disc tissue indicates that the use of agarose wells to construct engineered temporomandibular joint disc will be possible and practicable.

Intervertebral disc regeneration or repair with biomaterials and stem cell therapy--feasible or fiction?

PubMed

Chan, Samantha C W; Gantenbein-Ritter, Benjamin

2012-05-31

The "gold standard" for treatment of intervertebral disc herniations and degenerated discs is still spinal fusion, corresponding to the saying "no disc - no pain". Mechanical prostheses, which are currently implanted, do only have medium outcome success and have relatively high re-operation rates. Here, we discuss some of the biological intervertebral disc replacement approaches, which can be subdivided into at least two classes in accordance to the two different tissue types, the nucleus pulposus (NP) and the annulus fibrosus (AF). On the side of NP replacement hydrogels have been extensively tested in vitro and in vivo. However, these gels are usually a trade-off between cell biocompatibility and load-bearing capacity, hydrogels which fulfill both are still lacking. On the side of AF repair much less is known and the question of the anchoring of implants is still to be addressed. New hope for cell therapy comes from developmental biology investigations on the existence of intervertebral disc progenitor cells, which would be an ideal cell source for cell therapy. Also notochordal cells (remnants of the embryonic notochord) have been recently pushed back into focus since these cells have regenerative potential and can activate disc cells. Growth factor treatment and molecular therapies could be less problematic. The biological solutions for NP and AF replacement are still more fiction than fact. However, tissue engineering just scratched the tip of the iceberg, more satisfying solutions are yet to be added to the biomedical pipeline.

Disc size regulation in the brood cell building behavior of leaf-cutter bee, Megachile tsurugensis

NASA Astrophysics Data System (ADS)

Kim, Jong-Yoon

2007-12-01

The leaf-cutter bee, Megachile tsurugensis, builds a brood cell in a preexisting tunnel with leaf discs that she cuts in decreasing sizes and assembles them like a Russian matryoshka doll. By experimentally manipulating the brood cell, it was investigated how she regulates the size of leaf discs that fit in the brood cell’s internal volume. When the internal volume was artificially increased by removing a bulk of leaf discs, she decreased the leaf disc size, although increasing it would have made the leaf disc more fitting in the increased internal volume. As a reverse manipulation, when the internal volume was decreased by inserting a group of inner layers of preassembled leaf discs to a brood cell, she decreased the leaf disc size, so that the leaf disc could fit in the decreased internal volume. These results suggest that she uses at least two different mechanisms to regulate the disc size: the use of some internal memory about the degree of building work accomplished in the first and of sensory feedback of dimensional information at the construction site in the second manipulation, respectively. It was concluded that a stigmergic mechanism, an immediate sensory feedback from the brood cell changed by the building work, alone cannot explain the details of the bee’s behavior particularly with respect to her initial response to the first manipulation. For a more complete explanation of the behavior exhibited by the solitary bee, two additional behavioral elements, reinforcement of building activity and processing of dimensional information, were discussed along with stigmergy.

STAT, Wingless, and Nurf-38 determine the accuracy of regeneration after radiation damage in Drosophila.

PubMed

Verghese, Shilpi; Su, Tin Tin

2017-10-01

We report here a study of regeneration in Drosophila larval wing imaginal discs after damage by ionizing radiation. We detected faithful regeneration that restored a wing disc and abnormal regeneration that produced an extra wing disc. We describe a sequence of changes in cell number, location and fate that occur to produce an ectopic disc. We identified a group of cells that not only participate in ectopic disc formation but also recruit others to do so. STAT92E (Drosophila STAT3/5) and Nurf-38, which encodes a member of the Nucleosome Remodeling Factor complex, oppose each other in these cells to modulate the frequency of ectopic disc growth. The picture that emerges is one in which activities like STAT increase after radiation damage and fulfill essential roles in rebuilding the tissue. But such activities must be kept in check so that one and only one wing disc is regenerated.

Comparison of Ripening Processes in Intact Tomato Fruit and Excised Pericarp Discs 1

PubMed Central

Campbell, Alan D.; Huysamer, Marius; Stotz, Henrik U.; Greve, L. Carl; Labavitch, John M.

1990-01-01

Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L*a*b* color space, changing from green (a* < −5) to red (a* > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO2 production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a* = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a* = −5), appeared as discs turned yellow-red (a* = 5), and increased another eight-fold as discs became full red (a* value +20). Carboxymethyl-cellulase activity, low in extracts from green discs, increased about six-fold as discs changed from yellow (a* = 0) to red. PMID:16667893

Ultrastructure of inclusion bodies in annulus cells in the degenerating human intervertebral disc.

PubMed

Gruber, H E; Hanley, E N

2009-06-01

The rough endoplasmic reticulum (rER) of the cell has an architectural editing function that checks whether protein structure and three-dimensional assembly have occurred properly prior to export of newly synthesized material out of the cell. If these have been faulty, the material is retained within the rER as an inclusion body. Inclusion bodies have been identified previously in chondrocytes and osteoblasts in chondrodysplasias and osteogenesis imperfecta. Inclusion bodies in intervertebral disc cells, however, have only recently been recognized. Our objectives were to use transmission electron microscopy to analyze more fully inclusion bodies in the annulus pulposus and to study the extracellular matrix (ECM) surrounding cells containing inclusion bodies. ECM frequently encapsulated cells with inclusion bodies, and commonly contained prominent banded aggregates of Type VI collagen. Inclusion body material had several morphologies, including relatively smooth, homogeneous material, or a rougher, less homogeneous feature. Such findings expand our knowledge of the fine structure of the human disc cell and ECM during disc degeneration, and indicate the potential utility of ultrastructural identification of discs with intracellular inclusion bodies as a screening method for molecular studies directed toward identification of defective gene products in degenerating discs.

Imaging cell competition in Drosophila imaginal discs.

PubMed

Ohsawa, Shizue; Sugimura, Kaoru; Takino, Kyoko; Igaki, Tatsushi

2012-01-01

Cell competition is a process in which cells with higher fitness ("winners") survive and proliferate at the expense of less fit neighbors ("losers"). It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population. By advent of a genetic mosaic technique, which enables to generate fluorescently marked somatic clones in Drosophila imaginal discs, recent studies have presented some aspects of molecular mechanisms underlying cell competition. Now, with a live-imaging technique using ex vivo-cultured imaginal discs, we can dissect the spatiotemporal nature of competitive cell behaviors within multicellular communities. Here, we describe procedures and tips for live imaging of cell competition in Drosophila imaginal discs. Copyright © 2012 Elsevier Inc. All rights reserved.

Lead Neurotoxicity on Human Neuroblastoma Cell Line SH-SY5Y is Mediated via Transcription Factor EGR1/Zif268 Induced Disrupted in Scherophernia-1 Activation.

PubMed

You, Yuanyuan; Peng, Bo; Ben, Songbin; Hou, Weijian; Sun, Liguang; Jiang, Wei

2018-07-01

Lead (Pb 2+ ) is a well-known type of neurotoxin and chronic exposure to Pb 2+ induces cognition dysfunction. In this work, the potential role of early growth response gene 1 (EGR1) in the linkage of Pb 2+ exposure and disrupted in scherophernia-1 (DISC1) activity was investigated. Human neuroblastoma cell line SH-SY5Y was subjected to different concentrations of lead acetate (PbAc) to determine the effect of Pb 2+ exposure on the cell viability, apoptosis, and activity of EGR1 and DISC1. Then the expression of EGR1 in SH-SY5Y cells was knocked down with specific siRNA to assess the function of EGR1 in Pb 2+ induced activation of DISC1. The interaction between EGR1 and DISC1 was further validated with dual luciferase assay, Supershift electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP)-PCR. Administration of PbAc decreased cell viability and induced apoptosis in SH-SY5Y cells in a dose-dependent manner. Additionally, exposure to PbAc also up-regulated expression of EGR1 and DISC1 at all concentrations. Knockdown of EGR1 blocked the effect of PbAc on SH-SY5Y cells, indicating the central role of EGR1 in the function of Pb 2+ on activity of DISC1. Based on the results of dual luciferase assay, Supershift EMSA, and ChIP-PCR, EGR1 mediated the effect of Pb 2+ on DISC1 by directly bound to the promoter region of DISC1 gene. The current study elaborated the mechanism involved in the effect of Pb 2+ exposure on expression of DISC1 for the first time: EGR1 activated by Pb 2+ substitution of zinc triggered the transcription of DISC1 gene by directly binding to its promoter.

Physico-chemical characteristics of ZnO nanoparticles-based discs and toxic effect on human cervical cancer HeLa cells

NASA Astrophysics Data System (ADS)

Sirelkhatim, Amna; Mahmud, Shahrom; Seeni, Azman; Kaus, Noor Haida Mohd.; Sendi, Rabab

2014-10-01

In this study, we investigated physico-chemical properties of zinc oxide nanoparticles (ZnO NPs)-based discs and their toxicity on human cervical cancer HeLa cell lines. ZnO NPs (80 nm) were produced by the conventional ceramic processing method. FESEM analysis indicated dominant structure of nanorods with dimensions 100-500 nm in length, and 20-100 nm in diameter. The high content of ZnO nanorods in the discs probably played significant role in toxicity towards HeLa cells. Structural defects (oxygen vacancies and zinc/oxygen interstitials) were revealed by PL spectra peaks at 370-376 nm and 519-533 nm for the ZnO discs. The structural, optical and electrical properties of prepared sample have influenced the toxicological effects of ZnO discs towards HeLa cell lines via the generation of reactive oxygen species (ROS), internalization, membrane damage, and eventually cell death. The larger surface to volume area of the ZnO nanorods, combined with defects, stimulated enhanced toxicity via ROS generation hydrogen peroxide, hydroxyl radicals, and superoxide anion. The preliminary results confirmed the ZnO-disc toxicity on HeLa cells was significantly associated with the unique physicochemical properties of ZnO NPs and to our knowledge, this is the first cellular study for treatment of HeLa cells with ZnO discs made from 80 nm ZnO particles.

Notochordal cells in the adult intervertebral disc: new perspective on an old question.

PubMed

Risbud, Makarand V; Shapiro, Irving M

2011-01-01

The intervertebral disc is a tissue positioned between each of the vertebrae that accommodates applied biomechanical forces to the spine. The central compartment of the disc contains the nucleus pulposus (NP) which is enclosed by the annulus fibrosus and the endplate cartilage.The NP is derived from the notochord, a rod-like structure of mesodermal origin. Development of the notochord is tightly regulated by interactive transcription factors and target genes. Since a number of these molecules are unique they have be used for cell lineage and fate mapping studies of tissues of the intervertebral disc. These studies have shown that in a number of species including human, NP tissue retains notochordal cells throughout life. In the adult NP, there are present both large and small notochordal cells, as well as a progenitor cell population which can differentiate along the mesengenic pathway. Since tissue renewal in the intervertebral disc is dependent on the ability of these cells to commit to the NP lineage and undergo terminal differentiation, studies have been performed to assess which signaling pathways may regulate these activities. The notch signaling pathway is active in the intervertebral disc and is responsive to hypoxia, probably through HIF-1a. From a disease viewpoint, it is hypothesized that an oxemic shift, possibly mediated by alterations in the vascular supply to the tissues of the disc would be expected to lead to a failure in notochordal progenitor cell activation and a decrease in the number of differentiated cells. In turn, this would lead to decrements in function and enhancement of the effect of agents that are known to promote disc degeneration.

A 1q42 Deletion Involving DISC1, DISC2, and TSNAX in an Autism Spectrum Disorder

PubMed Central

Williams, Jaime M.; Beck, Tyler F.; Pearson, David M.; Proud, Monica B.; Cheung, Sau Wai; Scott, Daryl A.

2010-01-01

Individuals with autism spectrum disorders have impairments in social, communicative, and behavior development that are often accompanied by abnormalities in cognitive functioning, learning, attention, and sensory processing. In this report, we describe a 3-year-old male child with an autism spectrum disorder who carries a 2Mb deletion of chromosome 1q42. Array comparative genome hybridization revealed that this deletion involves at least three genes—DISC1, DISC2, and TSNAX—which have been found to be associated with neuropsychiatric disorders and are likely to play key roles in normal CNS development. Further studies revealed that the deletion was inherited from his unaffected mother. This suggests that other genetic and/or environmental factors, some of which may be sex specific, may modify the phenotypic effects of this deletion. While this case provides evidence for the potential role of DISC1, DISC2, and TSNAX in the development of autism spectrum disorders, it is equally clear that caution must be taken when providing families with prognostic information and genetic counseling regarding such deletions. PMID:19606485

A new in vivo animal model to create intervertebral disc degeneration characterized by MRI, radiography, CT/discogram, biochemistry, and histology.

PubMed

Zhou, HaoWei; Hou, ShuXun; Shang, WeiLin; Wu, WenWen; Cheng, Yao; Mei, Fang; Peng, BaoGan

2007-04-15

A new in vivo sheep model was developed that produced disc degeneration through the injection of 5-bromodeoxyuridine (BrdU) into the intervertebral disc. This process was studied using magnetic resonance imaging (MRI), radiography, CT/discogram, histology, and biochemistry. To develop a sheep model of intervertebral disc degeneration that more faithfully mimics the pathologic hallmarks of human intervertebral disc degeneration. Recent studies have shown age-related alterations in proteoglycan structure and organization in human intervertebral discs. An animal model that involves the use of age-related changes in disc cells can be beneficial over other more invasive degenerative models that involves directly damaging the matrix of disc tissue. Twelve sheep were injected with BrdU or vehicle (phosphate-buffered saline) into the central region of separate lumbar discs. Intact discs were used as controls. At the 2-, 6-, 10-, and 14-week time points, discs underwent MRI, radiography, histology, and biochemical analyses. A CT/discogram study was performed at the 14-week time point. MRI demonstrated a progressive loss of T2-weighted signal intensity at BrdU-injected discs over the 14-week study period. Radiograph findings included osteophyte and disc space narrowing formed by 10 weeks post-BrdU treatment. CT discography demonstrated internal disc disruption in several BrdU-treated discs at the 14-week time point. Histology showed a progressive loss of the normal architecture and cell density of discs from the 2-week time point to the 14-week time point. A progressive loss of cell proliferation capacity, water content, and proteoglycans was also documented. BrdU injection into the central region of sheep discs resulted in degeneration of intervertebral discs. This progressive, degenerative process was confirmed using MRI, histology, and by observing changes in biochemistry. Degeneration occurred in a manner that was similar to that observed in human disc degeneration.

Translation of an Engineered Nanofibrous Disc-like Angle Ply Structure for Intervertebral Disc Replacement in a Small Animal Model

PubMed Central

Martin, John T.; Milby, Andrew H.; Chiaro, Joseph A.; Kim, Dong Hwa; Hebela, Nader M.; Smith, Lachlan J.; Elliott, Dawn M.; Mauck, Robert L.

2015-01-01

Intervertebral disc degeneration has been implicated in the etiology of low back pain; however the current surgical strategies for treating symptomatic disc disease are limited. A variety of materials have been developed to replace disc components, including the nucleus pulposus (NP), the annulus fibrosus (AF), and their combination into disc-like engineered constructs. We have previously shown that layers of electrospun poly(ε-caprolactone) scaffold, mimicking the hierarchical organization of the native AF, have functional parity with native tissue. Likewise, we have combined these structures with cell-seeded hydrogels (as an NP replacement) to form disc-like angle ply structures (DAPS). The objective of this study was to develop a model for the evaluation of DAPS in vivo. Through a series of studies, we developed a surgical approach to replace the rat caudal disc with an acellular DAPS and then stabilize the motion segment by external fixation. We then optimized cell infiltration into DAPS by including sacrificial poly(ethylene oxide) layers interspersed throughout the angle-ply structure. Our findings illustrate that DAPS are stable in the caudal spine, are infiltrated by cells from the peri-implant space, and that infiltration is expedited by providing additional routes for cell migration. These findings establish a new in vivo platform in which to evaluate and optimize the design of functional disc replacements. PMID:24560621

Clinical Characterization of Patients With Autosomal Dominant Short Stature due to Aggrecan Mutations

PubMed Central

Gkourogianni, Alexandra; Andrew, Melissa; Tyzinski, Leah; Crocker, Melissa; Douglas, Jessica; Dunbar, Nancy; Fairchild, Jan; Funari, Mariana F. A.; Heath, Karen E.; Jorge, Alexander A. L.; Kurtzman, Tracey; LaFranchi, Stephen; Lalani, Seema; Lebl, Jan; Lin, Yuezhen; Los, Evan; Newbern, Dorothee; Nowak, Catherine; Olson, Micah; Popovic, Jadranka; Průhová, Štěpánka; Elblova, Lenka; Quintos, Jose Bernardo; Segerlund, Emma; Sentchordi, Lucia; Shinawi, Marwan; Stattin, Eva-Lena; Swartz, Jonathan; del Angel, Ariadna González; Cuéllar, Sinhué Diaz; Hosono, Hidekazu; Sanchez-Lara, Pedro A.; Hwa, Vivian; Baron, Jeffrey; Dauber, Andrew

2017-01-01

Context: Heterozygous mutations in the aggrecan gene (ACAN) cause autosomal dominant short stature with accelerated skeletal maturation. Objective: We sought to characterize the phenotypic spectrum and response to growth-promoting therapies. Patients and Methods: One hundred three individuals (57 females, 46 males) from 20 families with autosomal dominant short stature and heterozygous ACAN mutations were identified and confirmed using whole-exome sequencing, targeted next-generation sequencing, and/or Sanger sequencing. Clinical information was collected from the medical records. Results: Identified ACAN variants showed perfect cosegregation with phenotype. Adult individuals had mildly disproportionate short stature [median height, −2.8 standard deviation score (SDS); range, −5.9 to −0.9] and a history of early growth cessation. The condition was frequently associated with early-onset osteoarthritis (12 families) and intervertebral disc disease (9 families). No apparent genotype–phenotype correlation was found between the type of ACAN mutation and the presence of joint complaints. Childhood height was less affected (median height, −2.0 SDS; range, −4.2 to −0.6). Most children with ACAN mutations had advanced bone age (bone age − chronologic age; median, +1.3 years; range, +0.0 to +3.7 years). Nineteen individuals had received growth hormone therapy with some evidence of increased growth velocity. Conclusions: Heterozygous ACAN mutations result in a phenotypic spectrum ranging from mild and proportionate short stature to a mild skeletal dysplasia with disproportionate short stature and brachydactyly. Many affected individuals developed early-onset osteoarthritis and degenerative disc disease, suggesting dysfunction of the articular cartilage and intervertebral disc cartilage. Additional studies are needed to determine the optimal treatment strategy for these patients. PMID:27870580

The effects of dynamic loading on the intervertebral disc.

PubMed

Chan, Samantha C W; Ferguson, Stephen J; Gantenbein-Ritter, Benjamin

2011-11-01

Loading is important to maintain the balance of matrix turnover in the intervertebral disc (IVD). Daily cyclic diurnal assists in the transport of large soluble factors across the IVD and its surrounding circulation and applies direct and indirect stimulus to disc cells. Acute mechanical injury and accumulated overloading, however, could induce disc degeneration. Recently, there is more information available on how cyclic loading, especially axial compression and hydrostatic pressure, affects IVD cell biology. This review summarises recent studies on the response of the IVD and stem cells to applied cyclic compression and hydrostatic pressure. These studies investigate the possible role of loading in the initiation and progression of disc degeneration as well as quantifying a physiological loading condition for the study of disc degeneration biological therapy. Subsequently, a possible physiological/beneficial loading range is proposed. This physiological/beneficial loading could provide insight into how to design loading regimes in specific system for the testing of various biological therapies such as cell therapy, chemical therapy or tissue engineering constructs to achieve a better final outcome. In addition, the parameter space of 'physiological' loading may also be an important factor for the differentiation of stem cells towards most ideally 'discogenic' cells for tissue engineering purpose.

[Topography and mechanical property of goat temporomandibular joint disc cells].

PubMed

Bao, Guangjie; Kong, Nannan; Guo, Manli; Su, Xuelian; Kang, Hong

2015-08-01

This study is performed to investigate the cell topographies and biomechanical properties of two different types of temporomandibular joint (TMJ) discs from goats by using JPK Nano Wizard 3 biological atomic force microscopy (AFM). This process provides a guideline for selecting seed cells for TMJ disc tissue engineering. TMJ disc cells from primary goats were cultured by monolayer culture method. AFM was used to contact scan the topographies of the two types of TMJ disc cells under physiological environment. Approximately 20 chondrocyte-like and fibroblast-like cells were selected randomly to plot the force-versus-distance curves of the cytoplasm and nucleus. Young's modulus and adhesion were analyzed by JPK Data Processing. The triangle-shapednucleus of the chondrocyte-like cell occupied a large portion of the cell. Cytoskeleton was arranged dendritically on the surface. Pseudopodia were extended from cell edges. The spindle-shaped nucleus of the fibroblast-like cell occupied a significantly larger region compared with the cytoplasmic region. Cytoskeleton was arranged regularly. Cell edges were smooth with less pseudopodia extended. No difference was found in the surface roughness between the two types of cells. According to the force-versus-distance curves, the Young's moduli of the two types of cells were not statistically different (P>0.05), but differences were found in the cytoplasmic regions (P=0.047). No statistical difference was found in the adhesions between the two types of cells (P>0.05). The AFM topography and curves were compared and analyzed. The two types of TMJ disc cells exhibited significantly different topographies, but only slight difference in their mechanical abilities.

Catabolic Effects of Endothelial Cell-Derived Microparticles on Disc Cells: Implications in Intervertebral Disc Neovascularization and Degeneration

PubMed Central

Pohl, Pedro H. I.; Lozito, Thomas P.; Cuperman, Thais; Yurube, Takashi; Moon, Hong J.; Ngo, Kevin; Tuan, Rocky S.; Croix, Claudette St.; Sowa, Gwendolyn A.; Rodrigues, Luciano M. R.; Kang, James D.; Vo, Nam V.

2017-01-01

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 hours with 250μg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC-I. Live-cell imaging revealed uptake of EMPs by AF cells. RT-PCR analysis demonstrated increased mRNA expression of MMP-1 (50.3 fold), MMP-3 (4.5 fold) and MMP-13 (5.5 fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP-treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo-angiogenesis of degenerating disc. PMID:27246627

Notochord Cells in Intervertebral Disc Development and Degeneration

PubMed Central

McCann, Matthew R.; Séguin, Cheryle A.

2016-01-01

The intervertebral disc is a complex structure responsible for flexibility, multi-axial motion, and load transmission throughout the spine. Importantly, degeneration of the intervertebral disc is thought to be an initiating factor for back pain. Due to a lack of understanding of the pathways that govern disc degeneration, there are currently no disease-modifying treatments to delay or prevent degenerative disc disease. This review presents an overview of our current understanding of the developmental processes that regulate intervertebral disc formation, with particular emphasis on the role of the notochord and notochord-derived cells in disc homeostasis and how their loss can result in degeneration. We then describe the role of small animal models in understanding the development of the disc and their use to interrogate disc degeneration and associated pathologies. Finally, we highlight essential development pathways that are associated with disc degeneration and/or implicated in the reparative response of the tissue that might serve as targets for future therapeutic approaches. PMID:27252900

Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells.

PubMed

Kasra, Mehran; Goel, Vijay; Martin, James; Wang, Shea-Tien; Choi, Woosung; Buckwalter, Joseph

2003-07-01

The pathogenesis of vibration-induced disorders of intervertebral disc at the cellular level is largely unknown. The objective of this study was to establish a method to investigate the ranges of constructive and destructive hydrostatic loading frequencies and amplitudes in preventing or inducing extracellular disc matrix degradation. Using a hydraulic chamber, normal rabbit intervertebral disc cells were tested under dynamic hydrostatic loading. Monolayer cultures of disc outer annulus cells and 3-dimensional (3-D) alginate cultures of disc nucleus pulposus cells were tested. Effects of different loading amplitudes (3-D culture, 0-3 MPa; monolayer, 0-1.7 MPa) and frequencies (1-20 Hz) on disc collagen and protein metabolism were investigated by measuring 3H-proline-labeled proteins associated with the cells in the extracellular matrix and release of 3H-proline-labeled molecules into culture medium. High frequency and high amplitude hydrostatic stress stimulated collagen synthesis in cultures of outer annulus cells whereas the lower amplitude and frequency hydrostatic stress had little effect. For the same loading duration and repetition, neither treatment significantly affected the relative amount of protein released from the cell layers, indicating that protein degradation and stability were unaffected. In the 3-D nucleus culture, higher amplitude and frequency increased synthesis rate and lowered degradation. In this case, loading amplitude had a stronger influence on cell response than that of loading frequency. Considering the ranges of loading amplitude and frequency used in this study, short-term application of high loading amplitudes and frequencies was beneficial in stimulation of protein synthesis and reduction of protein degradation.

Engineered disc-like angle-ply structures for intervertebral disc replacement.

PubMed

Nerurkar, Nandan L; Sen, Sounok; Huang, Alice H; Elliott, Dawn M; Mauck, Robert L

2010-04-15

To develop a construction algorithm in which electrospun nanofibrous scaffolds are coupled with a biocompatible hydrogel to engineer a mesenchymal stem cell (MSC)-based disc replacement. To engineer a disc-like angle-ply structure (DAPS) that replicates the multiscale architecture of the intervertebral disc. Successful engineering of a replacement for the intervertebral disc requires replication of its mechanical function and anatomic form. Despite many attempts to engineer a replacement for ailing and degenerated discs, no prior study has replicated the multiscale hierarchical architecture of the native disc, and very few have assessed the mechanical function of formed neo-tissues. A new algorithm for the construction of a disc analogue was developed, using agarose to form a central nucleus pulposus (NP) and oriented electrospun nanofibrous scaffolds to form the anulus fibrosus region (AF). Bovine MSCs were seeded into both regions and biochemical, histologic, and mechanical maturation were evaluated with in vitro culture. We show that mechanical testing in compression and torsion, loading methods commonly used to assess disc mechanics, reveal equilibrium and time-dependent behaviors that are qualitatively similar to native tissue, although lesser in magnitude. Further, we demonstrate that cells seeded into both AF and NP regions adopt distinct morphologies that mirror those seen in native tissue, and that, in the AF region, this ordered community of cells deposit matrix that is organized in an angle-ply configuration. Finally, constructs demonstrate functional development with long-term in vitro culture. These findings provide a new approach for disc tissue engineering that replicates multi-scale form and function of the intervertebral disc, providing a foundation from which to build a multi-scale, biologic, anatomically and hierarchically relevant composite disc analogue for eventual disc replacement.

Biomaterials for intervertebral disc regeneration and repair.

PubMed

Bowles, Robert D; Setton, Lori A

2017-06-01

The intervertebral disc contributes to motion, weight bearing, and flexibility of the spine, but is susceptible to damage and morphological changes that contribute to pathology with age and injury. Engineering strategies that rely upon synthetic materials or composite implants that do not interface with the biological components of the disc have not met with widespread use or desirable outcomes in the treatment of intervertebral disc pathology. Here we review bioengineering advances to treat disc disorders, using cell-supplemented materials, or acellular, biologically based materials, that provide opportunity for cell-material interactions and remodeling in the treatment of intervertebral disc disorders. While a field still in early development, bioengineering-based strategies employing novel biomaterials are emerging as promising alternatives for clinical treatment of intervertebral disc disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Translation of an engineered nanofibrous disc-like angle-ply structure for intervertebral disc replacement in a small animal model.

PubMed

Martin, John T; Milby, Andrew H; Chiaro, Joseph A; Kim, Dong Hwa; Hebela, Nader M; Smith, Lachlan J; Elliott, Dawn M; Mauck, Robert L

2014-06-01

Intervertebral disc degeneration has been implicated in the etiology of low back pain; however, the current surgical strategies for treating symptomatic disc disease are limited. A variety of materials have been developed to replace disc components, including the nucleus pulposus (NP), the annulus fibrosus (AF) and their combination into disc-like engineered constructs. We have previously shown that layers of electrospun poly(ε-caprolactone) scaffold, mimicking the hierarchical organization of the native AF, can achieve functional parity with native tissue. Likewise, we have combined these structures with cell-seeded hydrogels (as an NP replacement) to form disc-like angle-ply structures (DAPS). The objective of this study was to develop a model for the evaluation of DAPS in vivo. Through a series of studies, we developed a surgical approach to replace the rat caudal disc with an acellular DAPS and then stabilized the motion segment via external fixation. We then optimized cell infiltration into DAPS by including sacrificial poly(ethylene oxide) layers interspersed throughout the angle-ply structure. Our findings illustrate that DAPS are stable in the caudal spine, are infiltrated by cells from the peri-implant space and that infiltration is expedited by providing additional routes for cell migration. These findings establish a new in vivo platform in which to evaluate and optimize the design of functional disc replacements. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Comparative effects of chlorhexidine and essential oils containing mouth rinse on stem cells cultured on a titanium surface.

PubMed

Park, Jun-Beom; Lee, Gil; Yun, Byeong Gon; Kim, Chang-Hyen; Ko, Youngkyung

2014-04-01

Chlorhexidine (CHX) and Listerine (LIS), an essential oil compound, are the two commonly used adjunctive agents for mechanical debridement, for reducing the bacterial load in the treatment of peri-implant inflammation. However, antimicrobial agents have been reported to be cytotoxic to the alveolar bone cells and gingival epithelial cells. The present study was performed to examine the effects of antiseptics CHX and LIS, on the morphology and proliferation of stem cells. Stem cells derived from the buccal fat pad were grown on machined titanium discs. Each disc was immersed in CHX or LIS for 30 sec, 1.5 min or 4.5 min. Cell morphology was evaluated with a confocal laser microscope and the viability of the cells was quantitatively analyzed with the cell counting kit-8 (CCK-8). The untreated cells attached to the titanium discs demonstrated well-organized actin cytoskeletons. No marked alterations in the cytoskeletal organization were observed in any of the treated groups. The treatment with CHX and LIS of the titanium discs decreased the viability of the cells grown on the treated discs (P<0.05). The stem cells derived from the buccal fat pad were sensitive to CHX and LIS, and a reduction in cellular viability was observed when these agents were applied to the discs for 30 sec. Further studies are required to determine the optimal application time and concentration of this antimicrobial agent for maximizing the reduction of the bacterial load and minimizing the cytotoxicity to the surrounding cells.

Catabolic effects of endothelial cell-derived microparticles on disc cells: Implications in intervertebral disc neovascularization and degeneration.

PubMed

Pohl, Pedro H I; Lozito, Thomas P; Cuperman, Thais; Yurube, Takashi; Moon, Hong J; Ngo, Kevin; Tuan, Rocky S; St Croix, Claudette; Sowa, Gwendolyn A; Rodrigues, Luciano M R; Kang, James D; Vo, Nam V

2016-08-01

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study, we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 h with 250 µg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC-I. Live-cell imaging revealed uptake of EMPs by AF cells. RT-PCR analysis demonstrated increased mRNA expression of MMP-1 (50.3-fold), MMP-3 (4.5-fold) and MMP-13 (5.5-fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP-treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo-angiogenesis of degenerating disc. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1466-1474, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Refining the application of direct embryogenesis in sugarcane: Effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency.

PubMed

Snyman, S J; Meyer, G M; Richards, J M; Haricharan, N; Ramgareeb, S; Huckett, B I

2006-10-01

A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.

Effects of Tobacco Smoking on the Degeneration of the Intervertebral Disc: A Finite Element Study

PubMed Central

Elmasry, Shady; Asfour, Shihab; de Rivero Vaccari, Juan Pablo; Travascio, Francesco

2015-01-01

Tobacco smoking is associated with numerous pathological conditions. Compelling experimental evidence associates smoking to the degeneration of the intervertebral disc (IVD). In particular, it has been shown that nicotine down-regulates both the proliferation rate and glycosaminoglycan (GAG) biosynthesis of disc cells. Moreover, tobacco smoking causes the constriction of the vascular network surrounding the IVD, thus reducing the exchange of nutrients and anabolic agents from the blood vessels to the disc. It has been hypothesized that both nicotine presence in the IVD and the reduced solute exchange are responsible for the degeneration of the disc due to tobacco smoking, but their effects on tissue homeostasis have never been quantified. In this study, a previously presented computational model describing the homeostasis of the IVD was deployed to investigate the effects of impaired solute supply and nicotine-mediated down-regulation of cell proliferation and biosynthetic activity on the health of the disc. We found that the nicotine-mediated down-regulation of cell anabolism mostly affected the GAG concentration at the cartilage endplate, reducing it up to 65% of the value attained in normal physiological conditions. In contrast, the reduction of solutes exchange between blood vessels and disc tissue mostly affected the nucleus pulposus, whose cell density and GAG levels were reduced up to 50% of their normal physiological levels. The effectiveness of quitting smoking on the regeneration of a degenerated IVD was also investigated, and showed to have limited benefit on the health of the disc. A cell-based therapy in conjunction with smoke cessation provided significant improvements in disc health, suggesting that, besides quitting smoking, additional treatments should be implemented in the attempt to recover the health of an IVD degenerated by tobacco smoking. PMID:26301590

Increased density of DISC1-immunoreactive oligodendroglial cells in fronto-parietal white matter of patients with paranoid schizophrenia.

PubMed

Bernstein, Hans-Gert; Jauch, Esther; Dobrowolny, Henrik; Mawrin, Christian; Steiner, Johann; Bogerts, Bernhard

2016-09-01

Profound white matter abnormalities have repeatedly been described in schizophrenia, which involve the altered expression of numerous oligodendrocyte-associated genes. Transcripts of the disrupted-in-schizophrenia 1 (DISC1) gene, a key susceptibility factor in schizophrenia, have recently been shown to be expressed by oligodendroglial cells and to negatively regulate oligodendrocyte differentiation and maturation. To learn more about the putative role(s) of oligodendroglia-associated DISC1 in schizophrenia, we analyzed the density of DISC1-immunoreactive oligodendrocytes in the fronto-parietal white matter in postmortem brains of patients with schizophrenia. Compared with controls (N = 12) and cases with undifferentiated/residual schizophrenia (N = 6), there was a significantly increased density of DISC1-expressing glial cells in paranoid schizophrenia (N = 12), which unlikely resulted from neuroleptic treatment. Pathophysiologically, over-expression of DISC1 protein(s) in white matter oligodendrocytes might add to the reduced levels of two myelin markers, 2',3'-cyclic-nucleotide 3'-phosphodiesterase and myelin basic protein in schizophrenia. Moreover, it might significantly contribute to cell cycle abnormalities as well as to deficits in oligodendroglial cell differentiation and maturation found in schizophrenia.

Uniform and accelerated degradation of pure iron patterned by Pt disc arrays

PubMed Central

Huang, Tao; Zheng, Yufeng

2016-01-01

Pure iron has been confirmed as a promising biodegradable metal. However, the degradation rate of pure iron should be accelerated to meet the clinical requirements. In this work, two different designs of platinum disc arrays, including sizes of Φ20 μm × S5 μm and Φ4 μm × S4 μm, have been coated on the surface of pure iron. Corrosion tests showed the platinum discs formed plenty of galvanic cells with the iron matrix which significantly accelerated the degradation of pure iron. Simultaneously, due to the designability of the shape, size as well as distribution of Pt discs, the degradation rate as well as degradation uniformity of pure iron can be effectively controlled by coating with platinum discs. The cytotoxicity test results unveiled that Pt discs patterned pure iron exhibited almost no toxicity to human umbilical vein endothelial cells, but a significant inhibition on proliferation of vascular smooth muscle cells. In addition, the hemolysis rate of Pt discs patterned pure iron was lower than 1%. Moreover, Pt discs also effectively reduced the number of adhered platelets. All these results indicated that Pt discs patterning is an effective way to accelerate degradation and improve biocompatibility of pure iron. PMID:27033380

Effect of hyaluronic acid on morphological changes to dentin surfaces and subsequent effect on periodontal ligament cell survival, attachment, and spreading.

PubMed

Mueller, Andrea; Fujioka-Kobayashi, Masako; Mueller, Heinz-Dieter; Lussi, Adrian; Sculean, Anton; Schmidlin, Patrick R; Miron, Richard J

2017-05-01

Hyaluronic acid (HA) is a natural constituent of connective tissues and plays an important role in their development, maintenance, and regeneration. Recently, HA has been shown to improve wound healing. However, no basic in vitro study to date has investigated its mode of action. Therefore, the purpose of this study was to examine morphological changes of dentin surfaces following HA coating and thereafter investigate the influence of periodontal ligament (PDL) cell survival, attachment, and spreading to dentin discs. HA was coated onto dentin discs utilizing either non-cross-linked (HA) or cross-linked (HA cl) delivery systems. Morphological changes to dentin discs were then assessed using scanning electron microscopy (SEM). Thereafter, human PDL cells were seeded under three in vitro conditions including (1) dilution of HA (1:100), (2) dilution of HA (1:10), and (3) HA coated directly to dentin discs. Samples were then investigated for PDL cell survival, attachment, and spreading using a live/dead assay, cell adhesion assay, and SEM imaging, respectively. While control dentin discs demonstrated smooth surfaces both at low and high magnification, the coating of HA altered surface texture of dentin discs by increasing surface roughness. HA cl further revealed greater surface texture/roughness likely due to the cross-linking carrier system. Thereafter, PDL cells were seeded on control and HA coated dentin discs and demonstrated a near 100 % survival rate for all samples demonstrating high biocompatibility of HA at dilutions of both 1:100 and 1:10. Interestingly, non-cross-linked HA significantly increased cell numbers at 8 h, whereas cross-linked HA improved cell spreading as qualitatively assessed by SEM. The results from the present study demonstrate that both carrier systems for HA were extremely biocompatible and demonstrated either improved cell numbers or cell spreading onto dentin discs. Future in vitro and animal research is necessary to further characterize the optimal delivery system of HA for improved clinical use. HA is a highly biocompatible material that may improve PDL cell attachment or spreading on dentin.

A preliminary in vitro study into the use of IL-1Ra gene therapy for the inhibition of intervertebral disc degeneration

PubMed Central

Le Maitre, Christine L; Freemont, Anthony J; Hoyland, Judith A

2006-01-01

Conventional therapies for low back pain (LBP) are purely symptomatic and do not target the cause of LBP, which in approximately 40% of cases is caused by degeneration of the intervertebral disc (DIVD). Targeting therapies to inhibit the process of degeneration would be a potentially valuable treatment for LBP. There is increasing evidence for a role for IL-1 in DIVD. A natural inhibitor of IL-1 exists, IL-1Ra, which would be an ideal molecular target for inhibiting IL-1-mediated effects involved in DIVD and LBP. In this study, the feasibility of ex vivo gene transfer of IL-1Ra to the IVD was investigated. Monolayer and alginate cultures of normal and degenerate human intervertebral disc (IVD) cells were infected with an adenoviral vector carrying the IL-1Ra gene (Ad-IL-1Ra) and protein production measured using an enzyme-linked immunosorbent assay. The ability of these infected cells to inhibit the effects of IL-1 was also investigated. In addition, normal and degenerate IVD cells infected with Ad-IL-1Ra were injected into degenerate disc tissue explants and IL-1Ra production in these discs was assessed. This demonstrated that both nucleus pulposus and annulus fibrosus cells infected with Ad-IL-1Ra produced elevated levels of IL-1Ra for prolonged time periods, and these infected cells were resistant to IL-1. When the infected cells were injected into disc explants, IL-1Ra protein expression was increased which was maintained for 2 weeks of investigation. This in vitro study has shown that the use of ex vivo gene transfer to degenerate disc tissue is a feasible therapy for the inhibition of IL-1-mediated events during disc degeneration. PMID:16436110

Collagen-derived N-acetylated proline-glycine-proline upregulates the expression of pro-inflammatory cytokines and extracellular matrix proteases in nucleus pulposus cells via the NF-κB and MAPK signaling pathways.

PubMed

Feng, Chencheng; He, Jinyue; Zhang, Yang; Lan, Minghong; Yang, Minghui; Liu, Huan; Huang, Bo; Pan, Yong; Zhou, Yue

2017-07-01

N-acetylated proline-glycine-proline (N-Ac-PGP) is a chemokine involved in inflammatory diseases and is found to accumulate in degenerative discs. N-Ac-PGP has been demonstrated to have a pro-inflammatory effect on human cartilage endplate stem cells. However, the effect of N-Ac-PGP on human intervertebral disc cells, especially nucleus pulposus (NP) cells, remains unknown. The purpose of this study was to investigate the effect of N-Ac-PGP on the expression of pro-inflammatory factors and extracellular matrix (ECM) proteases in NP cells and the molecular mechanism underlying this effect. Therefore, Milliplex assays were used to detect the levels of various inflammatory cytokines in conditioned culture medium of NP cells treated with N-Ac-PGP, including interleukin-1β (IL-1β), IL-6, IL-17, tumor necrosis factor-α (TNF-α) and C-C motif ligand 2 (CCL2). RT-qPCR was also used to determine the expression of pro-inflammatory cytokines and ECM proteases in the NP cells treated with N-Ac-PGP. Moreover, the role of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in mediating the effect of N-Ac-PGP on the phenotype of NP cells was investigated using specific signaling inhibitors. Milliplex assays showed that NP cells treated with N-Ac-PGP (10 and 100 µg/ml) secreted higher levels of IL-1β, IL-6, IL-17, TNF-α and CCL2 compared with the control. RT-qPCR assays showed that NP cells treated with N-Ac-PGP (100 µg/ml) had markedly upregulated expression of matrix metalloproteinase 3 (MMP3), MMP13, a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4), ADAMTS5, IL-6, CCL-2, CCL-5 and C-X-C motif chemokine ligand 10 (CXCL10). Moreover, N-Ac-PGP was shown to activate the MAPK and NF-κB signaling pathways in NP cells. MAPK and NF-κB signaling inhibitors suppressed the upregulation of proteases and pro-inflammatory cytokines in NP cells treated with N-Ac-PGP. In conclusion, N-Ac-PGP induces the expression of pro-inflammatory cytokines and matrix catabolic enzymes in NP cells via the NF-κB and MAPK signaling pathways. N-Ac-PGP is a novel therapeutic target for intervertebral disc degeneration.

Small Interfering RNA-Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells.

PubMed

Park, Jong-Beom; Park, Chanjoo

2017-10-01

In vitro cell culture model. To investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells. Synthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear. Rat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls. Serum deprivation increased apoptosis by 40.3% ( p <0.001), decreased proliferation by 45.3% ( p <0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures ( p <0.001). Finally, Fas siRNA-mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures ( p <0.05 for both). The observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.

Effects of air polishing and an amino acid buffered hypochlorite solution to dentin surfaces and periodontal ligament cell survival, attachment, and spreading.

PubMed

Schmidlin, Patrick R; Fujioka-Kobayashi, Masako; Mueller, Heinz-Dieter; Sculean, Anton; Lussi, Adrian; Miron, Richard J

2017-06-01

The aim of this study is to examine morphological changes of dentin surfaces following air polishing or amino acid buffered hypochlorite solution application and to assess their influence on periodontal ligament (PDL) cell survival, attachment, and spreading to dentin discs in vitro. Bovine dentin discs were treated with either (i) Classic, (ii) Plus, or (iii) Perio powder (EMS). Furthermore, Perisolv® a hypochlorite solution buffered with various amino acids was investigated. Untreated dentin discs served as controls. Morphological changes to dentin discs were assessed using scanning electron microscopy (SEM). Human PDL cells were seeded onto the respectively treated discs, and samples were then investigated for PDL cell survival, attachment, and spreading using a live/dead assay, adhesion assay, and SEM imaging, respectively. Both control and Perisolv®-rinsed dentin discs demonstrated smooth surfaces at low and high magnifications. The Classic powders demonstrated the thickest coating followed by the Powder Plus. The Perio powder demonstrated marked alterations of dentin discs by revealing the potential to open dentinal tubules even before rinsing. Seeding of PDL cells demonstrated an almost 100 % survival rate on all samples demonstrating very high biocompatibility for all materials. Significantly higher PDL cell numbers were observed on samples treated with the Perio powder and the Perisolv® solution (approximately 40 % more cells; p < 0.05). SEM imaging revealed the potential for PDL cells to attach and spread on all surfaces. The results from the present study demonstrate that cell survival and spreading of PDL cells on root surfaces is possible following either air polishing or application with Perisolv®. Future in vitro and animal testing is necessary to further characterize the beneficial effects of either system in a clinical setting. The use of air polishing or application with Perisolv amino acid buffered hypochlorite solution was effective in treating root surfaces and allowed for near 100 % PDL cell survival, attachment, and spreading onto all root surfaces.

Detection of extended-spectrum β-lactamase in Enterobacter spp.--evaluation of six phenotypic tests.

PubMed

Nogueira-Miranda, Keite da Silva; Palmeiro, Jussara Kasuko; Conte, Danieli; Maia, Fernanda Valverde; Reason, Iara Taborda de Messias; Monteiro, Cristina Leise; Dalla-Costa, Libera Maria

2012-02-01

Extended-spectrum β-lactamases (ESBL) are plasmid-mediated enzymes that hydrolyze cephalosporins and monobactams. The lack of a standard method to detect ESBL in Enterobacter spp. has led to underestimating its frequency. The aim of this study was to evaluate ESBL detection in Enterobacter spp. By the double-disk synergy test (DDST) and combined disk test (CDT) assay using cefepime, cefotaxime, and ceftazime as substrates for ESBL, plus AmpC inhibitors in different associations. A total of 83 Enterobacter spp. ESBL and 31 non-ESBL Enterobacter spp. were tested, and a cutoff point ≥3 mm was defined using a receiver operating characteristic (ROC) curve for combined disc methods. All tests showed 100% specificity. The sensitivity was 89.2% for DDST and CDT without AmpC inibitor, 90.4% in the combined disc test in Mueller-Hinton agar containing phenylboronic acid (CDT-PBAA), and 94% in the combined disc test in Mueller-Hinton agar containing cloxacillin (CDT-CLXA). Cefepime was the best substrate, mainly when AmpC inhibitors were not used. However, superior results were achieved when all cephalosporins were evaluated together. In conclusion, to improve ESBL detection in Enterobacter spp., some modifications in phenotypic tests are needed, such as to reduce the distance between the discs to 20 mm in DDST, to use a cutoff point for ≥3 mm on the CDT, and to include a cefepime disk or an inhibitor of AmpC in all tests.

Regional cell density distribution and oxygen consumption rates in porcine TMJ discs: an explant study.

PubMed

Kuo, J; Shi, C; Cisewski, S; Zhang, L; Kern, M J; Yao, H

2011-07-01

To determine the regional cell density distribution and basal oxygen consumption rates (based on tissue volume and cell number) of temporomandibular joint (TMJ) discs and further examine the impact of oxygen tension on these rates. TMJ discs from pigs aged 6-8 months were divided into five regions: anterior, intermediate, posterior, lateral and medial. The cell density was determined using confocal laser scanning microscopy. The change in oxygen tension was recorded while TMJ disc explants were cultured in sealed metabolism chambers. The volume based oxygen consumption rate of explants was determined by theoretical curve-fitting of the recorded oxygen tension data with the Michaelis-Menten equation. The rate on a per-cell basis was calculated based on the cell density measurements and volume based rate measured in another group of discs. The overall cell density [mean, 95% confidence interval (CI)] was 51.3 (21.3-81.3) × 10(6) cells/mL wet tissue. Along the anteroposterior axis, the anterior band had 25.5% higher cell density than the intermediate zone (P<0.02) and 29.1% higher than the posterior band (P<0.008). Along the mediolateral axes, the medial region had 26.2% higher cell density than the intermediate zone (P<0.04) and 25.4% higher than the lateral region (P<0.045). The overall volume and cell based maximum oxygen consumption rates were 1.44 (0.44-2.44) μmol/mL wet tissue/h and 28.7 (12.2-45.2)nmol/10(6)cells/h, respectively. The central regions (intermediate, lateral, and medial) had significantly higher volume based (P<0.02) and cell based (P<0.005) oxygen consumption rates than the anterior and posterior bands. At high oxygen tension, the oxygen consumption rate remained constant, but dropped as oxygen tension fell below 5%. The TMJ disc had higher cell density and oxygen consumption rates than articular cartilage reported in the literature. These results suggest that a steeper oxygen gradient may exist in the TMJ disc and may be vulnerable to pathological events that impede nutrient supply. Copyright © 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Regional Cell Density Distribution and Oxygen Consumption Rates in Porcine TMJ Discs: An Explant Study

PubMed Central

Kuo, Jonathan; Shi, Changcheng; Cisewski, Sarah; Zhang, Lixia; Kern, Michael J.; Yao, Hai

2011-01-01

Objective To determine the regional cell density distribution and basal oxygen consumption rates (based on tissue volume and cell number) of temporomandibular joint (TMJ) discs and further examine the impact of oxygen tension on these rates. Design TMJ discs from pigs aged 6–8 months were divided into five regions: anterior, intermediate, posterior, lateral and medial. The cell density was determined using confocal laser scanning microscopy. The change in oxygen tension was recorded while TMJ disc explants were cultured in sealed metabolism chambers. The volume based oxygen consumption rate of explants was determined by theoretical curve fitting of the recoded oxygen tension data with the Michaelis-Menten equation. The rate on a per-cell basis was calculated based on the cell density measurements and volume based rate measured in another group of discs. Results The overall cell density (mean, 95% CI) was 51.3(21.3–81.3)×106cells/mL wet tissue. Along the anteroposterior axis, the anterior band had 25.5% higher cell density than the intermediate zone (p<0.02) and 29.1% higher than the posterior band (p<0.008). Along the mediolateral axes, the medial region had 26.2% higher cell density than the intermediate zone (p<0.04) and 25.4% higher than the lateral region (p<0.045). The overall volume and cell based maximum oxygen consumption rates were 1.44(0.44–2.44) μmol/mL wet tissue/hr and 28.7(12.2–45.2) nmol/106 cells/hr, respectively. The central regions (intermediate, lateral, and medial) had significantly higher volume based (p<0.02) and cell based (p<0.005) oxygen consumption rates than the anterior and posterior bands. At high oxygen tension, the oxygen consumption rate remained constant, but dropped as oxygen tension fell below 5%. Conclusions The TMJ disc had higher cell density and oxygen consumption rates than articular cartilage reported in the literature. These results suggest that a steeper oxygen gradient may exist in the TMJ disc and may be vulnerable to pathological events that impede nutrient supply. PMID:21397032

Disabled infectious single cycle-herpes simplex virus (DISC-HSV) as a vector for immunogene therapy of cancer.

PubMed

Rees, Robert C; McArdle, Stephanie; Mian, Shahid; Li, Geng; Ahmad, Murrium; Parkinson, Richard; Ali, Selman A

2002-02-01

Disabled infectious single cycle-herpes simplex viruses (DISC-HSV) have been shown to be safe for use in humans and may be considered efficacious as vectors for immunogene therapy in cancer. Preclinical studies show that DISC-HSV is an efficient delivery system for cytokine genes and antigens. DISC-HSV infects a high proportion of cells, resulting in rapid gene expression for at least 72 h. The DISC-HSV-mGM-CSF vector, when inoculated into tumors, induces tumor regression in a high percentage of animals, concomitant with establishing a cytotoxic T-cell response, which is MHC class I restricted and directed against peptides of known tumor antigens. The inherent properties of DISC-HSV makes it a suitable vector for consideration in human immunogene therapy trials.

The Effects of Oxygen Level and Glucose Concentration on the Metabolism of Porcine TMJ Disc Cells

PubMed Central

Cisewski, Sarah E.; Zhang, Lixia; Kuo, Jonathan; Wright, Gregory J.; Wu, Yongren; Kern, Michael J.; Yao, Hai

2015-01-01

Objective To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells. Design TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 hours with 0, 1.5, 5, or 25mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-3H]proline and [35S]sulfate into the cells, respectively. Results TMJ disc cell viability significantly decreased (P<0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P<0.0001), while a decrease in glucose concentration significantly decreased viability (P<0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P<0.0001) and matrix synthesis (P<0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P<0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P<0.0001), ATP production (P=0.00015), and collagen (P=0.0002) and proteoglycan synthesis (P<0.0001). Conclusions Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply. PMID:26033165

The effects of oxygen level and glucose concentration on the metabolism of porcine TMJ disc cells.

PubMed

Cisewski, S E; Zhang, L; Kuo, J; Wright, G J; Wu, Y; Kern, M J; Yao, H

2015-10-01

To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells. TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 h with 0, 1.5, 5, or 25 mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-(3)H] proline and [(35)S] sulfate into the cells, respectively. TMJ disc cell viability significantly decreased (P < 0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P < 0.0001), while a decrease in glucose concentration significantly decreased viability (P < 0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P < 0.0001) and matrix synthesis (P < 0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P < 0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P < 0.0001), ATP production (P = 0.00015), and collagen (P = 0.0002) and proteoglycan synthesis (P < 0.0001). Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Missense mutation in DISC1 C-terminal coiled-coil has GSK3β signaling and sex-dependent behavioral effects in mice

PubMed Central

Dachtler, James; Elliott, Christina; Rodgers, R. John; Baillie, George S.; Clapcote, Steven J.

2016-01-01

Disrupted-in-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and affective disorders. The full-length DISC1 protein consists of an N-terminal ‘head’ domain and a C-terminal tail domain that contains several predicted coiled-coils, structural motifs involved in protein-protein interactions. To probe the in vivo effects of missense mutation of DISC1’s C-terminal tail, we tested mice carrying mutation D453G within a predicted α-helical coiled-coil region. We report that, relative to wild-type littermates, female DISC1D453G mice exhibited novelty-induced hyperlocomotion, an anxiogenic profile in the elevated plus-maze and open field tests, and reduced social exploration of unfamiliar mice. Male DISC1D453G mice displayed a deficit in passive avoidance, while neither males nor females exhibited any impairment in startle reactivity or prepulse inhibition. Whole brain homogenates showed normal levels of DISC1 protein, but decreased binding of DISC1 to GSK3β, decreased phospho-inhibition of GSK3β at serine 9, and decreased levels of β-catenin in DISC1D453G mice of either sex. Interrupted GSK3β signaling may thus be part of the mechanism underlying the behavioral phenotype associated with D453G, in common with the previously described N-terminal domain mutations Q31L and L100P in mice, and the schizophrenia risk-conferring variant R264Q in humans. PMID:26728762

A Histopathological Scheme for the Quantitative Scoring of Intervertebral Disc Degeneration and the Therapeutic Utility of Adult Mesenchymal Stem Cells for Intervertebral Disc Regeneration

PubMed Central

Shu, Cindy C.; Smith, Margaret M.; Smith, Susan M.; Dart, Andrew J.; Little, Christopher B.; Melrose, James

2017-01-01

The purpose of this study was to develop a quantitative histopathological scoring scheme to evaluate disc degeneration and regeneration using an ovine annular lesion model of experimental disc degeneration. Toluidine blue and Haematoxylin and Eosin (H&E) staining were used to evaluate cellular morphology: (i) disc structure/lesion morphology; (ii) proteoglycan depletion; (iii) cellular morphology; (iv) blood vessel in-growth; (v) cell influx into lesion; and (vi) cystic degeneration/chondroid metaplasia. Three study groups were examined: 5 × 5 mm lesion; 6 × 20 mm lesion; and 6 × 20 mm lesion plus mesenchymal stem cell (MSC) treatment. Lumbar intervertebral discs (IVDs) were scored under categories (i–vi) to provide a cumulative score, which underwent statistical analysis using STATA software. Focal proteoglycan depletion was associated with 5 × 5 mm annular rim lesions, bifurcations, annular delamellation, concentric and radial annular tears and an early influx of blood vessels and cells around remodeling lesions but the inner lesion did not heal. Similar features in 6 × 20 mm lesions occurred over a 3–6-month post operative period. MSCs induced a strong recovery in discal pathology with a reduction in cumulative histopathology degeneracy score from 15.2 to 2.7 (p = 0.001) over a three-month recovery period but no recovery in carrier injected discs. PMID:28498326

A Histopathological Scheme for the Quantitative Scoring of Intervertebral Disc Degeneration and the Therapeutic Utility of Adult Mesenchymal Stem Cells for Intervertebral Disc Regeneration.

PubMed

Shu, Cindy C; Smith, Margaret M; Smith, Susan M; Dart, Andrew J; Little, Christopher B; Melrose, James

2017-05-12

The purpose of this study was to develop a quantitative histopathological scoring scheme to evaluate disc degeneration and regeneration using an ovine annular lesion model of experimental disc degeneration. Toluidine blue and Haematoxylin and Eosin (H&E) staining were used to evaluate cellular morphology: (i) disc structure/lesion morphology; (ii) proteoglycan depletion; (iii) cellular morphology; (iv) blood vessel in-growth; (v) cell influx into lesion; and (vi) cystic degeneration/chondroid metaplasia. Three study groups were examined: 5 × 5 mm lesion; 6 × 20 mm lesion; and 6 × 20 mm lesion plus mesenchymal stem cell (MSC) treatment. Lumbar intervertebral discs (IVDs) were scored under categories (i-vi) to provide a cumulative score, which underwent statistical analysis using STATA software. Focal proteoglycan depletion was associated with 5 × 5 mm annular rim lesions, bifurcations, annular delamellation, concentric and radial annular tears and an early influx of blood vessels and cells around remodeling lesions but the inner lesion did not heal. Similar features in 6 × 20 mm lesions occurred over a 3-6-month post operative period. MSCs induced a strong recovery in discal pathology with a reduction in cumulative histopathology degeneracy score from 15.2 to 2.7 ( p = 0.001) over a three-month recovery period but no recovery in carrier injected discs.

Synovial joint formation requires local Ext1 expression and heparan sulfate production in developing mouse embryo limbs and spine.

PubMed

Mundy, Christina; Yasuda, Tadashi; Kinumatsu, Takashi; Yamaguchi, Yu; Iwamoto, Masahiro; Enomoto-Iwamoto, Motomi; Koyama, Eiki; Pacifici, Maurizio

2011-03-01

Heparan sulfate proteoglycans (HSPGs) regulate a number of major developmental processes, but their roles in synovial joint formation remain unknown. Here we created conditional mouse embryo mutants lacking Ext1 in developing joints by mating Ext1(f/f) and Gdf5-Cre mice. Ext1 encodes a subunit of the Ext1/Ext2 Golgi-associated protein complex responsible for heparan sulfate (HS) synthesis. The proximal limb joints did form in the Gdf5-Cre;Ext1(f/f) mutants, but contained an uneven articulating superficial zone that expressed very low lubricin levels. The underlying cartilaginous epiphysis was deranged as well and displayed random patterns of cell proliferation and matrillin-1 and collagen IIA expression, indicative of an aberrant phenotypic definition of the epiphysis itself. Digit joints were even more affected, lacked a distinct mesenchymal interzone and were often fused likely as a result of local abnormal BMP and hedgehog activity and signaling. Interestingly, overall growth and lengthening of long bones were also delayed in the mutants. To test whether Ext1 function is needed for joint formation at other sites, we examined the spine. Indeed, entire intervertebral discs, normally composed by nucleus pulposus surrounded by the annulus fibrosus, were often missing in Gdf5-Cre;Ext1(f/f) mice. When disc remnants were present, they displayed aberrant organization and defective joint marker expression. Similar intervertebral joint defects and fusions occurred in Col2-Cre;β-catenin(f/f) mutants. The study provides novel evidence that local Ext1 expression and HS production are needed to maintain the phenotype and function of joint-forming cells and coordinate local signaling by BMP, hedgehog and Wnt/β-catenin pathways. The data indicate also that defects in joint formation reverberate on, and delay, overall long bone growth. Copyright © 2011 Elsevier Inc. All rights reserved.

Development of a novel automated cell isolation, expansion, and characterization platform.

PubMed

Franscini, Nicola; Wuertz, Karin; Patocchi-Tenzer, Isabel; Durner, Roland; Boos, Norbert; Graf-Hausner, Ursula

2011-06-01

Implementation of regenerative medicine in the clinical setting requires not only biological inventions, but also the development of reproducible and safe method for cell isolation and expansion. As the currently used manual techniques do not fulfill these requirements, there is a clear need to develop an adequate robotic platform for automated, large-scale production of cells or cell-based products. Here, we demonstrate an automated liquid-handling cell-culture platform that can be used to isolate, expand, and characterize human primary cells (e.g., from intervertebral disc tissue) with results that are comparable to the manual procedure. Specifically, no differences could be observed for cell yield, viability, aggregation rate, growth rate, and phenotype. Importantly, all steps-from the enzymatic isolation of cells through the biopsy to the final quality control-can be performed completely by the automated system because of novel tools that were incorporated into the platform. This automated cell-culture platform can therefore replace entirely manual processes in areas that require high throughput while maintaining stability and safety, such as clinical or industrial settings. Copyright © 2011 Society for Laboratory Automation and Screening. Published by Elsevier Inc. All rights reserved.

Identification of a New Modulator of the Intercalated Disc in a Zebrafish Model of Arrhythmogenic Cardiomyopathy

PubMed Central

Asimaki, Angeliki; Kapoor, Sudhir; Plovie, Eva; Arndt, Anne Karin; Adams, Edward; Liu, ZhenZhen; James, Cynthia A.; Judge, Daniel P.; Calkins, Hugh; Churko, Jared; Wu, Joseph C.; MacRae, Calum A.; Kléber, André G.; Saffitz, Jeffrey E.

2015-01-01

Arrhythmogenic cardiomyopathy (ACM) is characterized by frequent cardiac arrhythmias. To elucidate the underlying mechanisms and discover potential chemical modifiers, we created a zebrafish model of ACM with cardiac myocyte–specific expression of the human 2057del2 mutation in the gene encoding plakoglobin. A high-throughput screen identified SB216763 as a suppressor of the disease phenotype. Early SB216763 therapy prevented heart failure and reduced mortality in the fish model. Zebrafish ventricular myocytes that expressed 2057del2 plakoglobin exhibited 70 to 80% reductions in INa and IK1 current densities, which were normalized by SB216763. Neonatal rat ventricular myocytes that expressed 2057del2 plakoglobin recapitulated pathobiological features seen in patients with ACM, all of which were reversed or prevented by SB216763. The reverse remodeling observed with SB216763 involved marked subcellular redistribution of plakoglobin, connexin 43, and Nav1.5, but without changes in their total cellular content, implicating a defect in protein trafficking to intercalated discs. In further support of this mechanism, we observed SB216763-reversible, abnormal subcellular distribution of SAP97 (a protein known to mediate forward trafficking of Nav1.5 and Kir2.1) in rat cardiac myocytes expressing 2057del2 plakoglobin and in cardiac myocytes derived from induced pluripotent stem cells from two ACM probands with plakophilin-2 mutations. These observations pinpoint aberrant trafficking of intercalated disc proteins as a central mechanism in ACM myocyte injury and electrical abnormalities. PMID:24920660

Prominent dominant negative effect of a mutant Fas molecule lacking death domain on cell-mediated induction of apoptosis.

PubMed

Yokota, Aya; Takeuchi, Emiko; Iizuka, Misao; Ikegami, Yuko; Takayama, Hajime; Shinohara, Nobukata

2005-01-01

Using a panel of transfectant B lymphoma cells expressing varying amounts of the mutant Fas together with the endogenous wild type Fas, semi-quantitative studies on the dominant negative effect of a murine mutant Fas molecule lacking death domain were carried out. In anti-Fas antibody-mediated induction of apoptosis, the mutant molecules exerted significant dominant-negative effect only when their expression level was comparable to or higher than that of wild type molecules, or when exposed to low amounts of the antibody. The inhibitory effect was accompanied by the failure in DISC formation in spite of Fas aggregation. When they were subjected to T cell-mediated Fas-based induction of apoptosis, however, the dominant negative effect was prominent such that the expression of even a small amount of the mutant molecules resulted in significant inhibition. Such a strong inhibitory effect explains the dominant phenotype of this type of mutant Fas molecules in ALPS heterozygous patients and also implies that the physiological effectors for Fas in vivo are cells, i.e., FasL-expressing activated T cells.

Age-related changes in expression of transforming growth factor-beta and receptors in cells of intervertebral discs.

PubMed

Matsunaga, Shunji; Nagano, Satoshi; Onishi, Toshiyuki; Morimoto, Norio; Suzuki, Shusaku; Komiya, Setsuro

2003-01-01

The authors conducted a study to determine age-related changes in expression of transforming growth factor (TGF)-beta1, -beta2, -beta3, and Type I and Type II receptors in various cells in the nucleus pulposus and anulus fibrosus. Immunolocalization of TGFbetas and Type I and II receptors was examined during the aging process of cervical intervertebral discs in senescence-accelerated mice (SAM). The TGFbeta family has important roles for cellular function of various tissues. Its role in disc aging, however, is unknown. Detailed information on the temporal and spatial localization of TGFbetas and their receptors in discs is required before discussing introduction of them clinically into the intervertebral disc. Three groups of five SAM each were used. The groups of SAM were age 8, 24, and 50 weeks, respectively. Hematoxylin and eosin staining and immunohistochemical study involving specific antibodies for TGFbeta1, -beta2, -beta3, and Types I and II TGF receptors were performed. Intervertebral discs exhibited degenerative change with advancing age. The TGFbetas and their receptors were present in the fibrocartilaginous cells within the anulus fibrosus and notochord-like cells within the nucleus pulposus of young mice. Expression of TGFbetas and Type I and Type II receptors changed markedly in the cells within the anulus fibrosus during the aging process. The TGFbetas and their receptors were present in cells within the nucleus pulposus and the anulus fibrosus of young mice, and their expression decreased with age.

Insulin and heparin co-immobilized 3D polyester fabrics for the cultivation of fibroblasts in low-serum media.

PubMed

Türkoğlu Saşmazel, Hilal; Aday, Sezin; Gümüşderelioğlu, Menemşe

2007-08-01

Insulin and/or heparin immobilized/co-immobilized non-woven polyester fabric (NWPF) discs were developed for the cultivation of L929 mouse fibroblasts in low-serum media. At first, NWPF discs were hydrolyzed to obtain a carboxylic acid group-introduced matrix (NWPF-hydrolyzed). Insulin and heparin co-immobilized NWPF (NWPF-insulin-heparin) was prepared by the grafting of PEO onto NWPF-hydrolyzed disc (NWPF-PEO), followed by the reaction first with insulin and then heparin. In the presence of spacer arm, PEO, the amount of immobilized insulin molecules significantly increased from 6.96 to 84.45 microg/cm(2). The amount of heparin bound to the NWPF-PEO (5.93 microg/cm(2)) was higher than that of the insulin immobilized surface (4.59 microg/cm(2)). Insulin and heparin immobilized NWPF discs were observed with fluorescence microscopy by labeling the insulin and heparin with 8-anilino-1-naphthalene sulfonic acid (ANS) or fluorescein isothiocyanate (FITC), respectively. L929 fibroblasts were used to check the cell adhesion and cell growth capabilities of modified NWPF discs in low-serum media (containing 5% fetal bovine serum). Optical photographs showed that after 2nd day of the culture, fibroblastic cells spread along the length of modified fibers, eventually filling the interfiber space. At the end of 6-day growth period, cell yield in the presence of immobilized heparin was a little bit higher than that of the immobilized insulin. Co-immobilized (insulin/heparin) NWPF discs did not accelerate the cell growth as well as insulin or heparin immobilized discs.

Genotoxicity testing of different types of beverages in the Drosophila wing Somatic Mutation And Recombination Test.

PubMed

Graf, U; Moraga, A A; Castro, R; Díaz Carrillo, E

1994-05-01

Five wines and one brandy of Spanish origin as well as three herbal teas and ordinary black tea were tested for genotoxicity in the wing Somatic Mutation And Recombination Test (SMART) which makes use of the two recessive wing cell markers multiple wing hairs (mwh) and flare (flr3) on the left arm of chromosome 3 of Drosophila melanogaster. 3-day-old larvae trans-heterozygous for these two markers were fed the beverages at different concentrations and for different feeding periods using Drosophila instant medium. Somatic mutations or mitotic recombinations induced in the cells of the wing imaginal discs give rise to mutant single or twin spots on the wing blade of the emerging adult flies showing either the mwh phenotype or/and the flr phenotype. One of the red wines showed a clear genotoxic activity that was not due to its ethanol content. Two herbal teas (Urtica dioica, Achillea millefolium) and black tea (Camellia sinensis) proved to be weakly genotoxic as well. Furthermore, it was shown that quercetin and rutin, two flavonols present in beverages of plant origin, also exhibited weak genotoxic activity in the somatic cells of Drosophila. These results demonstrate that Drosophila in vivo somatic assays can detect the genotoxicity of complex mixtures such as beverages. In particular, it is possible to administer these test materials in the same form as that in which they are normally consumed.

Identification of the Drosophila eIF4A gene as a target of the DREF transcription factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Hiroyuki; Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585; Yoshida, Hideki

2007-12-10

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. We have carried out a genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express full-length DREF in the eye imaginal discs and identified the eukaryotic initiation factor 4A (eIF4A) gene as a dominant suppressor of the DREF-induced rough eye phenotype. The eIF4A gene was here found to carry three DRE sequences, DRE1 (- 40 to - 47), DRE2 (- 48 to - 55), and DRE3 (- 267 to - 274) in its promoter region, these all being important for the eIF4A genemore » promoter activity in cultured Drosophila Kc cells and in living flies. Knockdown of DREF in Drosophila S2 cells decreased the eIF4A mRNA level and the eIF4A gene promoter activity. Furthermore, specific binding of DREF to genomic regions containing DRE sequences was demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. Band mobility shift assays using Kc cell nuclear extracts revealed that DREF could bind to DRE1 and DRE3 sequences in the eIF4A gene promoter in vitro, but not to the DRE2 sequence. The results suggest that the eIF4A gene is under the control of the DREF pathway and DREF is therefore involved in the regulation of protein synthesis.« less

A potential role for cell-based therapeutics in the treatment of intervertebral disc herniation.

PubMed

Ganey, Timothy M; Meisel, Hans Joerg

2002-10-01

Lower back pain and disc degeneration negatively affect quality of life and impose an enormous financial burden. An extensive body of scientific work has evolved that characterizes the disc, demonstrating spinal anatomy and morphology that contribute to risk and likely promote failure. Ultimately, matrix failure is responsible for mechanical failure, which in turn results in spinal compromise anatomically and subsequent pain. One intervening approach to breaking this sequence has been to repopulate the anatomy with autologous disc chondrocytes--cells capable of restoring the matrix and retaining the mechanical balance by which the disc functions. This strategy has been implemented both in patients and in animal models, and early results, although preliminary, support the premise as a positive approach.

Enhanced viability of corneal epithelial cells for efficient transport/storage using a structurally modified calcium alginate hydrogel.

PubMed

Wright, Bernice; Cave, Richard A; Cook, Joseph P; Khutoryanskiy, Vitaliy V; Mi, Shengli; Chen, Bo; Leyland, Martin; Connon, Che J

2012-05-01

Therapeutic limbal epithelial stem cells could be managed more efficiently if clinically validated batches were transported for 'on-demand' use. In this study, corneal epithelial cell viability in calcium alginate hydrogels was examined under cell culture, ambient and chilled conditions for up to 7 days. Cell viability improved as gel internal pore size increased, and was further enhanced with modification of the gel from a mass to a thin disc. Ambient storage conditions were optimal for supporting cell viability in gel discs. Cell viability in gel discs was significantly enhanced with increases in pore size mediated by hydroxyethyl cellulose. Our novel methodology of controlling alginate gel shape and pore size together provides a more practical and economical alternative to established corneal tissue/cell storage methods.

Effect of Air-Polishing on Titanium Surfaces, Biofilm Removal, and Biocompatibility: A Pilot Study

PubMed Central

Bennani, Vincent; Hwang, Linda; Tawse-Smith, Andrew; Dias, George J.; Cannon, Richard D.

2015-01-01

Purpose. The aims of this in vitro study were to evaluate morphological changes induced by glycine powder air-polishing on titanium surfaces, biofilm removal, and biocompatibility. Material and Methods. Titanium grade IV discs were allocated into two groups: (1) discs without biofilm and (2) discs for Streptococcus mutans biofilm formation. Discs in each group were further subdivided into (a) no treatment and (b) air-polishing treatment with glycine powder. Discs were characterized by scanning electron microscopy (SEM), electron-dispersive spectroscopy (EDS), and confocal microscopy. Bacterial biofilms were quantified using a crystal violet dye-binding assay. Biocompatibility was evaluated by measuring the coverage and viability of L929 fibroblast cells cultured on the discs. Results. Air-polishing increased the roughness of treated discs (P < 0.05). EDS analysis did not show significant differences in the chemical composition of treated and nontreated discs. The amount of residual biofilm on treated discs was 8.6-fold lower than untreated controls (P < 0.05). Coverage of treated discs by fibroblasts was half that of untreated discs (P < 0.05) although both groups had the same cell viability. Conclusions. Air-polishing removed a significant amount of biofilm from titanium surfaces. The “polishing” was accompanied by increased surface roughness, but there were no changes in chemical and elemental compositions, nor the biocompatibility. PMID:26881198

Glial cell migration in the eye disc.

PubMed

Silies, Marion; Yuva, Yeliz; Engelen, Daniel; Aho, Annukka; Stork, Tobias; Klämbt, Christian

2007-11-28

Any complex nervous system is made out of two major cell types, neurons and glial cells. A hallmark of glial cells is their pronounced ability to migrate. En route to their final destinations, glial cells are generally guided by neuronal signals. Here we show that in the developing visual system of Drosophila glial cell migration is largely controlled by glial-glial interactions and occurs independently of axonal contact. Differentiation into wrapping glia is initiated close to the morphogenetic furrow. Using single cell labeling experiments we identified six distinct glial cell types in the eye disc. The migratory glial population is separated from the wrapping glial cells by the so-called carpet cells, extraordinary large glial cells, each covering a surface area of approximately 10,000 epithelial cells. Subsequent cell ablation experiments demonstrate that the carpet glia regulates glial migration in the eye disc epithelium and suggest a new model underlying glial migration and differentiation in the developing visual system.

Development of gellan gum-based microparticles/hydrogel matrices for application in the intervertebral disc regeneration.

PubMed

Pereira, Diana Ribeiro; Silva-Correia, Joana; Caridade, Sofia Glória; Oliveira, Joao T; Sousa, Rui A; Salgado, Antonio J; Oliveira, Joaquim M; Mano, João F; Sousa, Nuno; Reis, Rui L

2011-10-01

Low back pain is one of the most reported medical conditions associated to intervertebral disc (IVD) degeneration. Nucleus pulposus (NP) is often regarded as the structure where IVD degeneration begins. Gellan gum (GG)-based hydrogels for acellular and cellular tissue engineering strategies have been developed for finding applications as NP substitutes. The innovative strategy is based on the reinforcement of the hydrogel matrix with biocompatible and biodegradable GG microparticles (MPs), which are expected to improve the mechanical properties, while allowing to tailor its degradation rate. In this study, several GG MP/hydrogel disc formulations were prepared by means of mixing high acyl GG (0.75% (w/v)) and low acyl GG (2% (w/v)) GG aqueous solutions at different ratios, namely, 75%:25% (v/v), 50%:50% (v/v), and 25%:75% (v/v), respectively. The GG MP size was measured using a stereo microscope, and their dispersion within the hydrogel matrix was evaluated by means of staining the MPs with Toluidine Blue-O. The developed GG MPs/hydrogel discs were physicochemically characterized by Fourier-transform infrared spectroscopy and (1)H-nuclear magnetic resonance spectroscopy. The swelling behavior and degradation rate were assessed by immersion in a phosphate buffer saline for 14 days. The morphology and mechanical behavior were investigated by scanning electron microscopy and dynamic mechanical analysis, respectively. The mechanical properties of the hydrogel disc were improved by mixing the gels with the MPs. In addition, the possible cytotoxicity of the leachables released by MPs/hydrogel discs was screened in vitro, using a mouse lung fibroblast cell line (L929 cells). To investigate the encapsulation efficacy of L929 cells into the GG MPs/hydrogel discs, cells were stained with DAPI blue/Texas Red-Phalloidin and observed by confocal microscopy, after 24, 48, and 72 h of culturing. A cell viability assay was also performed using Calcein AM staining. The cell culture studies demonstrated that MPs/hydrogel discs are noncytotoxic over L929 cells. It was also demonstrated that L929 cells can be successfully encapsulated into the GG MPs of different formulations, remaining viable after 72 h of culturing. This study showed that GG hydrogel matrices reinforced with cell-loaded MPs could be a candidate strategy for NP regeneration. © Mary Ann Liebert, Inc.

The effects of cyclic tensile strain on the organisation and expression of cytoskeletal elements in bovine intervertebral disc cells: an in vitro study.

PubMed

Li, S; Jia, X; Duance, V C; Blain, E J

2011-06-20

It is still relatively unclear how intervertebral disc (IVD) cells sense a mechanical stimulus and convert this signal into a biochemical response. Previous studies demonstrated that the cytoskeletal elements are mechano-responsive in many cell types and may contribute to mechano-signalling pathways. The objective of this study was to determine the response of cells from the outer annulus fibrosus (OAF) to physiological levels of cyclic tensile strain; further, cells from the nucleus pulposus (NP) were also subjected to an identical loading regime to compare biological responses across the IVD populations. We determined whether the organisation and expression of the major cytoskeletal elements and their associated accessory proteins are responsive to mechanical stimulation in these cells, and whether these changes correlated with either a catabolic or anabolic phenotype. OAF and NP cells from immature bovine IVD were seeded onto Flexcell® type I collagen coated plates. Cells were subjected to cyclic tensile strain (10 %, 1 Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy, RNA extracted for quantitative PCR and protein extracted for Western blotting analysis. F-actin reorganisation was evident in OAF and NP cells subjected to tensile strain; strain induced β-actin at the transcriptional and translational level in OAF cells. β-tubulin mRNA and protein synthesis increased in strained OAF cells, but vimentin expression was significantly inhibited. Cytoskeletal element organisation and expression were less responsive to strain in NP cells. Tensile strain increased type I collagen and differentially regulated extracellular matrix (ECM)-degrading enzymes' mRNA levels in OAF cells. Strain induced type II collagen transcription in NP cells, but had no effect on the transcription of any other genes analysed. Tensile strain induces different mechano-responses in the organisation and/or expression of cytoskeletal elements and on markers of IVD metabolism. Differential mechano-regulation of anabolic and catabolic ECM components in the OAF and NP populations reflects their respective mechanical environments in situ.

Myoblast cytonemes mediate Wg signaling from the wing imaginal disc and Delta-Notch signaling to the air sac primordium.

PubMed

Huang, Hai; Kornberg, Thomas B

2015-05-07

The flight muscles, dorsal air sacs, wing blades, and thoracic cuticle of the Drosophila adult function in concert, and their progenitor cells develop together in the wing imaginal disc. The wing disc orchestrates dorsal air sac development by producing decapentaplegic and fibroblast growth factor that travel via specific cytonemes in order to signal to the air sac primordium (ASP). Here, we report that cytonemes also link flight muscle progenitors (myoblasts) to disc cells and to the ASP, enabling myoblasts to relay signaling between the disc and the ASP. Frizzled (Fz)-containing myoblast cytonemes take up Wingless (Wg) from the disc, and Delta (Dl)-containing myoblast cytonemes contribute to Notch activation in the ASP. Wg signaling negatively regulates Dl expression in the myoblasts. These results reveal an essential role for cytonemes in Wg and Notch signaling and for a signal relay system in the myoblasts.

Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation.

PubMed

Wang, Feng; Gao, Zeng-Xin; Cai, Feng; Sinkemani, Arjun; Xie, Zhi-Yang; Shi, Rui; Wei, Ji-Nan; Wu, Xiao-Tao

2017-08-22

Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration.

Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation

PubMed Central

Sinkemani, Arjun; Xie, Zhi-Yang; Shi, Rui; Wei, Ji-Nan; Wu, Xiao-Tao

2017-01-01

Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration. PMID:28915712

A disc diffusion assay for detection of class A, B and OXA-48 carbapenemases in Enterobacteriaceae using phenyl boronic acid, dipicolinic acid and temocillin.

PubMed

van Dijk, K; Voets, G M; Scharringa, J; Voskuil, S; Fluit, A C; Rottier, W C; Leverstein-Van Hall, M A; Cohen Stuart, J W T

2014-04-01

Class A and B carbapenemases in Enterobacteriaceae may be detected using carbapenemase inhibition tests with boronic acid derivatives (BA) and dipicolinic acid (DPA)/EDTA, respectively. However, for OXA-48 (like) carbapenemases, no specific inhibitor is available. Because OXA-48 confers high-level temocillin resistance, a disc diffusion assay using temocillin as well as BA and DPA inhibition tests was evaluated for detection of class A, B and OXA-48 carbapenemases. The test collection included 128 well-characterized non-repeat Enterobacteriaceae isolates suspected of carbapenemase production; that is, with meropenem MICs ≥ 0.5 mg/L, including 99 carbapenemase producers (36 KPC, one GES, 31 MBL, four KPC plus VIM, 25 OXA-48, two OXA-162), and 29 ESBL and/or AmpC-producing isolates. PCR and sequencing of beta-lactamase genes was used as a reference test. Phenotypic carbapenemase detection was performed with discs (Rosco) containing meropenem (10 μg), temocillin (30 μg), meropenem + phenyl boronic acid (PBA), meropenem + DPA, meropenem + BA + DPA, and meropenem + cloxacillin (CL). Absence of synergy between meropenem and BA and/or DPA and a temocillin zone ≤10 mm was used to identify OXA-48. The sensitivity for identification of class A, B and OXA-48 carbapenemases was 95%, 90% and 100%, with 96-100% specificity. In non-Proteus species, the sensitivity for class B carbapenemase detection was 97%. All isolates without PBA or DPA synergy and a temocillin disc zone ≤10 mm were OXA-48 (like) positive. In conclusion, carbapenemase inhibition tests with PBA and DPA combined with a temocillin disc provide a reliable phenotypic confirmation method for class A, B and OXA-48 carbapenemases in Enterobacteriaceae. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Human cells derived from degenerate intervertebral discs respond differently to those derived from non-degenerate intervertebral discs following application of dynamic hydrostatic pressure.

PubMed

Le Maitre, Christine Lyn; Frain, Jennie; Fotheringham, Andrew P; Freemont, Anthony J; Hoyland, Judith Alison

2008-01-01

The intervertebral disc (IVD) is one of the body's most important load-bearing structures with the major mechanical force experienced in the nucleus pulposus (NP) being hydrostatic pressure (HP). Physiological levels of HP have an anabolic effect on IVD matrix metabolism in cells derived from non-degenerate animal and herniated IVD while excessive HP has a catabolic effect. However, no studies have investigated the response of non-degenerate and degenerate human disc cells derived from non-herniated discs to HP. Here we investigate the effect of physiological HP on such cells using a novel loading rig. Human IVD cells (both NP and AF) cultured in alginate were subjected to dynamic HP (0.8-1.7 MPa 0.5 Hz) for 2 h. Cell viability was assessed, RNA extracted and qRT-PCR for 18 s, c-fos, Sox-9, collagen type II, aggrecan and MMP-3 performed. Cell viability was unaffected by the loading regime. In non-degenerate NP cells, HP increased c-fos, aggrecan, Sox-9 and collagen type II (significantly so in the case of c-fos and aggrecan), but not MMP-3 gene expression. In contrast, application of HP to AF or degenerate NP cells had no effect on target gene expression. Our data shows that cells obtained from the healthy NP respond to dynamic HP by up-regulating genes indicative of healthy matrix homeostasis. However, responses differed in degenerate NP cells suggesting that an altered mechanotransduction pathway may be operational.

N-Cadherin Attenuates High Glucose-Induced Nucleus Pulposus Cell Senescence Through Regulation of the ROS/NF-κB Pathway.

PubMed

Hou, Gang; Zhao, Huiqing; Teng, Haijun; Li, Pei; Xu, Wenbin; Zhang, Junbin; Lv, Lulu; Guo, Zhiliang; Wei, Li; Yao, Hui; Xu, Yichun

2018-01-01

Diabetes mellitus (DM) is a potential etiology of disc degeneration. N-cadherin (N-CDH) helps maintain the cell viability, cell phenotype and matrix biosynthesis of nucleus pulposus (NP) cells. Here, we mainly aimed to investigate whether N-CDH can attenuate high glucose-induced NP cell senescence and its potential mechanism. Rat NP cells were cultured in a base culture medium and base culture medium with a 0.2 M glucose concentration. Recombinant lentiviral vectors were used to enhance N-CDH expression in NP cells. Senescence-associated β-galactosidase (SA-β-Gal) activity was measured by SA-β-Gal staining. NP cell proliferation was evaluated by CCK-8 assay. Telomerase activity and intracellular reactive oxygen species (ROS) content were tested by specific chemical kits according to the manufacturer's instructions. G0/G1 cell cycle arrest was evaluated by flow cytometry. Real-time PCR and Western blotting were used to analyze mRNA and protein expressions of senescence markers (p16 and p53) and matrix macromolecules (aggrecan and collagen II). Additionally, p-NF-κB expression was also analyzed by Western blotting to evaluate NF-κB pathway activity. High glucose significantly decreased N-CDH expression, increased ROS generation and NF-κB pathway activity, and promoted NP cell senescence, which was reflected in the increase in SA-β-Gal activity and senescence marker (p16 and p53) expression, compared to the control group. High glucose decreased telomerase activity and cell proliferation potency. However, N-CDH overexpression partially attenuated NP cell senescence, decreased ROS content and inhibited the activation of the NF-κB pathway under the high glucose condition. High glucose decreases N-CDH expression and promotes NP cell senescence. N-CDH overexpression can attenuate high glucose-induced NP cell senescence through the regulation of the ROS/ NF-κB pathway. This study suggests that N-CDH is a potential therapeutic target to slow DM-mediated disc NP degeneration. © 2018 The Author(s). Published by S. Karger AG, Basel.

SEM evaluation of human gingival fibroblasts growth onto CAD/CAM zirconia and veneering ceramic for zirconia

PubMed Central

Zizzari, Vincenzo; Borelli, Bruna; De Colli, Marianna; Tumedei, Margherita; Di Iorio, Donato; Zara, Susi; Sorrentino, Roberto; Cataldi, Amelia; Gherlone, Enrico Felice; Zarone, Fernando; Tetè, Stefano

2013-01-01

Summary Aim To evaluate the growth of Human Gingival Fibroblasts (HGFs) cultured onto sample discs of CAD/CAM zirconia and veneering ceramic for zirconia by means of Scanning Electron Microscope (SEM) analysis at different experimental times. Methods A total of 26 experimental discs, divided into 2 groups, were used: Group A) CAD/CAM zirconia (3Y-TZP) discs (n=13); Group B) veneering ceramic for zirconia discs (n=13). HGFs were obtained from human gingival biopsies, isolated and placed in culture plates. Subsequently, cells were seeded on experimental discs at 7,5×103/cm2 concentration and cultured for a total of 7 days. Discs were processed for SEM observation at 3h, 24h, 72h and 7 days. Results In Group A, after 3h, HGFs were adherent to the surface and showed a flattened profile. The disc surface covered by HGFs resulted to be wider in Group A than in Group B samples. At SEM observation, after 24h and 72h, differences in cell attachment were slightly noticeable between the groups, with an evident flattening of HGFs on both surfaces. All differences between Group A and group B became less significant after 7 days of culture in vitro. Conclusions SEM analysis of HGFs showed differences in terms of cell adhesion and proliferation, especially in the early hours of culture. Results showed a better adhesion and cell growth in Group A than in Group B, especially up to 72h in vitro. Differences decreased after 7 days, probably because of the rougher surface of CAD/CAM zirconia, promoting better cell adhesion, compared to the smoother surface of veneering ceramic. PMID:24611089

The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

PubMed Central

Le Maitre, Christine Lyn; Freemont, Anthony J; Hoyland, Judith Alison

2005-01-01

In this study, we investigated the hypotheses that in human intervertebral disc (IVD) degeneration there is local production of the cytokine IL-1, and that this locally produced cytokine can induce the cellular and matrix changes of IVD degeneration. Immunohistochemistry was used to localize five members of the IL-1 family (IL-1α, IL-1β, IL-1Ra (IL-1 receptor antagonist), IL-1RI (IL-1 receptor, type I), and ICE (IL-1β-converting enzyme)) in non-degenerate and degenerate human IVDs. In addition, cells derived from non-degenerate and degenerate human IVDs were challenged with IL-1 agonists and the response was investigated using real-time PCR for a number of matrix-degrading enzymes, matrix proteins, and members of the IL-1 family. This study has shown that native disc cells from non-degenerate and degenerate discs produced the IL-1 agonists, antagonist, the active receptor, and IL-1β-converting enzyme. In addition, immunopositivity for these proteins, with the exception of IL-1Ra, increased with severity of degeneration. We have also shown that IL-1 treatment of human IVD cells resulted in increased gene expression for the matrix-degrading enzymes (MMP 3 (matrix metalloproteinase 3), MMP 13 (matrix metalloproteinase 13), and ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs)) and a decrease in the gene expression for matrix genes (aggrecan, collagen II, collagen I, and SOX6). In conclusion we have shown that IL-1 is produced in the degenerate IVD. It is synthesized by native disc cells, and treatment of human disc cells with IL-1 induces an imbalance between catabolic and anabolic events, responses that represent the changes seen during disc degeneration. Therefore, inhibiting IL-1 could be an important therapeutic target for preventing and reversing disc degeneration. PMID:15987475

A Substance Exchanger-Based Bioreactor Culture of Pig Discs for Studying the Immature Nucleus Pulposus.

PubMed

Li, Pei; Gan, Yibo; Wang, Haoming; Xu, Yuan; Song, Lei; Wang, Liyuan; Ouyang, Bin; Zhou, Qiang

2017-11-01

Various research models have been developed to study the biology of disc cells. Recently, the adult disc nucleus pulposus (NP) has been well studied. However, the immature NP is underinvestigated due to a lack of a suitable model. This study aimed to establish an organ culture of immature porcine disc by optimizing culture conditions and using a self-developed substance exchanger-based bioreactor. Immature porcine discs were first cultured in the bioreactor for 7 days at various levels of glucose (low, medium, high), osmolarity (hypo-, iso-, hyper-) and serum (5, 10, 20%) to determine the respective optimal level. The porcine discs were then cultured under the optimized conditions in the novel bioreactor, and were compared with fresh discs at day 14. For high-glucose, iso-osmolarity, or 10% serum, cell viability, the gene expression profile (for anabolic genes and catabolic genes), and glycosaminoglycan (GAG) and hydroxyproline (HYP) contents were more favorable than for other levels of glucose, osmolarity, and serum. When the immature discs were cultured under the optimized conditions using the novel bioreactor for 14 days, the viability of the immature NP was maintained based on histology, cell viability, GAG and HYP contents, and matrix molecule expression. In conclusion, the viability of the immature NP in organ culture could be maintained under the optimized culture conditions (high-glucose, iso-osmolarity, and 10% serum) in the substance exchanger-based bioreactor. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Expression of receptors for putative anabolic growth factors in human intervertebral disc: implications for repair and regeneration of the disc.

PubMed

Le Maitre, Christine L; Richardson, Stephen M A; Baird, Pauline; Freemont, Anthony J; Hoyland, Judith A

2005-12-01

Low back pain (LBP) is a common, debilitating and economically important disorder. Current evidence implicates loss of intervertebral disc (IVD) matrix consequent upon 'degeneration' as a major cause of LBP. Degeneration of the IVD involves increases in degradative enzymes and decreases in the extracellular matrix (ECM) component in a process that is controlled by a range of cytokines and growth factors. Studies have suggested using anabolic growth factors to regenerate the normal matrix of the IVD, hence restoring disc height and reversing degenerative disc disease. However, for such therapies to be successful it is vital that the target cells (i.e. the disc cells) express the appropriate receptors. This immunohistochemical study has for the first time investigated the expression and localization of four potentially beneficial growth factor receptors (i.e. TGFbetaRII, BMPRII, FGFR3 and IGFRI) in non-degenerate and degenerate human IVDs. Receptor expression was quantified across regions of the normal and degenerate disc and showed that cells of the nucleus pulposus (NP) and inner annulus fibrosus (IAF) expressed significantly higher levels of the four growth factor receptors investigated. There were no significant differences between the four growth factor expression in non-degenerate and degenerate biopsies. However, expression of TGFbetaRII, FGFR3 and IGFRI, but not BMP RII, were observed in the ingrowing blood vessels that characterize part of the disease aetiology. In conclusion, this study has demonstrated the expression of the four growth factor receptors at similar levels in the chondrocyte-like cells of the NP and IAF in both non-degenerate and degenerate discs, implicating a role in normal disc homeostasis and suggesting that the application of these growth factors to the degenerate human IVD would stimulate matrix production. However, the expression of some of the growth factor receptors on ingrowing blood vessels might be problematic in a therapeutic approach. Copyright 2005 Pathological Society of Great Britain and Ireland.

Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye

PubMed Central

Morrison, Carolyn A.; Chen, Hao; Cook, Tiffany; Brown, Stuart

2018-01-01

Transcriptional regulators can specify different cell types from a pool of equivalent progenitors by activating distinct developmental programs. The Glass transcription factor is expressed in all progenitors in the developing Drosophila eye, and is maintained in both neuronal and non-neuronal cell types. Glass is required for neuronal progenitors to differentiate as photoreceptors, but its role in non-neuronal cone and pigment cells is unknown. To determine whether Glass activity is limited to neuronal lineages, we compared the effects of misexpressing it in neuroblasts of the larval brain and in epithelial cells of the wing disc. Glass activated overlapping but distinct sets of genes in these neuronal and non-neuronal contexts, including markers of photoreceptors, cone cells and pigment cells. Coexpression of other transcription factors such as Pax2, Eyes absent, Lozenge and Escargot enabled Glass to induce additional genes characteristic of the non-neuronal cell types. Cell type-specific glass mutations generated in cone or pigment cells using somatic CRISPR revealed autonomous developmental defects, and expressing Glass specifically in these cells partially rescued glass mutant phenotypes. These results indicate that Glass is a determinant of organ identity that acts in both neuronal and non-neuronal cells to promote their differentiation into functional components of the eye. PMID:29324767

Thermosensitive hydrogel made of ferulic acid-gelatin and chitosan glycerophosphate.

PubMed

Cheng, Yung-Hsin; Yang, Shu-Hua; Liu, Chia-Ching; Gefen, Amit; Lin, Feng-Huei

2013-02-15

Reactive oxygen species-induced oxidative stress is involved in apoptosis of nucleus pulposus (NP) cells that can alter cellular phenotype and accelerate disc degeneration. Ferulic acid (FA) possesses an excellent antioxidant and anti-inflammatory properties. In the study, we developed the thermosensitive FA-gelatin/chitosan/glycerol phosphate (FA-G/C/GP) hydrogel which was applied as a sustained release system of FA to treat NP cells from the damage caused by oxidative stress. The gelation temperature of the FA-G/C/GP hydrogel was 32.17 °C. NP cells submitted to oxidative stress promoted by H(2)O(2), and post-treated with FA-G/C/GP exhibited down-regulation of MMP-3 and up-regulation aggrecan and type II collagen in mRNA level. The sulfated-glycosaminoglycan production was increased and the apoptosis was inhibited in the post-treatment group. The results suggest that the thermosensitive FA-G/C/GP hydrogel can treat NP cells from the damage caused by oxidative stress and may apply in minimally invasive surgery for NP regeneration. Copyright © 2012 Elsevier Ltd. All rights reserved.

Interaction between DISC1 and CHL1 in regulation of neurite outgrowth.

PubMed

Ren, Jun; Zhao, Tian; Xu, Yiliang; Ye, Haihong

2016-10-01

Disrupted-in-schizophrenia 1 (DISC1), a gene susceptible for major mental illnesses, including schizophrenia, plays multiple roles in neural development, including neuronal proliferation, maturation, migration and neurite outgrowth. DISC1 regulates neurite length via interaction with several intracellular proteins, such as NDEL1, FEZ1 and dysbindin. However, the signal transduction mechanism upstream of DISC1 in regulating neurite outgrowth remains to be elucidated. Here we show that DISC1 interacts with the intracellular domain of close homolog of L1 (CHL1), a member of the L1 family of neural cell adhesion molecules. DISC1 and CHL1 proteins co-localize in growth cones of cortical neurons. Moreover, in neurite outgrowth assay, CHL1 rescues the inhibitory effect of DISC1 on the initial phase of neurite outgrowth. Considering the fact that CHL1 also plays crucial roles in neural development, and its coding gene is associated with schizophrenia, our findings indicate that DISC1 and CHL1 may engage in physical and functional interaction in neural development, supporting the notion that DISC1 regulates neurite outgrowth with a receptor belonging to the neural cell adhesion molecules, and disruption of such interaction may contribute to increased risk for schizophrenia. Copyright © 2016. Published by Elsevier B.V.

Interleukin 1-beta upregulates brain-derived neurotrophic factor, neurotrophin 3 and neuropilin 2 gene expression and NGF production in annulus cells.

PubMed

Gruber, H E; Hoelscher, G L; Bethea, S; Hanley, E N

2012-11-01

The relationship between disc cells, nerves and pain production in the intervertebral disc is poorly understood. Neurotrophins, signaling molecules involved in the survival, differentiation and migration of neurons, and neurite outgrowth, are expressed in non-neuronal tissues including the disc. We hypothesized that three-dimensional exposure of human disc cells to the proinflammatory cytokine IL-1ß in vitro would elevate neurotrophin gene expression levels and production of nerve growth factor (NGF). Cells isolated from Thompson grade III and IV discs were cultured for 14 days under control conditions or with addition of 10(2) pM IL-1ß; mRNA was isolated and conditioned media assayed for NGF content. IL-1ß exposure in three-dimensional culture significantly increased expression of neurotrophin 3, brain-derived neurotrophic factor, and neuropilin 2 compared to controls. IL-1ß-exposed cells showed significantly increased NGF production compared to controls. Findings support our hypothesis, expand previous data concerning expression of neurotrophins, and provide the first documented expression of neurotrophin 3 and neuropilin 2. Our results have direct translational relevance, because they address the primary clinical issue of low back pain and open the possibility of novel analgesic therapies using specific small-molecular antagonists to neurotrophins.

An immunohistochemical study of the triangular fibrocartilage complex of the wrist: regional variations in cartilage phenotype

PubMed Central

Milz, S; Sicking, B; Sprecher, C M; Putz, R; Benjamin, M

2007-01-01

The triangular fibrocartilage complex (TFCC) transmits load from the wrist to the ulna and stabilizes the distal radioulnar joint. Damage to it is a major cause of wrist pain. Although its basic structure is well established, little is known of its molecular composition. We have analysed the immunohistochemical labelling pattern of the extracellular matrix of the articular disc and the meniscal homologue of the TFCC in nine elderly individuals (age range 69–96 years), using a panel of monoclonal antibodies directed against collagens, glycosaminoglycans, proteoglycans and cartilage oligomeric matrix protein (COMP). Although many of the molecules (types I, III and VI collagen, chondroitin 4 sulphate, dermatan sulphate and keratan sulphate, the oversulphated epitope of chondroitin 6 sulphate, versican and COMP) were found in all parts of the TFCC, aggrecan, link protein and type II collagen were restricted to the articular disc and to entheses. They were thus not a feature of the meniscal homologue. The shift in tissue phenotype within the TFCC, from a fibrocartilaginous articular disc to a more fibrous meniscal homologue, correlates with biomechanical data suggesting that the radial region is stiff and subject to considerable stress concentration. The presence of aggrecan, link protein and type II collagen in the articular disc could explain why the TFCC is destroyed in rheumatoid arthritis, given that it has been suggested that autoimmunity to these antigens results in the destruction of articular cartilage. The differential distribution of aggrecan within the TFCC is likely to be reflected by regional differences in water content and mobility on the radial and ulnar side. This needs to be taken into account in the design of improved MRI protocols for visualizing this ulnocarpal complex of the wrist. PMID:17532798

A prenatal interruption of DISC1 function in the brain exhibits a lasting impact on adult behaviors, brain metabolism, and interneuron development.

PubMed

Deng, Dazhi; Jian, Chongdong; Lei, Ling; Zhou, Yijing; McSweeney, Colleen; Dong, Fengping; Shen, Yilun; Zou, Donghua; Wang, Yonggang; Wu, Yuan; Zhang, Limin; Mao, Yingwei

2017-10-17

Mental illnesses like schizophrenia (SCZ) and major depression disorder (MDD) are devastating brain disorders. The SCZ risk gene, disrupted in schizophrenia 1 ( DISC1 ), has been associated with neuropsychiatric conditions. However, little is known regarding the long-lasting impacts on brain metabolism and behavioral outcomes from genetic insults on fetal NPCs during early life. We have established a new mouse model that specifically interrupts DISC1 functions in NPCs in vivo by a dominant-negative DISC1 (DN-DISC1) with a precise temporal and spatial regulation. Interestingly, prenatal interruption of mouse Disc1 function in NPCs leads to abnormal depression-like deficit in adult mice. Here we took a novel unbiased metabonomics approach to identify brain-specific metabolites that are significantly changed in DN-DISC1 mice. Surprisingly, the inhibitory neurotransmitter, GABA, is augmented. Consistently, parvalbumin (PV) interneurons are increased in the cingulate cortex, retrosplenial granular cortex, and motor cortex. Interestingly, somatostatin (SST) positive and neuropeptide Y (NPY) interneurons are decreased in some brain regions, suggesting that DN-DISC1 expression affects the localization of interneuron subtypes. To further explore the cellular mechanisms that cause this change, DN-DISC1 suppresses proliferation and promotes the cell cycle exit of progenitors in the medial ganglionic eminence (MGE), whereas it stimulates ectopic proliferation of neighboring cells through cell non-autonomous effect. Mechanistically, it modulates GSK3 activity and interrupts Dlx2 activity in the Wnt activation. In sum, our results provide evidence that specific genetic insults on NSCs at a short period of time could lead to prolonged changes of brain metabolism and development, eventually behavioral defects.

Localised JAK/STAT Pathway Activation Is Required for Drosophila Wing Hinge Development

PubMed Central

Johnstone, Kirsty; Wells, Richard E.; Strutt, David; Zeidler, Martin P.

2013-01-01

Extensive morphogenetic remodelling takes place during metamorphosis from a larval to an adult insect body plan. These changes are particularly intricate in the generation of the dipteran wing hinge, a complex structure that is derived from an apparently simple region of the wing imaginal disc. Using the characterisation of original outstretched alleles of the unpaired locus as a starting point, we demonstrate the role of JAK/STAT pathway signalling in the process of wing hinge development. We show that differences in JAK/STAT signalling within the proximal most of three lateral folds present in the wing imaginal disc is required for fold morphology and the subsequent differentiation of the first and second auxiliary sclerites as well as the posterior notal wing process. Changes in these domains are consistent with the established fate map of the wing disc. We show that outstretched wing posture phenotypes arise from the loss of a region of Unpaired expression in the proximal wing fold and demonstrate that this results in a decrease in JAK/STAT pathway activity. Finally we show that reduction of JAK/STAT pathway activity within the proximal wing fold is sufficient to phenocopy the outstretched phenotype. Taken together, we suggest that localised Unpaired expression and hence JAK/STAT pathway activity, is required for the morphogenesis of the adult wing hinge, providing new insights into the link between signal transduction pathways, patterning and development. PMID:23741461

Dysregulation of YAP by the Hippo pathway is involved in intervertebral disc degeneration, cell contact inhibition, and cell senescence.

PubMed

Zhang, Cong; Wang, Feng; Xie, Zhiyang; Chen, Lu; Sinkemani, Arjun; Yu, Haomin; Wang, Kun; Mao, Lu; Wu, Xiaotao

2018-01-05

The Hippo pathway plays important roles in wound healing, tissue repair and regeneration, and in the treatment of degenerative diseases, by regulating cell proliferation and apoptosis in mammals. Intervertebral disc degeneration (IDD) is one of the major causes of low back pain, a widespread issue associated with a heavy economic burden. However, the mechanism underlying how the Hippo pathway regulates IDD is not well understood. Here, we demonstrate that the Hippo pathway is involved in natural IDD. Activation and dephosphorylation of yes-associated protein (YAP) were observed in younger rat discs, and decreased gradually with age. Surprisingly, Hippo pathway suppression was accompanied by overexpression of YAP, caused by acute disc injury, suggesting a limited ability for self-repair in IDD. We also demonstrated that YAP is inhibited by cell-to-cell contact via the Hippo pathway in vitro . Phosphorylation by large tumor suppressor kinases 1/2 (LATS1/2) led to cytoplasmic translocation and inactivation of YAP. YAP dephosphorylation was mainly localized in the nucleus and regulated by the Hippo pathway, whereas YAP dephosphorylation occurred in the cytoplasm and was associated with nucleus pulposus cell (NPC) senescence. Moreover, NPCs were transfected with shYAP and it accelerates the premature senescence of cells by interfered Hippo pathway through YAP. Therefore, our results indicate that the Hippo pathway plays an important role in maintaining the homeostasis of intervertebral discs and controlling NPC proliferation.

Vertebral Augmentation can Induce Early Signs of Degeneration in the Adjacent Intervertebral Disc: Evidence from a Rabbit Model.

PubMed

Feng, Zhiyun; Chen, Lunhao; Hu, Xiaojian; Yang, Ge; Wang, Yue; Chen, Zhong

2018-04-11

An experimental study. The aim of this study was to determine the effect of polymethylmethacrylate (PMMA) augmentation on the adjacent disc. Vertebral augmentation with PMMA reportedly may predispose the adjacent vertebra to fracture. The influence of PMMA augmentation on the adjacent disc, however, remains unclear. Using a retroperitoneal approach, PMMA augmentation was performed for 23 rabbits. For each animal, at least one vertebra was augmented with 0.2 to 0.3 mL PMMA. The disc adjacent to the augmented vertebra and a proximal control disc were studied using magnetic resonance (MR) imaging, histological and molecular level evaluation at 1, 3, and 6 months postoperatively. Marrow contact channels in the endplate were quantified in histological slices and number of invalid channels (those without erythrocytes inside) was rated. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to determine disc cell apoptosis. On MR images, the signal and height of the adjacent disc did not change 6 months after vertebral augmentation. Histological scores of the adjacent disc increased over time, particularly for the nucleus pulposus. The adjacent disc had greater nucleus degeneration score than the control disc at 3 months (5.7 vs. 4.5, P < 0.01) and 6 months (6.9 vs. 4.4, P < 0.001). There were more invalid marrow contact channels in the endplate of augmented vertebra than the control (43.3% vs. 11.1%, P < 0.01). mRNA of ADAMTS-5, MMP-13, HIF-1α, and caspase-3 were significantly upregulated in the adjacent disc at 3 and 6 months (P < 0.05 for all). In addition, there were more TUNEL-positive cells in the adjacent disc than in the control disc (43.4% vs. 24.0%, P < 0.05) at 6 months postoperatively. Vertebral augmentation can induce early degenerative signs in the adjacent disc, which may be due to impaired nutrient supply to the disc. N/A.

SUSCEPTIBILITY OF RESPIRATORY ISOLATES OF STREPTOCOCCUS PNEUMONIAE ISOLATED FROM CHILDREN HOSPITALIZED IN THE CLINICAL CENTER NIS.

PubMed

Dinić, Marina M; Mladenović Antić, Snezana; Kocić, Branislava; Stanković Dordević, Dobrila; Vrbić, Miodrag; Bogdanović, Milena

2016-01-01

Streptococcus pneumoniae is one of the most common causes of respiratory infections. The aim was to study the susceptibility to antimicrobial agents of respiratory isolates ofStreptococcus pneumoniae obtained from hospitalized children. A total of 190 respiratory pneumococcal isolates obtained from children aged from 0 to 14 years were isolated and identified by using standard microbiological methods. Susceptibility to oxacillin, erythromycin, clindamycin, tetracycline, cotrimoxazole, ofloxacin and rifampicin was tested by disc diffusion method. Minimal inhibitory concentrations for amoxicillin and ceftriaxone were determined by means of E test. The macrolide-resistant phenotype was detected by double disc diffusion test. All tested isolates were susceptible to amoxicillin and ceftriaxone. The minimal amoxicillin concentration inhibiting the growth of 50% of isolates and of 90% of isolates was 0.50 microg/ml and 1.0 microg/ml, respectively and the minimal ceftriaxone concentration inhibiting the growth of 50% of isolates and of 90% of isolates was 0.25 microg/ml and 0.50 microg/ml, respectively. Susceptibility to erythromycin and clindamycin was observed in 21.6% and 29.47% of isolates, respectively. The resistence to macrolides-M phenotype was detected in 10.07% of isolates and constitutive macrolide-lincosamide-streptogramin phenotype (constitutive MLS phenotype) was found in 89.93% of isolates. All tested isolates were susceptible to ofloxacin and rifampicin. Amoxicillin could be the therapy of choice in pediatric practice. The macrolides should not be recommended for the empirical therapy of pneumococcal respiratory tract infection in our local area.

Comparative Evaluation of Multiplex PCR and Routine Laboratory Phenotypic Methods for Detection of Carbapenemases among Gram Negative Bacilli.

PubMed

Solanki, Rachana; Vanjari, Lavanya; Subramanian, Sreevidya; B, Aparna; E, Nagapriyanka; Lakshmi, Vemu

2014-12-01

Carbapenem resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of Multiplex PCR for rapid detection of carbapenemase genes among carbapenem resistant Gram negative bacteria in comparison with the existing phenotypic methods like modified Hodge test (MHT), combined disc test (CDT) and automated methods. A total of 100 Carbapenem resistant clinical isolates, [Escherichia coli (25), Klebsiella pneumoniae (35) P. aeruginosa (18) and Acinetobacter baumannii (22)] were screened for the presence of carbapenemases (bla NDM-1, bla VIM , blaIMP and blaKPC genes) by phenotype methods such as the modified Hodge test (MHT) and combined disc test (CDT) and the molecular methods such as Multiplex PCR. Seventy of the 100 isolates were MHT positive while, 65 isolates were positive by CDT. All the CDT positive isolates with EDTA and APB were Metallo betalactamase (MBL) and K. pneumoniae carbapenemase (KPC) producers respectively. bla NDM-1 was present as a lone gene in 44 isolates. In 14 isolates bla NDM-1 gene was present with blaKPC gene, and in one isolate bla NDM-1 gene was present with blaVIM , gene. Only one E. coli isolate had a lone blaKPC gene. We didn't find bla IMP gene in any of the isolates. Neither of the genes could be detected in 35 isolates. Accurate detection of the genes related with carbapenemase production by Molecular methods like Multiplex PCR overcome the limitations of the phenotypic methods and Automated systems.

Computer Vision for High-Throughput Quantitative Phenotyping: A Case Study of Grapevine Downy Mildew Sporulation and Leaf Trichomes.

PubMed

Divilov, Konstantin; Wiesner-Hanks, Tyr; Barba, Paola; Cadle-Davidson, Lance; Reisch, Bruce I

2017-12-01

Quantitative phenotyping of downy mildew sporulation is frequently used in plant breeding and genetic studies, as well as in studies focused on pathogen biology such as chemical efficacy trials. In these scenarios, phenotyping a large number of genotypes or treatments can be advantageous but is often limited by time and cost. We present a novel computational pipeline dedicated to estimating the percent area of downy mildew sporulation from images of inoculated grapevine leaf discs in a manner that is time and cost efficient. The pipeline was tested on images from leaf disc assay experiments involving two F 1 grapevine families, one that had glabrous leaves (Vitis rupestris B38 × 'Horizon' [RH]) and another that had leaf trichomes (Horizon × V. cinerea B9 [HC]). Correlations between computer vision and manual visual ratings reached 0.89 in the RH family and 0.43 in the HC family. Additionally, we were able to use the computer vision system prior to sporulation to measure the percent leaf trichome area. We estimate that an experienced rater scoring sporulation would spend at least 90% less time using the computer vision system compared with the manual visual method. This will allow more treatments to be phenotyped in order to better understand the genetic architecture of downy mildew resistance and of leaf trichome density. We anticipate that this computer vision system will find applications in other pathosystems or traits where responses can be imaged with sufficient contrast from the background.

Genetic and functional studies of the intervertebral disc: a novel murine intervertebral disc model.

PubMed

Pelle, Dominic W; Peacock, Jacqueline D; Schmidt, Courtney L; Kampfschulte, Kevin; Scholten, Donald J; Russo, Scott S; Easton, Kenneth J; Steensma, Matthew R

2014-01-01

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.

Genetic and Functional Studies of the Intervertebral Disc: A Novel Murine Intervertebral Disc Model

PubMed Central

Pelle, Dominic W.; Peacock, Jacqueline D.; Schmidt, Courtney L.; Kampfschulte, Kevin; Scholten, Donald J.; Russo, Scott S.; Easton, Kenneth J.; Steensma, Matthew R.

2014-01-01

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration. PMID:25474689

Activin receptor inhibition by Smad2 regulates Drosophila wing disc patterning through BMP-response elements

PubMed Central

Peterson, Aidan J.; O'Connor, Michael B.

2013-01-01

Imaginal disc development in Drosophila requires coordinated cellular proliferation and tissue patterning. In our studies of TGFβ superfamily signaling components, we found that a protein null mutation of Smad2, the only Activin subfamily R-Smad in the fruit fly, produces overgrown wing discs that resemble gain of function for BMP subfamily signaling. The wing discs are expanded specifically along the anterior-posterior axis, with increased proliferation in lateral regions. The morphological defect is not observed in mutants for the TGFβ receptor baboon, and epistasis tests showed that baboon is epistatic to Smad2 for disc overgrowth. Rescue experiments indicate that Baboon binding, but not canonical transcription factor activity, of Smad2 is required for normal disc growth. Smad2 mutant discs generate a P-Mad stripe that is narrower and sharper than the normal gradient, and activation targets are correspondingly expressed in narrowed domains. Repression targets of P-Mad are profoundly mis-regulated, with brinker and pentagone reporter expression eliminated in Smad2 mutants. Loss of expression requires a silencer element previously shown to be controlled by BMP signaling. Epistasis experiments show that Baboon, Mad and Schnurri are required to mediate the ectopic silencer output in the absence of Smad2. Taken together, our results show that loss of Smad2 permits promiscuous Baboon activity, which represses genes subject to control by Mad-dependent silencer elements. The absence of Brinker and Pentagone in Smad2 mutants explains the compound wing disc phenotype. Our results highlight the physiological relevance of substrate inhibition of a kinase, and reveal a novel interplay between the Activin and BMP pathways. PMID:23293296

Motivation, characterization, and strategy for tissue engineering the temporomandibular joint disc.

PubMed

Detamore, Michael S; Athanasiou, Kyriacos A

2003-12-01

The purpose of this review is to serve as the standard point of reference in guiding researchers investigating the tissue engineering of the temporomandibular joint (TMJ) disc. Tissue engineering of the TMJ disc is in its infancy, and currently there exists a gap between the tissue engineering community and the TMJ characterization community. The primary goal is to help bridge that gap by consolidating the characterization studies here as a reference to researchers attempting to tissue engineer the TMJ disc. A brief review of TMJ anatomy is provided, along with a description of relevant pathology, current treatment, and a rationale for engineering the TMJ disc. The biochemical composition and organization of the disc are reviewed, including glycosaminoglycan (GAG) and collagen content. The collagen of the disc is almost exclusively type I and primarily runs anteroposteriorly through the center and in a ringlike fashion around the periphery. The GAG content is approximately an order of magnitude less than that of hyaline cartilage, and although the distribution is not entirely clear, it seems as though chondroitin and dermatan sulfate are by far the primary GAGs. Cellular characterization and mechanical properties under compression, tension, and shear are reviewed as well. The cells of the disc are not chondrocytes, but rather resemble fibrocytes and fibrochondrocytes and may be of the same lineage. Mechanically, the disc is certainly anisotropic and nonhomogeneous. Finally, a review of efforts in tissue engineering and cell culture studies of the disc is provided and we close with a description of the direction we envision/propose for successful tissue engineering of the TMJ disc.

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. II. Quantity and quality of light required

NASA Technical Reports Server (NTRS)

Van Volkenburgh, E.; Cleland, R. E.; Watanabe, M.

1990-01-01

The quantity and quality of light required for light-stimulated cell expansion in leaves of Phaseolus vulgaris L. have been determined. Seedlings were grown in dim red light (RL; 4 micromoles photons m-2 s-1) until cell division in the primary leaves was completed, then excised discs were incubated in 10 mM sucrose plus 10 mM KCl in a variety of light treatments. The growth response of discs exposed to continuous white light (WL) for 16 h was saturated at 100 micromoles m-2 s-1, and did not show reciprocity. Extensive, but not continuous, illumination was needed for maximal growth. The wavelength dependence of disc expansion was determined from fluence-response curves obtained from 380 to 730 nm provided by the Okazaki Large Spectrograph. Blue (BL; 460 nm) and red light (RL; 660 nm) were most effective in promoting leaf cell growth, both in photosynthetically active and inhibited leaf discs. Far-red light (FR; 730 nm) reduced the effectiveness of RL, but not BL, indicating that phytochrome and a separate blue-light receptor mediate expansion of leaf cells.

Evidence that the Dictyostelium STAT protein Dd-STATa plays a role in the differentiation of inner basal disc cells and identification of a promoter element essential for expression in these cells.

PubMed

Shimada, Nao; Maruo, Toshinari; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

2005-02-01

Dd-STATa, a Dictyostelium homolog of the metazoan STAT (signal transducers and activators of transcription) proteins, is necessary in the slug for correct entry into culmination. Dd-STATa-null mutant fails to culminate and its phenotype correlates with the loss of a funnel-shaped core region, the pstAB core region, which expresses both the ecmA and ecmB genes. To understand how the differentiation of pstAB core cells is regulated, we identified an EST that is expressed in the core cells of normal slugs but down-regulated in the Dd-STATa-null mutant. This EST, SSK348, encodes a close homolog of the Dictyostelium acetyl-CoA synthetase (ACS). A promoter fragment of the cognate gene, aslA (acetyl-CoA synthetase-like A), was fused to a lacZ reporter and the expression pattern determined. As expected from the behavior of the endogenous aslA gene, the aslA::lacZ fusion gene is not expressed in Dd-STATa-null slugs. In parental cells, the aslA promoter is first activated in the funnel-shaped core cells located at the slug anterior, the "pstAB core." During culmination, the pstAB core cells move down, through the prespore cells, to form the inner part of the basal disc. As the spore mass climbs the stalk, the aslA gene comes to be expressed in cells of the upper and lower cups, structures that cradle the spore head. Deletion and point mutation analyses of the promoter identified an AT-rich sequence that is necessary for expression in the pstAB core. This acts in combination with repressor regions that prevent ectopic aslA expression in the pre-stalk regions of slugs and the stalks of culminants. Thus, this study confirms that Dd-STATa is necessary for the differentiation of pstAB core cells, by showing that it is needed for the activation of the aslA gene. It also identifies aslA promoter elements that are likely to be regulated, directly or indirectly, by Dd-STATa.

Biological treatment strategies for disc degeneration: potentials and shortcomings

PubMed Central

Nerlich, Andreas G.; Boos, Norbert

2006-01-01

Recent advances in molecular biology, cell biology and material sciences have opened a new emerging field of techniques for the treatment of musculoskeletal disorders. These new treatment modalities aim for biological repair of the affected tissues by introducing cell-based tissue replacements, genetic modifications of resident cells or a combination thereof. So far, these techniques have been successfully applied to various tissues such as bone and cartilage. However, application of these treatment modalities to cure intervertebral disc degeneration is in its very early stages and mostly limited to experimental studies in vitro or in animal studies. We will discuss the potential and possible shortcomings of current approaches to biologically cure disc degeneration by gene therapy or tissue engineering. Despite the increasing number of studies examining the therapeutic potential of biological treatment strategies, a practicable solution to routinely cure disc degeneration might not be available in the near future. However, knowledge gained from these attempts might be applied in a foreseeable future to cure the low back pain that often accompanies disc degeneration and therefore be beneficial for the patient. PMID:16983559

Wnt signaling activates Shh signaling in early postnatal intervertebral discs, and re-activates Shh signaling in old discs in the mouse.

PubMed

Winkler, Tamara; Mahoney, Eric J; Sinner, Debora; Wylie, Christopher C; Dahia, Chitra Lekha

2014-01-01

Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease.

Social defeat interacts with Disc1 mutations in the mouse to affect behavior.

PubMed

Haque, F Nipa; Lipina, Tatiana V; Roder, John C; Wong, Albert H C

2012-08-01

DISC1 (Disrupted-in-schizophrenia 1) is a strong candidate susceptibility gene for psychiatric disease that was originally discovered in a family with a chromosomal translocation severing this gene. Although the family members with the translocation had an identical genetic mutation, their clinical diagnosis and presentation varied significantly. Gene-environment interactions have been proposed as a mechanism underlying the complex heritability and variable phenotype of psychiatric disorders such as major depressive disorder and schizophrenia. We hypothesized that gene-environment interactions would affect behavior in a mutant Disc1 mouse model. We examined the effect of chronic social defeat (CSD) as an environmental stressor in two lines of mice carrying different Disc1 point mutations, on behaviors relevant to psychiatric illness: locomotion in a novel open field (OF), pre-pulse inhibition (PPI) of the acoustic startle response, latent inhibition (LI), elevated plus maze (EPM), forced swim test (FST), sucrose consumption (SC), and the social interaction task for sociability and social novelty (SSN). We found that Disc1-L100P +/- and wild-type mice have similar anxiety responses to CSD, while Q31L +/- mice had a very different response. We also found evidence of significant gene-environment interactions in the OF, EPM and SSN. Copyright © 2012 Elsevier B.V. All rights reserved.

Linkage to 10q22 for maximum intraocular pressure and 1p32 for maximum cup-to-disc ratio in an extended primary open-angle glaucoma pedigree.

PubMed

Charlesworth, Jac C; Dyer, Thomas D; Stankovich, Jim M; Blangero, John; Mackey, David A; Craig, Jamie E; Green, Catherine M; Foote, Simon J; Baird, Paul N; Sale, Michèle M

2005-10-01

The purpose of this study was to identify genetic contributions to primary open-angle glaucoma (POAG) through investigations of two quantitative components of the POAG phenotype. Genome-wide multipoint variance-components linkage analyses of maximum recorded intraocular pressure (IOP) and maximum vertical cup-to-disc ratio were conducted on data from a single, large Australian POAG pedigree that has been found to segregate the myocilin Q368X mutation in some individuals. Multipoint linkage analysis of maximum recorded IOP produced a peak LOD score of 3.3 (P = 0.00015) near marker D10S537 on 10q22, whereas the maximum cup-to-disc ratio produced a peak LOD score of 2.3 (P = 0.00056) near markers D1S197 to D1S220 on 1p32. Inclusion of the myocilin Q368X mutation as a covariate provided evidence of an interaction between this mutation and the IOP and cup-to-disc ratio loci. Significant linkage has been identified for maximum IOP and suggestive linkage for vertical cup-to-disc ratio. Identification of genes contributing to the variance of these traits will enhance understanding of the pathophysiology of POAG as a whole.

Trisomy 15 mosaicism and uniparental disomy (UPD) in a liveborn infant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milunsky, J.M.; Wyandt, H.E.; Amos, J.A.

We describe a liveborn infant with UPD in association with trisomy 15 mosaicism. Third trimester amniocentesis was performed for suspected IUGR. Results revealed 46,XX/47,XX,+15. The infant initially had respiratory distress and fed poorly. Symmetrical growth retardation, craniofacial dysmorphism, excess nuchal folds, a heart murmur, hypermobile joints, minor limb abnormalities, absent spontaneous movement and an abnormal cry were noted. Further study showed complex heart defects, including VSD and PDA, a left choroid plexus cyst, 13 ribs bilaterally, abnormal optic discs, abnormal visual evoked potentials and abnormal auditory brain stem responses. The infant died at 6 weeks of life from cardio-respiratory complications.more » Blood chromosomes were normal, 46,XX in 100 cells. Parental blood chromosomes were normal. Skin biopsy revealed 46,XX/47,XX,+15 in 40/50 (80%) cells as did autopsy lung tissue. Molecular analysis of the infant`s blood revealed maternal uniparental heterodisomy for chromosome 15 in the 46,XX cell line. Microsatellite analysis demonstrated that the extra chromosome originated from a maternal meiosis I nondisjunction. To our knowledge, this is the first liveborn infant with mosaic trisomy 15 and UPD in the diploid cells. Trisomy 15, heretofore, has been regarded as nonviable, even in mosaic form. While maternal UPD is associated with the Prader-Willi syndrome phenotype, mosaicism for trisomy 15 has been reported only when confined to the placenta. UPD in this case generally complicated prediction of the phenotype and raises the question whether all cases with UPD 15 should have more than one tissue studied to determine undetected trisomy 15.« less

Occupational and genetic risk factors associated with intervertebral disc disease.

PubMed

Virtanen, Iita M; Karppinen, Jaro; Taimela, Simo; Ott, Jürg; Barral, Sandra; Kaikkonen, Kaisu; Heikkilä, Olli; Mutanen, Pertti; Noponen, Noora; Männikkö, Minna; Tervonen, Osmo; Natri, Antero; Ala-Kokko, Leena

2007-05-01

Cross-sectional epidemiologic study. To evaluate the interaction between known genetic risk factors and whole-body vibration for symptomatic intervertebral disc disease (IDD) in an occupational sample. Risk factors of IDD include, among others, whole-body vibration and heredity. In this study, the importance of a set of known genetic risk factors and whole-body vibration was evaluated in an occupational sample of train engineers and sedentary controls. Eleven variations in 8 genes (COL9A2, COL9A3, COL11A2, IL1A, IL1B, IL6, MMP-3, and VDR) were genotyped in 150 male train engineers with an average of 21-year exposure to whole-body vibration and 61 male paper mill workers with no exposure to vibration. Subjects were classified into IDD-phenotype and asymptomatic groups, based on the latent class analysis. The number of individuals belonging to the IDD-phenotype was significantly higher among train engineers (42% of train engineers vs. 17.5% of sedentary workers; P = 0.005). IL1A -889T allele represented a significant risk factor for the IDD-phenotype both in the single marker allelic association test (P = 0.043) and in the logistic regression analysis (P = 0.01). None of the other allele markers was significantly associated with symptoms when analyzed independently. However, for all the SNP markers considered, whole-body vibration represents a nominally significant risk factor. The results suggest that whole-body vibration is a risk factor for symptomatic IDD. Moreover, whole-body vibration had an additive effect with genetic risk factors increasing the likelihood of belonging to the IDD-phenotype group. Of the independent genetic markers, IL1A -889T allele had strongest association with IDD-phenotype.

Difference in Energy Metabolism of Annulus Fibrosus and Nucleus Pulposus Cells of the Intervertebral Disc

PubMed Central

Salvatierra, Jessica Czamanski; Yuan, Tai Yi; Fernando, Hanan; Castillo, Andre; Gu, Wei Yong; Cheung, Herman S.; Huant, C.-Y. Charles

2011-01-01

Low back pain is associated with intervertebral disc degeneration. One of the main signs of degeneration is the inability to maintain extracellular matrix integrity. Extracellular matrix synthesis is closely related to production of adenosine triphosphate (i.e. energy) of the cells. The intervertebral disc is composed of two major anatomical regions: annulus fibrosus and nucleus pulposus, which are structurally and compositionally different, indicating that their cellular metabolisms may also be distinct. The objective of this study was to investigate energy metabolism of annulus fibrosus and nucleus pulposus cells with and without dynamic compression, and examine differences between the two cell types. Porcine annulus and nucleus tissues were harvested and enzymatically digested. Cells were isolated and embedded into agarose constructs. Dynamically loaded samples were subjected to a sinusoidal displacement at 2 Hz and 15% strain for 4 h. Energy metabolism of cells was analyzed by measuring adenosine triphosphate content and release, glucose consumption, and lactate/nitric oxide production. A comparison of those measurements between annulus and nucleus cells was conducted. Annulus and nucleus cells exhibited different metabolic pathways. Nucleus cells had higher adenosine triphosphate content with and without dynamic loading, while annulus cells had higher lactate production and glucose consumption. Compression increased adenosine triphosphate release from both cell types and increased energy production of annulus cells. Dynamic loading affected energy metabolism of intervertebral disc cells, with the effect being greater in annulus cells. PMID:21625336

Fortifying the Bone-Implant Interface Part 1: An In Vitro Evaluation of 3D-Printed and TPS Porous Surfaces.

PubMed

MacBarb, Regina F; Lindsey, Derek P; Bahney, Chelsea S; Woods, Shane A; Wolfe, Mark L; Yerby, Scott A

2017-01-01

An aging society and concomitant rise in the incidence of impaired bone health have led to the need for advanced osteoconductive spinal implant surfaces that promote greater biological fixation ( e.g. for interbody fusion cages, sacroiliac joint fusion implants, and artificial disc replacements). Additive manufacturing, i.e. 3D-printing, may improve bone integration by generating biomimetic spinal implant surfaces that mimic bone morphology. Such surfaces may foster an enhanced cellular response compared to traditional implant surfacing processes. This study investigated the response of human osteoblasts to additive manufactured (AM) trabecular-like titanium implant surfaces compared to traditionally machined base material with titanium plasma spray (TPS) coated surfaces, with and without a nanocrystalline hydroxyapatite (HA) coating. For TPS-coated discs, wrought Ti6Al4V ELI was machined and TPS-coating was applied. For AM discs, Ti6Al4V ELI powder was 3D-printed to form a solid base and trabecular-like porous surface. The HA-coating was applied via a precipitation dip-spin method. Surface porosity, pore size, thickness, and hydrophilicity were characterized. Initial cell attachment, proliferation, alkaline phosphatase (ALP) activity, and calcium production of hFOB cells ( n =5 per group) were measured. Cells on AM discs exhibited expedited proliferative activity. While there were no differences in mean ALP expression and calcium production between TPS and AM discs, calcium production on the AM discs trended 48% higher than on TPS discs ( p =0.07). Overall, HA-coating did not further enhance results compared to uncoated TPS and AM discs. Results demonstrate that additive manufacturing allows for controlled trabecular-like surfaces that promote earlier cell proliferation and trends toward higher calcium production than TPS coating. Results further showed that nanocrystalline HA may not provide an advantage on porous titanium surfaces. Additive manufactured porous titanium surfaces may induce a more osteogenic environment compared to traditional TPS, and thus present as an attractive alternative to TPS-coating for orthopedic spinal implants.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF-5-deficient mice.

PubMed

Maier, Jennifer A; Harfe, Brian D

2011-11-15

The transition of the mouse embryonic notochord into nuclei pulposi was determined ("fate mapped") in vivo in growth and differentiating factor-5 (GDF-5)-null mice using the Shhcre and R26R alleles. To determine whether abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5-null mice. The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5-null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5-null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or result from progressive postnatal degeneration of nuclei pulposi. Gdf-5 messenger RNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5-null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24-week-old mice. Our Gdf-5 messenger RNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate-mapping experiments revealed that notochord cells in Gdf-5-null mice correctly form nuclei pulposi. Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5-null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF5-deficient mice

PubMed Central

Maier, Jennifer A.; Harfe, Brian D.

2011-01-01

Study Design The transition of the mouse embryonic notochord into nuclei pulposi was determined (“fate mapped”) in vivo in GDF-5 null mice using the Shhcre and R26R alleles. Objective To determine if abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5 null mice. Summary of Background Data The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5 null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5 null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or resulted from progressive postnatal degeneration of nuclei pulposi. Methods Gdf-5 mRNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5 null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24 week old mice. Results Our Gdf-5 mRNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate mapping experiments revealed that notochord cells in Gdf-5 null mice correctly form nuclei pulposi. Conclusion Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5 null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects. PMID:21278629

Evaluation of intervertebral disc regeneration with implantation of bone marrow mesenchymal stem cells (BMSCs) using quantitative T2 mapping: a study in rabbits.

PubMed

Cai, Feng; Wu, Xiao-Tao; Xie, Xin-Hui; Wang, Feng; Hong, Xin; Zhuang, Su-Yang; Zhu, Lei; Rui, Yun-Feng; Shi, Rui

2015-01-01

The aim of the study was to investigate the curative effects of transplantation of bone marrow mesenchymal stem cells (BMSCs) on intervertebral disc regeneration and to investigate the feasibility of the quantitative T2 mapping method for evaluating repair of the nucleus pulposus after implantation of BMSCs. Forty-eight New Zealand white rabbits were used to establish the lumber disc degenerative model by stabbing the annulus fibrosus and then randomly divided into four groups, i.e. two weeks afterwards, BMSCs or phosphate-buffered saline (PBS) were transplanted into degenerative discs (BMSCs group and PBS group), while the operated rabbits without implantation of BMSCs or PBS served as the sham group and the rabbits without operation were used as the control group. At weeks two, six and ten after operation, the T2 values and disc height indices (DHI) were calculated by magnetic resonance imaging (MRI 3.0 T), and the gene expressions of type II collagen (COL2) and aggrecan (ACAN) in degenerative discs were evaluated by real-time reverse transcription polymerase chain reaction (RT-PCR). T2 values for the nucleus pulposus were correlated with ACAN or COL2 expression by regression analysis. Cell clusters, disorganised fibres, interlamellar glycosaminoglycan (GAG) matrix and vascularisation were observed in lumber degenerative discs. BMSCs could be found to survive in intervertebral discs and differentiate into nucleus pulposus-like cells expressing COL2 and ACAN. The gene expression of COL2 and ACAN increased during ten weeks after transplantation as well as the T2 signal intensity and T2 value. The DHI in the BMSCs group decreased more slowly than that in PBS and sham groups. The T2 value correlated significantly with the gene expression of ACAN and COL2 in the nucleus pulposus. Transplantation of BMSCs was able to promote the regeneration of degenerative discs. Quantitative and non-invasive T2 mapping could be used to evaluate the regeneration of the nucleus pulposus with good sensitivity.

Intra-discal injection of autologous, hypoxic cultured bone marrow-derived mesenchymal stem cells in five patients with chronic lower back pain: a long-term safety and feasibility study.

PubMed

Elabd, Christian; Centeno, Christopher J; Schultz, John R; Lutz, Gregory; Ichim, Thomas; Silva, Francisco J

2016-09-01

Chronic low back pain due to disc degeneration represents a major social and economic burden worldwide. The current standard of care is limited to symptomatic relief and no current approved therapy promotes disc regeneration. Bone marrow-derived mesenchymal stem cells (MSCs) are easily accessible and well characterized. These MSCs are multipotent and exhibit great tissue regenerative potential including bone, cartilage, and fibrous tissue regeneration. The use of this cell-based biologic for treating protruding disc herniation and/or intervertebral disc degeneration is a promising therapeutic strategy, due to their known regenerative, immuno-modulatory and anti-inflammatory properties. Five patients diagnosed with degenerative disc disease received an intra-discal injection of autologous, hypoxic cultured, bone marrow-derived mesenchymal stem cells (15.1-51.6 million cells) as part of a previous study. These patients were re-consented to participate in this study in order to assess long-term safety and feasibility of intra-discal injection of autologous, hypoxic cultured, bone marrow-derived mesenchymal stem cells 4-6 years post mesenchymal stem cell infusion. The follow-up study consisted of a physical examination, a low back MRI, and a quality of life questionnaire. Patients' lower back MRI showed absence of neoplasms or abnormalities surrounding the treated region. Based on the physical examination and the quality of life questionnaire, no adverse events were reported due to the procedure or to the stem cell treatment 4-6 years post autologous, hypoxic cultured mesenchymal stem cell infusion. All patients self-reported overall improvement, as well as improvement in strength, post stem cell treatment, and four out of five patients reported improvement in mobility. This early human clinical data suggests the safety and feasibility of the clinical use of hypoxic cultured bone marrow-derived mesenchymal stem cells for the treatment of lower back pain due to degenerative disc disorders and support further studies utilizing hypoxic cultured bone marrow-derived stem cells. The overall improvements reported are encouraging, but a larger double-blind, controlled, randomized clinical study with significant number of patients and implementation of validated endpoint measurements are next steps in order to demonstrate efficacy of this cell-based biologic.

Tumor regression induced by intratumor therapy with a disabled infectious single cycle (DISC) herpes simplex virus (HSV) vector, DISC/HSV/murine granulocyte-macrophage colony-stimulating factor, correlates with antigen-specific adaptive immunity.

PubMed

Ali, Selman A; Lynam, June; McLean, Cornelia S; Entwisle, Claire; Loudon, Peter; Rojas, José M; McArdle, Stephanie E B; Li, Geng; Mian, Shahid; Rees, Robert C

2002-04-01

Direct intratumor injection of a disabled infectious single cycle HSV-2 virus encoding the murine GM-CSF gene (DISC/mGM-CSF) into established murine colon carcinoma CT26 tumors induced a significant delay in tumor growth and complete tumor regression in up to 70% of animals. Pre-existing immunity to HSV did not reduce the therapeutic efficacy of DISC/mGM-CSF, and, when administered in combination with syngeneic dendritic cells, further decreased tumor growth and increased the incidence of complete tumor regression. Direct intratumor injection of DISC/mGM-CSF also inhibited the growth of CT26 tumor cells implanted on the contralateral flank or seeded into the lungs following i.v. injection of tumor cells (experimental lung metastasis). Proliferation of splenocytes in response to Con A was impaired in progressor and tumor-bearer, but not regressor, mice. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-gamma, but not IL-4 cytokine production. Depletion of CD8(+) T cells from regressor splenocytes before in vitro stimulation with the relevant peptide abolished their cytolytic activity, while depletion of CD4(+) T cells only partially inhibited CTL generation. Tumor regression induced by DISC/mGM-CSF virus immunotherapy provides a unique model for evaluating the immune mechanism(s) involved in tumor rejection, upon which tumor immunotherapy regimes may be based.

Haemoglobin content modulated deformation dynamics of red blood cells on a compact disc.

PubMed

Kar, Shantimoy; Ghosh, Uddipta; Maiti, Tapas Kumar; Chakraborty, Suman

2015-12-21

We investigate the deformation characteristics of red blood cells (RBCs) on a rotating compact disc platform. Our study brings out the interplay between haemoglobin content and RBC deformability in a centrifugally actuated microfluidic environment. We reveal that RBC deformations follow the similar trend of principal stress distributed throughout the radial direction, rendering an insight into the mechano-physical processes involved. This study can be used as a diagnostic marker to determine haematological disorders in diseased blood samples tested on compact disc based microfluidic platforms.

Enhancing cell migration in shape-memory alginate-collagen composite scaffolds: In vitro and ex vivo assessment for intervertebral disc repair.

PubMed

Guillaume, Olivier; Naqvi, Syeda Masooma; Lennon, Kerri; Buckley, Conor Timothy

2015-04-01

Lower lumbar disc disorders pose a significant problem in an aging society with substantial socioeconomic consequences. Both inner tissue (nucleus pulposus) and outer tissue (annulus fibrosus) of the intervertebral disc are affected by such debilitating disorders and can lead to disc herniation and lower back pain. In this study, we developed an alginate-collagen composite porous scaffold with shape-memory properties to fill defects occurring in annulus fibrosus tissue of degenerated intervertebral discs, which has the potential to be administered using minimal invasive surgery. In the first part of this work, we assessed how collagen incorporation on preformed alginate scaffolds influences the physical properties of the final composite scaffold. We also evaluated the ability of annulus fibrosus cells to attach, migrate, and proliferate on the composite alginate-collagen scaffolds compared to control scaffolds (alginate only). In vitro experiments, performed in intervertebral disc-like microenvironmental conditions (low glucose and low oxygen concentrations), revealed that for alginate only scaffolds, annulus fibrosus cells agglomerated in clusters with limited infiltration and migration capacity. In comparison, for alginate-collagen scaffolds, annulus fibrosus cells readily attached and colonized constructs, while preserving their typical fibroblastic-like cell morphology with spreading behavior and intense cytoskeleton expression. In a second part of this study, we investigated the effects of alginate-collagen scaffold when seeded with bone marrow derived mesenchymal stem cells. In vitro, we observed that alginate-collagen porous scaffolds supported cell proliferation and extracellular matrix deposition (collagen type I), with secretion amplified by the local release of transforming growth factor-β3. In addition, when cultured in ex vivo organ defect model, alginate-collagen scaffolds maintained viability of transplanted mesenchymal stem cells for up to 5 weeks. Taken together, these findings illustrate the advantages of incorporating collagen as a means to enhance cell migration and proliferation in porous scaffolds which could be used to augment tissue repair strategies. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Genome-wide analysis of pain-, nerve- and neurotrophin -related gene expression in the degenerating human annulus

PubMed Central

2012-01-01

Background In spite of its high clinical relevance, the relationship between disc degeneration and low back pain is still not well understood. Recent studies have shown that genome-wide gene expression studies utilizing ontology searches provide an efficient and valuable methodology for identification of clinically relevant genes. Here we use this approach in analysis of pain-, nerve-, and neurotrophin-related gene expression patterns in specimens of human disc tissue. Control, non-herniated clinical, and herniated clinical specimens of human annulus tissue were studied following Institutional Review Board approval. Results Analyses were performed on more generated (Thompson grade IV and V) discs vs. less degenerated discs (grades I-III), on surgically operated discs vs. control discs, and on herniated vs. control discs. Analyses of more degenerated vs. less degenerated discs identified significant upregulation of well-recognized pain-related genes (bradykinin receptor B1, calcitonin gene-related peptide and catechol-0-methyltransferase). Nerve growth factor was significantly upregulated in surgical vs. control and in herniated vs. control discs. All three analyses also found significant changes in numerous proinflammatory cytokine- and chemokine-related genes. Nerve, neurotrophin and pain-ontology searches identified many matrix, signaling and functional genes which have known importance in the disc. Immunohistochemistry was utilized to confirm the presence of calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1 at the protein level in the human annulus. Conclusions Findings point to the utility of microarray analyses in identification of pain-, neurotrophin and nerve-related genes in the disc, and point to the importance of future work exploring functional interactions between nerve and disc cells in vitro and in vivo. Nerve, pain and neurotrophin ontology searches identified numerous changes in proinflammatory cytokines and chemokines which also have significant relevance to disc biology. Since the degenerating human disc is primarily an avascular tissue site into which disc cells have contributed high levels of proinflammatory cytokines, these substances are not cleared from the tissue and remain there over time. We hypothesize that as nerves grow into the human annulus, they encounter a proinflammatory cytokine-rich milieu which may sensitize nociceptors and exacerbate pain production. PMID:22963171

A t(1;11) translocation linked to schizophrenia and affective disorders gives rise to aberrant chimeric DISC1 transcripts that encode structurally altered, deleterious mitochondrial proteins

PubMed Central

Eykelenboom, Jennifer E.; Briggs, Gareth J.; Bradshaw, Nicholas J.; Soares, Dinesh C.; Ogawa, Fumiaki; Christie, Sheila; Malavasi, Elise L.V.; Makedonopoulou, Paraskevi; Mackie, Shaun; Malloy, Mary P.; Wear, Martin A.; Blackburn, Elizabeth A.; Bramham, Janice; McIntosh, Andrew M.; Blackwood, Douglas H.; Muir, Walter J.; Porteous, David J.; Millar, J. Kirsty

2012-01-01

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a risk factor for psychiatric illness through its disruption by a balanced chromosomal translocation, t(1;11)(q42.1;q14.3), that co-segregates with schizophrenia, bipolar disorder and depression. We previously reported that the translocation reduces DISC1 expression, consistent with a haploinsufficiency disease model. Here we report that, in lymphoblastoid cell lines, the translocation additionally results in the production of abnormal transcripts due to the fusion of DISC1 with a disrupted gene on chromosome 11 (DISC1FP1/Boymaw). These chimeric transcripts encode abnormal proteins, designated CP1, CP60 and CP69, consisting of DISC1 amino acids 1–597 plus 1, 60 or 69 amino acids, respectively. The novel 69 amino acids in CP69 induce increased α-helical content and formation of large stable protein assemblies. The same is predicted for CP60. Both CP60 and CP69 exhibit profoundly altered functional properties within cell lines and neurons. Both are predominantly targeted to mitochondria, where they induce clustering and loss of membrane potential, indicative of severe mitochondrial dysfunction. There is currently no access to neural material from translocation carriers to confirm these findings, but there is no reason to suppose that these chimeric transcripts will not also be expressed in the brain. There is thus potential for the production of abnormal chimeric proteins in the brains of translocation carriers, although at substantially lower levels than for native DISC1. The mechanism by which inheritance of the translocation increases risk of psychiatric illness may therefore involve both DISC1 haploinsufficiency and mitochondrial deficiency due to the effects of abnormal chimeric protein expression. GenBank accession numbers: DISC1FP1 (EU302123), Boymaw (GU134617), der 11 chimeric transcript DISC1FP1 exon 2 to DISC1 exon 9 (JQ650115), der 1 chimeric transcript DISC1 exon 4 to DISC1FP1 exon 4 (JQ650116), der 1 chimeric transcript DISC1 exon 6 to DISC1FP1 exon 3a (JQ650117). PMID:22547224

Expression of Genes Involved in Drosophila Wing Morphogenesis and Vein Patterning Are Altered by Spaceflight

NASA Technical Reports Server (NTRS)

Parsons-Wingerter, Patricia A.; Hosamani, Ravikumar; Bhattacharya, Sharmila

2015-01-01

Imaginal wing discs of Drosophila melanogaster (fruit fly) defined during embryogenesis ultimately result in mature wings of stereotyped (specific) venation patterning. Major regulators of wing disc development are the epidermal growth factor receptor (EGF), Notch, Hedgehog (Hh), Wingless (Wg), and Dpp signaling pathways. Highly stereotyped vascular patterning is also characteristic of tissues in other organisms flown in space such as the mouse retina and leaves of Arabidopsis thaliana. Genetic and other adaptations of vascular patterning to space environmental factors have not yet been systematically quantified, despite widespread recognition of their critical importance for terrestrial and microgravity applications. Here we report changes in gene expression with space flight related to Drosophila wing morphogenesis and vein patterning. In addition, genetically modified phenotypes of increasingly abnormal ectopic wing venation in the Drosophila wing1 were analyzed by NASA's VESsel GENeration Analysis (VESGEN) software2. Our goal is to further develop insightful vascular mappings associated with bioinformatic dimensions of genetic or other molecular phenotypes for correlation with genetic and other molecular profiling relevant to NASA's GeneLab and other Space Biology exploration initiatives.

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis

PubMed Central

Kranz, Dominique; Boutros, Michael

2014-01-01

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions. PMID:24442637

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis.

PubMed

Kranz, Dominique; Boutros, Michael

2014-02-03

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions.

Self-organization of vertebrate mesoderm based on simple boundary conditions.

PubMed

Green, Jeremy B A; Dominguez, Isabel; Davidson, Lance A

2004-11-01

Embryonic development requires cell movements whose coordination is robust and reproducible. A dramatic example is the primary body axis of vertebrates: despite perturbation, cells in prospective axial tissue coordinate their movements to make an elongated body axis. The spatial cues coordinating these movements are not known. We show here that cells deprived of preexisting spatial cues by physical dissociation and reaggregation nonetheless organize themselves into an axis. Activin-induced cells that are reaggregated into a flat disc initially round up into a ball before elongating perpendicular to the disc. Manipulations of the geometry of the disc and immunofluorescence micrography reveal that the edge of the disc provides a circumferential alignment zone. This finding indicates that physical boundaries provide alignment cues and that circumferential "hoop stress" drives the axial extrusion in a manner resembling late-involuting mesoderm of Xenopus and archenteron elongation in other deuterostome species such as sea urchins. Thus, a population of cells finds its own midline based on the form of the population's boundaries using an edge-aligning mechanism. This process provides a remarkably simple organizing principle that contributes to the reliability of embryonic development as a whole. (c) 2004 Wiley-Liss, Inc.

Anomalous optic discs in a patient with a Dandy-Walker cyst.

PubMed

Orcutt, J C; Bunt, A H

1982-03-01

A 19-month-old female infant with a Dandy-Walker cyst had anomalous optic discs, each of which appeared to divide to form an accessory optic nerve. The discs probably lie within the spectrum of anomalous discs including optic nerve aplasia and hypoplasia, megallopapillae, morning glory disc, optic disc dysplasia, and optic nerve colobomas. The association of anomalous optic discs with a Dandy-Walker cyst has not been previously recognized. The ocular and brain malformations in this patient likely occurred during the fourth to eighth week of gestation, when the retinal ganglion cell axons were penetrating the optic nerve, and the rhombic lips were enlarging in early cerebellar development. The etiology of these anomalies is not known; however, teratogens, sporadic events, and genetic disorders should be considered.

Lactoferricin enhances BMP7-stimulated anabolic pathways in intervertebral disc cells.

PubMed

Ellman, Michael B; Kim, Jaesung; An, Howard S; Chen, Di; Kc, Ranjan; Li, Xin; Xiao, Guozhi; Yan, Dongyao; Suh, Joon; van Wjnen, Andre J; Wang, James H-C; Kim, Su-Gwan; Im, Hee-Jeong

2013-07-25

Bone-morphogenetic protein-7 (BMP7) is a well-known anabolic and anti-catabolic growth factor on intervertebral disc (IVD) matrix and cell homeostasis. Similarly, Lactoferricin B (LfcinB) has recently been shown to have pro-anabolic, anti-catabolic, anti-oxidative and/or anti-inflammatory effects in bovine disc cells in vitro. In this study, we investigated the potential benefits of using combined peptide therapy with LfcinB and BMP7 for intervertebral disc matrix repair and to understand cellular and signaling mechanisms controlled by these factors. We studied the effects of BMP7 and LfcinB as individual treatments and combined therapy on bovine nucleus pulposus (NP) cells by assessing proteoglycan (PG) accumulation and synthesis, and the gene expression of matrix protein aggrecan and transcription factor SOX-9. We also analyzed the role of Noggin, a BMP antagonist, in IVD tissue and examined its effect after stimulation with LfcinB. To understand the molecular mechanisms by which LfcinB synergizes with BMP7, we investigated the ERK-SP1 axis as a downstream intracellular signaling regulator involved in BMP7 and LfcinB-mediated activities. Treatment of bovine NP cells cultured in alginate with LfcinB plus BMP7 synergistically stimulates PG synthesis and accumulation in part by upregulation of aggrecan gene expression. The synergism results from LfcinB-mediated activation of Sp1 and SMAD signaling pathways by (i) phosphorylation of SMAD 1/5/8; (ii) downregulation of SMAD inhibitory factors [i.e., noggin and SMAD6 (inhibitory SMAD)]; and (iii) upregulation of SMAD4 (universal co-SMAD). These data indicate that LfcinB-suppression of Noggin may eliminate the negative feedback of BMP7, thereby maximizing biological activity of BMP7 and ultimately shifting homeostasis to a pro-anabolic state in disc cells. We propose that combination growth factor therapy using BMP7 and LfcinB may be beneficial for treatment of disc degeneration. Copyright © 2013 Elsevier B.V. All rights reserved.

Assessment of methicillin resistant Staphylococcus Aureus detection methods: analytical comparative study.

PubMed

Ibrahim, Omer Mohammed Ali; Bilal, Naser Eldin; Osman, Omran Fadl; Magzoub, Magzoub Abbas

2017-01-01

The heterogeneous expression of methicillin resistance in Staphylococcus aureus (MRSA) affects the efficiency of tests available to detect it. The objective of this study was to assess four phenotypic tests used to detect MRSA. This is an analytical comparative study conducted among sudanese patients during period from May 2012 to July 2014, Staphylococcus aureus strains were isolated and identified by conventional methods, and then confirmed by PCR detection of coagulase gene. PCR detection of mecA gene was used as a gold standard to assess oxacillin resistance screen agar base (ORSAB), oxacillin disc, cefoxitin disc (at different temperatures and incubation periods) and MRSA-latex agglutination test. S.aureus ATCC 25923 was used as control. Sensitivity and specificity were calculated. MRSA- latex agglutination was the most accurate test; it showed 100% of both sensitivity and specificity, followed by cefoxitin disc with sensitivity of 98.48% and specificity of 100%. However, both of oxacillin disc and oxacillin resistance screen agar base showed less accurate results, and were affected by incubation periods. Oxacillin disc after 24 h incubation both at 30°C and 35°C showed sensitivity and specificity values of 87.88% and 96.23%, respectively. However, after 48h incubation the test at 30°C showed sensitivity and specificity values of 89.39%, and 94.34%, respectively. At 35°C (48h) it showed values of 89.39%, 92.45% respectively. Specificity of ORSAB was more than oxacillin disc at 35°C after 24h incubation 98.11% and 96.23%, respectively. MRSA- latex agglutination and cefoxitin disc diffusion tests are recommended for routine detection of MRSA.

Tumorigenic Properties of Drosophila Epithelial Cells Mutant for lethal giant larvae.

PubMed

Calleja, Manuel; Morata, Ginés; Casanova, Jordi

2016-08-01

Mutations in Drosophila tumor suppressor genes (TSGs) lead to the formation of invasive tumors in the brain and imaginal discs. Here we studied the tumorigenic properties of imaginal discs mutant for the TSG gene lethal giant larvae (lgl). lgl mutant cells display the characteristic features of mammalian tumor cells: they can proliferate indefinitely, induce additional tracheogenesis (an insect counterpart of vasculogenesis) and invade neighboring tissues. Lgl mutant tissues exhibit high apoptotic levels, which lead to the activation of the Jun-N-Terminal Kinase (JNK) pathway. We propose that JNK is a key factor in the acquisition of these tumorigenic properties; it promotes cell proliferation and induces high levels of Mmp1 and confers tumor cells capacity to invade wild-type tissue. Noteworthy, lgl RNAi-mediated down-regulation does not produce similar transformations in the central nervous system (CNS), thereby indicating a fundamental difference between the cells of developing imaginal discs and those of differentiated organs. We discuss these results in the light of the "single big-hit origin" of some human pediatric or developmental cancers. Down-regulation of lgl in imaginal discs is sufficient to enhance tracheogenesis and to promote invasion and colonization of other larval structures including the CNS. Developmental Dynamics 245:834-843, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Lesser devil rays Mobula cf. hypostoma from Venezuela are almost twice their previously reported maximum size and may be a new sub-species.

PubMed

Ehemann, N R; González-González, L V; Trites, A W

2017-03-01

Three rays opportunistically obtained near Margarita Island, Venezuela, were identified as lesser devil rays Mobula cf. hypostoma, but their disc widths were between 207 and 230 cm, which is almost double the reported maximum disc width of 120 cm for this species. These morphometric data suggest that lesser devil rays are either larger than previously recognized or that these specimens belong to an unknown sub-species of Mobula in the Caribbean Sea. Better data are needed to describe the distribution, phenotypic variation and population structure of this poorly known species. © 2017 The Fisheries Society of the British Isles.

A Systematic Review of the Safety and Efficacy of Mesenchymal Stem Cells for Disc Degeneration: Insights and Future Directions for Regenerative Therapeutics

PubMed Central

Yim, Rita Lok-Hay; Lee, Juliana Tsz-Yan; Bow, Cora H.; Meij, Björn; Leung, Victor; Cheung, Kenneth M.C.; Vavken, Patrick

2014-01-01

Intervertebral disc degeneration is associated with low-back pain. Mesenchymal stem cells (MSCs) have been used to “regenerate” the disc. The aim of this study was to perform a systematic review of comparative controlled studies that have assessed the safety and efficacy of using MSCs for disc regeneration. Literature databases were extensively searched. Trial design, subject-type, MSC sources, injection method, disc assessment, outcome intervals, and complication events were assessed. Validity of each study was performed. Twenty-four animal studies were included with 20.8% of the studies reporting randomization of groups. Trials in humans fulfilling inclusion criteria were not noted. The studies represented 862 discs that were injected with MSCs and 1,603 discs as controls. All three types of MSCs (ie, bone marrow, synovial, and adipose tissues) showed successful inhibition of disc degeneration. Bone-marrow-derived MSCs demonstrated superior quality of repair compared with other non-MSC treatments. A 2.7% overall complication rate was noted, whereby complications were noted only in rabbits. Overall, evidence suggested that MSCs increased disc space height in the majority of animal models. This is the first systematic review to assess the safety and efficacy of MSCs for the treatment of disc degeneration. Short-term MSC transplantation is safe and effective; however, additional, larger, and higher-quality studies are needed to assess the long-term safety and efficacy. Inconsistencies in methodological design and outcome parameters prevent any robust conclusions. Human-based clinical trials are needed. Recommendations are further made to improve efficacy, reduce potential complications, and standardize techniques for future studies. PMID:25050446

Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

PubMed

Wu, Yue; Zhuang, Yuan; Han, Min; Xu, Tian; Deng, Kejing

2009-10-20

Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

3D-Printed ABS and PLA Scaffolds for Cartilage and Nucleus Pulposus Tissue Regeneration.

PubMed

Rosenzweig, Derek H; Carelli, Eric; Steffen, Thomas; Jarzem, Peter; Haglund, Lisbet

2015-07-03

Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds were compared for cell ingrowth, viability, and tissue generation. Primary articular chondrocytes and nucleus pulposus (NP) cells were cultured on ABS and PLA scaffolds for three weeks. Both cell types proliferated well, showed high viability, and produced ample amounts of proteoglycan and collagen type II on both scaffolds. NP generated more matrix than chondrocytes; however, no difference was observed between scaffold types. Mechanical testing revealed sustained scaffold stability. This study demonstrates that chondrocytes and NP cells can proliferate on both ABS and PLA scaffolds printed with a simplistic, inexpensive desktop 3D printer. Moreover, NP cells produced more proteoglycan than chondrocytes, irrespective of thermoplastic type, indicating that cells maintain individual phenotype over the three-week culture period. Future scaffold designs covering larger pore sizes and better mimicking native tissue structure combined with more flexible or resorbable materials may provide implantable constructs with the proper structure, function, and cellularity necessary for potential cartilage and disc tissue repair in vivo.

Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

PubMed Central

Zhang, Shan-Shan; Shaw, Robin M.

2016-01-01

With each heartbeat, billions of cardiomyocytes work in concert to propagate the electrical excitation needed to effectively circulate blood. Regulated expression and timely delivery of connexin proteins to form gap junctions at the specialized cell – cell contact region, known as the intercalated disc, is essential to ventricular cardiomyocyte coupling. We focus this review on several regulatory mechanisms that have been recently found to govern the lifecycle of connexin 43 (Cx43), the short-lived and most abundantly expressed connexin in cardiac ventricular muscle. The Cx43 lifecycle begins with gene expression, followed by oligomerization into hexameric channels, and then cytoskeletal-based transport toward the disc region. Once delivered, hemichannels interact with resident disc proteins and are organized to effect intercellular coupling. We highlight recent studies exploring regulation of Cx43 localization to the intercalated disc, with emphasis on alternatively translated Cx43 isoforms and cytoskeletal transport machinery that together regulate Cx43 gap junction coupling between cardiomyocytes. PMID:24460200

Repurposing Blu-ray movie discs as quasi-random nanoimprinting templates for photon management

NASA Astrophysics Data System (ADS)

Smith, Alexander J.; Wang, Chen; Guo, Dongning; Sun, Cheng; Huang, Jiaxing

2014-11-01

Quasi-random nanostructures have attracted significant interests for photon management purposes. To optimize such patterns, typically very expensive fabrication processes are needed to create the pre-designed, subwavelength nanostructures. While quasi-random photonic nanostructures are abundant in nature (for example, in structural coloration), interestingly, they also exist in Blu-ray movie discs, an already mass-produced consumer product. Here we uncover that Blu-ray disc patterns are surprisingly well suited for light-trapping applications. While the algorithms in the Blu-ray industrial standard were developed with the intention of optimizing data compression and error tolerance, they have also created quasi-random arrangement of islands and pits on the final media discs that are nearly optimized for photon management over the solar spectrum, regardless of the information stored on the discs. As a proof-of-concept, imprinting polymer solar cells with the Blu-ray patterns indeed increases their efficiencies. Simulation suggests that Blu-ray patterns could be broadly applied for solar cells made of other materials.

Fortifying the Bone-Implant Interface Part 1: An In Vitro Evaluation of 3D-Printed and TPS Porous Surfaces

PubMed Central

Lindsey, Derek P.; Bahney, Chelsea S.; Woods, Shane A.; Wolfe, Mark L.; Yerby, Scott A.

2017-01-01

Background An aging society and concomitant rise in the incidence of impaired bone health have led to the need for advanced osteoconductive spinal implant surfaces that promote greater biological fixation (e.g. for interbody fusion cages, sacroiliac joint fusion implants, and artificial disc replacements). Additive manufacturing, i.e. 3D-printing, may improve bone integration by generating biomimetic spinal implant surfaces that mimic bone morphology. Such surfaces may foster an enhanced cellular response compared to traditional implant surfacing processes. Methods This study investigated the response of human osteoblasts to additive manufactured (AM) trabecular-like titanium implant surfaces compared to traditionally machined base material with titanium plasma spray (TPS) coated surfaces, with and without a nanocrystalline hydroxyapatite (HA) coating. For TPS-coated discs, wrought Ti6Al4V ELI was machined and TPS-coating was applied. For AM discs, Ti6Al4V ELI powder was 3D-printed to form a solid base and trabecular-like porous surface. The HA-coating was applied via a precipitation dip-spin method. Surface porosity, pore size, thickness, and hydrophilicity were characterized. Initial cell attachment, proliferation, alkaline phosphatase (ALP) activity, and calcium production of hFOB cells (n=5 per group) were measured. Results Cells on AM discs exhibited expedited proliferative activity. While there were no differences in mean ALP expression and calcium production between TPS and AM discs, calcium production on the AM discs trended 48% higher than on TPS discs (p=0.07). Overall, HA-coating did not further enhance results compared to uncoated TPS and AM discs. Conclusions Results demonstrate that additive manufacturing allows for controlled trabecular-like surfaces that promote earlier cell proliferation and trends toward higher calcium production than TPS coating. Results further showed that nanocrystalline HA may not provide an advantage on porous titanium surfaces. Clinical Relevance Additive manufactured porous titanium surfaces may induce a more osteogenic environment compared to traditional TPS, and thus present as an attractive alternative to TPS-coating for orthopedic spinal implants. PMID:28765799

Maintenance of “stem cell” features of cartilage cell sub-populations during in vitro propagation

PubMed Central

2013-01-01

Background The discovery of mesenchymal stem cells (MSCs) or MSC-like cells in cartilage tissue does not tie in well with the established view that MSCs derive from a perivascular niche. The presence of MSCs may raise concerns about specificity and application safety, particularly in terms of the regulatory site. The aim of the present study was to investigate the benefits or possible risks of the MSC-like properties of cells isolated from cartilage in the context of autologous chondrocyte implantation. Methods Chondrocytic cells were isolated from cartilage or intervertebral disc tissue. Flow cytometry was used to analyze the expression of cell surface antigens. MSC-like cells were either enriched or depleted by means of magnetic cell sorting (MACS) involving the monoclonal antibodies W5C5/SUSD2 and W8B2/MSCA-1. We addressed the issues of prolonged expansion of such cells as well as the influence of culture medium as a trigger for selecting a single cell type. Established protocols were used to study in vitro differentiation. In addition to histological and biochemical assessment, the acquired phenotypes were also evaluated on the mRNA transcript level. Results In the studied cells, we found strongly analogous expression of antigens typically expressed on MSCs, including CD49e, CD73, CD90, CD105, CD140b and CD166. The expression of W5C5 and W8B2 antigens in cartilage cell sub-populations did not correlate with multi-potency. We demonstrated that a chondroid precursor, but not a bona fide multipotent mesenchymal, cell type can be obtained under established in vitro culture conditions. The culture media used for expansion influenced the cell phenotype. Conclusions The risk of adverse adipose or osseous differentiation is not posed by expanded chondrocyte cultures, even after enrichment of putative MSC-like cell populations by MACS. It is possible that this limited “stemness” in chondrocytes, expanded for use in ACI, may instead be beneficial as it allows re-differentiation under appropriate conditions despite prolonged times in culture. PMID:23363653

Effects of surface finishing conditions on the biocompatibility of a nickel-chromium dental casting alloy.

PubMed

McGinley, Emma Louise; Coleman, David C; Moran, Gary P; Fleming, Garry J P

2011-07-01

To assess the effects of surface finishing condition (polished or alumina particle air abraded) on the biocompatibility of direct and indirect exposure to a nickel-chromium (Ni-Cr) d.Sign®10 dental casting alloy on oral keratinocytes. Biocompatibility was performed by assessing cellular viability and morphology, metabolic activity, cellular toxicity and presence of inflammatory cytokine markers. Discs of d.Sign®10 were cast, alumina particle air abraded and half were polished before surface roughness was determined by profilometry. Biocompatibility was assessed by placing the discs directly or indirectly (with immersion solutions) into contact with TR146 monolayers. Metal ion release was determined by ICP-MS. Cell viability was assessed by trypan blue dye exclusion, metabolic activity by XTT and cellular toxicity by LDH. Inflammatory cytokine analysis was performed using sandwich ELISAs. The mean polished Ra value was significantly reduced (P<0.001) compared with the alumina particle air abraded discs but metal ion release was significantly increased for the polished discs. Significant reductions in cell density of polished compared with alumina particle air abraded discs was observed following direct or indirect exposure. A significant reduction in metabolic activity, increase in cellular toxicity and an increase in the presence of inflammatory cytokine markers was highlighted for the polished relative to the alumina particle air abraded discs at 24h. Finishing condition of the Ni-Cr dental alloy investigated has important clinical implications. The approach of employing cell density and morphology, metabolic activity, cellular toxicity levels and inflammatory marker responses to TR146 epithelial cells combined with ICP-MS afforded the authors an increased insight into the complex processes dental alloys undergo in the oral environment. Copyright © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Biological Analysis of Simvastatin-releasing Chitosan Scaffold as a Cell-free System for Pulp-dentin Regeneration.

PubMed

Soares, Diana G; Anovazzi, Giovanna; Bordini, Ester Alves F; Zuta, Uxua O; Silva Leite, Maria Luísa A; Basso, Fernanda G; Hebling, Josimeri; de Souza Costa, Carlos A

2018-06-01

The improvement of biomaterials capable of driving the regeneration of the pulp-dentin complex mediated by resident cells is the goal of regenerative dentistry. In the present investigation, a chitosan scaffold (CHSC) that released bioactive concentrations of simvastatin (SIM) was tested, aimed at the development of a cell-free tissue engineering system. First, we performed a dose-response assay to select the bioactive dose of SIM capable of inducing an odontoblastic phenotype in dental pulp cells (DPCs); after which we evaluated the synergistic effect of this dosage with the CHSC/DPC construct. SIM at 1.0 μmol/L (CHSC-SIM1.0) and 0.5 μmol/L were incorporated into the CHSC, and cell viability, adhesion, and calcium deposition were evaluated. Finally, we assessed the biomaterials in an artificial pulp chamber/3-dimensional culture model to simulate the cell-free approach in vitro. SIM at 0.1 μmol/L was selected as the bioactive dose. This drug was capable of strongly inducing an odontoblastic phenotype on the DPC/CHSC construct. The incorporation of SIM into CHSC had no deleterious effect on cell viability and adhesion to the scaffold structure. CHSC-SIM1.0 led to significantly higher calcium-rich matrix deposition on scaffold/dentin disc assay compared with the control (CHSC). This biomaterial induced the migration of DPCs from a 3-dimensional culture to its surface as well as stimulated significantly higher expressions of alkaline phosphatase, collagen type 1 alpha 1, dentin matrix acidic phosphoprotein 1, and dentin sialophosphoprotein on 3-dimensional-cultured DPCs than on those in contact with CHSC. CHSC-SIM1.0 scaffold was capable of increasing the chemotaxis and regenerative potential of DPCs. Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Juvenile porcine temporomandibular joint: Three different cartilaginous structures?

PubMed

Tabeian, Hessam; Bakker, Astrid D; de Vries, Teun J; Zandieh-Doulabi, Behrouz; Lobbezoo, Frank; Everts, Vincent

2016-12-01

The temporomandibular joint (TMJ) consists of three cartilaginous structures: the fossa, disc, and condyle. In juvenile idiopathic arthritis (JIA), inflammation of the TMJ leads to destruction of the condyle, but not of the fossa or the disc. Such a different effect of inflammation might be related to differences in matrix composition of the cartilaginous structures. The matrix composition of the three TMJ structures was analyzed in juvenile porcine samples and in an in vitro system of cells isolated from each anatomical structure embedded in 3% agarose gels. The matrix of all three anatomical structures of the TMJ contained collagen type I and its gene expression was maintained after isolation. The condyle and the fossa stained positive for collagen type II and proteoglycans, but the condyle contained considerably more collagen type II and proteoglycans than the fossa. The disc contained neither collagen type II protein nor expression of its gene, and the disc did not stain positive for proteoglycans. Aggrecan gene expression was lower in the disc compared to condyle and fossa cell-isolates. In general, the cell-isolates in vitro closely mimicked the characteristic features found in the tissue. The collagen type II content of the condyle clearly distinguished this cartilaginous structure from the disc and fossa. Since autoimmunity against collagen type II is associated with JIA, the relatively abundant presence of this type of collagen in the condyle might provide an explanation why primarily this cartilaginous structure of the TMJ is affected in JIA patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Effect of Basic Fibroblast Growth Factor and Transforming Growth Factor-Β1 Combined with Bone Marrow Mesenchymal Stem Cells on the Repair of Degenerated Intervertebral Discs in Rat Models].

PubMed

Jiang, Chao; Li, Da-peng; Zhang, Zhi-jian; Shu, Hao-ming; Hu, Lang; Li, Zheng-nan; Huang, Yong-hui

2015-08-01

To evaluate the effects of the combination of basic fibroblast growth factor (bFGF), transforming growth factor-Β1 (TGF-Β1), bone marrow mesenchymal stem cells (BMSCs), and temperature-responsive chitosan hydrogel (TCH) gel on the repair of degenerative intervertebral disc in rat models. Rat models of intervertebral disc degeneration were established by acupuncture. The degenerative effects were observed under magnetic resonance imaging (MRI). The BMSCs was cultured in vitro and then transfected by adenovirus with enhanced green fluorescent protein to make it carry the gene of enhanced green fluorescent protein,which functioned as fluorescence labeling. The SD rat models of intervertebral disc degeneration were divided into four groups: group A, treated with the combination of bFGF, TGF-Β1,BMSCs,and TCH gel; group B, treated with the combination of BMSCs and TCH gel;group C, treated with the combination of bFGF,TGF-Β1, and TCH gel;and group D, treated with PBS buffer solution. After the corresponding reagents were injected into the degenerative intervertebral discs of each group, the rats were cultivated for another four weeks and then the repair effects of the intervertebral discs were observed under MRI. Furthermore,the intervertebral discs of each group were taken out and observed by HE and Masson staining. The nucleus pulposus was aspirated and the expressions of aggrecan,collagen 2,Sox-9,and collagen I of nucleus pulposus of each group were tested by reverse transcription polymerase chain reaction and Western blot. The transplanted BMSCs survived in the intervertebral disc and differentiated into nucleus pulposus-like cells. MRI showed that:the signal intensity of the nucleus pulposus of group A was much higher than that of the rest groups, the signal intensity of group B was higher than that of group C, and the signal intensity of group D was the lowest,in which the dura mater spinalis was in compression and the spinal cord changed in beaded shape. The differences of the Pfirrmann grading among the four groups had statistical significance (P<0.05). The results of the HE and Masson stains showed:the intervertebral disc of group A was well-structured,the quantity of nucleus pulposus cells was larger than that of the other three groups,and the boundary between the nucleus pulposus and the annulus fibrosus was clearly defined;the quantity of the nucleus pulposus cells of group B was larger than that of group C, and the broken annulus fibrosus was not observed in group B, while the broken annulus fibrosus could be observed in group C; and, the nucleus pulposus cells of group D were replaced by fibrous tissue. The results of the reverse transcription polymerase chain reaction and Western blot tests showed that,in terms of the expressions of aggrecan,collagen 2 and Sox-9,group A was the highest, followed by group B,group C,and group D (P<0.05); in terms of the expression of collagen 1,there was no obvious difference among these four groups (P>0.05). The transplanted BMSCs can survive in the degenerative intervertebral disc and differentiate into nucleus pulposus-like cells. The combination of bFGF, TGF-Β1, BMSCs,and TCH gel has obvious repair effect on the degenerative intervertebral discs. The effect of the combination of BMSCs and TCH gel on transplantation therapy of the degenerative intervertebral discs is better than that of the combination of bFGF, TGF-Β1 and TCH gel but worse than that of the combination of bFGF, TGF-Β1, BMSCs, and TCH gel.

Long Term Ex Vivo Culture and Live Imaging of Drosophila Larval Imaginal Discs.

PubMed

Tsao, Chia-Kang; Ku, Hui-Yu; Lee, Yuan-Ming; Huang, Yu-Fen; Sun, Yi Henry

Continuous imaging of live tissues provides clear temporal sequence of biological events. The Drosophila imaginal discs have been popular experimental subjects for the study of a wide variety of biological phenomena, but long term culture that allows normal development has not been satisfactory. Here we report a culture method that can sustain normal development for 18 hours and allows live imaging. The method is validated in multiple discs and for cell proliferation, differentiation and migration. However, it does not support disc growth and cannot support cell proliferation for more than 7 to 12 hr. We monitored the cellular behavior of retinal basal glia in the developing eye disc and found that distinct glia type has distinct properties of proliferation and migration. The live imaging provided direct proof that wrapping glia differentiated from existing glia after migrating to the anterior front, and unexpectedly found that they undergo endoreplication before wrapping axons, and their nuclei migrate up and down along the axons. UV-induced specific labeling of a single carpet glia also showed that the two carpet glia membrane do not overlap and suggests a tiling or repulsion mechanism between the two cells. These findings demonstrated the usefulness of an ex vivo culture method and live imaging.

Frequency response of pig intervertebral disc cells subjected to dynamic hydrostatic pressure.

PubMed

Kasra, Mehran; Merryman, W David; Loveless, Kristen N; Goel, Vijay K; Martin, James D; Buckwalter, Joseph A

2006-10-01

The pathogenesis of vibration-induced disorders of intervertebral disc at the cellular level is largely unknown. Dynamic loads with frequencies close to that of the in vivo human spine resonant frequency (4-6 Hz) have a destructive effect, which may induce extracellular disc matrix (ECM) degradation. To investigate this issue, three-dimensional (3D) alginate cultures of normal pig intervertebral disc nucleus and inner annulus cells were tested under dynamic hydrostatic loading. Alginate cultures of each region were divided into six groups; five groups were exposed to cyclic hydrostatic pressures of frequencies 1, 3, 5, 8, and 10 Hz with the same amplitude (1 MPa), and group 6 was the control group (no loading). Cultures of different groups were loaded for 3 days (30 min daily) in a hydraulic chamber. Effects of loading frequency on disc collagen and protein metabolism were investigated by measuring 3H-proline-labeled proteins associated with the cells in the extracellular matrix and release of 3H-proline-labeled molecules into culture medium. The results indicated a poor synthesis rate and more degradation near the 5 Hz frequency. The repeatability of experiments was verified by performing two experiments with the same protocol. Both experiments indicated that a threshold frequency of around 5 Hz disrupted protein metabolism. Copyright (c) 2006 Orthopaedic Research Society.

PEG modulated release of etanidazole from implantable PLGA/PDLA discs.

PubMed

Wang, Fangjing; Lee, Timothy; Wang, Chi-Hwa

2002-09-01

In this work, etanidazole (one type of hypoxic radiosensitizer) is encapsulated into spray dried poly(D),L-lactide-co-glycolide) (PLGA) microspheres and then compressed into discs for controlled release applications. Etanidazole is characterized by intracellular glutathione depletion and glutathione transferases inhibition, thereby enhancing sensitivity to radiation. It is also cytotoxic to tumor cells and can chemosensitize some alkylating agents by activating their tumor cell killing capabilities. We observed the release characteristics of etanidazole in the dosage forms of microspheres and discs, subjected to different preparation conditions. The release characteristics, morphology changes, particle size, and encapsulation efficiency of microspheres are also investigated. The release rate of etanidazole from implantable discs (13 mm in diameter, 1 mm in thickness, fabricated by a press) is much lower than microspheres due to the reduced specific surface. After the initial burst of 1% release for the first day, the cumulative release within the first week is less than 2% until a secondary burst of release (caused by polymer degradation) occurs after one month. Some key preparation conditions such as drug loadings, disc thickness and diameter, and compression pressure can affect the initial burst of etanidazole from the discs. However, none of them can significantly make the release more uniform. In contrast, the incorporation of polyethylene glycol (PEG) can greatly enhance the release rate of discs and also reduces the secondary burst effect, thereby achieving a sustained release for about 2 months.

Irregular chiasm-C-roughest, a member of the immunoglobulin superfamily, affects sense organ spacing on the Drosophila antenna by influencing the positioning of founder cells on the disc ectoderm.

PubMed

Venugopala Reddy, G; Reiter, C; Shanbhag, S; Fischbach, K F; Rodrigues, V

1999-10-01

We describe a role for Irregular chiasmC-roughest (IrreC-rst), an immunoglobulin (Ig) superfamily member, in patterning sense organs on the Drosophila antenna. IrreC-rst protein is initially expressed homogeneously on apical profiles of ectodermal cells in regions of the antennal disc. During specification of founder cells (FCs), the intracellular protein distribution changes and becomes concentrated in regions where specific intercellular contacts presumably occur. Loss of function mutations as well as misexpression of irreC-rst results in an altered arrangement of FCs within the disc compared to wildtype. Sense organ development occurs normally, although spacing is affected. Unlike its role in interommatidial spacing, irreC-rst does not affect apoptosis during antennal development. We propose that IrreC-rst affects the spatial relationship between sensory and ectodermal cells during FC delamination.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy.

PubMed

Rosa, Adalberto Luiz; Crippa, Grasiele Edilaine; de Oliveira, Paulo Tambasco; Taba, Mario; Lefebvre, Louis-Philippe; Beloti, Marcio Mateus

2009-05-01

This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 microm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

[Biology and mechanobiology of the intervertebral disc].

PubMed

González Martínez, Emilio; García-Cosamalón, José; Cosamalón-Gan, Iván; Esteban Blanco, Marta; García-Suarez, Olivia; Vega, José A

The intervertebral disc (IVD) is noted for its low cell content, and being the largest avascular structure of human body. The low amount of cells in the disc have to adapt to an anaerobic metabolism with low oxygen pressure and acidic pH. Apart from surviving in an adverse microenvironment, they are exposed to a high level of mechanical stress. The biological adaptation of cells to acidosis and hyperosmolarity conditions are regulated by mechanoproteins, which are responsible for converting a mechanical signal into a cellular response, thus modifying its gene expression. Mechanobiology helps us to better understand the pathophysiology of IVD and its potential biological repair. Copyright © 2016 Sociedad Española de Neurocirugía. Publicado por Elsevier España, S.L.U. All rights reserved.

3D surface reconstruction and visualization of the Drosophila wing imaginal disc at cellular resolution

NASA Astrophysics Data System (ADS)

Bai, Linge; Widmann, Thomas; Jülicher, Frank; Dahmann, Christian; Breen, David

2013-01-01

Quantifying and visualizing the shape of developing biological tissues provide information about the morphogenetic processes in multicellular organisms. The size and shape of biological tissues depend on the number, size, shape, and arrangement of the constituting cells. To better understand the mechanisms that guide tissues into their final shape, it is important to investigate the cellular arrangement within tissues. Here we present a data processing pipeline to generate 3D volumetric surface models of epithelial tissues, as well as geometric descriptions of the tissues' apical cell cross-sections. The data processing pipeline includes image acquisition, editing, processing and analysis, 2D cell mesh generation, 3D contourbased surface reconstruction, cell mesh projection, followed by geometric calculations and color-based visualization of morphological parameters. In their first utilization we have applied these procedures to construct a 3D volumetric surface model at cellular resolution of the wing imaginal disc of Drosophila melanogaster. The ultimate goal of the reported effort is to produce tools for the creation of detailed 3D geometric models of the individual cells in epithelial tissues. To date, 3D volumetric surface models of the whole wing imaginal disc have been created, and the apicolateral cell boundaries have been identified, allowing for the calculation and visualization of cell parameters, e.g. apical cross-sectional area of cells. The calculation and visualization of morphological parameters show position-dependent patterns of cell shape in the wing imaginal disc. Our procedures should offer a general data processing pipeline for the construction of 3D volumetric surface models of a wide variety of epithelial tissues.

Optic nerve and retinal features in uveitis associated with juvenile systemic granulomatous disease (Blau syndrome).

PubMed

Carreño, Ester; Guly, Catherine M; Chilov, Michael; Hinchcliffe, Annie; Arostegui, Juan I; Lee, Richard W J; Dick, Andrew D; Ramanan, Athimalaipet V

2015-05-01

To determine whether patients with juvenile systemic granulomatous disease (JSGD) (Blau syndrome) and uveitis have a characteristic ocular phenotype. Clinical and imaging data were collected retrospectively from patients attending the Regional Combined Paediatric Rheumatology and Ocular Inflammatory Service, Bristol Eye Hospital. General demographic information, laterality of the uveitis, age at onset, anatomical classification and course of the uveitis, clinical phenotype and specific NOD2 mutation were recorded for each patient. Seventeen eyes from nine patients (five males; four females) were included in the study. Mean age at the disease onset was 15 months, range 1-84 months. Eight patients had bilateral uveitis. Anterior uveitis was present in five eyes, intermediate uveitis in two eyes, and there were 10 eyes with panuveitis, manifesting as multifocal choroiditis. Appearance of optic disc included indistinct disc margins in six eyes, optic nerve head pallor in six eyes, optic disc vessel sheathing in four eyes, and there was peripapillary hypo/hyperpigmentation in 13 eyes accompanied with characteristic peripapillary nodular excrescences. Among NOD2 mutations, the p.R334W was the most commonly detected (n: four cases), and three patients carried novel variants, the p.E338D and p.D390V variants in one patient, and the p.H520Y and p.Q809K variants in two different patients. Chronic bilateral panuveitis and a nodular peripapillary appearance in childhood onset uveitis are characteristic features of JSGD, which support the need for an appropriate genetic NOD2 analysis. © 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Creation of an injectable in situ gelling native extracellular matrix for nucleus pulposus tissue engineering.

PubMed

Wachs, Rebecca A; Hoogenboezem, Ella N; Huda, Hammad I; Xin, Shangjing; Porvasnik, Stacy L; Schmidt, Christine E

2017-03-01

Disc degeneration is the leading cause of low back pain and is often characterized by a loss of disc height, resulting from cleavage of chondroitin sulfate proteoglycans (CSPGs) present in the nucleus pulposus. Intact CSPGs are critical to water retention and maintenance of the nucleus osmotic pressure. Decellularization of healthy nucleus pulposus tissue has the potential to serve as an ideal matrix for tissue engineering of the disc because of the presence of native disc proteins and CSPGs. Injectable in situ gelling matrices are the most viable therapeutic option to prevent damage to the anulus fibrosus and future disc degeneration. The purpose of this research was to create a gentle decellularization method for use on healthy nucleus pulposus tissue explants and to develop an injectable formulation of this matrix to enable therapeutic use without substantial tissue disruption. Porcine nuclei pulposi were isolated, decellularized, and solubilized. Samples were assessed to determine the degree of cell removal, matrix maintenance, gelation ability, cytotoxic residuals, and native cell viability. Nuclei pulposi were decellularized using serial detergent, buffer, and enzyme treatments. Decellularized nuclei pulposi were solubilized, neutralized, and buffered. The efficacy of decellularization was assessed by quantifying DNA removal and matrix preservation. An elution study was performed to confirm removal of cytotoxic residuals. Gelation kinetics and injectability were quantified. Long-term in vitro experiments were performed with nucleus pulposus cells to ensure cell viability and native matrix production within the injectable decellularized nucleus pulposus matrices. This work resulted in the creation of a robust acellular matrix (>96% DNA removal) with highly preserved sulfated glycosaminoglycans (>47%), and collagen content and microstructure similar to native nucleus pulposus, indicating preservation of disc components. Furthermore, it was possible to create an injectable formulation that gelled in situ within 45 minutes and formed fibrillar collagen with similar diameters to native nucleus pulposus. The processing did not result in any remaining cytotoxic residuals. Solubilized decellularized nucleus pulposus samples seeded with nucleus pulposus cells maintained robust viability (>89%) up to 21 days of culture in vitro, with morphology similar to native nucleus pulposus cells, and exhibited significantly enhanced sulfated glycosaminoglycans production over 21 days. A gentle decellularization of porcine nucleus pulposus followed by solubilization enabled the creation of an injectable tissue-specific matrix that is well tolerated in vitro by nucleus pulposus cells. These matrices have the potential to be used as a minimally invasive nucleus pulposus therapeutic to restore disc height. Copyright © 2016 Elsevier Inc. All rights reserved.

Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs

PubMed Central

Choi, Kyung-Suk; Harfe, Brian D.

2011-01-01

The vertebrae notochord is a transient rod-like structure that produces secreted factors that are responsible for patterning surrounding tissues. During later mouse embryogenesis, the notochord gives rise to the middle part of the intervertebral disc, called the nucleus pulposus. Currently, very little is known about the molecular mechanisms responsible for forming the intervertebral discs. Here we demonstrate that hedgehog signaling is required for formation of the intervertebral discs. Removal of hedgehog signaling in the notochord and nearby floorplate resulted in the formation of an aberrant notochord sheath that normally surrounds this structure. In the absence of the notochord sheath, small nuclei pulposi were formed, with most notochord cells dispersed throughout the vertebral bodies during embryogenesis. Our data suggest that the formation of the notochord sheath requires hedgehog signaling and that the sheath is essential for maintaining the rod-like structure of the notochord during early embryonic development. As notochord cells form nuclei pulposi, we propose that the notochord sheath functions as a “wrapper” around the notochord to constrain these cells along the vertebral column. PMID:21606373

Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs.

PubMed

Choi, Kyung-Suk; Harfe, Brian D

2011-06-07

The vertebrae notochord is a transient rod-like structure that produces secreted factors that are responsible for patterning surrounding tissues. During later mouse embryogenesis, the notochord gives rise to the middle part of the intervertebral disc, called the nucleus pulposus. Currently, very little is known about the molecular mechanisms responsible for forming the intervertebral discs. Here we demonstrate that hedgehog signaling is required for formation of the intervertebral discs. Removal of hedgehog signaling in the notochord and nearby floorplate resulted in the formation of an aberrant notochord sheath that normally surrounds this structure. In the absence of the notochord sheath, small nuclei pulposi were formed, with most notochord cells dispersed throughout the vertebral bodies during embryogenesis. Our data suggest that the formation of the notochord sheath requires hedgehog signaling and that the sheath is essential for maintaining the rod-like structure of the notochord during early embryonic development. As notochord cells form nuclei pulposi, we propose that the notochord sheath functions as a "wrapper" around the notochord to constrain these cells along the vertebral column.

Distribution of cardiac sodium channels in clusters potentiates ephaptic interactions in the intercalated disc.

PubMed

Hichri, Echrak; Abriel, Hugues; Kucera, Jan P

2018-02-15

It has been proposed that ephaptic conduction, relying on interactions between the sodium (Na + ) current and the extracellular potential in intercalated discs, might contribute to cardiac conduction when gap junctional coupling is reduced, but this mechanism is still controversial. In intercalated discs, Na + channels form clusters near gap junction plaques, but the functional significance of these clusters has never been evaluated. In HEK cells expressing cardiac Na + channels, we show that restricting the extracellular space modulates the Na + current, as predicted by corresponding simulations accounting for ephaptic effects. In a high-resolution model of the intercalated disc, clusters of Na + channels that face each other across the intercellular cleft facilitate ephaptic impulse transmission when gap junctional coupling is reduced. Thus, our simulations reveal a functional role for the clustering of Na + channels in intercalated discs, and suggest that rearrangement of these clusters in disease may influence cardiac conduction. It has been proposed that ephaptic interactions in intercalated discs, mediated by extracellular potentials, contribute to cardiac impulse propagation when gap junctional coupling is reduced. However, experiments demonstrating ephaptic effects on the cardiac Na + current (I Na ) are scarce. Furthermore, Na + channels form clusters around gap junction plaques, but the electrophysiological significance of these clusters has never been investigated. In patch clamp experiments with HEK cells stably expressing human Na v 1.5 channels, we examined how restricting the extracellular space modulates I Na elicited by an activation protocol. In parallel, we developed a high-resolution computer model of the intercalated disc to investigate how the distribution of Na + channels influences ephaptic interactions. Approaching the HEK cells to a non-conducting obstacle always increased peak I Na at step potentials near the threshold of I Na activation and decreased peak I Na at step potentials far above threshold (7 cells, P = 0.0156, Wilcoxon signed rank test). These effects were consistent with corresponding control simulations with a uniform Na + channel distribution. In the intercalated disc computer model, redistributing the Na + channels into a central cluster of the disc potentiated ephaptic effects. Moreover, ephaptic impulse transmission from one cell to another was facilitated by clusters of Na + channels facing each other across the intercellular cleft when gap junctional coupling was reduced. In conclusion, our proof-of-principle experiments demonstrate that confining the extracellular space modulates cardiac I Na , and our simulations reveal the functional role of the aggregation of Na + channels in the perinexus. These findings highlight novel concepts in the physiology of cardiac excitation. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Construction Strategy and Progress of Whole Intervertebral Disc Tissue Engineering.

PubMed

Yang, Qiang; Xu, Hai-wei; Hurday, Sookesh; Xu, Bao-shan

2016-02-01

Degenerative disc disease (DDD) is the major cause of low back pain, which usually leads to work absenteeism, medical visits and hospitalization. Because the current conservative procedures and surgical approaches to treatment of DDD only aim to relieve the symptoms of disease but not to regenerate the diseased disc, their long-term efficiency is limited. With the rapid developments in medical science, tissue engineering techniques have progressed markedly in recent years, providing a novel regenerative strategy for managing intervertebral disc disease. However, there are as yet no ideal methods for constructing tissue-engineered intervertebral discs. This paper reviews published reports pertaining to intervertebral disc tissue engineering and summarizes data concerning the seed cells and scaffold materials for tissue-engineered intervertebral discs, construction of tissue-engineered whole intervertebral discs, relevant animal experiments and effects of mechanics on the construction of tissue-engineered intervertebral disc and outlines the existing problems and future directions. Although the perfect regenerative strategy for treating DDD has not yet been developed, great progress has been achieved in the construction of tissue-engineered intervertebral discs. It is believed that ongoing research on intervertebral disc tissue engineering will result in revolutionary progress in the treatment of DDD. © 2016 Chinese Orthopaedic Association and John Wiley & Sons Australia, Ltd.

Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Koteswara Rao, G; Bag, Indira; Bhadra, Utpal; Pal-Bhadra, Manika

2016-03-01

Histone modulations have been implicated in various cellular and developmental processes where in Drosophila Mof is involved in acetylation of H4K16. Reduction in the size of larval imaginal discs is observed in the null mutants of mof with increased apoptosis. Deficiency involving Hid, Reaper and Grim [H99] alleviated mof (RNAi) induced apoptosis in the eye discs. mof (RNAi) induced apoptosis leads to activation of caspases which is suppressed by over expression of caspase inhibitors like P35 and Diap1 clearly depicting the role of caspases in programmed cell death. Also apoptosis induced by knockdown of mof is rescued by JNK mutants of bsk and tak1 indicating the role of JNK in mof (RNAi) induced apoptosis. The adult eye ablation phenotype produced by ectopic expression of Hid, Rpr and Grim, was restored by over expression of Mof. Accumulation of Mof at the Diap1 promoter 800 bp upstream of the transcription start site in wild type larvae is significantly higher (up to twofolds) compared to mof (1) mutants. This enrichment coincides with modification of histone H4K16Ac indicating an induction of direct transcriptional up regulation of Diap1 by Mof. Based on these results we propose that apoptosis triggered by mof (RNAi) proceeds through a caspase-dependent and JNK mediated pathway.

Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage

PubMed Central

Wei, Yiyong; Zhou, Bin; Bernhard, Jonathan; Robinson, Samuel; Burapachaisri, Aonnicha; Guo, X. Edward

2017-01-01

Standard isotropic culture fails to recapitulate the spatiotemporal gradients present during native development. Cartilage grown from human mesenchymal stem cells (hMSCs) is poorly organized and unstable in vivo. We report that human cartilage with physiologic organization and in vivo stability can be grown in vitro from self-assembling hMSCs by implementing spatiotemporal regulation during induction. Self-assembling hMSCs formed cartilage discs in Transwell inserts following isotropic chondrogenic induction with transforming growth factor β to set up a dual-compartment culture. Following a switch in the basal compartment to a hypertrophic regimen with thyroxine, the cartilage discs underwent progressive deep-zone hypertrophy and mineralization. Concurrent chondrogenic induction in the apical compartment enabled the maintenance of functional and hyaline cartilage. Cartilage homeostasis, chondrocyte maturation, and terminal differentiation markers were all up-regulated versus isotropic control groups. We assessed the in vivo stability of the cartilage formed under different induction regimens. Cartilage formed under spatiotemporal regulation in vitro resisted endochondral ossification, retained the expression of cartilage markers, and remained organized following s.c. implantation in immunocompromised mice. In contrast, the isotropic control groups underwent endochondral ossification. Cartilage formed from hMSCs remained stable and organized in vivo. Spatiotemporal regulation during induction in vitro recapitulated some aspects of native cartilage development, and potentiated the maturation of self-assembling hMSCs into stable and organized cartilage resembling the native articular cartilage. PMID:28228529

Hippocampal place cell and inhibitory neuron activity in disrupted-in-schizophrenia-1 mutant mice: implications for working memory deficits

PubMed Central

Mesbah-Oskui, Lia; Georgiou, John; Roder, John C

2015-01-01

Background: Despite the prevalence of working memory deficits in schizophrenia, the neuronal mechanisms mediating these deficits are not fully understood. Importantly, deficits in spatial working memory are identified in numerous mouse models that exhibit schizophrenia-like endophenotypes. The hippocampus is one of the major brain regions that actively encodes spatial location, possessing pyramidal neurons, commonly referred to as ‘place cells’, that fire in a location-specific manner. This study tests the hypothesis that mice with a schizophrenia-like endophenotype exhibit impaired encoding of spatial location in the hippocampus. Aims: To characterize hippocampal place cell activity in mice that exhibit a schizophrenia-like endophenotype. Methods: We recorded CA1 place cell activity in six control mice and six mice that carry a point mutation in the disrupted-in-schizophrenia-1 gene (Disc1-L100P) and have previously been shown to exhibit deficits in spatial working memory. Results: The spatial specificity and stability of Disc1-L100P place cells were similar to wild-type place cells. Importantly, however, Disc1-L100P place cells exhibited a higher propensity to increase their firing rate in a single, large location of the environment, rather than multiple smaller locations, indicating a generalization in their spatial selectivity. Alterations in the signaling and numbers of CA1 putative inhibitory interneurons and decreased hippocampal theta (5–12 Hz) power were also identified in the Disc1-L100P mice. Conclusions: The generalized spatial selectivity of Disc1-L100P place cells suggests a simplification of the ensemble place codes that encode individual locations and subserve spatial working memory. Moreover, these results suggest that deficient working memory in schizophrenia results from an impaired ability to uniquely code the individual components of a memory sequence. PMID:27280123

Effects of radial compression on a novel simulated intervertebral disc-like assembly using bone marrow-derived mesenchymal stem cell cell-sheets for annulus fibrosus regeneration.

PubMed

See, Eugene Yong-Shun; Toh, Siew Lok; Goh, James Cho-Hong

2011-10-01

The aim of this study was to develop a tissue engineering approach in regenerating the annulus fibrosus (AF) as part of an overall strategy to produce a tissue-engineered intervertebral disc (IVD) replacement. To determine whether a rehabilitative simulation regime on bone marrow–derived mesenchymal stem cell cell-sheet is able to aid the regeneration of the AF. No previous study has used bone marrow–derived mesenchymal stem cell cell-sheets simulated by a rehabilitative regime to regenerate the AF. The approach was to use bone marrow–derived stem cells to form cell-sheets and incorporating them onto silk scaffolds to simulate the native lamellae of the AF. The in vitro experimental model used to study the efficacy of such a system was made up of the tissue engineering AF construct wrapped around a silicone disc to form a simulated IVD-like assembly. The assembly was cultured within a custom-designed bioreactor that provided a compressive mechanical stimulation onto the silicone disc. The silicone nucleus pulposus would bulge radially and compress the simulated AF to mimic the physiological conditions. The simulated IVD-like assembly was compressed using a rehabilitative regime that lasted for 4 weeks at 0.25 Hz, for 15 minutes each day. With the rehabilitative regime, the cell-sheets remained viable but showed a decrease in cell numbers and viability. Gene expression analysis showed significant upregulation of IVD-related genes and there was an increased ratio of collagen type II to collagen type I found within the extracellular matrix. The results suggested that a rehabilitative regime caused extensive remodeling to take place within the simulated IVD-like assembly, producing extracellular matrix similar to that found in the inner AF.

Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells.

PubMed

Strassburg, Sandra; Hodson, Nigel W; Hill, Patrick I; Richardson, Stephen M; Hoyland, Judith A

2012-01-01

Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.

Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

PubMed

Porcheron, P; Morinière, M; Coudouel, N; Oberlander, H

1991-01-01

Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.

Organotypic three-dimensional assays based on human leiomyoma–derived matrices

PubMed Central

Dourado, Mauricio Rocha; Sundquist, Elias; Apu, Ehsanul Hoque; Alahuhta, Ilkka; Tuomainen, Katja; Vasara, Jenni; Al-Samadi, Ahmed

2018-01-01

Alongside cancer cells, tumours exhibit a complex stroma containing a repertoire of cells, matrix molecules and soluble factors that actively crosstalk between each other. Recognition of this multifaceted concept of the tumour microenvironment (TME) calls for authentic TME mimetics to study cancer in vitro. Traditionally, tumourigenesis has been investigated in non-human, three-dimensional rat type I collagen containing organotypic discs or by means of mouse sarcoma-derived gel, such as Matrigel®. However, the molecular compositions of these simplified assays do not properly simulate human TME. Here, we review the main properties and benefits of using human leiomyoma discs and their matrix Myogel for in vitro assays. Myoma discs are practical for investigating the invasion of cancer cells, as are cocultures of cancer and stromal cells in a stiff, hypoxic TME mimetic. Myoma discs contain soluble factors and matrix molecules commonly present in neoplastic stroma. In Transwell, IncuCyte, spheroid and sandwich assays, cancer cells move faster and form larger colonies in Myogel than in Matrigel®. Additionally, Myogel can replace Matrigel® in hanging-drop and tube-formation assays. Myogel also suits three-dimensional drug testing and extracellular vesicle interactions. To conclude, we describe the application of our myoma-derived matrices in 3D in vitro cancer assays. This article is part of the discussion meeting issue ‘Extracellular vesicles and the tumour microenvironment’. PMID:29158312

Organotypic three-dimensional assays based on human leiomyoma-derived matrices.

PubMed

Salo, Tuula; Dourado, Mauricio Rocha; Sundquist, Elias; Apu, Ehsanul Hoque; Alahuhta, Ilkka; Tuomainen, Katja; Vasara, Jenni; Al-Samadi, Ahmed

2018-01-05

Alongside cancer cells, tumours exhibit a complex stroma containing a repertoire of cells, matrix molecules and soluble factors that actively crosstalk between each other. Recognition of this multifaceted concept of the tumour microenvironment (TME) calls for authentic TME mimetics to study cancer in vitro Traditionally, tumourigenesis has been investigated in non-human, three-dimensional rat type I collagen containing organotypic discs or by means of mouse sarcoma-derived gel, such as Matrigel ® However, the molecular compositions of these simplified assays do not properly simulate human TME. Here, we review the main properties and benefits of using human leiomyoma discs and their matrix Myogel for in vitro assays. Myoma discs are practical for investigating the invasion of cancer cells, as are cocultures of cancer and stromal cells in a stiff, hypoxic TME mimetic. Myoma discs contain soluble factors and matrix molecules commonly present in neoplastic stroma. In Transwell, IncuCyte, spheroid and sandwich assays, cancer cells move faster and form larger colonies in Myogel than in Matrigel ® Additionally, Myogel can replace Matrigel ® in hanging-drop and tube-formation assays. Myogel also suits three-dimensional drug testing and extracellular vesicle interactions. To conclude, we describe the application of our myoma-derived matrices in 3D in vitro cancer assays.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'. © 2017 The Authors.

Dynamic pressurization induces transition of notochordal cells to a mature phenotype while retaining production of important patterning ligands from development

PubMed Central

2013-01-01

Introduction Notochordal cells (NCs) pattern aneural and avascular intervertebral discs (IVDs), and their disappearance, is associated with onset of IVD degeneration. This study induced and characterized the maturation of nucleus pulposus (NP) tissue from a gelatinous NC-rich structure to a matrix-rich structure populated by small NP cells using dynamic pressurization in an ex vivo culture model, and also identified soluble factors from NCs with therapeutic potential. Methods Porcine NC-rich NP tissue was cultured and loaded with hydrostatic pressure (0.5 to 2 MPa at 0.1 Hz for 2 hours) either Daily, for 1 Dose, or Control (no pressurization) groups for up to eight days. Cell phenotype and tissue maturation was characterized with measurements of cell viability, cytomorphology, nitric oxide, metabolic activity, matrix composition, gene expression, and proteomics. Results Daily pressurization induced transition of NCs to small NP cells with 73.8%, 44%, and 28% NCs for Control, 1 Dose and Daily groups, respectively (P < 0.0002) and no relevant cell death. Dynamic loading matured NP tissue by significantly increasing metabolic activity and accumulating Safranin-O-stained matrix. Load-induced maturation was also apparent from the significantly decreased glycolytic, cytoskeletal (Vimentin) and stress-inducible (HSP70) proteins assessed with proteomics. Loading increased the production of bioactive proteins Sonic Hedgehog (SHH) and Noggin, and maintained Semaphorin3A (Sema3A). Discussion NP tissue maturation was induced from dynamic hydrostatic pressurization in a controlled ex vivo environment without influence from systemic effects or surrounding structures. NCs transitioned into small nonvacuolated NP cells probably via differentiation as evidenced by high cell viability, lack of nitric oxide and downregulation of stress-inducible and cytoskeletal proteins. SHH, Sema3A, and Noggin, which have patterning and neurovascular-inhibiting properties, were produced in both notochordal and matured porcine NP. Results therefore provide an important piece of evidence suggesting the transition of NCs to small NP cells is a natural part of aging and not the initiation of degeneration. Bioactive candidates identified from young porcine IVDs may be isolated and harnessed for therapies to target discogenic back pain. PMID:24427812

Dynamic pressurization induces transition of notochordal cells to a mature phenotype while retaining production of important patterning ligands from development.

PubMed

Purmessur, Devina; Guterl, Clare C; Cho, Samuel K; Cornejo, Marisa C; Lam, Ying W; Ballif, Bryan A; Laudier, James C Iatridis; Iatridis, James C

2013-01-01

Notochordal cells (NCs) pattern aneural and avascular intervertebral discs (IVDs), and their disappearance, is associated with onset of IVD degeneration. This study induced and characterized the maturation of nucleus pulposus (NP) tissue from a gelatinous NC-rich structure to a matrix-rich structure populated by small NP cells using dynamic pressurization in an ex vivo culture model, and also identified soluble factors from NCs with therapeutic potential. Porcine NC-rich NP tissue was cultured and loaded with hydrostatic pressure (0.5 to 2 MPa at 0.1 Hz for 2 hours) either Daily, for 1 Dose, or Control (no pressurization) groups for up to eight days. Cell phenotype and tissue maturation was characterized with measurements of cell viability, cytomorphology, nitric oxide, metabolic activity, matrix composition, gene expression, and proteomics. Daily pressurization induced transition of NCs to small NP cells with 73.8%, 44%, and 28% NCs for Control, 1 Dose and Daily groups, respectively (P < 0.0002) and no relevant cell death. Dynamic loading matured NP tissue by significantly increasing metabolic activity and accumulating Safranin-O-stained matrix. Load-induced maturation was also apparent from the significantly decreased glycolytic, cytoskeletal (Vimentin) and stress-inducible (HSP70) proteins assessed with proteomics. Loading increased the production of bioactive proteins Sonic Hedgehog (SHH) and Noggin, and maintained Semaphorin3A (Sema3A). NP tissue maturation was induced from dynamic hydrostatic pressurization in a controlled ex vivo environment without influence from systemic effects or surrounding structures. NCs transitioned into small nonvacuolated NP cells probably via differentiation as evidenced by high cell viability, lack of nitric oxide and downregulation of stress-inducible and cytoskeletal proteins. SHH, Sema3A, and Noggin, which have patterning and neurovascular-inhibiting properties, were produced in both notochordal and matured porcine NP. Results therefore provide an important piece of evidence suggesting the transition of NCs to small NP cells is a natural part of aging and not the initiation of degeneration. Bioactive candidates identified from young porcine IVDs may be isolated and harnessed for therapies to target discogenic back pain.

Unique glycosignature for intervertebral disc and articular cartilage cells and tissues in immaturity and maturity.

PubMed

Collin, E C; Kilcoyne, M; White, S J; Grad, S; Alini, M; Joshi, L; Pandit, A S

2016-03-11

In this study, on/off markers for intervertebral disc (IVD) and articular cartilage (AC) cells (chondrocytes) and distinct glycoprofiles of cell and tissue-types were identified from immaturity to maturity. Three and eleven month-old ovine IVD and AC tissues were histochemically profiled with a panel of lectins and antibodies. Relationships between tissue and cell types were analysed by hierarchical clustering. Chondroitin sulfate (CS) composition of annulus fibrosus (AF), nucleus pulposus (NP) and AC tissues was determined by HPLC analysis. Clear on/off cell type markers were identified, which enabled the discrimination of chondrocytes, AF and NP cells. AF and NP cells were distinguishable using MAA, SNA-I, SBA and WFA lectins, which bound to both NP cells and chondrocytes but not AF cells. Chondrocytes were distinguished from NP and AF cells with a specific binding of LTA and PNA lectins to chondrocytes. Each tissue showed a unique CS composition with a distinct switch in sulfation pattern in AF and NP tissues upon disc maturity while cartilage maintained the same sulfation pattern over time. In conclusion, distinct glycoprofiles for cell and tissue-types across age groups were identified in addition to altered CS composition and sulfation patterns for tissue types upon maturity.

A role for the PDZ-binding domain of the coxsackie B virus and adenovirus receptor (CAR) in cell adhesion and growth.

PubMed

Excoffon, Katherine J D Ashbourne; Hruska-Hageman, Alesia; Klotz, Michael; Traver, Geri L; Zabner, Joseph

2004-09-01

The coxsackie and adenovirus receptor (CAR) plays a role in viral infection, maintenance of the junction adhesion complex in polarized epithelia, and modulation of cellular growth properties. As a viral receptor, the C-terminus appears to play no role indicating that the major function of CAR is to tether the virus to the cell. By contrast, the C-terminus is known to play a role in cellular localization and probably has a significant function in CAR-mediated adhesion and cell growth properties. We hypothesized that the CAR PDZ (PSD-95/Disc-large/ZO-1) binding motif interacts with PDZ-domain-containing proteins to modulate the cellular phenotype. CAR was modified by deleting the last four amino acids (CARDeltaGSIV) and evaluated for cell-cell adhesion in polarized primary human airway epithelia and growth characteristics in stably transfected L-cells. Although ablation of the CAR PDZ-binding motif did not affect adenoviral infection, it did have a significant effect both on cell-cell adhesion and on cell growth. Expression of CARDeltaGSIV failed to increase the transepithelial resistance in polarized epithelia to the same degree as wild-type CAR and failed to act as a growth modulator in L-cells. Furthermore, we provide evidence for three new CAR interacting partners, including MAGI-1b, PICK1 and PSD-95. CAR appears to interact with several distinct PDZ-domain-containing proteins and may exert its biological function through these interactions.

Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels.

PubMed

Richardson, Stephen M; Hughes, Nesta; Hunt, John A; Freemont, Anthony J; Hoyland, Judith A

2008-01-01

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, interest in methods focused on biological repair has increased. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD. Here, we have studied the temperature-sensitive hydrogel chitosan-glycerophosphate (C/Gp), seeded with human MSCs and cultured for 4 weeks in standard medium. We have analysed the gene and protein expression profile of the MSCs and compared it to that of both nucleus pulposus (NP) cells and articular chondrocytes cultured in C/Gp. Gene expression analysis for chondrocytic-cell marker genes demonstrated differentiation of MSCs to a phenotype which showed similarities to both articular chondrocytes and NP cells. Conventional PCR demonstrated a lack of expression of osteogenic marker genes and the hypertrophic marker gene type X collagen. MSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. These results therefore suggest that MSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.

Growth promoting in vitro effect of synthetic cyclic RGD-peptides on human osteoblast-like cells attached to cancellous bone.

PubMed

Magdolen, Ursula; Auernheimer, Jörg; Dahmen, Claudia; Schauwecker, Johannes; Gollwitzer, Hans; Tübel, Jutta; Gradinger, Reiner; Kessler, Horst; Schmitt, Manfred; Diehl, Peter

2006-06-01

In tissue engineering, the application of biofunctional compounds on biomaterials such as integrin binding RGD-peptides has gained growing interest. Anchorage-dependent cells like osteoblasts bind to these peptides thus ameliorating the integration of a synthetic implant. In case sterilized bone grafts are used as substitutes for reconstruction of bone defects, the ingrowth of the implanted bone is often disturbed because of severe pretreatment such as irradiation or autoclaving, impairing the biological and mechanical properties of the bone. We report for the first time on the in vitro coating of the surface of freshly resected, cleaned bone discs with synthetic, cyclic RGD-peptides. For this approach, two different RGD-peptides were used, one containing two phosphonate anchors, the other peptide four of these binding moieties to allow efficient association of these reactive RGD-peptides to the inorganic bone matrix. Human osteoblast-like cells were cultured on RGD-coated bone discs and the adherence and growth of the cells were analyzed. Coating of bone discs with RGD-peptides did not improve the adhesion rate of osteoblast-like cells to the discs but significantly (up to 40%) accelerated growth of these cells within 8 days after attachment. This effect points to pretreatment of bone implants, especially at the critical interface area between the implanted bone and the non-resected residual bone structure, before re-implantation in order to stimulate and enhance osteointegration of a bone implant.

Expression and regulation of neurotrophins in the nondegenerate and degenerate human intervertebral disc

PubMed Central

Purmessur, Devina; Freemont, Anthony J; Hoyland, Judith A

2008-01-01

Introduction The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been identified in the human intervertebral disc (IVD) and have been implicated in the mechanisms associated with nerve ingrowth and nociception in degeneration of the IVD. The aim of the current study was to investigate an association between neurotrophin expression in the IVD and the severity of disc degeneration, including the effect of disc-related proinflammatory cytokines on neurotrophin and neuropeptide expression in cells derived from the human IVD. Methods Immunohistochemical analysis was performed to examine the expression of NGF, BDNF and their high-affinity receptors Trk-A and Trk-B in human IVD samples, divided into three categories: non-degenerate, moderate degeneration and severe degeneration. In order to study the effect of disc-related cytokines on neurotrophin/neuropeptide gene expression, nucleus pulposus cells derived from non-degenerate and degenerate IVD samples were seeded in alginate and were stimulated with either IL-1β or TNFα for 48 hours. RNA was extracted, cDNA was synthesised and quantitative real-time PCR was performed to examine the expression of NGF, BDNF and substance P. Results Immunohistochemistry showed expression of NGF and BDNF in the native chondrocyte-like cells in all regions of the IVD and in all grades of degeneration. Interestingly only BDNF significantly increased with the severity of degeneration (P < 0.05). Similar expression was observed for Trk-A and Trk-B, although no association with disease severity was demonstrated. In cultured human nucleus pulposus cells, stimulation with IL-1β led to significant increases in NGF and BDNF gene expression (P < 0.05). Treatment with TNFα was associated with an upregulation of substance P expression only. Conclusion Our findings show that both the annulus fibrosus and nucleus pulposus cells of the IVD express the neurotrophins NGF and BDNF, factors that may influence and enhance innervation and pain in the degenerate IVD. Expression of Trk-A and Trk-B by cells of the nondegenerate and degenerate IVD suggests an autocrine role for neurotrophins in regulation of disc cell biology. Furthermore, modulation of neurotrophin expression by IL-1β and modulation of substance P expression by TNFα, coupled with their increased expression in the degenerate IVD, highlights novel roles for these cytokines in regulating nerve ingrowth in the degenerate IVD and associated back pain. PMID:18727839

Orthopedic Health: Targeting Musculoskeletal Pain

MedlinePlus

... problems. Using tiny, biodegradable fibers seeded with adult stem cells, we've made a prototype disc which is remarkably similar to the natural disc. We will be testing it in the laboratory shortly to see how it performs. "We're close to a touchdown" ...

Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

NASA Astrophysics Data System (ADS)

Restrepo, Simon; Basler, Konrad

2016-08-01

Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.

Biomaterial Property Effects on Platelets and Macrophages: An in Vitro Study.

PubMed

Fernandes, Kelly R; Zhang, Yang; Magri, Angela M P; Renno, Ana C M; van den Beucken, Jeroen J J P

2017-12-11

The purpose of this study was to evaluate the effects of surface properties of bone implants coated with hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) on platelets and macrophages upon implant installation and compare them to grit-blasted Ti and Thermanox used as a control. Surface properties were characterized using scanning electron microscopy, profilometry, crystallography, Fourier transform infrared spectroscopy, and coating stability. For platelets, platelet adherence and morphology were assessed. For macrophages, morphology, proliferation, and polarization were evaluated. Surface characterization showed similar roughness of ∼2.5 μm for grit-blasted Ti discs, both with and without coating. Coating stability assessment showed substantial dissolution of HA and β-TCP coatings. Platelet adherence was significantly higher for grit-blasted Ti, Ti-HA, and Ti-β-TCP coatings compared to that of cell culture control Thermanox. Macrophage cultures revealed a decreased proliferation on both HA and β-TCP coated discs compared to both Thermanox and grit-blasted Ti. In contrast, secretion of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine TGF-β were marginal for grit-blasted Ti and Thermanox, while a coating-dependent increased secretion of pro- and anti-inflammatory cytokines was observed for HA and β-TCP coatings. The results demonstrated a significantly upregulated pro-inflammatory and anti-inflammatory cytokine secretion and marker gene expression of macrophages on HA and β-TCP coatings. Furthermore, HA induced an earlier M1 macrophage polarization but more M2 phenotype potency than β-TCP. In conclusion, our data showed that material surface affects the behaviors of first cell types attached to implants. Due to the demonstrated crucial roles of platelets and macrophages in bone healing and implant integration, this information will greatly aid the design of metallic implants for a higher rate of success in patients.

Development and Translation of a Tissue-Engineered Disc in a Preclinical Rodent Model

DTIC Science & Technology

2014-12-01

annulus fibrosus tissue into full 3D Disc-like Angle Ply Structures (DAPS), inclusive of a hyaluronic acid hydrogel seeded with adult stem cells, that...AF constructs surrounding an engineered nucleus pulposus (NP) composed of a hyaluronic acid (HA) hydrogel. Measure the disc structural mechanics in...exposure to TGF-ß3 improves the functional properties of MSC-seeded photocrosslinked hyaluronic acid hydrogels by authors Minwook Kim, Isaac E

Developments in intervertebral disc disease research: pathophysiology, mechanobiology, and therapeutics.

PubMed

Weber, Kathryn T; Jacobsen, Timothy D; Maidhof, Robert; Virojanapa, Justin; Overby, Chris; Bloom, Ona; Quraishi, Shaheda; Levine, Mitchell; Chahine, Nadeen O

2015-03-01

Low back pain is a leading cause of disability worldwide and the second most common cause of physician visits. There are many causes of back pain, and among them, disc herniation and intervertebral disc degeneration are the most common diagnoses and targets for intervention. Currently, clinical treatment outcomes are not strongly correlated with diagnoses, emphasizing the importance for characterizing more completely the mechanisms of degeneration and their relationships with symptoms. This review covers recent studies elucidating cellular and molecular changes associated with disc mechanobiology, as it relates to degeneration and regeneration. Specifically, we review findings on the biochemical changes in disc diseases, including cytokines, chemokines, and proteases; advancements in disc disease diagnostics using imaging modalities; updates on studies examining the response of the intervertebral disc to injury; and recent developments in repair strategies, including cell-based repair, biomaterials, and tissue engineering. Findings on the effects of the omega-6 fatty acid, linoleic acid, on nucleus pulposus tissue engineering are presented. Studies described in this review provide greater insights into the pathogenesis of disc degeneration and may define new paradigms for early or differential diagnostics of degeneration using new techniques such as systemic biomarkers. In addition, research on the mechanobiology of disease enriches the development of therapeutics for disc repair, with potential to diminish pain and disability associated with disc degeneration.

Total disc replacement using a tissue-engineered intervertebral disc in vivo: new animal model and initial results

PubMed Central

Gebhard, Harry; Bowles, Robby; Dyke, Jonathan; Saleh, Tatianna; Doty, Stephen; Bonassar, Lawrence; Härtl, Roger

2010-01-01

Study type: Basic science Introduction: Chronic back pain due to degenerative disc disease (DDD) is among the most important medical conditions causing morbidity and significant health care costs. Surgical treatment options include disc replacement or fusion surgery, but are associated with significant short- and long-term risks.1 Biological tissue-engineering of human intervertebral discs (IVD) could offer an important alternative.2 Recent in vitro data from our group have shown successful engineering and growth of ovine intervertebral disc composites with circumferentially aligned collagen fibrils in the annulus fibrosus (AF) (Figure 1).3 Figure 1 Tissue-engineered composite disc a Experimental steps to generate composite tissue-engineered IVDs3 b Example of different AF formulations on collagen alignment in the AF. Second harmonic generation and two-photon excited fluorescence images of seeded collagen gels (for AF) of 1 and 2.5 mg/ml over time. At seeding, cells and collagen were homogenously distributed in the gels. Over time, AF cells elongated and collagen aligned parallel to cells. Less contraction and less alignment is noted after 3 days in the 2.5 mg/mL gel. c Imaging-based creation of a virtual disc model that will serve as template for the engineered disc. Total disc dimensions (AF and NP) were retrieved from micro-computer tomography (CT) (left images), and nucleus pulposus (NP) dimensions alone were retrieved from T2-weighted MRI images (right images). Merging of MRI and micro-CT models revealed a composite disc model (middle image)—Software: Microview, GE Healthcare Inc., Princeton, NJ; and slicOmatic v4.3, TomoVision, Montreal, Canada. d Flow chart describing the process for generating multi-lamellar tissue engineered IVDs. IVDs are produced by allowing cell-seeded collagen layers to contract around a cell-seeded alginate core (NP) over time Objective: The next step is to investigate if biological disc implants survive, integrate, and restore function to the spine in vivo. A model will be developed that allows efficient in vivo testing of tissue-engineered discs of various compositions and characteristics. Methods: Athymic rats were anesthetized and a dorsal approach was chosen to perform a microsurgical discectomy in the rat caudal spine (Fig. 2,Fig. 3). Control group I (n = 6) underwent discectomy only, Control group II (n = 6) underwent discectomy, followed by reimplantation of the autologous disc. Two treatment groups (group III, n = 6, 1 month survival; group IV, n = 6, 6 months survival) received a tissue-engineered composite disc implant. The rodents were followed clinically for signs of infection, pain level and wound healing. X-rays and magnetic resonance imaging (MRI) were assessed postoperatively and up to 6 months after surgery (Fig. 6,Fig. 7). A 7 Tesla MRI (Bruker) was implemented for assessment of the operated level as well as the adjacent disc (hydration). T2-weighted sequences were interpreted by a semiquantitative score (0 = no signal, 1 = weak signal, 2 = strong signal and anatomical features of a normal disc). Histology was performed with staining for proteoglycans (Alcian blue) and collagen (Picrosirius red) (Fig. 4,Fig. 5). Figure 2 Disc replacement surgery a Operative situs with native disc that has been disassociated from both adjacent vertebrae b Native disc (left) and tissue-engineered implant (right) c Implant in situ before wound closureAF: Annulus fi brosus, nP: nucleus pulposus, eP: endplate, M: Muscle, T: Tendon, s: skin, art: artery, GP: Growth plate, B: Bone Figure 3 Disc replacement surgery. Anatomy of the rat caudal disc space a Pircrosirius red stained axial cut of native disc space b Saffranin-O stained sagittal cut of native disc space Figure 4 Histologies of three separate motion segments from three different rats. Animal one = native IVD, Animal two = status after discectomy, Animal three = tissue-engineered implant (1 month) a–c H&E (overall tissue staining for light micrsocopy) d–f Alcian blue (proteoglycans) g–i Picrosirius red (collagen I and II) Figure 5 Histology from one motion segment four months after implantation of a bio-engineered disc construct a Picrosirius red staining (collagen) b Polarized light microscopy showing collagen staining and collagen organization in AF region c Increased Safranin-O staining (proteoglycans) in NP region of the disc implant d Higher magnification of figure 5c: Integration between implanted tissue-engineered total disc replacement and vertebral body bone Figure 6 MRI a Disc space height measurements in flash/T1 sequence (top: implant (714.0 micrometer), bottom: native disc (823.5 micrometer) b T2 sequence, red circle surrounding the implant NP Figure 7 7 Tesla MRI imaging of rat tail IVDs showing axial images (preliminary pilot data) a Diffusion tensor imaging (DTI) on two explanted rat tail discs in Formalin b Higher magnification of a, showing directional alignment of collagen fibers (red and green) when compared to the color ball on top which maps fibers' directional alignment (eg, fibers directing from left to right: red, from top to bottom: blue) c Native IVD in vivo (successful imaging of top and bottom of the IVD (red) d Gradient echo sequence (GE) showing differentiation between NP (light grey) and AF (dark margin) e GE of reimplanted tail IVD at the explantation level f T1Rho sequence demonstrating the NP (grey) within the AF (dark margin), containing the yellow marked region of interest for value acquisition (preliminary data are consistent with values reported in the literature). g T2 image of native IVD in vivo for monitoring of hydration (white: NP) Results: The model allowed reproducible and complete discectomies as well as disc implantation in the rat tail spine without any surgical or postoperative complications. Discectomy resulted in immediate collapse of the disc space. Preliminary results indicate that disc space height was maintained after disc implantation in groups II, III and IV over time. MRI revealed high resolution images of normal intervertebral discs in vivo. Eight out of twelve animals (groups III and IV) showed a positive signal in T2-weighted images after 1 month (grade 0 = 4, grade 1 = 4, grade 2 = 4). Positive staining was seen for collagen as well as proteoglycans at the site of disc implantation after 1 month in each of the six animals with engineered implants (group III). Analysis of group IV showed positive T2 signal in five out of six animals and disc-height preservation in all animals after 6 months. Conclusions: This study demonstrates for the first time that tissue-engineered composite IVDs with circumferentially aligned collagen fibrils survive and integrate with surrounding vertebral bodies when placed in the rat spine for up to 6 months. Tissue-engineered composite IVDs restored function to the rat spine as indicated by maintenance of disc height and vertebral alignment. A significant finding was that maintenance of the composite structure in group III was observed, with increased proteoglycan staining in the nucleus pulposus region (Figure 4d–f). Proteoglycan and collagen matrix as well as disc height preservation and positive T2 signals in MRI are promising parameters and indicate functionality of the implants. PMID:23637671

Total disc replacement using a tissue-engineered intervertebral disc in vivo: new animal model and initial results.

PubMed

Gebhard, Harry; Bowles, Robby; Dyke, Jonathan; Saleh, Tatianna; Doty, Stephen; Bonassar, Lawrence; Härtl, Roger

2010-08-01

 Basic science Introduction:  Chronic back pain due to degenerative disc disease (DDD) is among the most important medical conditions causing morbidity and significant health care costs. Surgical treatment options include disc replacement or fusion surgery, but are associated with significant short- and long-term risks.1 Biological tissue-engineering of human intervertebral discs (IVD) could offer an important alternative.2 Recent in vitro data from our group have shown successful engineering and growth of ovine intervertebral disc composites with circumferentially aligned collagen fibrils in the annulus fibrosus (AF) (Figure 1).3 Figure 1 Tissue-engineered composite disc a Experimental steps to generate composite tissue-engineered IVDs3b Example of different AF formulations on collagen alignment in the AF. Second harmonic generation and two-photon excited fluorescence images of seeded collagen gels (for AF) of 1 and 2.5 mg/ml over time. At seeding, cells and collagen were homogenously distributed in the gels. Over time, AF cells elongated and collagen aligned parallel to cells. Less contraction and less alignment is noted after 3 days in the 2.5 mg/mL gel. c Imaging-based creation of a virtual disc model that will serve as template for the engineered disc. Total disc dimensions (AF and NP) were retrieved from micro-computer tomography (CT) (left images), and nucleus pulposus (NP) dimensions alone were retrieved from T2-weighted MRI images (right images). Merging of MRI and micro-CT models revealed a composite disc model (middle image)-Software: Microview, GE Healthcare Inc., Princeton, NJ; and slicOmatic v4.3, TomoVision, Montreal, Canada. d Flow chart describing the process for generating multi-lamellar tissue engineered IVDs. IVDs are produced by allowing cell-seeded collagen layers to contract around a cell-seeded alginate core (NP) over time Objective:  The next step is to investigate if biological disc implants survive, integrate, and restore function to the spine in vivo. A model will be developed that allows efficient in vivo testing of tissue-engineered discs of various compositions and characteristics.  Athymic rats were anesthetized and a dorsal approach was chosen to perform a microsurgical discectomy in the rat caudal spine (Fig. 2,Fig. 3). Control group I (n = 6) underwent discectomy only, Control group II (n = 6) underwent discectomy, followed by reimplantation of the autologous disc. Two treatment groups (group III, n = 6, 1 month survival; group IV, n = 6, 6 months survival) received a tissue-engineered composite disc implant. The rodents were followed clinically for signs of infection, pain level and wound healing. X-rays and magnetic resonance imaging (MRI) were assessed postoperatively and up to 6 months after surgery (Fig. 6,Fig. 7). A 7 Tesla MRI (Bruker) was implemented for assessment of the operated level as well as the adjacent disc (hydration). T2-weighted sequences were interpreted by a semiquantitative score (0 = no signal, 1 = weak signal, 2 = strong signal and anatomical features of a normal disc). Histology was performed with staining for proteoglycans (Alcian blue) and collagen (Picrosirius red) (Fig. 4,Fig. 5). Figure 2 Disc replacement surgery a Operative situs with native disc that has been disassociated from both adjacent vertebrae b Native disc (left) and tissue-engineered implant (right) c Implant in situ before wound closureAF: Annulus fi brosus, nP: nucleus pulposus, eP: endplate, M: Muscle, T: Tendon, s: skin, art: artery, GP: Growth plate, B: BoneFigure 3 Disc replacement surgery. Anatomy of the rat caudal disc space a Pircrosirius red stained axial cut of native disc space b Saffranin-O stained sagittal cut of native disc spaceFigure 4 Histologies of three separate motion segments from three different rats. Animal one = native IVD, Animal two = status after discectomy, Animal three = tissue-engineered implant (1 month) a-c H&E (overall tissue staining for light micrsocopy) d-f Alcian blue (proteoglycans) g-i Picrosirius red (collagen I and II)Figure 5 Histology from one motion segment four months after implantation of a bio-engineered disc construct a Picrosirius red staining (collagen) b Polarized light microscopy showing collagen staining and collagen organization in AF region c Increased Safranin-O staining (proteoglycans) in NP region of the disc implant d Higher magnification of figure 5c: Integration between implanted tissue-engineered total disc replacement and vertebral body boneFigure 6 MRI a Disc space height measurements in flash/T1 sequence (top: implant (714.0 micrometer), bottom: native disc (823.5 micrometer) b T2 sequence, red circle surrounding the implant NPFigure 7 7 Tesla MRI imaging of rat tail IVDs showing axial images (preliminary pilot data) a Diffusion tensor imaging (DTI) on two explanted rat tail discs in Formalin b Higher magnification of a, showing directional alignment of collagen fibers (red and green) when compared to the color ball on top which maps fibers' directional alignment (eg, fibers directing from left to right: red, from top to bottom: blue) c Native IVD in vivo (successful imaging of top and bottom of the IVD (red) d Gradient echo sequence (GE) showing differentiation between NP (light grey) and AF (dark margin) e GE of reimplanted tail IVD at the explantation level f T1Rho sequence demonstrating the NP (grey) within the AF (dark margin), containing the yellow marked region of interest for value acquisition (preliminary data are consistent with values reported in the literature). g T2 image of native IVD in vivo for monitoring of hydration (white: NP) Results:  The model allowed reproducible and complete discectomies as well as disc implantation in the rat tail spine without any surgical or postoperative complications. Discectomy resulted in immediate collapse of the disc space. Preliminary results indicate that disc space height was maintained after disc implantation in groups II, III and IV over time. MRI revealed high resolution images of normal intervertebral discs in vivo. Eight out of twelve animals (groups III and IV) showed a positive signal in T2-weighted images after 1 month (grade 0 = 4, grade 1 = 4, grade 2 = 4). Positive staining was seen for collagen as well as proteoglycans at the site of disc implantation after 1 month in each of the six animals with engineered implants (group III). Analysis of group IV showed positive T2 signal in five out of six animals and disc-height preservation in all animals after 6 months.  This study demonstrates for the first time that tissue-engineered composite IVDs with circumferentially aligned collagen fibrils survive and integrate with surrounding vertebral bodies when placed in the rat spine for up to 6 months. Tissue-engineered composite IVDs restored function to the rat spine as indicated by maintenance of disc height and vertebral alignment. A significant finding was that maintenance of the composite structure in group III was observed, with increased proteoglycan staining in the nucleus pulposus region (Figure 4d-f). Proteoglycan and collagen matrix as well as disc height preservation and positive T2 signals in MRI are promising parameters and indicate functionality of the implants.

Conditioned Medium Derived from Notochordal Cell-Rich Nucleus Pulposus Tissue Stimulates Matrix Production by Canine Nucleus Pulposus Cells and Bone Marrow-Derived Stromal Cells

PubMed Central

de Vries, Stefan A.H.; Potier, Esther; van Doeselaar, Marina; Meij, Björn P.; Tryfonidou, Marianna A.

2015-01-01

Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect. Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×106 cells/mL) and NPCs+BMSCs (6×106 cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10 ng/mL transforming growth factor-β1 (TGF-β1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR. Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone. Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed. PMID:25370929

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

PubMed Central

Cafferty, Patrick; Xie, Xiaojun; Browne, Kristen; Auld, Vanessa J.

2009-01-01

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals. PMID:19590493

Influence of optic disc size on the diagnostic performance of macular ganglion cell complex and peripapillary retinal nerve fiber layer analyses in glaucoma.

PubMed

Cordeiro, Daniela Valença; Lima, Verônica Castro; Castro, Dinorah P; Castro, Leonardo C; Pacheco, Maria Angélica; Lee, Jae Min; Dimantas, Marcelo I; Prata, Tiago Santos

2011-01-01

To evaluate the influence of optic disc size on the diagnostic accuracy of macular ganglion cell complex (GCC) and conventional peripapillary retinal nerve fiber layer (pRNFL) analyses provided by spectral domain optical coherence tomography (SD-OCT) in glaucoma. Eighty-two glaucoma patients and 30 healthy subjects were included. All patients underwent GCC (7 × 7 mm macular grid, consisting of RNFL, ganglion cell and inner plexiform layers) and pRNFL thickness measurement (3.45 mm circular scan) by SD-OCT. One eye was randomly selected for analysis. Initially, receiver operating characteristic (ROC) curves were generated for different GCC and pRNFL parameters. The effect of disc area on the diagnostic accuracy of these parameters was evaluated using a logistic ROC regression model. Subsequently, 1.5, 2.0, and 2.5 mm(2) disc sizes were arbitrarily chosen (based on data distribution) and the predicted areas under the ROC curves (AUCs) and sensitivities were compared at fixed specificities for each. Average mean deviation index for glaucomatous eyes was -5.3 ± 5.2 dB. Similar AUCs were found for the best pRNFL (average thickness = 0.872) and GCC parameters (average thickness = 0.824; P = 0.19). The coefficient representing disc area in the ROC regression model was not statistically significant for average pRNFL thickness (-0.176) or average GCC thickness (0.088; P ≥ 0.56). AUCs for fixed disc areas (1.5, 2.0, and 2.5 mm(2)) were 0.904, 0.891, and 0.875 for average pRNFL thickness and 0.834, 0.842, and 0.851 for average GCC thickness, respectively. The highest sensitivities - at 80% specificity for average pRNFL (84.5%) and GCC thicknesses (74.5%) - were found with disc sizes fixed at 1.5 mm(2) and 2.5 mm(2). Diagnostic accuracy was similar between pRNFL and GCC thickness parameters. Although not statistically significant, there was a trend for a better diagnostic accuracy of pRNFL thickness measurement in cases of smaller discs. For GCC analysis, an inverse effect was observed.

Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity

PubMed Central

Torres-Oliva, Montserrat; Schneider, Julia; Wiegleb, Gordon

2018-01-01

Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb) regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells) and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells. PMID:29360820

Lactoferricin Enhances BMP7-Stimulated Anabolic Pathways in Intervertebral Disc Cells

PubMed Central

Ellman, Michael B; Kim, Jaesung; An, Howard S; Chen, Di; Kc, Ranjan; Li, Xin; Xiao, Guozhi; Yan, Dongyao; Suh, Joon; van Wijnen, Andre J.; Wang, James H-C; Kim, Su-Gwan; Im, Hee-Jeong

2013-01-01

Bone-morphogenetic protein-7 (BMP7) is a well-known anabolic and anti-catabolic growth factor on intervertebral (IVD) matrix and cell homeostasis. Similarly, lactoferricin B (LfcinB) has recently been shown to have pro-anabolic, anti-catabolic, anti-oxidative and/or anti-inflammatory effects in bovine disc cells in vitro. In this study, we investigated the potential benefits of using combined peptide therapy with LfcinB and BMP7 for intervertebral disc (IVD) matrix repair and to understand cellular and signaling mechanisms controlled by these factors. We studied the effects of BMP7 and LfcinB as individual treatments and combined therapy on bovine nucleus pulposus (NP) cells by assessing proteoglycan (PG) accumulation and synthesis, and the expression of matrix protein aggrecan and transcription factor SOX-9. We also analyzed the role of noggin, a BMP antagonist, in IVD tissue and examined its effect after stimulation with LfcinB. To understand the molecular mechanisms by which LfcinB synergizes with BMP7, we investigated the ERK-SP1 axis as a downstream intracellular signaling regulator involved in BMP7 and LfcinB-mediated activities. Treatment of bovine NP cells cultured in alginate with LfcinB plus BMP7 synergistically stimulates PG synthesis and accumulation in part by upregulation of aggrecan gene expression. The synergism results from LfcinB-mediated activation of Sp1 and SMAD signaling pathways by (i) phosphorylation of SMAD 1/5/8; (ii) downregulation of SMAD inhibitory factors [i.e., noggin (BMP receptor antagonist) and SMAD6 (inhibitory SMAD)]; and (iii) upregulation of SMAD4 (universal co-SMAD). These data indicate that LfcinB-suppression of noggin may eliminate the negative feedback of BMP7, thereby maximizing biological activity of BMP7 and ultimately shifting homeostasis to a pro-anabolic state in disc cells. We propose that combination growth factor therapy using BMP7 and LfcinB may be beneficial for treatment of disc degeneration. PMID:23644135

DISC1 Protein Regulates γ-Aminobutyric Acid, Type A (GABAA) Receptor Trafficking and Inhibitory Synaptic Transmission in Cortical Neurons.

PubMed

Wei, Jing; Graziane, Nicholas M; Gu, Zhenglin; Yan, Zhen

2015-11-13

Association studies have suggested that Disrupted-in-Schizophrenia 1 (DISC1) confers a genetic risk at the level of endophenotypes that underlies many major mental disorders. Despite the progress in understanding the significance of DISC1 at neural development, the mechanisms underlying DISC1 regulation of synaptic functions remain elusive. Because alterations in the cortical GABA system have been strongly linked to the pathophysiology of schizophrenia, one potential target of DISC1 that is critically involved in the regulation of cognition and emotion is the GABAA receptor (GABAAR). We found that cellular knockdown of DISC1 significantly reduced GABAAR-mediated synaptic and whole-cell current, whereas overexpression of wild-type DISC1, but not the C-terminal-truncated DISC1 (a schizophrenia-related mutant), significantly increased GABAAR currents in pyramidal neurons of the prefrontal cortex. These effects were accompanied by DISC1-induced changes in surface GABAAR expression. Moreover, the regulation of GABAARs by DISC1 knockdown or overexpression depends on the microtubule motor protein kinesin 1 (KIF5). Our results suggest that DISC1 exerts an important effect on GABAergic inhibitory transmission by regulating KIF5/microtubule-based GABAAR trafficking in the cortex. The knowledge gained from this study would shed light on how DISC1 and the GABA system are linked mechanistically and how their interactions are critical for maintaining a normal mental state. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

PubMed Central

Iessi, Elisabetta; Zischler, Luciana; Etringer, Aurélie; Bergeret, Marion; Morlé, Aymeric; Jacquemin, Guillaume; Morizot, Alexandre; Shirley, Sarah; Lalaoui, Najoua; Elifio-Esposito, Selene L.; Fais, Stefano; Garrido, Carmen; Solary, Eric; Micheau, Olivier

2015-01-01

Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells. PMID:26010871

Identification of a new stem cell population that generates Drosophila flight muscles.

PubMed

Gunage, Rajesh D; Reichert, Heinrich; VijayRaghavan, K

2014-08-18

How myoblast populations are regulated for the formation of muscles of different sizes is an essentially unanswered question. The large flight muscles of Drosophila develop from adult muscle progenitor (AMP) cells set-aside embryonically. The thoracic segments are all allotted the same small AMP number, while those associated with the wing-disc proliferate extensively to give rise to over 2500 myoblasts. An initial amplification occurs through symmetric divisions and is followed by a switch to asymmetric divisions in which the AMPs self-renew and generate post-mitotic myoblasts. Notch signaling controls the initial amplification of AMPs, while the switch to asymmetric division additionally requires Wingless, which regulates Numb expression in the AMP lineage. In both cases, the epidermal tissue of the wing imaginal disc acts as a niche expressing the ligands Serrate and Wingless. The disc-associated AMPs are a novel muscle stem cell population that orchestrates the early phases of adult flight muscle development.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ziemba, Stamatina E.; McCabe, Michael J.; Rosenspire, Allen J.

Genetically susceptible rodents exposed to low burdens of inorganic mercury (Hg{sup 2+}) develop autoimmune disease. Previous studies have shown that low, noncytotoxic levels of Hg{sup 2+} inhibit Fas-mediated apoptosis in T cells. These results suggest that inhibition of the Fas death receptor pathway potentially contributes to autoimmune disease after Hg{sup 2+} exposure, as a consequence of disruption of peripheral tolerance. The formation of active death inducing signaling complexes (DISC) following CD95/Fas receptor oligomerization is a primary step in the Fas-mediated apoptotic pathway. Other recent studies have shown that Hg{sup 2+} at concentrations that inhibit apoptosis also inhibit formation of activemore » DISC, suggesting that inhibition of DISC is the mechanism responsible for Hg{sup 2+}-mediated inhibition of apotosis. Preassociated Fas receptors have been implicated as key elements necessary for the production of functional DISC. We present evidence in this study showing that low and nontoxic concentrations of Hg{sup 2+} induce the dissociation of preassembled Fas receptor complexes in Jurkat T cells. Thus, this Hg{sup 2+}-induced event should subsequently decrease the amount of preassembled Fas available for DISC formation, potentially resulting in the attenuation of Fas-mediated apoptosis in T lymphocytes.« less

Insulin released from titanium discs with insulin coatings-Kinetics and biological activity.

PubMed

Malekzadeh, B Ö; Ransjo, M; Tengvall, P; Mladenovic, Z; Westerlund, A

2017-10-01

Local administration of insulin from a titanium surface has been demonstrated to increase bone formation in non-diabetic rats. The authors hypothesized that insulin was released from the titanium surface and with preserved biological activity after the release. Thus, in the present in vitro study, human recombinant insulin was immobilized onto titanium discs, and the insulin release kinetics was evaluated using Electro-chemiluminescence immunoassay. Neutral Red uptake assay and mineralization assay were used to evaluate the biological effects of the released insulin on human osteoblast-like MG-63 cells. The results confirmed that insulin was released from titanium surfaces during a six-week period. Etching the disc prior to insulin coating, thickening of the insulin coating and incubation of the discs in serum-enriched cell culture medium increased the release. However, longer storage time decreased the release of insulin. Furthermore, the released insulin had retained its biological activity, as demonstrated by the significant increase in cell number and a stimulated mineralization process, upon exposure to released insulin. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1847-1854, 2017. © 2016 Wiley Periodicals, Inc.

Macular Ganglion Cell Imaging Study: Covariate Effects on the Spectral Domain Optical Coherence Tomography for Glaucoma Diagnosis.

PubMed

Jeong, Jae Hoon; Choi, Yun Jeong; Park, Ki Ho; Kim, Dong Myung; Jeoung, Jin Wook

2016-01-01

To evaluate the effect of multiple covariates on the diagnostic performance of the Cirrus high-definition optical coherence tomography (HD-OCT) for glaucoma detection. A prospective case-control study was performed and included 173 recently diagnosed glaucoma patients and 63 unaffected individuals from the Macular Ganglion Cell Imaging Study. Regression analysis of receiver operating characteristic were conducted to evaluate the influence of age, spherical equivalent, axial length, optic disc size, and visual field index on the macular ganglion cell-inner plexiform layer (GCIPL) and peripapillary retinal nerve fiber layer (RNFL) measurements. Disease severity, as measured by visual field index, had a significant effect on the diagnostic performance of all Cirrus HD-OCT parameters. Age, axial length and optic disc size were significantly associated with diagnostic accuracy of average peripapillary RNFL thickness, whereas axial length had a significant effect on the diagnostic accuracy of average GCIPL thickness. Diagnostic performance of the Cirrus HD-OCT may be more accurate in the advanced stages of glaucoma than at earlier stages. A smaller optic disc size was significantly associated with improved the diagnostic ability of average RNFL thickness measurements; however, GCIPL thickness may be less affected by age and optic disc size.

Proteome-wide association studies identify biochemical modules associated with a wing-size phenotype in Drosophila melanogaster.

PubMed

Okada, Hirokazu; Ebhardt, H Alexander; Vonesch, Sibylle Chantal; Aebersold, Ruedi; Hafen, Ernst

2016-09-01

The manner by which genetic diversity within a population generates individual phenotypes is a fundamental question of biology. To advance the understanding of the genotype-phenotype relationships towards the level of biochemical processes, we perform a proteome-wide association study (PWAS) of a complex quantitative phenotype. We quantify the variation of wing imaginal disc proteomes in Drosophila genetic reference panel (DGRP) lines using SWATH mass spectrometry. In spite of the very large genetic variation (1/36 bp) between the lines, proteome variability is surprisingly small, indicating strong molecular resilience of protein expression patterns. Proteins associated with adult wing size form tight co-variation clusters that are enriched in fundamental biochemical processes. Wing size correlates with some basic metabolic functions, positively with glucose metabolism but negatively with mitochondrial respiration and not with ribosome biogenesis. Our study highlights the power of PWAS to filter functional variants from the large genetic variability in natural populations.

Neuroglian stabilizes epithelial structure during Drosophila oogenesis.

PubMed

Wei, Jun; Hortsch, Michael; Goode, Scott

2004-08-01

The vertebrate L1 family of cell adhesion molecules (CAMs) and their fly homolog, Neuroglian, are members of the immunoglobulin (Ig) superfamily of CAMs. In general, Ig CAMs have been found to play critical roles in mediating axon guidance. One Ig CAM, NCAM, has also been implicated in maintaining epithelial integrity and suppressing metastatic dissemination of tumor cells. Other Ig CAMs, such as Nrg, are also expressed in epithelia. We thus tested the hypothesis that, like NCAM, Nrg might also be required for maintaining epithelial integrity and for inhibiting tumor invasion. We used the Drosophila follicular epithelium to determine the function of Nrg in vivo in maintaining epithelial structure, and in regulating the motility of migrating border cells and invasive tumorous follicle cells. Nrg(167) is expressed on the lateral membrane of follicle cells. Loss of Nrg(167) causes border cells to delay delamination and causes other follicle cells to delaminate inappropriately. The delaminated cells have aberrant epithelial polarity manifested as severe mislocalization of apical and basal membrane proteins, and uniform localization of lateral membrane proteins. Furthermore, loss of Nrg(167) dramatically enhances the invasive phenotype associated with loss of Discs Large, a neoplastic tumor suppressor. These results indicate that Nrg(167) stabilizes epithelial polarity by regulating junctional adhesion and function in normal and tumorous epithelia. Our data also suggest that Ig superfamily members have significant functional redundancy in maintaining epithelial polarity, with individual members playing subtle, unique roles during epithelial morphogenesis. Copyright 2004 Wiley-Liss, Inc.

Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

PubMed Central

Xu, Yu; Huang, Lulin; Li, Jing; Zhang, Qi; Fei, Ping; Zhu, Xiong; Tai, Zhengfu; Ma, Shi; Gong, Bo; Li, Yun; Zang, Weizhou; Zhu, Xianjun; Zhao, Peiquan

2014-01-01

Purpose Familial exudative vitreoretinopathy (FEVR) is a group of inherited blinding eye diseases characterized by defects in the development of the retinal vessels. Recent studies have identified genetic variants in tetraspanin 12 (TSPAN12) as a cause of FEVR. The purpose of this study was to identify novel TSPAN12 mutations in Chinese patients with FEVR and to describe the associated phenotypes. Methods Mutation screening was performed by directly sequencing PCR products of genomic DNA with primers designed to amplify the seven coding exons and adjacent intronic regions of the FEVR-causing gene TSPAN12. Clinical phenotypes of the patients with TSPAN12 mutations were documented. Wild-type and mutant TSPAN12 proteins were assayed for the Norrin-β-catenin signaling pathway with luciferase reporter assays. Results Three novel heterozygous mutations in TSPAN12 were identified: c.566G>A (p.C189Y), c.177delC (p.Y59fsX67), and c.C254T (p.T85M). All three mutations involved highly conserved residues and were not present in 200 normal individuals. Ocular phenotypes included increased ramification of the peripheral retinal vessels, a peripheral avascular zone, inferotemporal dragging of the optic disc and macula, and retinal folds. The probands showed relatively severe retinopathy, whereas the other family members were often asymptomatic. In SuperTopFlash (STF) cell line transfection studies, C189Y, Y59fsX67, and T85M mutants failed to induce luciferase reporter activity in response to Norrin. Conclusions We found three novel TSPAN12 mutations in Chinese patients with autosomal dominant FEVR, and suggest that TSPAN12 mutations cause FEVR. The phenotypes associated with the TSPAN12 mutations showed extensive variation in disease severity among members of the same family, which implied the complexity of FEVR mutations and phenotypes. PMID:25352738

A Systematic Review of Mesenchymal Stem Cells in Spinal Cord Injury, Intervertebral Disc Repair and Spinal Fusion.

PubMed

Khan, Shujhat; Mafi, Pouya; Mafi, Reza; Khan, Wasim

2018-01-01

Spinal surgery presents a challenge for both neurosurgery and orthopaedic surgery. Due to the heterogeneous differentiation potential of mesenchymal stem cells, there is much interest in the treatment of spine surgery. Animal and human trials focussing on the efficacy of mesenchymal stem cells in spinal cord injury, spine fusion and disc degeneration were included in this systematic review. Published articles up to January 2016 from MEDLINE, PubMed and Ovid were used by searching for specific terms. Of the 2595 articles found, 53 met the selection criteria and were included for analysis (16 on spinal cord injury, 28 on intervertebral disc repair and 9 on spinal fusion). Numerous studies reported better results when the mesenchymal stem cells were used in co-culture with other cells or used in scaffolds. Mesenchymal stem cells were also found to have an immune-modulatory role, which can improve surgical outcome. This systematic review suggests that mesenchymal stem cells can be used safely and effectively for these spinal surgery treatments. Whilst, in certain studies, mesenchymal stem cells did not necessarily show improved results from existing treatments, they provide an alternative option. This can reduce morbidity that arises from current surgical treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Essential basal cytonemes take up Hedgehog in the Drosophila wing imaginal disc.

PubMed

Chen, Weitao; Huang, Hai; Hatori, Ryo; Kornberg, Thomas B

2017-09-01

Morphogen concentration gradients that extend across developmental fields form by dispersion from source cells. In the Drosophila wing disc, Hedgehog (Hh) produced by posterior compartment cells distributes in a concentration gradient to adjacent cells of the anterior compartment. We monitored Hh:GFP after pulsed expression, and analyzed the movement and colocalization of Hh, Patched (Ptc) and Smoothened (Smo) proteins tagged with GFP or mCherry and expressed at physiological levels from bacterial artificial chromosome transgenes. Hh:GFP moved to basal subcellular locations prior to release from posterior compartment cells that express it, and was taken up by basal cytonemes that extend to the source cells. Hh and Ptc were present in puncta that moved along the basal cytonemes and formed characteristic apical-basal distributions in the anterior compartment cells. The basal cytonemes required diaphanous , SCAR , N euroglian and S ynaptobrevin , and both the Hh gradient and Hh signaling declined under conditions in which the cytonemes were compromised. These findings show that in the wing disc, Hh distributions and signaling are dependent upon basal release and uptake, and on cytoneme-mediated movement. No evidence for apical dispersion was obtained. © 2017. Published by The Company of Biologists Ltd.

Ganglion cell-inner plexiform layer and retinal nerve fiber layer thickness according to myopia and optic disc area: a quantitative and three-dimensional analysis.

PubMed

Seo, Sam; Lee, Chong Eun; Jeong, Jae Hoon; Park, Ki Ho; Kim, Dong Myung; Jeoung, Jin Wook

2017-03-11

To determine the influences of myopia and optic disc size on ganglion cell-inner plexiform layer (GCIPL) and peripapillary retinal nerve fiber layer (RNFL) thickness profiles obtained by spectral domain optical coherence tomography (OCT). One hundred and sixty-eight eyes of 168 young myopic subjects were recruited and assigned to one of three groups according to their spherical equivalent (SE) values and optic disc area. All underwent Cirrus HD-OCT imaging. The influences of myopia and optic disc size on the GCIPL and RNFL thickness profiles were evaluated by multiple comparisons and linear regression analysis. Three-dimensional surface plots of GCIPL and RNFL thickness corresponding to different combinations of myopia and optic disc size were constructed. Each of the quadrant RNFL thicknesses and their overall average were significantly thinner in high myopia compared to low myopia, except for the temporal quadrant (all Ps ≤0.003). The average and all-sectors GCIPL were significantly thinner in high myopia than in moderate- and/or low-myopia (all Ps ≤0.002). The average OCT RNFL thickness was correlated significantly with SE (0.81 μm/diopter, P < 0.001), axial length (-1.44 μm/mm, P < 0.001), and optic disc area (5.35 μm/mm 2 , P < 0.001) by linear regression analysis. As for the OCT GCIPL parameters, average GCIPL thickness showed a significant correlation with SE (0.84 μm/diopter, P < 0.001) and axial length (-1.65 μm/mm, P < 0.001). There was no significant correlation of average GCIPL thickness with optic disc area. Three-dimensional curves showed that larger optic discs were associated with increased average RNFL thickness and that more-myopic eyes were associated with decreased average GCIPL and RNFL thickness. Myopia can significantly affect GCIPL and RNFL thickness profiles, and optic disc size has a significant influence on RNFL thickness. The current OCT maps employed in the evaluation of glaucoma should be analyzed in consideration of refractive status and optic disc size.

Expression of Mutant Human DISC1 in Mice Supports Abnormalities in Differentiation of Oligodendrocytes

PubMed Central

Katsel, Pavel; Tan, Weilun; Abazyan, Bagrat; Davis, Kenneth L; Ross, Christopher; Pletnikov, Mikhail V; Haroutunian, Vahram

2011-01-01

Abnormalities in oligodendrocyte (OLG) differentiation and OLG gene expression deficit have been described in schizophrenia (SZ). Recent studies revealed a critical requirement for Disrupted-in-Schizophrenia 1 (DISC1) in neural development. Transgenic mice with forebrain restricted expression of mutant human DISC1 (ΔhDISC1) are characterized by neuroanatomical and behavioral abnormalities reminiscent of some features of SZ. We sought to determine whether the expression of ΔhDISC1 may influence the development of OLGs in this mouse model. OLG- and cell cycle-associated gene and protein expression were characterized in the forebrain of ΔhDISC1 mice during different stages of neurodevelopment (E15 and P1 days) and in adulthood. The results suggest that the expression of ΔhDISC1 exerts a significant influence on oligodendrocyte differentiation and function, evidenced by premature OLG differentiation and increased proliferation of their progenitors. Additional findings showed that neuregulin 1 and its receptors may be contributing factors to the observed upregulation of OLG genes. Thus, OLG function may be perturbed by mutant hDISC1 in a model system that provides new avenues for studying aspects of the pathogenesis of SZ. PMID:21605958

Linear glandular trichomes of Helianthus (Asteraceae): morphology, localization, metabolite activity and occurrence

PubMed Central

Aschenbrenner, Anna-Katharina; Horakh, Silke; Spring, Otmar

2013-01-01

Capitate glandular trichomes of sunflower are well investigated, but detailed studies are lacking for the linear glandular trichomes (LGT), a second type of physiologically active plant hair present on the surface of sunflowers. Light, fluorescence and scanning electron microscopy as well as histochemical staining were used to investigate the structure and metabolite deposition of LGT. Consisting of 6–11 linearly arranged cells, LGT were found on the surface of most plant organs of Helianthus annuus. They were associated with the leaf vascular system, and also occurred along petioles, stems and the abaxial surface of chaffy bracts, ray and disc florets. The highest density was found on the abaxial surface of phyllaries. Phenotypically similar LGT were common in all species of the genus, but also occurred in most other genera of the Helianthinae so far screened. Brownish and fluorescent metabolites of an as yet unknown chemical structure, together with terpenoids, were produced and stored in apical cells of LGT. The deposition of compounds gradually progressed from the tip cell to the basal cells of older trichomes. This process was accompanied by nucleus degradation in metabolite-accumulating cells. The localization of these trichomes on prominent plant parts of the apical bud and the capitulum combined with the accumulation of terpenoids and other as yet unknown compounds suggests a chemo-ecological function of the LGT in plant–insect or plant–herbivore interaction.

Regression Analysis of Optical Coherence Tomography Disc Variables for Glaucoma Diagnosis.

PubMed

Richter, Grace M; Zhang, Xinbo; Tan, Ou; Francis, Brian A; Chopra, Vikas; Greenfield, David S; Varma, Rohit; Schuman, Joel S; Huang, David

2016-08-01

To report diagnostic accuracy of optical coherence tomography (OCT) disc variables using both time-domain (TD) and Fourier-domain (FD) OCT, and to improve the use of OCT disc variable measurements for glaucoma diagnosis through regression analyses that adjust for optic disc size and axial length-based magnification error. Observational, cross-sectional. In total, 180 normal eyes of 112 participants and 180 eyes of 138 participants with perimetric glaucoma from the Advanced Imaging for Glaucoma Study. Diagnostic variables evaluated from TD-OCT and FD-OCT were: disc area, rim area, rim volume, optic nerve head volume, vertical cup-to-disc ratio (CDR), and horizontal CDR. These were compared with overall retinal nerve fiber layer thickness and ganglion cell complex. Regression analyses were performed that corrected for optic disc size and axial length. Area-under-receiver-operating curves (AUROC) were used to assess diagnostic accuracy before and after the adjustments. An index based on multiple logistic regression that combined optic disc variables with axial length was also explored with the aim of improving diagnostic accuracy of disc variables. Comparison of diagnostic accuracy of disc variables, as measured by AUROC. The unadjusted disc variables with the highest diagnostic accuracies were: rim volume for TD-OCT (AUROC=0.864) and vertical CDR (AUROC=0.874) for FD-OCT. Magnification correction significantly worsened diagnostic accuracy for rim variables, and while optic disc size adjustments partially restored diagnostic accuracy, the adjusted AUROCs were still lower. Axial length adjustments to disc variables in the form of multiple logistic regression indices led to a slight but insignificant improvement in diagnostic accuracy. Our various regression approaches were not able to significantly improve disc-based OCT glaucoma diagnosis. However, disc rim area and vertical CDR had very high diagnostic accuracy, and these disc variables can serve to complement additional OCT measurements for diagnosis of glaucoma.

Evaluation of different phenotypic tests for detection of metallo-β-lactamases in imipenem-resistant Pseudomonas aeruginosa.

PubMed

Sachdeva, Rohit; Sharma, Babita; Sharma, Rajni

2017-01-01

Pseudomonas aeruginosa causes a wide spectrum of infections including bacteremia, pneumonia, urinary tract infection, etc., Metallo-beta-lactamase (MBL) producing P. aeruginosa is an emerging threat and cause of concern as they have emerged as one of the most feared resistance mechanisms. This study was designed to know the prevalence of MBL production in P. aeruginosa and to evaluate the four phenotypic tests for detection of MBL production in imipenem-resistant clinical isolates of P. aeruginosa . Totally, 800 isolates of P. aeruginosa isolated from various clinical samples were evaluated for carbapenem resistance and MBL production. All imipenem-resistant strains were tested for carabapenemase production by modified Hodge test. Screening for MBL production was done by double-disc synergy test and combined disc test (CDT). Confirmation of MBL production was done by the E-test (Ab BioDisk, Solna, Sweden). Out of the 800 isolates of P. aeruginosa , 250 isolates were found resistant to imipenem. Based on the results of E-test, 147 (18.37%) isolates of P. aeruginosa were positive for MBL production. The CDT has the highest sensitivity and specificity for the detection of MBL production as compared to other tests. The results of this study are indicative that MBL production is an important mechanism of carbapenem resistance among P. aeruginosa . Use of simple screening test like CDT will be crucial step toward large-scale monitoring of these emerging resistant determinants. Phenotypic test for MBL production has to be standardized, and all the isolates should be routinely screened for MBL production.

Evaluation of different phenotypic tests for detection of metallo-β-lactamases in imipenem-resistant Pseudomonas aeruginosa

PubMed Central

Sachdeva, Rohit; Sharma, Babita; Sharma, Rajni

2017-01-01

PURPOSE: Pseudomonas aeruginosa causes a wide spectrum of infections including bacteremia, pneumonia, urinary tract infection, etc., Metallo-beta-lactamase (MBL) producing P. aeruginosa is an emerging threat and cause of concern as they have emerged as one of the most feared resistance mechanisms. This study was designed to know the prevalence of MBL production in P. aeruginosa and to evaluate the four phenotypic tests for detection of MBL production in imipenem-resistant clinical isolates of P. aeruginosa. METHODS: Totally, 800 isolates of P. aeruginosa isolated from various clinical samples were evaluated for carbapenem resistance and MBL production. All imipenem-resistant strains were tested for carabapenemase production by modified Hodge test. Screening for MBL production was done by double-disc synergy test and combined disc test (CDT). Confirmation of MBL production was done by the E-test (Ab BioDisk, Solna, Sweden). RESULTS: Out of the 800 isolates of P. aeruginosa, 250 isolates were found resistant to imipenem. Based on the results of E-test, 147 (18.37%) isolates of P. aeruginosa were positive for MBL production. The CDT has the highest sensitivity and specificity for the detection of MBL production as compared to other tests. CONCLUSION: The results of this study are indicative that MBL production is an important mechanism of carbapenem resistance among P. aeruginosa. Use of simple screening test like CDT will be crucial step toward large-scale monitoring of these emerging resistant determinants. Phenotypic test for MBL production has to be standardized, and all the isolates should be routinely screened for MBL production. PMID:28966485

Expression of a putative grapevine hexose transporter in tobacco alters morphogenesis and assimilate partitioning.

PubMed

Leterrier, Marina; Atanassova, Rossitza; Laquitaine, Laurent; Gaillard, Cécile; Coutos-Thévenot, Pierre; Delrot, Serge

2003-04-01

Tobacco plants were transformed by leaf disc regeneration with the VvHT1 (Vitis vinifera hexose transporter 1) cDNA under the control of the constitutive CaMV 35S promoter in a sense or antisense orientation. Among the 20 sense plants and 10 antisense plants obtained, two sense plants showed a mutant phenotype when grown in vitro, with stunted growth and an increase in the (leaves+stem)/roots dry weight ratio. The rate of [(3)H]-glucose uptake in leaf discs from these plants was decreased to 25% of the value measured in control plants. The amount of VvHT1 transgene and of host monosaccharide transporter MST transcripts in the leaves were studied by RNA gel blot analysis. The VvHT1 transcripts were usually present, but the amount of MST transcripts was the lowest in the plants that exhibited the most marked phenotype. Although the phenotype was lost when the plants were transferred from in vitro to greenhouse conditions, it was found again in vitro in the progeny obtained by self-pollination or by back-cross. The data show that VvHT1 sense expression resulted in unidirectional post-transcriptional gene inactivation of MST in some of the transformants, with dramatic effects on growth. They provide the first example of plants modified for hexose transport by post-transcriptional gene silencing. Some of the antisense plants also showed reduced expression of MST, and decreased growth. These results indicate that, like the sucrose transporters, hexose transporters play an important role in assimilate transport and in morphogenesis.

Running Exercise Alleviates Pain and Promotes Cell Proliferation in a Rat Model of Intervertebral Disc Degeneration

PubMed Central

Luan, Shuo; Wan, Qing; Luo, Haijie; Li, Xiao; Ke, Songjian; Lin, Caina; Wu, Yuanyuan; Wu, Shaoling; Ma, Chao

2015-01-01

Chronic low back pain accompanied by intervertebral disk degeneration is a common musculoskeletal disorder. Physical exercise, which is clinically recommended by international guidelines, has proven to be effective for degenerative disc disease (DDD) patients. However, the mechanism underlying the analgesic effects of physical exercise on DDD remains largely unclear. The results of the present study showed that mechanical withdrawal thresholds of bilateral hindpaw were significantly decreased beginning on day three after intradiscal complete Freund’s adjuvant (CFA) injection and daily running exercise remarkably reduced allodynia in the CFA exercise group beginning at day 28 compared to the spontaneous recovery group (controls). The hindpaw withdrawal thresholds of the exercise group returned nearly to baseline at the end of experiment, but severe pain persisted in the control group. Histological examinations performed on day 70 revealed that running exercise restored the degenerative discs and increased the cell densities of the annulus fibrosus (AF) and nucleus pulposus (NP). Furthermore, immunofluorescence labeling revealed significantly higher numbers of 5-bromo-2-deoxyuridine (BrdU)-positive cells in the exercise group on days 28, 42, 56 and 70, which indicated more rapid proliferation compared to the control at the corresponding time points. Taken together, these results suggest that running exercise might alleviate the mechanical allodynia induced by intradiscal CFA injection via disc repair and cell proliferation, which provides new evidence for future clinical use. PMID:25607736

Effectiveness of disinfection therapies and promotion of osteoblast growth on osseotite and nanotite implant surfaces.

PubMed

Lubin, Judith; Hernandez, Maria A; Drukteinis, Saulius E; Parker, William B; Murray, Peter E

2014-08-01

To evaluate the effectiveness of 4 procedures to disinfect implant surfaces intentionally inoculated with bacteria and afterward to evaluate osteoblast viability to the disinfected implant surfaces. Eighty-eight commercially pure Osseotite and Nanotite titanium implant discs were inoculated with Porphyromonas gingivalis. The implant surfaces were disinfected with EDTA, tetracycline, citric acid, or neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. The implant discs were then placed in cultures of osteoblast cells. Osseotite implant discs were easier to disinfect compared with the Nanotite implant discs. Citric acid and tetracycline were the most effective solutions for the disinfection of P. gingivalis from the Osseotite implant discs. The Nanotite implant discs were the most difficult to disinfect, likely because of their chemical and physical properties. Citric acid and tetracycline were most effective for disinfecting the Osseotite implant discs, and further clinical research is needed to verify these effects in vivo. The Nd:YAG laser was the weakest disinfection method, and it is not recommended for disinfecting implant surfaces until its effectiveness is improved.

The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines.

PubMed

Peel, D J; Johnson, S A; Milner, M J

1990-01-01

We have examined the ultrastructure of cellular vesicles in primary cultures of wing imaginal disc cells of Drosophila melanogaster. These cells maintain the apico-basal polarity characteristic of epithelial cells. The apical surfaces secrete extracellular material into the lumen of the vesicle from plasma membrane plaques at the tip of microvilli. During the course of one passage, cells from the established cell lines grow to confluence and then aggregate into discrete condensations joined by aligned bridges of cells. Cells in these aggregates are tightly packed, and there appears to be a loss of the epithelial polarity characteristic of the vesicle cells. Elongated cell extensions containing numerous microtubules are found in aggregates, and we suggest that these may be epithelial feet involved in the aggregation process. Virus particles are commonly found both within the nucleus and the cytoplasm of cells in the aggregates.

Hyaline cartilage cells outperform mandibular condylar cartilage cells in a TMJ fibrocartilage tissue engineering application.

PubMed

Wang, L; Lazebnik, M; Detamore, M S

2009-03-01

To compare temporomandibular joint (TMJ) condylar cartilage cells in vitro to hyaline cartilage cells cultured in a three-dimensional (3D) environment for tissue engineering of mandibular condylar cartilage. Mandibular condylar cartilage and hyaline cartilage cells were harvested from pigs and cultured for 6 weeks in polyglycolic acid (PGA) scaffolds. Both types of cells were treated with glucosamine sulfate (0.4 mM), insulin-like growth factor-I (IGF-I) (100 ng/ml) and their combination. At weeks 0 and 6, cell number, glycosaminoglycan (GAG) and collagen content were determined, types I and II collagen were visualized by immunohistochemistry and GAGs were visualized by histology. Hyaline cartilage cells produced from half an order to a full order of magnitude more GAGs and collagen than mandibular condylar cartilage cells in 3D culture. IGF-I was a highly effective signal for biosynthesis with hyaline cartilage cells, while glucosamine sulfate decreased cell proliferation and biosynthesis with both types of cells. In vitro culture of TMJ condylar cartilage cells produced a fibrous tissue with predominantly type I collagen, while hyaline cartilage cells formed a fibrocartilage-like tissue with types I and II collagen. The combination of IGF and glucosamine had a synergistic effect on maintaining the phenotype of TMJ condylar cells to generate both types I and II collagen. Given the superior biosynthetic activity by hyaline cartilage cells and the practical surgical limitations of harvesting cells from the TMJ of a patient requiring TMJ reconstruction, cartilage cells from elsewhere in the body may be a potentially better alternative to cells harvested from the TMJ for TMJ tissue engineering. This finding may also apply to other fibrocartilages such as the intervertebral disc and knee meniscus in applications where a mature cartilage cell source is desired.

In vivo effects of bupivacaine and gadobutrol on the intervertebral disc following discoblock and discography: a histological analysis.

PubMed

Strube, Patrick; Pfitzner, Berit M; Streitparth, Florian; Hartwig, Tony; Putzier, Michael

2017-01-01

The aim of the present study was to histologically compare chondrotoxicity in surgically harvested intervertebral discs (IVDs) of patients following discoblock, discography, or no preoperative intervention. Thirty patients (IVD degeneration Modic ≥ 2°, Pfirrmann 3° or 4°) at L4/5 or L5/S1 who were planned for anterior lumbar interbody fusion were randomly assigned to three groups (open MRI: group DG - discography with gadobutrol; group DB - discoblock with bupivacaine at 4 weeks prior to surgery; group C - no intervention). The intervertebral discs were histologically evaluated and compared using ANOVA and Bonferroni tests for cell count, apoptosis, and proliferation. A reduced cell count (groups DG vs. DB vs. C: 14.9 ± 7.1, 9.2 ± 3.8, and 16.6 ± 5.2 cells/mm 2 , respectively; p ANOVA  = 0.016), increased apoptosis (groups DG vs. DB vs. C: 34.9 ± 10.2, 47.4 ± 16.3, 32.6 ± 12.2 %, respectively; p ANOVA  = 0.039) and increased cell proliferation (post hoc pDB vs. DG or C p < 0.001; for 3-7 cell monoclonal cell nests: groups DG vs. DB vs. C: 2.4 ± 1, 3.9 ± 1, 2.2 ± 1.1, respectively; p intervention x nest size  = 0.006) were found in the IVDs of patients in group DB. This in vivo study suggests that chondrotoxic effects occur in IVD cells after the intradiscal injection of bupivacaine but not after gadobutrol administration. • Local bupivacaine administration to intervertebral discs leads to cell toxicity and proliferation. • Gadobutrol demonstrated no significant effect on cell count, apoptosis, or cell proliferation. • In vivo cytotoxicity was demonstrated histologically in humans for the first time. • Addition/administration of bupivacaine during discographies must be judged critically.

Nitrogen-doped carbonaceous catalysts for gas-diffusion cathodes for alkaline aluminum-air batteries

NASA Astrophysics Data System (ADS)

Davydova, E. S.; Atamanyuk, I. N.; Ilyukhin, A. S.; Shkolnikov, E. I.; Zhuk, A. Z.

2016-02-01

Cobalt tetramethoxyphenyl porphyrin and polyacrylonitrile - based catalysts for oxygen reduction reaction were synthesized and characterized by means of SEM, TEM, XPS, BET, limited evaporation method, rotating disc and rotating ring-disc electrode methods. Half-cell and Al-air cell tests were carried out to determine the characteristics of gas-diffusion cathodes. Effect of active layer thickness and its composition on the characteristics of the gas-diffusion cathodes was investigated. Power density of 300 mW cm-2 was achieved for alkaline Al-air cell with an air-breathing polyacrylonitrile-based cathode.

Mechanical Vibrations Reduce the Intervertebral Disc Swelling and Muscle Atrophy from Bed Rest

NASA Technical Reports Server (NTRS)

Holguin, Nilsson; Muir, Jesse; Evans, Harlan J.; Qin, Yi-Xian; Rubin, Clinton; Wagshul, Mark; Judex, Stefan

2007-01-01

Loss of functional weight bearing, such as experienced during space flight or bed rest (BR), distorts intervertebral disc (IVD) and muscle morphology. IVDs are avascular structures consisting of cells that may derive their nutrition and waste removal from the load induced fluid flow into and out of the disc. A diurnal cycle is produced by forces related to weight bearing and muscular activity, and comprised of a supine and erect posture over a 24 hr period. A diurnal cycle will include a disc volume change of approx. 10-13%. However, in space there are little or no diurnal changes because of the microgravity, which removes the gravitational load and compressive forces to the back muscles. The BR model and the etiology of the disc swelling and muscle atrophy could provide insight into those subjects confined to bed for chronic disease/injury and aging. We hypothesize that extremely low-magnitude, high frequency mechanical vibrations will abate the disc degeneration and muscle loss associated with long-term BR.

Are animal models useful for studying human disc disorders/degeneration?

PubMed Central

Eisenstein, Stephen M.; Ito, Keita; Little, Christopher; Kettler, A. Annette; Masuda, Koichi; Melrose, James; Ralphs, Jim; Stokes, Ian; Wilke, Hans Joachim

2007-01-01

Intervertebral disc (IVD) degeneration is an often investigated pathophysiological condition because of its implication in causing low back pain. As human material for such studies is difficult to obtain because of ethical and government regulatory restriction, animal tissue, organs and in vivo models have often been used for this purpose. However, there are many differences in cell population, tissue composition, disc and spine anatomy, development, physiology and mechanical properties, between animal species and human. Both naturally occurring and induced degenerative changes may differ significantly from those seen in humans. This paper reviews the many animal models developed for the study of IVD degeneration aetiopathogenesis and treatments thereof. In particular, the limitations and relevance of these models to the human condition are examined, and some general consensus guidelines are presented. Although animal models are invaluable to increase our understanding of disc biology, because of the differences between species, care must be taken when used to study human disc degeneration and much more effort is needed to facilitate research on human disc material. PMID:17632738

Developmental Regulation of Nucleolus Size during Drosophila Eye Differentiation

PubMed Central

Baker, Nicholas E.

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals. PMID:23472166

Developmental regulation of nucleolus size during Drosophila eye differentiation.

PubMed

Baker, Nicholas E

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.

PubMed

Kepler, Christopher K; Markova, Dessislava Z; Koerner, John D; Mendelis, Joseph; Chen, Chiu-Ming; Vaccaro, Alexander R; Risbud, Makarand V; Albert, Todd J; Anderson, D Greg

2015-08-15

Laboratory study. To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1β, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1β, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways. 4.

An investigation into the effect of surface roughness of stainless steel on human umbilical vein endothelial cell gene expression.

PubMed

McLucas, E; Moran, M T; Rochev, Y; Carroll, W M; Smith, T J

2006-01-01

The surface properties of vascular devices dictate the initial postimplantation reactions that occur and thus the efficacy of the implantation procedure. Over the last number of years, a number of different stent designs have emerged and stents are generally polished to a mirror finish during the manufacturing procedure. This study sought to investigate the effect of stainless steel surface roughness on endothelial cell gene expression using an appropriate cell culture in vitro assay system. Stainless steel discs were roughened by shot blasting or polished by mechanical polishing. The surface roughness of the treated and untreated discs was determined by atomic force microscopy (AFM). Cells were seeded on collagen type 1 gels and left to attach for 24 h. Stainless steel discs of varying roughness were then placed in contact with the cells and incubated for 24 h. RNA extractions and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was then performed to determine the expression levels of candidate genes in the treated cells compared to suitable control cells. E-selectin and vascular cellular adhesion molecule (VCAM-1) were found to be significantly up-regulated in cells incubated with polished and roughened samples, indicating endothelial cell activation and inflammation. This study indicates that the surface roughness of stainless steel is an important surface property in the development of vascular stents.

In vivo performance of an acellular disc-like angle ply structure (DAPS) for total disc replacement in a small animal model.

PubMed

Martin, John T; Kim, Dong Hwa; Milby, Andrew H; Pfeifer, Christian G; Smith, Lachlan J; Elliott, Dawn M; Smith, Harvey E; Mauck, Robert L

2017-01-01

Total intervertebral disc replacement with a biologic engineered disc may be an alternative to spinal fusion for treating end-stage disc disease. In previous work, we developed disc-like angle ply structures (DAPS) that replicate the structure and function of the native disc and a rat tail model to evaluate DAPS in vivo. Here, we evaluated a strategy in which, after in vivo implantation, endogenous cells could colonize the acellular DAPS and form an extracellular matrix organized by the DAPS topographical template. To do so, acellular DAPS were implanted into the caudal spines of rats and evaluated over 12 weeks by mechanical testing, histology, and microcomputed tomography. An external fixation device was used to stabilize the implant site and various control groups were included to evaluate the effect of immobilization. There was robust tissue formation within the DAPS after implantation and compressive mechanical properties of the implant matched that of the native motion segment. Immobilization provided a stable site for fibrous tissue formation after either a discectomy or a DAPS implantation, but bony fusion eventually resulted, with segments showing intervertebral bridging after long-term implantation, a process that was accelerated by the implanted DAPS. Thus, while compressive mechanical properties were replicated after DAPS implantation, methods to actively prevent fusion must be developed. Future work will focus on limiting fusion by remobilizing the motion segment after a period of integration, delivering pro-chondrogenic factors, and pre-seeding DAPS with cells prior to implantation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:23-31, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

The Drosophila T-box transcription factor Midline functions within the Notch–Delta signaling pathway to specify sensory organ precursor cell fates and regulates cell survival within the eye imaginal disc

PubMed Central

Das, Sudeshna; Chen, Q. Brent; Saucier, Joseph D.; Drescher, Brandon; Zong, Yan; Morgan, Sarah; Forstall, John; Meriwether, Andrew; Toranzo, Randy; Leal, Sandra M.

2014-01-01

We report that the T-box transcription factor Midline (Mid), an evolutionary conserved homolog of the vertebrate Tbx20 protein, functions within the Notch–Delta signaling pathway essential for specifying the fates of sensory organ precursor cells. This complements an established history of research showing that Mid regulates the cell-fate specification of diverse cell types within the developing heart, epidermis and central nervous system. Tbx20 has been detected in diverse neuronal and epithelial cells of embryonic eye tissues in both mice and humans. However, the mechanisms by which either Mid or Tbx20 function to regulate cell-fate specification or other critical aspects of eye development including cell survival have not yet been elucidated. We have also gathered preliminary evidence suggesting that Mid may play an indirect, but vital role in selecting SOP cells within the third-instar larval eye disc by regulating the expression of the proneural gene atonal. During subsequent pupal stages, Mid specifies SOP cell fates as a member of the Notch–Delta signaling hierarchy and is essential for maintaining cell viability within by inhibiting apoptotic pathways. We present several new hypotheses that seek to understand the role of Mid in regulating developmental processes downstream of the Notch receptor that are critical for specifying unique cell fates, patterning the adult eye and maintaining cellular homeostasis during eye disc morphogenesis. PMID:23962751

Macular Ganglion Cell Imaging Study: Covariate Effects on the Spectral Domain Optical Coherence Tomography for Glaucoma Diagnosis

PubMed Central

Jeong, Jae Hoon; Choi, Yun Jeong; Park, Ki Ho; Kim, Dong Myung

2016-01-01

Purpose To evaluate the effect of multiple covariates on the diagnostic performance of the Cirrus high-definition optical coherence tomography (HD-OCT) for glaucoma detection. Methods A prospective case-control study was performed and included 173 recently diagnosed glaucoma patients and 63 unaffected individuals from the Macular Ganglion Cell Imaging Study. Regression analysis of receiver operating characteristic were conducted to evaluate the influence of age, spherical equivalent, axial length, optic disc size, and visual field index on the macular ganglion cell-inner plexiform layer (GCIPL) and peripapillary retinal nerve fiber layer (RNFL) measurements. Results Disease severity, as measured by visual field index, had a significant effect on the diagnostic performance of all Cirrus HD-OCT parameters. Age, axial length and optic disc size were significantly associated with diagnostic accuracy of average peripapillary RNFL thickness, whereas axial length had a significant effect on the diagnostic accuracy of average GCIPL thickness. Conclusions Diagnostic performance of the Cirrus HD-OCT may be more accurate in the advanced stages of glaucoma than at earlier stages. A smaller optic disc size was significantly associated with improved the diagnostic ability of average RNFL thickness measurements; however, GCIPL thickness may be less affected by age and optic disc size. PMID:27490718

A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes.

PubMed

Kim, Young-Il; Ryu, Taewoo; Lee, Judong; Heo, Young-Shin; Ahnn, Joohong; Lee, Seung-Jae; Yoo, OokJoon

2010-01-25

Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF) animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines). We screened approximately 15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future investigations of the complicated relationships between apoptosis and autophagy.

Expression of matrix metalloproteinase genes during basement membrane degradation in the metamorphosis of Bombyx mori.

PubMed

Kawasaki, Hideki; Manickam, Asaithambi; Shahin, Rima; Ote, Manabu; Iwanaga, Masashi

2018-01-05

The present study was conducted to clarify the involvement of the basement membrane (BM) in insect metamorphosis through analysis of the expression profile of two types of metalloproteinase (MMP and ADAMTS) genes in several organs, their ecdysone involvement, and the histological change of BM. BM was observed around wing sac and in the wing cavity and around fat bodies at the W0 stage but disappeared after the W3 stage, and wing discs evaginated and fat body cells scattered after the W3 stage. The disappearance of the BM of midgut and silk glands was not observed after the W3 stage, but degenerated epithelium cells in the midgut and shrunken cells in the silk gland were observed after the W3 stage. BmMMP1 showed a peak at P0 in the wing discs, fat bodies, midgut, and silk gland. BmMMP2 showed a broad peak around pupation in the wing discs, fat bodies, midgut, and silk gland. BmADAMTS-1 showed enhanced expression at W2 in the wing discs, fat bodies, midgut, and hemocyte, while BmADAMTS-L showed enhanced expression at W3 in the fat bodies, midgut, silk gland, and hemocyte. After pupation, they showed a different expression in different organs. All of four genes were induced by 20-hydroxyecdysone in wing discs in vitro. The present results suggested the involvement of MMPs and ADAMTS in the BM digestion and the morphogenesis of organs during Bombyx metamorphosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Bactericidal effect of photocatalytically-active nanostructured TiO2 surfaces on biofilms of the early oral colonizer, Streptococcus oralis.

PubMed

Westas, Emma; Hayashi, Mariko; Cecchinato, Francesca; Wennerberg, Ann; Andersson, Martin; Jimbo, Ryo; Davies, Julia R

2017-08-01

This study evaluated the photocatalytic bactericidal effect of nanostructured anatase-rich titanium dioxide (TiO 2 ) on microbial biofilms. Commercially pure titanium discs were spin-coated with photocatalytic TiO 2 nanoparticles (P25). Uncoated discs were used as control (CTRL). Half of the CTRL and half of the P25-coated surfaces were coated with purified saliva (SAL) to give four different groups (CTRL, CTRL + SAL, P25 and P25 + SAL). Streptococcus oralis were allowed to form biofilms on the discs for 18 h and non-adherent cells were rinsed off. Bacterial viability was assessed at time 0 with Live/Dead BacLight staining and epifluorescence microscopy. The remaining discs were divided into a non-UV group and UVA-irradiated (+UV) group (irradiation time, 6 or 24 h). Thereafter, viability was assessed as above. Viability at time 0 was high and no dead cells were seen on any of the surfaces, even after 24 h, in the absence of UVA. However, after 24 h of exposure, the proportion of viable cells was reduced by 40% on the P25 discs compared to 0 and 6 h, and this effect was enhanced with a salivary pellicle. Members of mixed species biofilms differ in their susceptibility to the bactericidal effect of the surfaces tested and further investigations are needed to optimize the conditions. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2321-2328, 2017. © 2017 Wiley Periodicals, Inc.

The role of cyclooxygenase-2 and inflammatory cytokines in pain induction of herniated lumbar intervertebral disc.

PubMed

Miyamoto, H; Saura, R; Harada, T; Doita, M; Mizuno, K

2000-04-01

Lumbar disc herniation (LDH) is the disease which is the major cause of radiculopathy. In terms of the pathogenesis of disease, it is reported that prostaglandinE2 (PGE2) plays an important role to induce radiculopathy. Arachidonate cascade, which is the process of PGE2 synthesis, is mainly regulated by two kinds of enzymes, phospholipaseA2 (PLA2) and cyclooxy genase (COX). Previously, PLA2 was recognized as the rate-limiting enzyme of this cascade, and some authors reported the clinical significance of PLA2 at the site of LDH concerning the radicular pain. Recently, COX was elucidated to consist of 2 types of isoform, a constitutive form of COX-1 and an inducible form of COX-2. COX-2 has been focused as a key enzyme to regulate PGE2 synthesis and plays an important role in inflammation, because COX-2 was induced in many types of cells by the stimulation of inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha). However, it is not fully discussed whether or not, COX-2 is induced in lumbar disc tissue and if it plays a significant role in the pathogenesis of LDH. To clarify the role of COX-2 in the pathomechanism of radiculopathy of LDH, we have investigated the expression of COX-2, IL-1 beta and TNF alpha in herniated lumbar disc tissue. Immunohistologically, they were detected in the cytosol of chondrocytes constituting the disc tissue. RT-PCR showed that herniated lumbar disc-derived cells expressed mRNA of COX-2, IL-1 beta and TNF alpha in the presence of inflammatory cytokines in vitro. The disc-derived cells also produced much PGE2 by stimulating of inflammatory cytokines at the same time and this PGE2 production was distinctly suppressed by a selective inhibitor of COX-2, 6-methoxy-2-naphtyl acetic acids (6MNA). These results suggest that COX-2 and inflammatory cytokines might play a causative role in the radiculopathy of LDH through upregulating PGE2 synthesis.

Adaptations in rod outer segment disc membranes in response to environmental lighting conditions.

PubMed

Rakshit, Tatini; Senapati, Subhadip; Parmar, Vipul M; Sahu, Bhubanananda; Maeda, Akiko; Park, Paul S-H

2017-10-01

The light-sensing rod photoreceptor cell exhibits several adaptations in response to the lighting environment. While adaptations to short-term changes in lighting conditions have been examined in depth, adaptations to long-term changes in lighting conditions are less understood. Atomic force microscopy was used to characterize the structure of rod outer segment disc membranes, the site of photon absorption by the pigment rhodopsin, to better understand how photoreceptor cells respond to long-term lighting changes. Structural properties of the disc membrane changed in response to housing mice in constant dark or light conditions and these adaptive changes required output from the phototransduction cascade initiated by rhodopsin. Among these were changes in the packing density of rhodopsin in the membrane, which was independent of rhodopsin synthesis and specifically affected scotopic visual function as assessed by electroretinography. Studies here support the concept of photostasis, which maintains optimal photoreceptor cell function with implications in retinal degenerations. Copyright © 2017 Elsevier B.V. All rights reserved.

[Characteristics of hydroxyapatite/PMMA nanocomposites for provisional restoration and its biocompatibility with human gingival fibroblasts].

PubMed

Zhang, Jing-chao; Mo, An-chun; Li, Ji-dong; Wang, Xue-jiang; Li, Yu-bao

2014-05-01

To formulate hydroxyapatite (HA)/polymethyl methacrylate (PMMA) composites with improved cytocompatibility for provisional restoration. Nanocomposites with 20 wt%, 30 wt%, 40 wt%, and 50 wt% HA/PMMA (H/P) were developed and examined using X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM). Human gingival fibroblasts were cultured on those HA/PMMA discs and investigated by fluorescent staining on 24 h and MTT assay at 1 d, 3 d, 5 d and 7 d. Chemical integration of HA/PMMA interface was confirmed by XPS. Typical fusiform cells with adhesion spots were detected on 40 wt% and 50 wt% H/P discs. MTT results showed insignificant differences in cell growth between 40 wt% H/P and pure titanium (Ti, P > 0.05), while the other H/P discs showed significantly lower cell growth than pure Ti (P < 0.05). 40 wt% H/P might be a promising candidate for provisional dental implant restoration and for esthetic gingival contour.

Overexpression of Shox2 Leads to Congenital Dysplasia of the Temporomandibular Joint in Mice

PubMed Central

Li, Xihai; Liang, Wenna; Ye, Hongzhi; Weng, Xiaping; Liu, Fayuan; Liu, Xianxiang

2014-01-01

Our previous study reported that inactivation of Shox2 led to dysplasia and ankylosis of the temporomandibular joint (TMJ), and that replacing Shox2 with human Shox partially rescued the phenotype with a prematurely worn out articular disc. However, the mechanisms of Shox2 activity in TMJ development remain to be elucidated. In this study, we investigated the molecular and cellular basis for the congenital dysplasia of TMJ in Wnt1-Cre; pMes-stop Shox2 mice. We found that condyle and glenoid fossa dysplasia occurs primarily in the second week after the birth. The dysplastic TMJ of Wnt1-Cre; pMes-stop Shox2 mice exhibits a loss of Collagen type I, Collagen type II, Ihh and Gli2. In situ zymography and immunohistochemistry further demonstrate an up-regulation of matrix metalloproteinases (MMPs), MMP9 and MMP13, accompanied by a significantly increased cell apoptosis. In addition, the cell proliferation and expressions of Sox9, Runx2 and Ihh are no different in the embryonic TMJ between the wild type and mutant mice. Our results show that overexpression of Shox2 leads to the loss of extracellular matrix and the increase of cell apoptosis in TMJ dysplasia by up-regulating MMPs and down-regulating the Ihh signaling pathway. PMID:25062348

GLYX-13 Ameliorates Schizophrenia-Like Phenotype Induced by MK-801 in Mice: Role of Hippocampal NR2B and DISC1

PubMed Central

Zhou, Dongsheng; Lv, Dan; Wang, Zhen; Zhang, Yanhua; Chen, Zhongming; Wang, Chuang

2018-01-01

Background: Evidence supports that the hypofunction of N-methyl-D-aspartate receptor (NMDAR) and downregulation of disrupted-in-schizophrenia 1 (DISC1) contribute to the pathophysiology of schizophrenia. N-Methyl D-aspartate receptor subtype 2B (NR2B)-containing NMDAR are associated with cognitive dysfunction in schizophrenia. GLYX-13 is an NMDAR glycine-site functional partial agonist and cognitive enhancer that does not induce psychotomimetic side effects. However, it remains unclear whether NR2B plays a critical role in the GLYX-13-induced alleviation of schizophrenia-like behaviors in mice. Methods: The effect of GLYX-13 was tested by observing changes in locomotor activity, novel object recognition ability, and prepulse inhibition (PPI) induced by dizocilpine (known as MK-801) in mice. Lentivirus-mediated NR2B knockdown in the hippocampus was assessed to confirm the role of NR2B in GLYX-13 pathophysiology, using Western blots and immunohistochemistry. Results: The systemic administration of GLYX-13 (0.5 and 1 mg/kg, i.p.) ameliorates MK-801 (0.5 mg/kg, i.p.)-induced hyperlocomotion, deficits in memory, and PPI in mice. Additionally, GLYX-13 normalized the MK-801-induced alterations in signaling molecules, including NR2B and DISC1 in the hippocampus. Furthermore, we found that NR2B knockdown produced memory and PPI deficits without any changes in locomotor activity. Notably, DISC1 levels significantly decreased by NR2B knockdown. However, the effective dose of GLYX-13 did not alleviate the memory and PPI dysfunctions or downregulation of DISC1 induced by NR2B knockdown. Conclusion: Our results suggest GLYX-13 as a candidate for schizophrenia treatment, and NR2B and DISC1 in the hippocampus may account for the molecular mechanisms of GLYX-13. PMID:29695955

Epithelial-mesenchymal transition in colonies of rhesus monkey embryonic stem cells: a model for processes involved in gastrulation.

PubMed

Behr, Rüdiger; Heneweer, Carola; Viebahn, Christoph; Denker, Hans-Werner; Thie, Michael

2005-01-01

Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc-like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial-mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E-cadherin and ZO-1 in the lower layer. In support of this, we found a concomitant 10-fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.

A New Bone Substitute Developed from 3D-Prints of Polylactide (PLA) Loaded with Collagen I: An In Vitro Study.

PubMed

Ritz, Ulrike; Gerke, Rebekka; Götz, Hermann; Stein, Stefan; Rommens, Pol Maria

2017-11-29

Although a lot of research has been performed, large segmental bone defects caused by trauma, infection, bone tumors or revision surgeries still represent big challenges for trauma surgeons. New and innovative bone substitutes are needed. Three-dimensional (3D) printing is a novel procedure to create 3D porous scaffolds that can be used for bone tissue engineering. In the present study, solid discs as well as porous cage-like 3D prints made of polylactide (PLA) are coated or filled with collagen, respectively, and tested for biocompatibility and endotoxin contamination. Microscopic analyses as well as proliferation assays were performed using various cell types on PLA discs. Stromal-derived factor (SDF-1) release from cages filled with collagen was analyzed and the effect on endothelial cells tested. This study confirms the biocompatibility of PLA and demonstrates an endotoxin contamination clearly below the FDA (Food and Drug Administration) limit. Cells of various cell types (osteoblasts, osteoblast-like cells, fibroblasts and endothelial cells) grow, spread and proliferate on PLA-printed discs. PLA cages loaded with SDF-1 collagen display a steady SDF-1 release, support cell growth of endothelial cells and induce neo-vessel formation. These results demonstrate the potential for PLA scaffolds printed with an inexpensive desktop printer in medical applications, for example, in bone tissue engineering.

A New Bone Substitute Developed from 3D-Prints of Polylactide (PLA) Loaded with Collagen I: An In Vitro Study

PubMed Central

Gerke, Rebekka; Götz, Hermann; Rommens, Pol Maria

2017-01-01

Although a lot of research has been performed, large segmental bone defects caused by trauma, infection, bone tumors or revision surgeries still represent big challenges for trauma surgeons. New and innovative bone substitutes are needed. Three-dimensional (3D) printing is a novel procedure to create 3D porous scaffolds that can be used for bone tissue engineering. In the present study, solid discs as well as porous cage-like 3D prints made of polylactide (PLA) are coated or filled with collagen, respectively, and tested for biocompatibility and endotoxin contamination. Microscopic analyses as well as proliferation assays were performed using various cell types on PLA discs. Stromal-derived factor (SDF-1) release from cages filled with collagen was analyzed and the effect on endothelial cells tested. This study confirms the biocompatibility of PLA and demonstrates an endotoxin contamination clearly below the FDA (Food and Drug Administration) limit. Cells of various cell types (osteoblasts, osteoblast-like cells, fibroblasts and endothelial cells) grow, spread and proliferate on PLA-printed discs. PLA cages loaded with SDF-1 collagen display a steady SDF-1 release, support cell growth of endothelial cells and induce neo-vessel formation. These results demonstrate the potential for PLA scaffolds printed with an inexpensive desktop printer in medical applications, for example, in bone tissue engineering. PMID:29186036

Drosophila S2 cells secrete wingless on exosome-like vesicles but the wingless gradient forms independently of exosomes.

PubMed

Beckett, Karen; Monier, Solange; Palmer, Lucy; Alexandre, Cyrille; Green, Hannah; Bonneil, Eric; Raposo, Graca; Thibault, Pierre; Le Borgne, Roland; Vincent, Jean-Paul

2013-01-01

Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless-expressing Drosophila S2 cells, Wingless is present on exosome-like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless-containing exosome-like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome-like vesicles. Using these exosome markers and a cell-based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless-expressing cells. © 2012 John Wiley & Sons A/S.

H2O2/HCl and heat-treated Ti-6Al-4V stimulates pre-osteoblast proliferation and differentiation.

PubMed

Shi, Geng-sheng; Ren, Ling-fei; Wang, Lin-zhi; Lin, Hai-sheng; Wang, Sha-bin; Tong, Yong-qing

2009-09-01

The purpose of the present study was to evaluate the bioactivity of chemical treatment of titanium alloy (Ti-6Al-4V) in vitro. Smooth-surface discs of Ti-6Al-4V were used in this study. Sandblasted, dual acid-etched and H(2)O(2)/HCl heat-treated discs were set as test group, and sandblasted, dual acid-etched discs as control group. SEM and XRD analysis revealed a porous anatase gel layer on rough surface in the test group and a rough surface in the control group. Mouse pre-osteoblasts (MC3T3-E1 cells) were cultured on these 2 group discs, and then cell proliferation and differentiation were examined 4 days, 7 days, and 14 days after cell seeding. Cell proliferation was greatly stimulated at all time points when cultured in test group (P < .05). The alkaline phosphatase (ALP) activity and osteocalcin (OC) production were much higher in the test group compared with the control group at every time point investigated (P < .05). Furthermore, in the test group, the expressions of alkaline phosphatase-2, osteocalcin, and collagen type I alpha 1 mRNAs were significantly up-regulated as compared with those in the control group (P < .05 or P < .01). The results suggested that H(2)O(2)/HCl and heat-treatment might facilitate better integration of Ti-6Al-4V implants with bone.

Intercellular signaling pathways active during intervertebral disc growth, differentiation, and aging.

PubMed

Dahia, Chitra Lekha; Mahoney, Eric J; Durrani, Atiq A; Wylie, Christopher

2009-03-01

Intervertebral discs at different postnatal ages were assessed for active intercellular signaling pathways. To generate a spatial and temporal map of the signaling pathways active in the postnatal intervertebral disc (IVD). The postnatal IVD is a complex structure, consisting of 3 histologically distinct components, the nucleus pulposus, fibrous anulus fibrosus, and endplate. These differentiate and grow during the first 9 weeks of age in the mouse. Identification of the major signaling pathways active during and after the growth and differentiation period will allow functional analysis using mouse genetics and identify targets for therapy for individual components of the disc. Antibodies specific for individual cell signaling pathways were used on cryostat sections of IVD at different postnatal ages to identify which components of the IVD were responding to major classes of intercellular signal, including sonic hedgehog, Wnt, TGFbeta, FGF, and BMPs. We present a spatial/temporal map of these signaling pathways during growth, differentiation, and aging of the disc. During growth and differentiation of the disc, its different components respond at different times to different intercellular signaling ligands. Most of these are dramatically downregulated at the end of disc growth.

An Automatic Lab-on-Disc System for Blood Typing.

PubMed

Chang, Yaw-Jen; Fan, Yi-Hua; Chen, Shia-Chung; Lee, Kuan-Hua; Lou, Liao-Yong

2018-04-01

A blood-typing assay is a critical test to ensure the serological compatibility of a donor and an intended recipient prior to a blood transfusion. This article presents a lab-on-disc blood-typing system to conduct a total of eight assays for a patient, including forward-typing tests, reverse-typing tests, and irregular-antibody tests. These assays are carried out in a microfluidic disc simultaneously. A blood-typing apparatus was designed to automatically manipulate the disc. The blood type can be determined by integrating the results of red blood cell (RBC) agglutination in the microchannels. The experimental results of our current 40 blood samples show that the results agree with those examined in the hospital. The accuracy reaches 97.5%.

Serotonergic transmission at Merkel discs: modulation by exogenously applied chemical messengers and involvement of Ih currents.

PubMed

Chang, Weipang; Kanda, Hirosato; Ikeda, Ryo; Ling, Jennifer; Gu, Jianguo G

2017-05-01

The Merkel disc is a main type of tactile end organ consisting of Merkel cells and Aβ-afferent endings that responds to tactile stimulation with slowly adapting type 1 (SA1) afferent impulses. Our recent study has shown that Merkel discs in whisker hair follicles are serotonergic synapses using endogenous serotonin to transmit tactile signals from Merkel cells to Aβ-afferent endings. In this study, we hypothesize that tactile sensitivity of Merkel discs can be modulated by chemical messengers. We tested this hypothesis by determining whether and how SA1 responses of mouse whisker hair follicles may be affected by exogenously applied chemical messengers. We found that SA1 responses were potentiated by serotonin at low concentration (10 μM) but almost completely occluded by serotonin at high concentration (2 mM). In contrast, SA1 responses were not significantly affected by ATP and its metabolically stable analog α,β-methylene-ATP, glutamate, γ-aminobutyric acid (GABA), and histamine. SA1 responses were also not affected by antagonists for P2X receptors, ionotropic glutamate receptors, and ionotropic GABA and glycine receptors. Whole-cell patch-clamp recordings reconfirm the presence of both ionotropic and metabotropic 5-HT receptors on afferent neurons and their terminals innervating whisker hair follicles. All whisker afferent neurons expressed hyperpolarization-activated inward currents (I h ), which are potentiated by serotonin through the activation of metabotropic 5-HT receptors. Taken together, the findings substantiate the serotonergic mechanism of tactile transmission at Merkel discs and identify the involvement of I h currents in postsynaptic excitatory actions of serotonin. In addition, the findings do not favor any significant involvement of ATP, glutamate, histamine, GABA, or glycine in tactile transmission at the Merkel discs of whisker hair follicles. © 2017 International Society for Neurochemistry.

Intervertebral disc-derived stem cells: implications for regenerative medicine and neural repair.

PubMed

Erwin, W Mark; Islam, Diana; Eftekarpour, Eftekhar; Inman, Robert D; Karim, Muhammad Zia; Fehlings, Michael G

2013-02-01

An in vitro and in vivo evaluation of intervertebral disc (IVD)-derived stem/progenitor cells. To determine the chondrogenic, adipogenic, osteogenic, and neurogenic differentiation capacity of disc-derived stem/progenitor cells in vitro and neurogenic differentiation in vivo. Tissue repair strategies require a source of appropriate cells that could be used to replace dead or damaged cells and tissues such as stem cells. Here we examined the potential use of IVD-derived stem cells in regenerative medicine approaches and neural repair. Nonchondrodystrophic canine IVD nucleus pulposus (NP) cells were used to generate stem/progenitor cells (NP progenitor cells [NPPCs]) and the NPPCs were differentiated in vitro into chondrogenic, adipogenic, and neurogenic lineages and in vivo into the neurogenic lineage. NPPCs were compared with bone marrow-derived mesenchymal (stromal) stem cells in terms of the expression of stemness genes. The expression of the neural crest marker protein 0 and the Brachyury gene were evaluated in NP cells and NPPCs. NPPCs contain stem/progenitor cells and express "stemness" genes such as Sox2, Oct3/4, Nanog, CD133, Nestin, and neural cell adhesion molecule but differ from mesenchymal (stromal) stem cells in the higher expression of the Nanog gene by NPPCs. NPPCs do not express protein 0 or the Brachyury gene both of which are expressed by the totality of IVD NP cells. The percentage of NPPCs within the IVD is 1% of the total as derived by colony-forming assay. NPPCs are capable of differentiating along chondrogenic, adipogenic, and neurogenic lineages in vitro and into oligodendrocyte, neuron, and astroglial specific precursor cells in vivo within the compact myelin-deficient shiverer mouse. We propose that the IVD NP represents a regenerative niche suggesting that the IVD could represent a readily accessible source of precursor cells for neural repair and regeneration.

Biomarkers reflect differences in osteoarthritis phenotypes of the lumbar spine: the Johnston County Osteoarthritis Project.

PubMed

Goode, A P; Nelson, A E; Kraus, V B; Renner, J B; Jordan, J M

2017-10-01

To determine differences in biomarker levels between radiographic phenotypes of facet joint osteoarthritis (FOA) only, spine OA only ((disc space narrowing (DSN) and vertebral osteophytes (OST)) or the combination of FOA and spine OA. A cross-sectional analysis of data from 555 participants in the Johnston County Osteoarthritis Project was performed. Lumbar spine levels were graded by severity (OST and DSN) and presence (FOA) of degeneration. Biomarkers included hyaluronan (HA) and type II collagen (CTX-II). Adjusted risk ratios (aRRR) were estimated using multinomial regression, with adjustment for age, race, sex, body mass index (BMI), and radiographic OA (knee, hip, hand). Interactions were tested between sex, race and low back symptoms. FOA only was present in 22.4%, 14.5% had spine OA only, and 34.6% had the combination of FOA and spine OA. Compared to the reference group of neither FOA or spine OA, a one unit higher ln HA level was associated with 31% higher relative risk ratio (RRR = 1.31 (95% 1.03, 1.67)) of having FOA only, while, a one unit higher lnuCTX-II level was associated with 84% higher relative risk ratio (RRR = 1.84 (95% CI 1.19, 2.84)) of having spine OA only. No significant interactions were identified. Interestingly, OA affecting the synovial facet joint was associated with a marker of inflammation (HA). Spine OA, affecting intervertebral discs that contain collagen type II, was associated with a marker reflecting collagen type II degradation (CTX-II). These findings suggest that biomarkers may reflect the different pathophysiologic processes of lumbar spine OA phenotypes. Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Fluid flow and convective transport of solutes within the intervertebral disc.

PubMed

Ferguson, Stephen J; Ito, Keita; Nolte, Lutz P

2004-02-01

Previous experimental and analytical studies of solute transport in the intervertebral disc have demonstrated that for small molecules diffusive transport alone fulfils the nutritional needs of disc cells. It has been often suggested that fluid flow into and within the disc may enhance the transport of larger molecules. The goal of the study was to predict the influence of load-induced interstitial fluid flow on mass transport in the intervertebral disc. An iterative procedure was used to predict the convective transport of physiologically relevant molecules within the disc. An axisymmetric, poroelastic finite-element structural model of the disc was developed. The diurnal loading was divided into discrete time steps. At each time step, the fluid flow within the disc due to compression or swelling was calculated. A sequentially coupled diffusion/convection model was then employed to calculate solute transport, with a constant concentration of solute being provided at the vascularised endplates and outer annulus. Loading was simulated for a complete diurnal cycle, and the relative convective and diffusive transport was compared for solutes with molecular weights ranging from 400 Da to 40 kDa. Consistent with previous studies, fluid flow did not enhance the transport of low-weight solutes. During swelling, interstitial fluid flow increased the unidirectional penetration of large solutes by approximately 100%. Due to the bi-directional temporal nature of disc loading, however, the net effect of convective transport over a full diurnal cycle was more limited (30% increase). Further study is required to determine the significance of large solutes and the timing of their delivery for disc physiology.

Antibiotic susceptibility of methicillin-resistant staphylococci (MRS) of food origin: A comparison of agar disc diffusion method and a commercially available miniaturized test.

PubMed

Buzón-Durán, Laura; Capita, Rosa; Alonso-Calleja, Carlos

2018-06-01

Methicillin-resistant staphylococci (MRS) are a major concern to public and animal health. Thirty MRS (Staphylococcus aureus, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. lentus, S. lugdunensis, S. sciuri, and S. xylosus) isolates from meat and poultry preparations were tested for antimicrobial susceptibility to 11 antimicrobials (belonging to seven different categories) of clinical significance using both the standard agar disc diffusion method and a commercially available miniaturized system (Sensi Test Gram-positive). It is worth stressing that 16 isolates (53.33%) exhibited an extensively drug-resistant phenotype (XDR). The average number of resistances per strain was 4.67. These results suggest that retail meat and poultry preparations are a likely vehicle for the transmission of multi-drug resistant MRS. Resistance to erythromycin was the commonest finding (76.67% of strains), followed by tobramycin, ceftazidime (66.67%), ciprofloxacin (56.67%) and fosfomycin (53.33%). An agreement (kappa coefficient) of 0.64 was found between the two testing methods. Using the agar disc diffusion as the reference method, the sensitivity, specificity and accuracy of the miniaturized test were 98.44%, 69.44% and 83.33%, respectively. Most discrepancies between the two methods were due to isolates that were susceptible according to the disc diffusion method but resistant according to the miniaturized test (false positives). Copyright © 2017. Published by Elsevier Ltd.

The Drosophila T-box transcription factor Midline functions within the Notch-Delta signaling pathway to specify sensory organ precursor cell fates and regulates cell survival within the eye imaginal disc.

PubMed

Das, Sudeshna; Chen, Q Brent; Saucier, Joseph D; Drescher, Brandon; Zong, Yan; Morgan, Sarah; Forstall, John; Meriwether, Andrew; Toranzo, Randy; Leal, Sandra M

2013-01-01

We report that the T-box transcription factor Midline (Mid), an evolutionary conserved homolog of the vertebrate Tbx20 protein, functions within the Notch-Delta signaling pathway essential for specifying the fates of sensory organ precursor (SOP) cells. These findings complement an established history of research showing that Mid regulates the cell-fate specification of diverse cell types within the developing heart, epidermis and central nervous system. Tbx20 has been detected in unique neuronal and epithelial cells of embryonic eye tissues in both mice and humans. However, the mechanisms by which either Mid or Tbx20 function to regulate cell-fate specification or other critical aspects of eye development including cell survival have not yet been elucidated. We have also gathered preliminary evidence suggesting that Mid may play an indirect, but vital role in selecting SOP cells within the third-instar larval eye disc by regulating the expression of the proneural gene atonal. During subsequent pupal stages, Mid specifies SOP cell fates as a member of the Notch-Delta signaling hierarchy and is essential for maintaining cell viability by inhibiting apoptotic pathways. We present several new hypotheses that seek to understand the role of Mid in regulating developmental processes downstream of the Notch receptor that are critical for specifying unique cell fates, patterning the adult eye and maintaining cellular homeostasis during eye disc morphogenesis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharjee, Rajesh; Xiang, Wenpei; Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People's Republic of China

2012-06-22

Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1more » (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC complex upon the binding of TNF to TNFR1. In conclusion, our study shows that cAMP prevents TNF + ActD-induced apoptosis in rat hepatocytes by inhibiting DISC complex formation.« less

Activated macrophage-like THP-1 cells modulate anulus fibrosus cell production of inflammatory mediators in response to cytokines.

PubMed

Kim, Joo Han; Studer, Rebecca K; Sowa, Gwendolyn A; Vo, Nam Viet; Kang, James D

2008-10-01

Anulus fibrosus (AF) cells obtained from patients undergoing surgery were cocultured with macrophage-like cells and production of inflammatory mediators was analyzed by quantitative assay. To investigate the role of macrophages in AF cell production of inflammatory mediators by cytokines stimulation. Discogenic pain caused by anular disruption is an important cause of low back pain and recent studies show the presence of macrophages in symptomatic discs but not in normal and aging discs. We hypothesize that macrophages play a major role in development of symptomatic disc. Human AF cells were cocultured with phorbol myristate acetate stimulated macrophage-like THP-1 cells. The conditioned medium from cells cultured alone or in coculture was assayed for cytokines by Enzyme-linked immunosorbent assay and nitric oxide (NO) by the Greiss method. Using the same outcome measures, comparisons of cell response to cytokines were made among macrophage-like cells, naïve AF cells, and macrophage exposed AF cells. RESULTS.: Tumor necrosis factor (TNF)-alpha, interleukin (IL)-8, IL-6, and NO (TNF-alpha: 1.45 +/- 0.29 ng/mL, IL-8: 97.02 +/- 7.94 ng/mL, IL-6: 33.40 +/- 3.55 ng/mL, NO: 8.42 +/- 0.78 micromol/L) were secreted in much greater amounts by cells maintained in coculture compared to macrophages (TNF-alpha: 0.78 +/- 0.12 ng/mL, IL-8: 58.04 +/- 4.44 ng/mL, IL-6: 0.14 +/- 0.03 ng/mL, NO: 0.30 +/- 0.08 micromol/L) or AF cells cultured alone. In addition, IL-6 secretion from AF cells in response to TNF-alpha was up-regulated by coculture, however, IL-6 secretion in response to IL-1 beta was downregulated in a dose-dependent manner. Coculture with macrophages also up-regulated AF cell secretion of IL-8 dose-dependently and downregulated NO to TNF-alpha or IL-1beta stimulation. We conclude that exposure to macrophages, as can be expected after anular injury, can result in enhanced response to local inflammation. Although changes were observed in all inflammatory mediators after macrophage exposure, the most significant change was observed in IL-6 and IL-8, implicating these mediators in development of symptomatic disc.

Prediction of glycosaminoglycan synthesis in intervertebral disc under mechanical loading.

PubMed

Gao, Xin; Zhu, Qiaoqiao; Gu, Weiyong

2016-09-06

The loss of glycosaminoglycan (GAG) content is a major biochemical change during intervertebral disc (IVD) degeneration. Abnormal mechanical loading is one of the major factors causing disc degeneration. In this study, a multiscale mathematical model was developed to quantify the effect of mechanical loading on GAG synthesis. This model was based on a recently developed cell volume dependent GAG synthesis theory that predicts the variation of GAG synthesis rate of a cell under the influence of mechanical stimuli, and the biphasic theory that describes the deformation of IVD under mechanical loading. The GAG synthesis (at the cell level) was coupled with the mechanical loading (at the tissue level) via a cell-matrix unit approach which established a relationship between the variation of cell dilatation and the local tissue dilatation. This multiscale mathematical model was used to predict the effect of static load (creep load) on GAG synthesis in bovine tail discs. The predicted results are in the range of experimental results. This model was also used to investigate the effect of static (0.2MPa) and diurnal loads (0.1/0.3MPa and 0.15/0.25MPa in 12/12 hours shift with an average of 0.2MPa over a cycle) on GAG synthesis. It was found that static load and diurnal loads have different effects on GAG synthesis in a diurnal cycle, and the diurnal load effects depend on the amplitude of the load. The model is important to understand the effect of mechanical loading at the tissue level on GAG synthesis at the cellular level, as well as to optimize the mechanical loading in growing engineered tissue. Copyright © 2016 Elsevier Ltd. All rights reserved.

Control of dental-derived induced pluripotent stem cells through modified surfaces for dental application.

PubMed

Choi, Hyunmin; Park, Kyu-Hyung; Lee, Ah-Reum; Mun, Chin Hee; Shin, Yong Dae; Park, Yong-Beom; Park, Young-Bum

2017-07-01

The aim of this study is to investigate the behaviour of iPSc derived from dental stem cells in terms of initial adhesion, differentiation potential on differently surface-treated titanium disc. iPSc derived from human gingival fibroblasts (hGFs) were established using 4-reprogramming factors transduction with Sendai virus. The hGF-iPSc established in this study exhibited the morphology and growth properties similar to human embryonic stem (ES) cells and expressed pluripotency makers. Alkaline Phosphatase (AP) staining, Embryoid Body (EB) formation and in vitro differentiation and karyotyping further confirmed pluripotency of hGF-iPSc. Then, hGF-iPSc were cultured on machined- and Sandblasted and acid etched (SLA)-treated titanium discs with osteogenic induction medium and their morphological as well as quantitative changes according to different surface types were investigated using Alizrin Red S staining, Scanning electron microscopy (SEM), Flow cytometry and RT-PCR. Time-dependent and surface-dependent morphological changes as well as quantitative change in osteogenic differentiation of hGF-iPSc were identified and osteogenic gene expression of hGF-iPSc cultured on SLA-treated titanium disc found to be greater than machined titanium disc, suggesting the fate of hGF-iPSc may be determined by the characteristics of surface to which hGF-iPSc first adhere. iPSc derived from dental stem cell can be one of the most promising and practical cell sources for personalized regenerative dentistry and their morphological change as well as quantitative change in osteogenic differentiation according to different surface types may be further utilized for future clinical application incorporated with dental implant.

Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation

PubMed Central

Zhu, Jinjin; Palliyil, Sneha; Ran, Chen; Kumar, Justin P.

2017-01-01

Paired box 6 (Pax6) is considered to be the master control gene for eye development in all seeing animals studied so far. In vertebrates, it is required not only for lens/retina formation but also for the development of the CNS, olfactory system, and pancreas. Although Pax6 plays important roles in cell differentiation, proliferation, and patterning during the development of these systems, the underlying mechanism remains poorly understood. In the fruit fly, Drosophila melanogaster, Pax6 also functions in a range of tissues, including the eye and brain. In this report, we describe the function of Pax6 in Drosophila eye-antennal disc development. Previous studies have suggested that the two fly Pax6 genes, eyeless (ey) and twin of eyeless (toy), initiate eye specification, whereas eyegone (eyg) and the Notch (N) pathway independently regulate cell proliferation. Here, we show that Pax6 controls eye progenitor cell survival and proliferation through the activation of teashirt (tsh) and eyg, thereby indicating that Pax6 initiates both eye specification and proliferation. Although simultaneous loss of ey and toy during early eye-antennal disc development disrupts the development of all head structures derived from the eye-antennal disc, overexpression of N or tsh in the absence of Pax6 rescues only antennal and head epidermis development. Furthermore, overexpression of tsh induces a homeotic transformation of the fly head into thoracic structures. Taking these data together, we demonstrate that Pax6 promotes development of the entire eye-antennal disc and that the retinal determination network works to repress alternative tissue fates, which ensures proper development of adult head structures. PMID:28584125

Therapeutic effects of naringin on degenerative human nucleus pulposus cells for discogenic low back pain.

PubMed

Li, Nianhu; Whitaker, Camden; Xu, Zhanwang; Heggeness, Michael; Yang, Shang-You

2016-10-01

Over half the population of the world will suffer from moderate or severe low back pain (LBP) during their life span. Studies have shown that naringin, a major flavonoid in grapefruit and an active compound extracted from a Chinese herbal medicine (Rhizoma Drynariae) possesses many pharmacological effects. The aim of this study was to evaluate the influence of naringin on the growth of degenerative human nucleus pulposus (NP) cells, and its repair effects on protein and gene expressions of the cells. This was an in vitro investigation of the human NP cells isolated from degenerated intervertebral discs that were interacted with various concentrated of naringin. This study was exempted by the institutional Human Subjects Committee-2, University of Kansas School of Medicine-Wichita. Degenerative human NP cells were isolated from intervertebral discs of patients with discogenic LBP and cultured at 37°C with 5% CO 2 . The proliferation of NP cells was determined following treatment with various concentrations of naringin. The protein expressions of tumor necrosis factor-α (TNF-α) and Bone morphogenetic protein 2 (BMP-2) were tested using enzyme-linked immunosorbent assay. Aggrecan and type II collagen levels were measured by immunohistological staining. Further examination of the gene expression of aggrecan, Sox6, and MMP3 was performed after intervention with naringin for 3 days. The human NP cells were successfully propagated in culture and stained positive with toluidine blue staining. Naringin effectively enhanced the cell proliferation at an optimal concentration of 20 µg/mL. Naringin treatment resulted in significant inhibition of TNF-α, but elevated protein expressions of BMP-2, collagen II, and aggrecan. Naringin also increased disc matrix gene activity including aggrecan and Sox6, and decreased the gene expression of MMP3. Naringin effectively promotes the proliferation of degenerative human NP cells and improves the recuperation of the cells from degeneration by increasing expression of aggrecan, BMP-2, and Sox6 while inhibiting the expression of TNF-α and MMP3. This study suggests that naringin may represent an alternative therapeutic agent for disc degeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

A distinct X-linked syndrome involving joint contractures, keloids, large optic cup-to-disc ratio, and renal stones results from a filamin A (FLNA) mutation.

PubMed

Lah, Melissa; Niranjan, Tejasvi; Srikanth, Sujata; Holloway, Lynda; Schwartz, Charles E; Wang, Tao; Weaver, David D

2016-04-01

We further evaluated a previously reported family with an apparently undescribed X-linked syndrome involving joint contractures, keloids, an increased optic cup-to-disc ratio, and renal stones to elucidate the genetic cause. To do this, we obtained medical histories and performed physical examination on 14 individuals in the family, five of whom are affected males and three are obligate carrier females. Linkage analysis was performed on all but one individual and chromosome X-exome sequencing was done on two affected males. The analysis localized the putative gene to Xq27-qter and chromosome X-exome sequencing revealed a mutation in exon 28 (c.4726G>A) of the filamin A (FLNA) gene, predicting that a conserved glycine had been replaced by arginine at amino acid 1576 (p.G1576R). Segregation analysis demonstrated that all known carrier females tested were heterozygous (G/A), all affected males were hemizygous for the mutation (A allele) and all normal males were hemizygous for the normal G allele. The data and the bioinformatic analysis indicate that the G1576R mutation in the FLNA gene is very likely pathogenic in this family. The syndrome affecting the family shares phenotypic overlap with other syndromes caused by FLNA mutations, but appears to be a distinct phenotype, likely representing a unique genetic syndrome. © 2016 Wiley Periodicals, Inc.

High-Resolution Metabolic Phenotyping of Genetically and Environmentally Diverse Potato Tuber Systems. Identification of Phenocopies

PubMed Central

Roessner, Ute; Willmitzer, Lothar; Fernie, Alisdair R.

2001-01-01

We conducted a comprehensive metabolic phenotyping of potato (Solanum tuberosum L. cv Desiree) tuber tissue that had been modified either by transgenesis or exposure to different environmental conditions using a recently developed gas chromatography-mass spectrometry profiling protocol. Applying this technique, we were able to identify and quantify the major constituent metabolites of the potato tuber within a single chromatographic run. The plant systems that we selected to profile were tuber discs incubated in varying concentrations of fructose, sucrose, and mannitol and transgenic plants impaired in their starch biosynthesis. The resultant profiles were then compared, first at the level of individual metabolites and then using the statistical tools hierarchical cluster analysis and principal component analysis. These tools allowed us to assign clusters to the individual plant systems and to determine relative distances between these clusters; furthermore, analyzing the loadings of these analyses enabled identification of the most important metabolites in the definition of these clusters. The metabolic profiles of the sugar-fed discs were dramatically different from the wild-type steady-state values. When these profiles were compared with one another and also with those we assessed in previous studies, however, we were able to evaluate potential phenocopies. These comparisons highlight the importance of such an approach in the functional and qualitative assessment of diverse systems to gain insights into important mediators of metabolism. PMID:11706160

Dpp signaling inhibits proliferation in the Drosophila wing by Omb-dependent regional control of bantam.

PubMed

Zhang, Xubo; Luo, Dan; Pflugfelder, Gert O; Shen, Jie

2013-07-01

The control of organ growth is a fundamental aspect of animal development but remains poorly understood. The morphogen Dpp has long been considered as a general promoter of cell proliferation during Drosophila wing development. It is an ongoing debate whether the Dpp gradient is required for the uniform cell proliferation observed in the wing imaginal disc. Here, we investigated how the Dpp signaling pathway regulates proliferation during wing development. By systematic manipulation of Dpp signaling we observed that it controls proliferation in a region-specific manner: Dpp, via omb, promoted proliferation in the lateral and repressed proliferation in the medial wing disc. Omb controlled the regional proliferation rate by oppositely regulating transcription of the microRNA gene bantam in medial versus lateral wing disc. However, neither the Dpp nor Omb gradient was essential for uniform proliferation along the anteroposterior axis.

Characterization and human gingival fibroblasts biocompatibility of hydroxyapatite/PMMA nanocomposites for provisional dental implant restoration

NASA Astrophysics Data System (ADS)

Zhang, Jingchao; Liao, Juan; Mo, Anchun; Li, Yubao; Li, Jidong; Wang, Xuejiang

2008-11-01

The aim of this study was to determine nHA/PMMA composites (H/P) in an optimal ratio with improved cytocompatibility as well as valid physical properties for provisional dental implant restoration. 20 wt.%, 30 wt.%, 40 wt.% and 50 wt.% H/P were developed and characterized using XPS, bending strength test and SEM. Human gingival fibroblasts cultured in extracts or directly on sample discs were investigated by fluorescent staining and MTT assay. Chemical integration in nHA/PMMA interface was indicated by XPS. Typical fusiform cells with adhesion spots were detected on H/P discs. MTT results also indicated higher cell viability in 30 wt.% and 40 wt.% H/P discs ( P < 0.05). We conclude that nHA addition to PMMA enhances cytocompatibility and the optimal nHA/PMMA ratio for provisional fixed crowns (PFC) is 0.4:1.

The effects of a magnetic field on planetary migration in laminar and turbulent discs

NASA Astrophysics Data System (ADS)

Comins, Megan L.; Romanova, Marina M.; Koldoba, Alexander V.; Ustyugova, Galina V.; Blinova, Alisa A.; Lovelace, Richard V. E.

2016-07-01

We investigate the migration of low-mass planets (1, 5 and 20 M⊕) in accretion discs threaded with a magnetic field using 2D magnetohydrodynamic code in polar coordinates. We observed that, in the case of a strong azimuthal magnetic field where the plasma parameter is β ˜ 2-4, density waves at the magnetic resonances exert a positive torque on the planet and may slow down or reverse its migration. However, when the magnetic field is weaker (I.e. the plasma parameter β is relatively large), then non-axisymmetric density waves excited by the planet lead to growth of the radial component of the field and, subsequently, to development of the magnetorotational instability, such that the disc becomes turbulent. Migration in a turbulent disc is stochastic, and the migration direction may change as such. To understand migration in a turbulent disc, both the interaction between a planet and individual turbulent cells, as well as the interaction between a planet and ordered density waves, have been investigated.

Effects of 15(S)-15-methyl prostaglandin F2 alpha methyl ester-containing silastic discs in male rats.

PubMed

Kimball, F A; Frielink, R D; Porteus, S E

1978-01-01

Silicone rubber discs containing 15(S)-15-methyl prostaglandin F2 alpha ester (15-Me-PGF2 alpha) in the matrix were implanted in the left side of the scrotums of Sprague-Dawley rats. The effect of 1% and 2% drug concentration was examined for 10, 20, or 28 days and compared with the effects of Silastic discs containing no prostaglandin. The discs containing prostaglandin reduced mean testicular and accessory gland weights. Histologically the testes and epididymides showed decreased or absent spermatogenic elements and hypertrophy of the interstitial cell masses in comparison with other cells. Implanted prostaglandin significantly depressed serum testosterone, luteinizing hormone, and follicle-stimulating hormone (FSH) concentrations when 15-Me-PGF2 alpha plasma concentrations exceeded 2 ng/ml. Hormone concentrations returned to control values as drug concentrations declined. FSH concentrations significantly exceeded control values 10 and 20 days after implantation, when prostaglandin concentration was nondetectable. The acute suppression of all three hormones suggest that 15-Me-PGF2 alpha either may act directly on the tests to suppress testosterone production or may suppress testosterone production or may suppress gonadotropin secretion, resulting in depressed testosterone output.

Cervical intradural disc herniation.

PubMed

Iwamura, Y; Onari, K; Kondo, S; Inasaka, R; Horii, H

2001-03-15

A case report of anterior en bloc resected cervical intradural disc herniation and a review of the literature. To discuss the pathogenesis of cervical intradural disc herniation. Including this study case, only 17 cases of cervical intradural disc herniation have been reported. There have been few detailed reports concerning the pathogenesis of cervical intradural disc herniation. A cervical intradural disc herniation at C6-C7, with localized hypertrophy and segmentally ossified posterior longitudinal ligament, is reported in a 45-year-old man who had Brown-Sequard syndrome diagnosed on neurologic examination. Neuroradiologic, operative, and histologic findings, particularly the pathology of the anterior en bloc resected posterior vertebral portion of C6 and C7, were evaluated for discussion of the pathogenesis. Adhesion of dura mater and hypertrophic posterior longitudinal ligament was observed around a perforated portion of the herniated disc, and histologic study showed irregularity in fiber alignment accompanied by scattered inflammatory cell infiltration and hypertrophy in the posterior longitudinal ligament. The cervical intradural disc herniation was removed successfully and followed by C5-Th1 anterior interbody fusion with fibular strut graft. Neurologic recovery was complete except for minor residual sensory disturbance in the leg 7 years after the surgery. Cervical intradural disc herniation is an extremely rare condition. The pathogenesis remains obscure. Only 16 cases have been reported in the literature, and there has been little discussion concerning the local pathology of the herniated portion. The pathogenesis of the disease in the patient reported here was considered to be the adhesion and fragility of dura mater and posterior longitudinal ligament. This was caused by hypertrophy, with chronic inflammation and ossification of the posterior longitudinal ligament sustaining chronic mechanical irritation to the dura mater, leading to perforation of the herniated disc by an accidental force.

Proximal—distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development

PubMed Central

Cohen, Stephen M.; Jürgens, Gerd

1989-01-01

Limb development in the Drosophila embryo requires a pattern-forming system to organize positional information along the proximal–distal axis of the limb. This system must function in the context of the well characterized anterior–posterior and dorsal–ventral pattern-forming systems that are required to organize the body plan of the embryo. By genetic criteria the Distal-less gene appears to play a central role in limb development. Lack-of-function Distal-less mutations cause the deletion of a specific subset of embryonic peripheral sense organs that represent the evolutionary remnants of larval limbs. Distal-less activity is also required in the imaginal discs for the development of adult limbs. This requirement is cell autonomous and region specific within the developing limb primordium. Production of genetically mosaic imaginal discs, in which clones of cells lack Distal-less activity, indicates the existence of an organized proximal–distal positional information in very young imaginal disc primordia. We suggest that this graded positional information may depend on the activity of the Distal-less gene. Images PMID:16453891

Recombinant fibronectin fragment III8-10/polylactic acid hybrid nanofibers enhance the bioactivity of titanium surface.

PubMed

Guillem-Marti, Jordi; Boix-Lemonche, Gerard; Gugutkov, Dencho; Ginebra, Maria-Pau; Altankov, George; Manero, Jose M

2018-04-01

To develop a nanofiber (NF)-based biomimetic coating on titanium (Ti) that mimics the complex spatiotemporal organization of the extracellular matrix (ECM). Recombinant cell attachment site (CAS) of fibronectin type III8-10 domain was co-electrospun with polylactic acid (PLA) and covalently bound on polished Ti discs. Osteoblast-like SaOS-2 cells were used to evaluate their complex bioactivity. A significant increase of cell spreading was found on CAS/PLA hybrid NFs, followed by control pure PLA NFs and bare Ti discs. Cell proliferation showed similar trend being about twice higher on CAS/PLA NFs. The significantly increased ALP activity at day 21 indicated an enhanced differentiation of SaOS-2 cells. Coating of Ti implants with hybrid CAS/PLA NFs may improve significantly their osseointegration potential.

Gene Activated Titanium Surfaces Promote In Vitro Osteogenesis

PubMed Central

Atluri, Keerthi; Lee, Joun; Seabold, Denise; Elangovan, Satheesh; Salem, Aliasger K.

2016-01-01

Commercially pure titanium (CpTi) and its alloys possess favorable mechanical and biological properties for use as implants in orthopedics and dentistry. However, failures in osseointegration still exist and are common in select individuals with risk factors such as smoking. Therefore, in this study, a proposal was made to enhance the potential of CpTi discs for osseointegration by coating their surfaces with nanoplexes comprising polyethyleneimine (PEI) and plasmid DNA encoding bone morphogenetic protein-2 (pBMP-2). The nanoplexes were characterized for size and surface charge at a range of N/P ratios. CpTi discs were surface characterized for morphology and composition before and after nanoplex coating using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and X-ray powder diffraction (XRD). The cytotoxicity and transfection ability of CpTi discs coated with nanoplexes of varying N/P ratios in human bone marrow derived mesenchymal stem cells (BMSCs) was measured via MTS assays and flow cytometry, respectively. The CpTi discs coated with nanoplexes prepared at an N/P ratio of 10 (N/P-10) were considered optimal, resulting in 75% cell viability and 14% transfection efficiency. ELISA results demonstrated a significant enhancement in BMP-2 protein secretion by BMSCs 7 days post-treatment with CpTi discs coated with PEI/pBMP-2 nanoplexes (N/P-10), compared to the controls. Real time PCR data demonstrated that the BMSCs treated with PEI/pBMP-2 nanoplex coated CpTi discs resulted in an enhancement of runx-2, alkaline phosphatase and osteocalcin gene expressions on day 7, post-treatment. In addition, these BMSCs demonstrated enhanced calcium deposition on day 30 post-treatment as determined by qualitative (alizarin red staining) and quantitative (atomic absorption spectroscopy) assays. Thus, from all the above data it can be concluded that PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs have the potential to induce osteogenesis and enhance osseointegration. PMID:27706263

Transgenic Muscle-Specific Nor-1 Expression Regulates Multiple Pathways That Effect Adiposity, Metabolism, and Endurance

PubMed Central

Pearen, Michael A.; Goode, Joel M.; Fitzsimmons, Rebecca L.; Eriksson, Natalie A.; Thomas, Gethin P.; Cowin, Gary J.; Wang, S.-C. Mary; Tuong, Zewen K.

2013-01-01

The mRNA encoding Nor-1/NR4A3 is rapidly and strikingly induced by β2-adrenergic signaling in glycolytic and oxidative skeletal muscle. In skeletal muscle cells, Nor-1 expression is important for the regulation of oxidative metabolism. Transgenic skeletal muscle-specific expression of activated Nor-1 resulted in the acquisition of an endurance phenotype, an increase in type IIA/X oxidative muscle fibers, and increased numbers of mitochondria. In the current study, we used dual-energy x-ray absorptiometry and magnetic resonance imaging analysis to demonstrate decreased adiposity in transgenic (Tg) Nor-1 mice relative to that in wild-type littermates. Furthermore, the Tg-Nor-1 mice were resistant to diet-induced weight gain and maintained fasting glucose at normoglycemic levels. Expression profiling and RT-quantitative PCR analysis revealed significant increases in genes involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation, and glycogen synthesis, in concordance with the lean phenotype. Moreover, expression profiling identified several Z-disc and sarcomeric binding proteins that modulate fiber type phenotype and endurance, eg, α-actinin-3. In addition, we demonstrated that the Tg-Nor-1 mouse line has significantly higher glycogen content in skeletal muscle relative to that in wild-type littermates. Finally, we identified a decreased NAD+/NADH ratio with a concordant increase in peroxisome proliferator-activated receptor γ coactivator-1α1 protein/mRNA expression. Increased NADH was associated with an induction of the genes involved in the malate-aspartate shuttle and a decrease in the glycerol 3-phosphate shuttle, which maximizes aerobic ATP production. In conclusion, skeletal muscle-specific Nor-1 expression regulates genes and pathways that regulate adiposity, muscle fiber type metabolic capacity, and endurance. PMID:24065705

Phenotypic characteristics of early Wolfram syndrome

PubMed Central

2013-01-01

Background Wolfram Syndrome (WFS:OMIM 222300) is an autosomal recessive, progressive, neurologic and endocrinologic degenerative disorder caused by mutations in the WFS1 gene, encoding the endoplasmic reticulum (ER) protein wolframin, thought to be involved in the regulation of ER stress. This paper reports a cross section of data from the Washington University WFS Research Clinic, a longitudinal study to collect detailed phenotypic data on a group of young subjects in preparation for studies of therapeutic interventions. Methods Eighteen subjects (ages 5.9–25.8, mean 14.2 years) with genetically confirmed WFS were identified through the Washington University International Wolfram Registry. Examinations included: general medical, neurologic, ophthalmologic, audiologic, vestibular, and urologic exams, cognitive testing and neuroimaging. Results Seventeen (94%) had diabetes mellitus with the average age of diabetes onset of 6.3 ± 3.5 years. Diabetes insipidus was diagnosed in 13 (72%) at an average age of 10.6 ± 3.3 years. Seventeen (94%) had optic disc pallor and defects in color vision, 14 (78%) had hearing loss and 13 (72%) had olfactory defects, eight (44%) had impaired vibration sensation. Enuresis was reported by four (22%) and nocturia by three (17%). Of the 11 tested for bladder emptying, five (45%) had elevated post-void residual bladder volume. Conclusions WFS causes multiple endocrine and neurologic deficits detectable on exam, even early in the course of the disease. Defects in olfaction have been underappreciated. The proposed mechanism of these deficits in WFS is ER stress-induced damage to neuronal and hormone-producing cells. This group of subjects with detailed clinical phenotyping provides a pool for testing proposed treatments for ER stress. Longitudinal follow-up is necessary for establishing the natural history and identifying potential biomarkers of progression. PMID:23981289

Programmed Death 1 Regulates Memory Phenotype CD4 T Cell Accumulation, Inhibits Expansion of the Effector Memory Phenotype Subset and Modulates Production of Effector Cytokines

PubMed Central

Charlton, Joanna J.; Tsoukatou, Debbie; Mamalaki, Clio; Chatzidakis, Ioannis

2015-01-01

Memory phenotype CD4 T cells are found in normal mice and arise through response to environmental antigens or homeostatic mechanisms. The factors that regulate the homeostasis of memory phenotype CD4 cells are not clear. In the present study we demonstrate that there is a marked accumulation of memory phenotype CD4 cells, specifically of the effector memory (TEM) phenotype, in lymphoid organs and tissues of mice deficient for the negative co-stimulatory receptor programmed death 1 (PD-1). This can be correlated with decreased apoptosis but not with enhanced homeostatic turnover potential of these cells. PD-1 ablation increased the frequency of memory phenotype CD4 IFN-γ producers but decreased the respective frequency of IL-17A-producing cells. In particular, IFN-γ producers were more abundant but IL-17A producing cells were more scarce among PD-1 KO TEM-phenotype cells relative to WT. Transfer of peripheral naïve CD4 T cells suggested that accumulated PD-1 KO TEM-phenotype cells are of peripheral and not of thymic origin. This accumulation effect was mediated by CD4 cell-intrinsic mechanisms as shown by mixed bone marrow chimera experiments. Naïve PD-1 KO CD4 T cells gave rise to higher numbers of TEM-phenotype lymphopenia-induced proliferation memory cells. In conclusion, we provide evidence that PD-1 has an important role in determining the composition and functional aspects of memory phenotype CD4 T cell pool. PMID:25803808

Application of reaction-diffusion models to cell patterning in Xenopus retina. Initiation of patterns and their biological stability.

PubMed

Shoaf, S A; Conway, K; Hunt, R K

1984-08-07

We have examined the behavior of two reaction-diffusion models, originally proposed by Gierer & Meinhardt (1972) and by Kauffman, Shymko & Trabert (1978), for biological pattern formation. Calculations are presented for pattern formation on a disc (approximating the geometry of a number of embryonic anlagen including the frog eye rudiment), emphasizing the sensitivity of patterns to changes in initial conditions and to perturbations in the geometry of the morphogen-producing space. Analysis of the linearized equations from the models enabled us to select appropriate parameters and disc size for pattern growth. A computer-implemented finite element method was used to solve the non-linear model equations reiteratively. For the Gierer-Meinhardt model, initial activation (varying in size over two orders of magnitude) of one point on the disc's edge was sufficient to generate the primary gradient. Various parts of the disc were removed (remaining only as diffusible space) from the morphogen-producing cycle to investigate the effects of cells dropping out of the cycle due to cell death or malfunction (single point removed) or differentiation (center removed), as occur in the Xenopus eye rudiment. The resulting patterns had the same general shape and amplitude as normal gradients. Nor did a two-fold increase in disc size affect the pattern-generating ability of the model. Disc fragments bearing their primary gradient patterns were fused (with gradients in opposite directions, but each parallel to the fusion line). The resulting patterns generated by the model showed many similarities to results of "compound eye" experiments in Xenopus. Similar patterns were obtained with the model of Kauffman's group (1978), but we found less stability of the pattern subject to simulations of central differentiation. However, removal of a single point from the morphogen cycle (cell death) did not result in any change. The sensitivity of the Kauffman et al. model to shape perturbations is not surprising since the model was originally designed to use shape and increasing size during growth to generate a sequence of transient patterns. However, the Gierer-Meinhardt model is remarkably stable even when subjected to a wide range of perturbations in the diffusible space, thus allowing it to cope with normal biological variability, and offering an exciting range of possibilities for reaction-diffusion models as mechanisms underlying the spatial patterns of tissue structures.

Glycosylated chicken ZP2 accumulates in the egg coat of immature oocytes and remains localized to the germinal disc region of mature eggs.

PubMed

Nishio, Shunsuke; Kohno, Yoshinori; Iwata, Yuki; Arai, Mayumi; Okumura, Hiroki; Oshima, Kenzi; Nadano, Daita; Matsuda, Tsukasa

2014-11-01

Vertebrate eggs are surrounded by an egg coat, which is a specific extracellular egg matrix consisting of several glycoproteins with a conserved zona pellucida (ZP) domain. Two mammalian egg coat subunits, ZP2 and ZP3, have been suggested to act as sperm receptors. In bird eggs, however, ZP2 has never been identified in the egg coat of mature oocytes and ovulated eggs. Here we report that chicken ZP2 is expressed in immature small follicles and remains as an egg-coat component locally in the germinal disc region of mature eggs. RT-PCR analysis indicated marked expression of the ZP2 and ZP4 genes in the granulosa cells of immature white follicles, whereas the ZP3 and ZPD genes showed marked expression in the cells of maturing yellow follicles. ZP2 was identified in the egg coat isolated from immature follicles as a heavily N-glycosylated glycoprotein of ∼200 kDa, which was enzymatically converted to a 70-kDa deglycosylated form. Immunoblotting and immunohistological analyses showed that ZP2 was localized around the germinal disc region of mature follicles. ZP2 was accumulated in the egg coat of immature white follicles at the earlier stages of oocyte development and became a minor component in the egg coat of maturing yellow follicles, except for the germinal disc region. Localization of ZP2 in the germinal disc region of mature eggs, where sperm bind to the egg coat at high density, suggests some role for ZP2 in the preferential binding and penetration of sperm in the germinal disc region of bird eggs. © 2014 by the Society for the Study of Reproduction, Inc.

Impaired intervertebral disc development and premature disc degeneration in mice with notochord-specific deletion of CCN2.

PubMed

Bedore, Jake; Sha, Wei; McCann, Matthew R; Liu, Shangxi; Leask, Andrew; Séguin, Cheryle A

2013-10-01

Currently, our ability to treat intervertebral disc (IVD) degeneration is hampered by an incomplete understanding of disc development and aging. The specific function of matricellular proteins, including CCN2, during these processes remains an enigma. The aim of this study was to determine the tissue-specific localization of CCN proteins and to characterize their role in IVD tissues during embryonic development and age-related degeneration by using a mouse model of notochord-specific CCN2 deletion. Expression of CCN proteins was assessed in IVD tissues from wild-type mice beginning on embryonic day 15.5 to 17 months of age. Given the enrichment of CCN2 in notochord-derived tissues, we generated notochord-specific CCN2-null mice to assess the impact on the IVD structure and extracellular matrix composition. Using a combination of histologic evaluation and magnetic resonance imaging (MRI), IVD health was assessed. Loss of the CCN2 gene in notochord-derived cells disrupted the formation of IVDs in embryonic and newborn mice, resulting in decreased levels of aggrecan and type II collagen and concomitantly increased levels of type I collagen within the nucleus pulposus. CCN2-knockout mice also had altered expression of CCN1 (Cyr61) and CCN3 (Nov). Mirroring its role during early development, notochord-specific CCN2 deletion accelerated age-associated degeneration of IVDs. Using a notochord-specific gene targeting strategy, this study demonstrates that CCN2 expression by nucleus pulposus cells is essential to the regulation of IVD development and age-associated tissue maintenance. The ability of CCN2 to regulate the composition of the intervertebral disc suggests that it may represent an intriguing clinical target for the treatment of disc degeneration. Copyright © 2013 by the American College of Rheumatology.

A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc.

PubMed

Brown, Joanna R; Schwartz, Cindi L; Heumann, John M; Dawson, Scott C; Hoenger, Andreas

2016-04-01

Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications. Copyright © 2016 Elsevier Inc. All rights reserved.

Chemical Fluxes in Cellular Steady States Measured by Fluorescence Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Qian, Hong; Elson, Elliot L.

Genetically, identical cells adopt phenotypes that have different structures, functions, and metabolic properties. In multi-cellular organisms, for example, tissue-specific phenotypes distinguish muscle cells, liver cells, fibroblasts, and blood cells that differ in biochemical functions, geometric forms, and interactions with extracellular environments. Tissue-specific cells usually have different metabolic functions such as synthesis of distinct spectra of secreted proteins, e.g., by liver or pancreatic cells, or of structural proteins, e.g., muscle vs. epithelial cells. But more importantly, a phenotype should include a dynamic aspect: different phenotypes can have distinctly different dynamic functions such as contraction of muscle cells and locomotion of leukocytes. The phenotypes of differentiated tissue cells are typically stable, but they can respond to changes in external conditions, e.g., as in the hypertrophy of muscle cells in response to extra load [1] or the phenotypic shift of fibroblasts to myofibroblasts as part of the wound healing response [2]. Cells pass through sequences of phenotypes during development and also undergo malignant phenotypic transformations as occur in cancer and heart disease.

Loss of the Drosophila cell polarity regulator Scribbled promotes epithelial tissue overgrowth and cooperation with oncogenic Ras-Raf through impaired Hippo pathway signaling

PubMed Central

2011-01-01

Background Epithelial neoplasias are associated with alterations in cell polarity and excessive cell proliferation, yet how these neoplastic properties are related to one another is still poorly understood. The study of Drosophila genes that function as neoplastic tumor suppressors by regulating both of these properties has significant potential to clarify this relationship. Results Here we show in Drosophila that loss of Scribbled (Scrib), a cell polarity regulator and neoplastic tumor suppressor, results in impaired Hippo pathway signaling in the epithelial tissues of both the eye and wing imaginal disc. scrib mutant tissue overgrowth, but not the loss of cell polarity, is dependent upon defective Hippo signaling and can be rescued by knockdown of either the TEAD/TEF family transcription factor Scalloped or the transcriptional coactivator Yorkie in the eye disc, or reducing levels of Yorkie in the wing disc. Furthermore, loss of Scrib sensitizes tissue to transformation by oncogenic Ras-Raf signaling, and Yorkie-Scalloped activity is required to promote this cooperative tumor overgrowth. The inhibition of Hippo signaling in scrib mutant eye disc clones is not dependent upon JNK activity, but can be significantly rescued by reducing aPKC kinase activity, and ectopic aPKC activity is sufficient to impair Hippo signaling in the eye disc, even when JNK signaling is blocked. In contrast, warts mutant overgrowth does not require aPKC activity. Moreover, reducing endogenous levels of aPKC or increasing Scrib or Lethal giant larvae levels does not promote increased Hippo signaling, suggesting that aPKC activity is not normally rate limiting for Hippo pathway activity. Epistasis experiments suggest that Hippo pathway inhibition in scrib mutants occurs, at least in part, downstream or in parallel to both the Expanded and Fat arms of Hippo pathway regulation. Conclusions Loss of Scrib promotes Yorkie/Scalloped-dependent epithelial tissue overgrowth, and this is also important for driving cooperative tumor overgrowth with oncogenic Ras-Raf signaling. Whether this is also the case in human cancers now warrants investigation since the cell polarity function of Scrib and its capacity to restrain oncogene-mediated transformation, as well as the tissue growth control function of the Hippo pathway, are conserved in mammals. PMID:21955824

Induction of appropriate Th-cell phenotypes: cellular decision-making in heterogeneous environments.

PubMed

van den Ham, H-J; Andeweg, A C; de Boer, R J

2013-11-01

Helper T (Th)-cell differentiation is a key event in the development of the adaptive immune response. By the production of a range of cytokines, Th cells determine the type of immune response that is raised against an invading pathogen. Th cells can adopt many different phenotypes, and Th-cell phenotype decision-making is crucial in mounting effective host responses. This review discusses the different Th-cell phenotypes that have been identified and how Th cells adopt a particular phenotype. The regulation of Th-cell phenotypes has been studied extensively using mathematical models, which have explored the role of regulatory mechanisms such as autocrine cytokine signalling and cross-inhibition between self-activating transcription factors. At the single cell level, Th responses tend to be heterogeneous, but corrections can be made soon after T-cell activation. Although pathogens and the innate immune system provide signals that direct the induction of Th-cell phenotypes, these instructive mechanisms could be easily subverted by pathogens. We discuss that a model of success-driven feedback would select the most appropriate phenotype for clearing a pathogen. Given the heterogeneity in the induction phase of the Th response, such a success-driven feedback loop would allow the selection of effective Th-cell phenotypes while terminating incorrect responses. © 2013 John Wiley & Sons Ltd.

Lactoferricin mediates anabolic and anti-catabolic effects in the intervertebral disc.

PubMed

Kim, Jae-Sung; Ellman, Michael B; An, Howard S; Yan, Dongyao; van Wijnen, Andre J; Murphy, Gillian; Hoskin, David W; Im, Hee-Jeong

2012-04-01

Lactoferricin (LfcinB) antagonizes biological effects mediated by angiogenic and catabolic growth factors, in addition to pro-inflammatory cytokines and chemokines in human endothelial cells and tumor cells. However, the effect of LfcinB on intervertebral disc (IVD) cell metabolism has not yet been investigated. Using bovine nucleus pulposus (NP) cells, we analyzed the effect of LfcinB on proteoglycan (PG) accumulation, PG synthesis, and anabolic gene expression. We assessed expression of genes for matrix-degrading enzymes such as matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family), as well as their endogenous inhibitors, tissue inhibitor of metalloproteases (TIMPs). In order to understand the specific molecular mechanisms by which LfcinB exerts its biological effects, we investigated intracellular signaling pathways in NP cells. LfcinB increased PG accumulation mainly via PG synthesis in a dose-dependent manner. Simultaneously, LfcinB dose-dependently downregulated catabolic enzymes. LfcinB's anti-catabolic effects were further demonstrated by a dose-dependent increase in multiple TIMP family members. Our results demonstrate that ERK and/or p38 mitogen-activated protein kinase pathways are the key signaling cascades that exert the biological effects of LfcinB in NP cells, regulating transcription of aggrecan, SOX-9, TIMP-1, TIMP-2, TIMP-3, and iNOS. Our results suggest that LfcinB has anabolic and potent anti-catabolic biological effects on bovine IVD cells that may have considerable promise in the treatment of disc degeneration in the future. Copyright © 2011 Wiley Periodicals, Inc.

Accuracy of different bioinformatics methods in detecting antibiotic resistance and virulence factors from Staphylococcus aureus whole genome sequences.

PubMed

Mason, Amy; Foster, Dona; Bradley, Phelim; Golubchik, Tanya; Doumith, Michel; Gordon, N Claire; Pichon, Bruno; Iqbal, Zamin; Staves, Peter; Crook, Derrick; Walker, A Sarah; Kearns, Angela; Peto, Tim

2018-06-20

Background : In principle, whole genome sequencing (WGS) can predict phenotypic resistance directly from genotype, replacing laboratory-based tests. However, the contribution of different bioinformatics methods to genotype-phenotype discrepancies has not been systematically explored to date. Methods : We compared three WGS-based bioinformatics methods (Genefinder (read-based), Mykrobe (de Bruijn graph-based) and Typewriter (BLAST-based)) for predicting presence/absence of 83 different resistance determinants and virulence genes, and overall antimicrobial susceptibility, in 1379 Staphylococcus aureus isolates previously characterised by standard laboratory methods (disc diffusion, broth and/or agar dilution and PCR). Results : 99.5% (113830/114457) of individual resistance-determinant/virulence gene predictions were identical between all three methods, with only 627 (0.5%) discordant predictions, demonstrating high overall agreement (Fliess-Kappa=0.98, p<0.0001). Discrepancies when identified were in only one of the three methods for all genes except the cassette recombinase, ccrC(b ). Genotypic antimicrobial susceptibility prediction matched laboratory phenotype in 98.3% (14224/14464) cases (2720 (18.8%) resistant, 11504 (79.5%) susceptible). There was greater disagreement between the laboratory phenotypes and the combined genotypic predictions (97 (0.7%) phenotypically-susceptible but all bioinformatic methods reported resistance; 89 (0.6%) phenotypically-resistant, but all bioinformatics methods reported susceptible) than within the three bioinformatics methods (54 (0.4%) cases, 16 phenotypically-resistant, 38 phenotypically-susceptible). However, in 36/54 (67%), the consensus genotype matched the laboratory phenotype. Conclusions : In this study, the choice between these three specific bioinformatic methods to identify resistance-determinants or other genes in S. aureus did not prove critical, with all demonstrating high concordance with each other and phenotypic/molecular methods. However, each has some limitations and therefore consensus methods provide some assurance. Copyright © 2018 American Society for Microbiology.

Warts phosphorylates Mud to promote Pins-mediated mitotic spindle orientation in Drosophila independent of Yorkie

PubMed Central

Dewey, Evan B.; Sanchez, Desiree; Johnston, Christopher A.

2015-01-01

SUMMARY Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. Here we describe a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. Our results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily-conserved cell proliferation pathway. PMID:26592339

DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2

PubMed Central

Kwan, Julian; Sczaniecka, Anna; Heidary Arash, Emad; Nguyen, Liem; Chen, Chia-Chun; Ratkovic, Srdjana; Klezovitch, Olga; Attisano, Liliana; McNeill, Helen; Emili, Andrew; Vasioukhin, Valeri

2016-01-01

Disruption of apical–basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5−/− tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz. Mechanistically, we show that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms. PMID:28087714

Warts phosphorylates mud to promote pins-mediated mitotic spindle orientation in Drosophila, independent of Yorkie.

PubMed

Dewey, Evan B; Sanchez, Desiree; Johnston, Christopher A

2015-11-02

Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. Here, we describe a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. Our results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily conserved cell proliferation pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

Low prevalence of metallo-beta-lactamase in Pseudomonas aeruginosa isolated from a tertiary burn care center in Tehran.

PubMed

Lari, Abdolaziz Rastegar; Azimi, Leila; Soroush, Setareh; Taherikalani, Morovat

2015-09-01

Production of metallo-beta-lactamase (MBL) is one of the main mechanisms for resistance in carbapenem antibiotics. Detection of MBL-producer Pseudomonas aeruginosa is crucial in preventing its spread to other gram-negative bacteria. The aim of this study was to evaluate combination disc (CD) for identification of MBL-producer P. aeruginosa by polymerase chain reaction (PCR). A total of 255 imipenem resistant P. aeruginosa were collected from burn patients. Antibiotic susceptibility testing was conducted after purification and identification. Double-disc synergy test (DDST) with EDTA and combination disc test (CDT) with dipicolinic acid were performed for phenotypic detection of MBL and the PCR was carried out for blaVIM, blaIMP, blaNDM-1, blaSPM-1 genes. DDST with EDTA was negative in all cases, but 161 isolates were positive in CDT with dipicolinic acid. Further, blaVIM and blaIMP were detected in five and four strains, respectively. None of the isolates were positive for BlaNDM-1 and blaSPM-1 . The results of this study showed that the prevalence of MBL is low in imipenem resistance P. aeruginosa and that other mechanisms could be involved in resistance to imipenem in this bacterium. © The Author(s) 2015.

Beta1 integrin inhibits apoptosis induced by cyclic stretch in annulus fibrosus cells via ERK1/2 MAPK pathway.

PubMed

Zhang, Kai; Ding, Wei; Sun, Wei; Sun, Xiao-jiang; Xie, You-zhuan; Zhao, Chang-qing; Zhao, Jie

2016-01-01

Low back pain is associated with intervertebral disc degeneration (IVDD) due to cellular loss through apoptosis. Mechanical factors play an important role in maintaining the survival of the annulus fibrosus (AF) cells and the deposition of extracellular matrix. However, the mechanisms that excessive mechanical forces lead to AF cell apoptosis are not clear. The present study was to look for how AF cells sense mechanical changes. In vivo experiments, the involvement of mechanoreceptors in apoptosis was examined by RT-PCR and/or immunoblotting in the lumbar spine of rats subjected to unbalanced dynamic and static forces. In vitro experiments, we investigated apoptotic signaling pathways in untransfected and transfected AF cells with the lentivirus vector for rat β1 integrin overexpression after cyclic stretch. Apoptosis in AF cells was assessed using flow cytometry, Hoechst 33258 nuclear staining. Western blotting was used to analyze expression of β1 integrin and caspase-3 and ERK1/2 MAPK signaling molecules. In the rat IVDD model, unbalanced dynamic and static forces induced apoptosis of disc cells, which corresponded to decreased expression of β1 integrin. Cyclic stretch-induced apoptosis in rat AF cells correlated with the activation of caspase-3 and with decreased levels of β1 integrin and the phosphorylation levels of ERK1/2 activation level. However, the overexpression of β1 integrin in AF cells ameliorated cyclic stretch-induced apoptosis and decreased caspase-3 activation. Furthermore, ERK1/2-specific inhibitor promotes apoptosis in vector β1-infected AF cells. These results suggest that the disruption of β1 integrin signaling may underlie disc cell apoptosis induced by mechanical stress. Further work is necessary to fully elucidate the pathophysiological mechanisms that underlie IVDD caused by unbalanced dynamic and static forces.

Propionibacterium acnes infected intervertebral discs cause vertebral bone marrow lesions consistent with Modic changes.

PubMed

Dudli, Stefan; Liebenberg, Ellen; Magnitsky, Sergey; Miller, Steve; Demir-Deviren, Sibel; Lotz, Jeffrey C

2016-08-01

Modic type I change (MC1) are vertebral bone marrow lesions adjacent to degenerated discs that are specific for discogenic low back pain. The etiopathogenesis is unknown, but occult discitis, in particular with Propionibacteria acnes (P. acnes), has been suggested as a possible etiology. If true, antibiotic therapy should be considered for patients with MC1. However, this hypothesis is controversial. While some studies report up to 40% infection rate in herniated discs, others fail to detect infected discs and attribute reports of positive cultures to contamination during sampling procedure. Irrespective of the clinical controversy, whether it is biologically plausible for P. acnes to cause MC1 has never been investigated. Therefore, the objective of this study was to test if P. acnes can proliferate within discs and cause reactive changes in the adjacent bone marrow. P. acnes was aseptically isolated from a symptomatic human L4/5 disc with MC1 and injected into rat tail discs. We demonstrate proliferation of P. acnes and up-regulation of IL-1 and IL-6 within three days of inoculation. At day-7, disc degeneration was apparent along with fibrotic endplate erosion. TNF-α immunoreactivity was enhanced within the effected endplates along with cellular infiltrates. The bone marrow appeared normal. At day-14, endplates and trabecular bone close to the disc were almost completely resorbed and fibrotic tissue extended into the bone marrow. T-cells and TNF-α immunoreactivity were identified at the disc/marrow junction. On MRI, bone marrow showed MC1-like changes. In conclusion, P. acnes proliferate within the disc, induce degeneration, and cause MC1-like changes in the adjacent bone marrow. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1447-1455, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy

PubMed Central

2013-01-01

Background In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. Results We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Conclusions Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities. PMID:23937664

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy.

PubMed

Benz, Peter M; Merkel, Carla J; Offner, Kristin; Abeßer, Marco; Ullrich, Melanie; Fischer, Tobias; Bayer, Barbara; Wagner, Helga; Gambaryan, Stepan; Ursitti, Jeanine A; Adham, Ibrahim M; Linke, Wolfgang A; Feller, Stephan M; Fleming, Ingrid; Renné, Thomas; Frantz, Stefan; Unger, Andreas; Schuh, Kai

2013-08-12

In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.

Genetic Interaction of Centrosomin and Bazooka in Apical Domain Regulation in Drosophila Photoreceptor

PubMed Central

Chen, Geng; Rogers, Alicia K.; League, Garrett P.; Nam, Sang-Chul

2011-01-01

Background Cell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes' role in photoreceptor morphogenesis. Methodology/Principal Findings Here, we have found a genetic interaction between baz and centrosomin (cnn). Cnn is a core protein for centrosome which is a major microtubule-organizing center. We analyzed the effect of the cnn mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn's gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs). Conclusions/Significance These results strongly suggest that the interaction of Baz and Cnn is essential for apical domain and AJ modulation during photoreceptor morphogenesis, but not for the initial photoreceptor differentiation in the Drosophila photoreceptor. PMID:21253601

Culture of human anulus fibrosus cells on polyamide nanofibers: extracellular matrix production.

PubMed

Gruber, Helen E; Hoelscher, Gretchen; Ingram, Jane A; Hanley, Edward N

2009-01-01

Studies were approved by the authors' Human Subjects Institutional Review Board. Human anulus cells were tested for growth and extracellular matrix (ECM) production in vitro. To investigate cell attachment, cell proliferation, and ECM production of human intervertebral disc anulus cells seeded onto randomly oriented electrospun polyamide nanofibers. Because nanofibrillar matrices have the potential to promote microenvironments, which may mimic in vivo conditions and resemble connective tissue, their utilization opens new avenues for cell-based tissue engineering applications for disc cells. Anulus cells were isolated from 4 cervical spine surgical disc specimens, expanded, and seeded into either routine plastic culture (control) or a nanofiber surface of randomly oriented electrospun polyamide nanofibers (Ultra-Web-coated culture dish, Corning) with a positive charge or without a charge. Cells were cultured for 9 days, digital images captured, cells harvested, embedded in paraffin, and examined for production of extracellular matrix (ECM). Additional anulus cultures were tested to quantitatively assess total proteoglycan production and cell proliferation under control or nanofiber cultures. Cells attached well and exhibited cell extensions within the nanofiber layers; cells on the charged nanofiber surface deposited greater amounts of chondroitin sulfate than of type II collagen than cells cultured on the uncharged nanofiber surface. Results showed that culture of anulus cells on nanofibers was permissive for secretion and assembly of type II collagen and chondroitin sulfate. Significantly greater total proteoglycan formation was present after culture on the nanofiber with added charge conditions {control, 0.6116 microg/mL +/- 0.186 [4] [mean +/- sem(n)] vs. 1.201 +/- 0.2509 [4], P < 0.05}. Cell proliferation, however, did not differ among treatment groups. Culture of anulus cells on nanofibers was found to be permissive for secretion and assembly of type II collagen and chondroitin sulfate, and culture on nanofibers with added charge significantly increased total proteoglycan production. These novel findings point to the need for further examination of nanofibrillar 3D culture of anulus cells for tissue engineering applications.

Various Surface Treatments to Implant Provisional Restorations and Their Effect on Epithelial Cell Adhesion: A Comparative In Vitro Study.

PubMed

Luchinskaya, Darya; Du, Rong; Owens, David M; Tarnow, Dennis; Bittner, Nurit

2017-02-01

The aim of this in vitro study was to investigate the ability of epithelial cells to attach to or proliferate on various mechanical or chemical surface treatments of an implant provisional material. Polyethyl methacrylate discs 10 mm in diameter and ∼0.2 to 0.75 mm in width were used in the study. Experimental discs were treated with either a mechanical (pumice, varnish for shine, or high polishing) or a chemical agent (alcohol, chlorhexidine, or steam) to provide cleaning and/or polishing. Using primary human epidermal keratinocytes, experiments were performed to test the adhesion or proliferation of cells on the discs with various surface treatments. Scanning electron microscope analysis, rhodamine staining, and cell counting using a hemocytometer corroborated all findings and illustrated that the highest cell adhesion was found to be in the smooth surface treatment groups and the poorest adhesion was found to be in the rough surface groups and chemical treatment group. Within the limitations of this study, the following clinical protocol is recommended for finishing, polishing, and disinfecting implant provisional restorations: coarse, medium, fine pumice → high polishing (if desired) → steam. It is recommended to avoid applying varnish in the perimucosal area near the epithelium. This study could establish the most appropriate way to handle provisional restorations in the peri-implant sulcus for improved soft tissue health, esthetics, and long-term stability.

Cytocompatible antifungal acrylic resin containing silver nanoparticles for dentures

PubMed Central

Acosta-Torres, Laura Susana; Mendieta, Irasema; Nuñez-Anita, Rosa Elvira; Cajero-Juárez, Marcos; Castaño, Víctor M

2012-01-01

Background Inhibition of Candida albicans on denture resins could play a significant role in preventing the development of denture stomatitis. The safety of a new dental material with antifungal properties was analyzed in this work. Methods Poly(methyl methacrylate) [PMMA] discs and PMMA-silver nanoparticle discs were formulated, with the commercial acrylic resin, Nature-CrylTM, used as a control. Silver nanoparticles were synthesized and characterized by ultraviolet-visible spectroscopy, dispersive Raman spectroscopy, and transmission electron microscopy. The antifungal effect was assessed using a luminescent microbial cell viability assay. Biocompatibility tests were carried out using NIH-3T3 mouse embryonic fibroblasts and a Jurkat human lymphocyte cell line. Cells were cultured for 24 or 72 hours in the presence or absence of the polymer formulations and analyzed using three different tests, ie, cellular viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell proliferation by enzyme-linked immunosorbent assay BrdU, and genomic DNA damage (Comet assay). Finally, the samples were evaluated mechanically, and the polymer-bearing silver nanoparticles were analyzed microscopically to evaluate dispersion of the nanoparticles. Results The results show that PMMA-silver nanoparticle discs significantly reduce adherence of C. albicans and do not affect metabolism or proliferation. They also appear not to cause genotoxic damage to cells. Conclusion The present work has developed a new biocompatible antifungal PMMA denture base material. PMID:22969297

The Drosophila DOCK family protein Sponge is required for development of the air sac primordium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morishita, Kazushge; Anh Suong, Dang Ngoc; Yoshida, Hideki

Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, we examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sacmore » primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway. - Highlights: • Spg mainly localizes in the air sac primordium in wing imaginal discs. • Spg plays a critical role in air sac primordium development. • Spg positively regulates the ERK signal cascade.« less

GDP-mannose-4,6-dehydratase (GMDS) Deficiency Renders Colon Cancer Cells Resistant to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor- and CD95-mediated Apoptosis by Inhibiting Complex II Formation*

PubMed Central

Moriwaki, Kenta; Shinzaki, Shinichiro; Miyoshi, Eiji

2011-01-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through binding to TRAIL receptors, death receptor 4 (DR4), and DR5. TRAIL has potential therapeutic value against cancer because of its selective cytotoxic effects on several transformed cell types. Fucosylation of proteins and lipids on the cell surface is a very important posttranslational modification that is involved in many cellular events. Recently, we found that a deficiency in GDP-mannose-4,6-dehydratase (GMDS) rendered colon cancer cells resistant to TRAIL-induced apoptosis, resulting in tumor development and metastasis by escape from tumor immune surveillance. GMDS is an indispensable regulator of cellular fucosylation. In this study, we investigated the molecular mechanism of inhibition of TRAIL signaling by GMDS deficiency. DR4, but not DR5, was found to be fucosylated; however, GMDS deficiency inhibited both DR4- and DR5-mediated apoptosis despite the absence of fucosylation on DR5. In addition, GMDS deficiency also inhibited CD95-mediated apoptosis but not the intrinsic apoptosis pathway induced by anti-cancer drugs. Binding of TRAIL and CD95 ligand to their cognate receptors primarily leads to formation of a complex comprising the receptor, FADD, and caspase-8, referred to as the death-inducing signaling complex (DISC). GMDS deficiency did not affect formation of the primary DISC or recruitment to and activation of caspase-8 on the DISC. However, formation of secondary FADD-dependent complex II, comprising caspase-8 and cFLIP, was significantly inhibited by GMDS deficiency. These results indicate that GMDS regulates the formation of secondary complex II from the primary DISC independent of direct fucosylation of death receptors. PMID:22027835

Osteophyte formation and matrix mineralization in a TMJ osteoarthritis mouse model are associated with ectopic hedgehog signaling

PubMed Central

Bechtold, Till E.; Saunders, Cheri; Decker, Rebekah S.; Um, Hyo-Bin; Cottingham, Naiga; Salhab, Imad; Kurio, Naito; Billings, Paul C.; Pacifici, Maurizio; Nah, Hyun-Duck; Koyama, Eiki

2016-01-01

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa’s articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis. PMID:26945615

Virtual tissue engineering and optic pathways: plotting the course of the axons in the retinal nerve fiber layer.

PubMed

Carreras, Francisco Javier; Medina, Javier; Ruiz-Lozano, Mariola; Carreras, Ignacio; Castro, Juan Luis

2014-04-17

As part of a larger project on virtual tissue engineering of the optic pathways, we describe the conditions that guide axons extending from the retina to the optic nerve head and formulate algorithms that meet such conditions. To find the entrance site on the optic nerve head of each axon, we challenge the fibers to comply with current models of axonal pathfinding. First, we build a retinal map using a single type of retinal ganglion cell (RGC) using density functions from the literature. Dendritic arbors are equated to receptive fields. Shape and size of retinal surface and optic nerve head (ONH) are defined. A computer model relates each soma to the corresponding entry point of its axon into the optic disc. Weights are given to the heuristics that guide the preference entry order in the nerve. Retinal ganglion cells from the area centralis saturate the temporal section of the disc. Retinal ganglion cells temporal to the area centralis curve their paths surrounding the fovea; some of these cells enter the disc centrally rather than peripherally. Nasal regions of the disc receive mixed axons from the far periphery of the temporal hemiretina, together with axons from the nasal half. The model plots the course of the axon using Bezier curves and compares them with clinical data, for a coincidence level of 86% or higher. Our model is able to simulate basic data of the early optic pathways including certain singularities and to mimic mechanisms operating during development, such as timing and fasciculation. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Molecular cloning, sequence analysis, prokaryotic expression, and function prediction of foot-specific peroxidase in Hydra magnipapillata Chinese strain.

PubMed

Pan, H C; Yang, H Q; Zhao, F X; Qian, X C

2014-08-28

The cDNA sequence of foot-specific peroxidase PPOD1 from the Chinese strain of Hydra magnipapillata was cloned by reverse transcription-polymerase chain reaction. The cDNA sequence contained a coding region with an 873-bp open reading frame, a 31-bp 5'-untranslated region, and a 36-bp 3'-untranslated region. The structure prediction results showed that PPOD1 contains 10.34% of α-helix, 38.62% of extended strand, 12.41% of β-turn, and 38.62% of random coil. The structural core was α-helix at the N terminus. The GenBank protein blast server showed that PPOD1 contains 2 fascin-like domains. In addition, high-level PPOD1 activity was only present in the ectodermal epithelial cells located on the edge of the adhesive face of the basal disc, and that these cells extended lamellipodia and filopodia when the basal disc was tightly attached to a glass slide. The fascin-like domains of Hydra PPOD1 might contribute to the bundling of the actin filament of these cells, and hence, the formation of filopodia. In conclusion, these cells might play an important role in strengthening the adsorbability of the basal disc to substrates.

Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure.

PubMed

Neidlinger-Wilke, Cornelia; Würtz, Karin; Urban, Jill P G; Börm, Wolfgang; Arand, Markus; Ignatius, Anita; Wilke, Hans-Joachim; Claes, Lutz E

2006-08-01

Intervertebral disc structures are exposed to wide ranges of intradiscal hydrostatic pressure during different loading exercises and are at their minimum during lying or relaxed sitting and at maximum during lifting weights with a round back. We hypothesize that these different loading magnitudes influence the intervertebral disc (IVD) by alteration of disc matrix turnover depending on their magnitudes. Therefore the aim of this study was to assess changes in gene expression of human nucleus cells after the application of low hydrostatic pressure (0.25 MPa) and high hydrostatic pressure (2.5 MPa). IVD cells isolated from the nucleus of human (n = 18) and bovine (n = 24 from four animals) disc biopsies were seeded into three-dimensional collagen type-I matrices and exposed to the different loading magnitudes by specially developed pressure chambers. The lower pressure range (0.25 MPa, 30 min, 0.1 Hz) was applied with a recently published device by using an external compression cylinder. For the application of higher loads (2.5 MPa, 30 min, 0.1 Hz) the cell-loaded collagen gels were sealed into sterile bags with culture medium and stimulated in a newly developed water-filled compression cylinder by using a loading frame. These methods allowed the comparison of loading regimes in a wide physiological range under an equal three-dimensional culture conditions. Cells were harvested 24 h after the end of stimulation and changes in the expression of genes known to influence IVD matrix turnover (collagen-I, collagen-II, aggrecan, MMP1, MMP2, MMP3, MMP13) were analyzed by real-time RT-PCR. A Wilcoxon signed-rank test(1) and a Wilcoxon 2-sample test(2) were performed to detect differences between the stimulated and control samples(1) and differences between low and high hydrostatic pressure(2). Multiple testing was considered by adjusting the p value appropriately. Both regimes of hydrostatic pressure influenced gene expression in nucleus cells with opposite tendencies for the matrix forming proteins aggrecan and collagen type-I in response to the two different pressure magnitudes: Low hydrostatic-pressure (0.25 MPa) tended to increase collagen-I and aggrecan expression of human nucleus cells (P < 0.05) but only to a small degree. High hydrostatic pressure (2.5 MPa) tended to decrease gene expression of all anabolic proteins with significant effects on aggrecan expression of nucleus cells (P = 0.004). Low hydrostatic pressure had no influence on the expression of matrix metalloproteinases (MMP1, MMP2, MMP3 and MMP13). In contrast, high hydrostatic pressure tended to increase the expression of MMP1, MMP3 and MMP13 of human nucleus cells with high individual-individual variations. The decreased expression of aggrecan (P = 0.008) and collagen type II (P = 0.023) and the increased MMP3 expression (P = 0.008) in response to high hydrostatic pressure could be confirmed in additional experiments with bovine nucleus cells. These results suggest that hydrostatic pressure as one of the physiological stimuli of the IVD may influence matrix turnover in a magnitude dependent way. Low hydrostatic pressure (0.25 MPa) has quite small influences with a tendency to anabolic effects, whereas high hydrostatic pressure (2.5 MPa) tends to decrease the matrix protein expression with a tendency to increase some matrix-turnover enzymes. Therefore, hydrostatic pressure may regulate disc matrix turnover in a dose-dependent way.

High-rate lithium thionyl chloride cells

NASA Technical Reports Server (NTRS)

Goebel, F.

1982-01-01

A high-rate C cell with disc electrodes was developed to demonstrate current rates which are comparable to other primary systems. The tests performed established the limits of abuse beyond which the cell becomes hazardous. Tests include: impact, shock, and vibration tests; temperature cycling; and salt water immersion of fresh cells.

Kinetic Self-Assembly of DNA Tiles and Bricks

DTIC Science & Technology

2016-08-26

research, and educa- tion. This is achieved by freeze drying cell-free systems into paper and other porous substrates to create materials with the...different toehold switches on paper . Experiments were performed by freeze drying the recombinant PT7 expression system onto paper discs, along with linear...DNA encoding specific switch RNAs. The paper discs were rehy- drated with or without the complementary RNA trigger 24 hr after drying and then

Developmental mechanisms of intervertebral disc and vertebral column formation.

PubMed

Lawson, Lisa Y; Harfe, Brian D

2017-11-01

The vertebral column consists of repeating units of ossified vertebrae that are adjoined by fibrocartilagenous intervertebral discs. These structures form from the embryonic notochord and somitic mesoderm. In humans, congenital malformations of the vertebral column include scoliosis, kyphosis, spina bifida, and Klippel Feil syndrome. In adulthood, a common malady affecting the vertebral column includes disc degeneration and associated back pain. Indeed, recent reports estimate that low back pain is the number one cause of disability worldwide. Our review provides an overview of the molecular mechanisms underlying vertebral column morphogenesis and intervertebral disc development and maintenance, with an emphasis on what has been gleaned from recent genetic studies in mice. The aim of this review is to provide a developmental framework through which vertebral column formation can be understood so that ultimately, research scientists and clinicians alike can restore disc health with appropriately designed gene and cell-based therapies. WIREs Dev Biol 2017, 6:e283. doi: 10.1002/wdev.283 For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

Notochord to Nucleus Pulposus Transition.

PubMed

Lawson, Lisa; Harfe, Brian D

2015-10-01

A tissue that commonly deteriorates in older vertebrates is the intervertebral disc, which is located between the vertebrae. Age-related changes in the intervertebral discs are thought to cause most cases of back pain. Back pain affects more than half of people over the age of 65, and the treatment of back pain costs 50-100 billion dollars per year in the USA. The normal intervertebral disc is composed of three distinct regions: a thick outer ring of fibrous cartilage called the annulus fibrosus, a gel-like material that is surrounded by the annulus fibrosus called the nucleus pulposus, and superior and inferior cartilaginous end plates. The nucleus pulposus has been shown to be critical for disc health and function. Damage to this structure often leads to disc disease. Recent reports have demonstrated that the embryonic notochord, a rod-like structure present in the midline of vertebrate embryos, gives rise to all cell types found in adult nuclei pulposi. The mechanism responsible for the transformation of the notochord into nuclei pulposi is unknown. In this review, we discuss potential molecular and physical mechanisms that may be responsible for the notochord to nuclei pulposi transition.

The BTB-zinc Finger Transcription Factor Abrupt Acts as an Epithelial Oncogene in Drosophila melanogaster through Maintaining a Progenitor-like Cell State

PubMed Central

Turkel, Nezaket; Sahota, Virender K.; Bolden, Jessica E.; Goulding, Karen R.; Doggett, Karen; Willoughby, Lee F.; Blanco, Enrique; Martin-Blanco, Enrique; Corominas, Montserrat; Ellul, Jason; Aigaki, Toshiro; Richardson, Helena E.; Brumby, Anthony M.

2013-01-01

The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state. PMID:23874226

A Phenotypic Cell-Binding Screen Identifies a Novel Compound Targeting Triple-Negative Breast Cancer.

PubMed

Chen, Luxi; Long, Chao; Youn, Jonghae; Lee, Jiyong

2018-06-11

We describe a "phenotypic cell-binding screen" by which therapeutic candidate targeting cancer cells of a particular phenotype can be isolated without knowledge of drug targets. Chemical library beads are incubated with cancer cells of the phenotype of interest in the presence of cancer cells lacking the phenotype of interest, and then the beads bound to only cancer cells of the phenotype of interest are selected as hits. We have applied this screening strategy in discovering a novel compound (LC129-8) targeting triple-negative breast cancer (TNBC). LC129-8 displayed highly specific binding to TNBC in cancer cell lines and patient-derived tumor tissues. LC129-8 exerted anti-TNBC activity by inducing apoptosis, inhibiting proliferation, reversing epithelial-mesenchymal transition, downregulating cancer stem cell activity and blocking in vivo tumor growth.

K6 linked polyubiquitylation of FADD by CHIP prevents death inducing signaling complex formation suppressing cell death.

PubMed

Seo, Jinho; Lee, Eun-Woo; Shin, Jihye; Seong, Daehyeon; Nam, Young Woo; Jeong, Manhyung; Lee, Seon-Hyeong; Lee, Cheolju; Song, Jaewhan

2018-05-23

Fas-associated death domain (FADD) is an adaptor protein recruiting complexes of caspase 8 to death ligand receptors to induce extrinsic apoptotic cell death in response to a TNF superfamily member. Although, formation of the complex of FADD and caspase 8 upon death stimuli has been studied in detail, posttranslational modifications fine-tuning these processes have yet to be identified. Here we revealed that K6-linked polyubiquitylation of FADD on lysines 149 and 153 mediated by C terminus HSC70-interacting protein (CHIP) plays an important role in preventing formation of the death inducing signaling complex (DISC), thus leading to the suppression of cell death. Cells depleted of CHIP showed higher sensitivity toward death ligands such as FasL and TRAIL, leading to upregulation of DISC formation composed of a death receptor, FADD, and caspase 8. CHIP was able to bind to FADD, induce K6-linked polyubiquitylation of FADD, and suppress DISC formation. By mass spectrometry, lysines 149 and 153 of FADD were found to be responsible for CHIP-mediated FADD ubiquitylation. FADD mutated at these sites was capable of more potent cell death induction as compared with the wild type and was no longer suppressed by CHIP. On the other hand, CHIP deficient in E3 ligase activity was not capable of suppressing FADD function and of FADD ubiquitylation. CHIP depletion in ME-180 cells induced significant sensitization of these cells toward TRAIL in xenograft analyses. These results imply that K6-linked ubiquitylation of FADD by CHIP is a crucial checkpoint in cytokine-dependent extrinsic apoptosis.

A radiopaque electrospun scaffold for engineering fibrous musculoskeletal tissues: Scaffold characterization and in vivo applications.

PubMed

Martin, John T; Milby, Andrew H; Ikuta, Kensuke; Poudel, Subash; Pfeifer, Christian G; Elliott, Dawn M; Smith, Harvey E; Mauck, Robert L

2015-10-01

Tissue engineering strategies have emerged in response to the growing prevalence of chronic musculoskeletal conditions, with many of these regenerative methods currently being evaluated in translational animal models. Engineered replacements for fibrous tissues such as the meniscus, annulus fibrosus, tendons, and ligaments are subjected to challenging physiologic loads, and are difficult to track in vivo using standard techniques. The diagnosis and treatment of musculoskeletal conditions depends heavily on radiographic assessment, and a number of currently available implants utilize radiopaque markers to facilitate in vivo imaging. In this study, we developed a nanofibrous scaffold in which individual fibers included radiopaque nanoparticles. Inclusion of radiopaque particles increased the tensile modulus of the scaffold and imparted radiation attenuation within the range of cortical bone. When scaffolds were seeded with bovine mesenchymal stem cells in vitro, there was no change in cell proliferation and no evidence of promiscuous conversion to an osteogenic phenotype. Scaffolds were implanted ex vivo in a model of a meniscal tear in a bovine joint and in vivo in a model of total disc replacement in the rat coccygeal spine (tail), and were visualized via fluoroscopy and microcomputed tomography. In the disc replacement model, histological analysis at 4 weeks showed that the scaffold was biocompatible and supported the deposition of fibrous tissue in vivo. Nanofibrous scaffolds that include radiopaque nanoparticles provide a biocompatible template with sufficient radiopacity for in vivo visualization in both small and large animal models. This radiopacity may facilitate image-guided implantation and non-invasive long-term evaluation of scaffold location and performance. The healing capacity of fibrous musculoskeletal tissues is limited, and injury or degeneration of these tissues compromises the standard of living of millions in the US. Tissue engineering repair strategies for the intervertebral disc, meniscus, tendon and ligament have progressed from in vitro to in vivo evaluation using a variety of animal models, and the clinical application of these technologies is imminent. The composition of most scaffold materials however does not allow for visualization by methods available to clinicians (e.g., radiography), and thus it is not possible to assess their performance in situ. In this work, we describe a radiopaque nanofibrous scaffold that can be visualized radiographically in both small and large animal models and serve as a framework for the development of an engineered fibrous tissue. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Insights into the dynamics of hind leg development in honey bee (Apis mellifera L.) queen and worker larvae - A morphology/differential gene expression analysis

PubMed Central

Santos, Carolina Gonçalves; Hartfelder, Klaus

2015-01-01

Phenotypic plasticity is a hallmark of the caste systems of social insects, expressed in their life history and morphological traits. These are best studied in bees. In their co-evolution with angiosperm plants, the females of corbiculate bees have acquired a specialized structure on their hind legs for collecting pollen. In the highly eusocial bees (Apini and Meliponini), this structure is however only present in workers and absent in queens. By means of histological sections and cell proliferation analysis we followed the developmental dynamics of the hind legs of queens and workers in the fourth and fifth larval instars. In parallel, we generated subtractive cDNA libraries for hind leg discs of queen and worker larvae by means of a Representational Difference Analysis (RDA). From the total of 135 unique sequences we selected 19 for RT-qPCR analysis, where six of these were confirmed as differing significantly in their expression between the two castes in the larval spinning stage. The development of complex structures such as the bees’ hind legs, requires diverse patterning mechanisms and signaling modules, as indicated by the set of differentially expressed genes related with cell adhesion and signaling pathways. PMID:26500430

Challenges In Early Glaucoma Detection.

PubMed

Dervisevic, Edita; Pavljasevic, Suzana; Dervisevic, Almir; Kasumovic, Sanja Sefic

2016-06-01

Glaucoma is the most common optic neuropathy which is characterized by progressive loss of retinal ganglion cells, the excavation of the optic nerve head, associated with defects in the visual field. It is not a disease, but the final result of united and yet completely unidentified cellular and subcellular processes and effects of many factors responsible for changes in retinal ganglion cells leading to their accelerated apoptosis. This is a prospective-retrospective, comparative, randomized clinical trial that included 150 patients, 97 were female and 53 male. The age of patients ranged from 18 to 80 years. The highest degree of myopia in category of tilted optic discs had patients with large disc (4.05 + -0.65). Values of the degree of myopia have linearly declined in relation to the size of the oblique disc. The analysis of the results revealed that the subjects who had a higher degree of myopia associated with glaucoma had frequent parapapillar atrophy of alpha and beta zones. The highest percentage of subjects with parapapillar changes were in the group of patients who had other than glaucoma and myopia (62%), then in the group of patients with glaucoma only (56%). Previous studies on the relationship between myopia and open-angle glaucoma are based on the results of observational studies. However, according to recent findings, based on the available studies, the systematic approach to estimate the association between myopia and glaucoma does not exist. Disc Damage Likelihood Scale (DDLS) is a new system for assessing glaucomatous damage of the optic disc which strongly correlates with the degree of visual field loss.

Regulation of CCN2/CTGF Expression in the Nucleus Pulposus of the Intervertebral Disc: Role of Smad and AP1 Signaling

PubMed Central

Tran, Cassie M.; Markova, Dessislava; Smith, Harvey E.; Susarla, Bala; Ponnappan, Ravi Kumar; Anderson, D Greg; Symes, Aviva; Shapiro, Irving M.; Risbud, Makarand V.

2011-01-01

Objective To investigate TGFβ regulation of CTGF expression in cells of the nucleus pulposus. Methods Real Time RT-PCR and Western blot analysis was used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effect of Smad2/3/7 and AP1on TGFβ mediated CTGF promoter activity. Results CTGF expression was lower in the neonatal disc compared with the skeletally mature rat disc. An increase in CTGF expression and promoter activity was observed in nucleus pulposus cells after TGFβ treatment. Deletion analysis indicated that promoter constructs lacking smad and AP1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3-MH2 domain alone increased CTGF activity. Further evidence of Smad3 and AP1 involvement was seen when DN-Smad3, SiRNA-Smad3, smad7 and DN-AP1 suppressed TGFβ mediated activation of the CTGF promoter. When either Smad3 or AP1 sites were mutated, CTGF promoter induction by TGFβ was suppressed. We also observed a decrease in expression of CTGF in discs of Smad3 null mice compared to the wild type. Analysis of human nucleus pulposus indicated a trend of increasing CTGF and TGFβ expression in the degenerate state. Conclusion TGFβ, through Smad3 and AP1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerate disc. PMID:20222112

Merkel cells transduce and encode tactile stimuli to drive Aβ-afferent impulses

PubMed Central

Ikeda, Ryo; Cha, Myeounghoon; Ling, Jennifer; Jia, Zhanfeng; Coyle, Dennis; Gu, Jianguo G.

2014-01-01

SUMMARY Sensory systems for detecting tactile stimuli have evolved from touch-sensing nerves in invertebrates to complicated tactile end-organs in mammals. Merkel discs are tactile end-organs consisting of Merkel cells and Aβ-afferent nerve endings, and are localized in fingertips, whisker hair follicles and other touch-sensitive spots. Merkel discs transduce touch into slowly adapting impulses to enable tactile discrimination, but their transduction and encoding mechanisms remain unknown. Using rat whisker hair follicles, we show that Merkel cells rather than Aβ-afferent nerve endings are primary sites of tactile transduction, and identify the Piezo2 ion channel as the Merkel cell mechanical transducer. Piezo2 transduces tactile stimuli into Ca2+-action potentials in Merkel cells, which drive Aβ-afferent nerve endings to fire slowly adapting impulses. We further demonstrate that Piezo2 and Ca2+-action potentials in Merkel cells are required for behavioral tactile responses. Our findings provide insights into how tactile end-organs function and have clinical implications for tactile dysfunctions. PMID:24746027

[Effects of hydrogen peroxide-containing bleaching on the growth of Streptococcus mutans biofilm on enamel disc surface].

PubMed

Zheng, Chun-yan; Pan, Jie; Wang, Zu-hua; Wang, Yang

2014-02-18

To evaluate the effects of a commercial bleaching agent containing 35% (mass fraction) hydrogen peroxide on the growth of Streptococcus mutans biofilm on enamel disc surface. A total of 20 enamel disks were made from human extracted teeth and the enamel surfaces were kept intact. The discs were autocalved and randomly divided into two groups: bleaching group and control group. Each group contained 10 discs. For bleaching group, the enamel discs were whitened by commercial 35% hydrogen peroxide according to the instruction (Beyond(TM) Professional Dental Whitening Kit, Beyond Technology, TX,USA ); no treatment for control group. All the discs were kept in sterile human saliva for 3.5 hours, and then the mixture of brain heart infusion broth (BHI) medium and Streptococcus mutans were added. The discs and Streptococcus mutans were incubated together in BHI medium with 5% CO(2) (volume fraction), at 37 °C. After 3, 7, 14, 21 and 28 d's incubation, two discs of each group were taken out and the biofilms on the enamel surfaces were evaluated by using conventional bacteria counts and confocal laser scanning microscope (CLSM). The bacteria in the biofilm on one disc enamel surface were analyzed by plating on BHIS agar and the colony-forming units were counted. The biofilm on the other disc surface was stained using a two-colour fluorescent dye kit (Bacerial Viability Kit L-7012) for CLSM. The vital bacteria counts of vital cells in the 3, 7, and 14 d's biofilms of the bleaching group were significantly fewer than those of the control group. Especially in the 3 days' biofilm on the whitened surface, the vital bacteria counts [(3 595 ± 2 903) μm(2) vs. (89 155 ± 65 963) μm(2),t = 8.71,P = 0.00] and proportion of vital bacteria [(26.0% ± 16.4%) vs.(92.2% ± 10.9%), t = 19.93, P = 0.00] were significantly fewer than those of the control. While, for the 21d's biofilm, the vital bacteria counts and the percentage of the vital cells of the bleaching group were more than those of the control group significantly [(66 262 ± 23 772) μm(2) vs. (51 184 ± 20 502) μm(2), t = 2.59, P = 0.012]. The hydrogen peroxide-containing bleaching agent may inhibit the growth of Streptococcus mutans biofilm for about 3 weeks; but after 3 weeks, it seems that the bleached surface will increase the growth of biofilm. Whether the whitening therapy will increase caries susceptibility of the bleached surface needs further research.

Dynamics of phenotypic switching of bacterial cells with temporal fluctuations in pressure

NASA Astrophysics Data System (ADS)

Nepal, Sudip; Kumar, Pradeep

2018-05-01

Phenotypic switching is one of the mechanisms by which bacteria thrive in ever changing environmental conditions around them. Earlier studies have shown that the application of steady high hydrostatic pressure leads to stochastic switching of mesophilic bacteria from a cellular phenotype having a normal cell cycle to another phenotype lacking cell division. Here, we have studied the dynamics of this phenotypic switching with fluctuating periodic pressure using a set of experiments and a theoretical model. Our results suggest that the phenotypic switching rate from high-pressure phenotype to low-pressure phenotype in the reversible regime is larger as compared to the switching rate from low-pressure phenotype to high-pressure phenotype. Furthermore, we find that even though the cell division and elongation are presumably regulated by a large number of genes the underlying physics of the dynamics of stochastic switching at high pressure is captured reasonably well by a simple two-state model.

The Effects of Glucosamine Sulfate on Intervertebral Disc Annulus Fibrosus Cells in Vitro

PubMed Central

Sowa, Gwendolyn; Coelho, J. Paulo; Jacobs, Lloydine; Komperda, Kasey; Sherry, Nora; Vo, Nam; Preuss, Harry; Balk, Judith; Kang, Jame

2014-01-01

Background context Glucosamine has gained widespread use among patients, despite inconclusive efficacy data. Inconsistency in the clinical literature may be related to lack of understanding of the effects of glucosamine on the intervertebral disc, and therefore, improper patient selection. Purpose The goal of our study was to investigate the effects of glucosamine on intervertebral disc cells in vitro under the physiological conditions of inflammation and mechanical loading. Study Design Controlled in vitro laboratory setting Methods Intervertebral disc cells isolated from the rabbit annulus fibrosus were exposed to glucosamine sulfate in the presence and absence of interleukin-1beta and tensile strain. Outcome measures included gene expression, measurement of total glycosaminoglycans, new proteoglycan synthesis, prostaglandin E2 production, and matrix metalloproteinase activity. The study was funded by NIH/NCCAM and the authors have no conflicts of interest. Results Under conditions of inflammatory stimulation alone, glucosamine demonstrated a dose dependent effect in decreasing inflammatory and catabolic mediators and increasing anabolic genes. However, under conditions of mechanical stimulation, although inflammatory gene expression was decreased, PGE2 was not. In addition, MMP-3 gene expression was increased and aggrecan expression decreased, both of which would have a detrimental effect on matrix homeostasis. Consistent with this, measurement of total glycosaminoglycans and new proteoglycan synthesis demonstrated detrimental effects of glucosamine under all conditions tested. Conclusions These results may in part help to explain the conflicting reports of efficacy, as there is biological plausibility for a therapeutic effect under conditions of predominate inflammation, but not under conditions where mechanical loading is present or in which matrix synthesis is needed. PMID:24361347

Laser-modified titanium surfaces enhance the osteogenic differentiation of human mesenchymal stem cells.

PubMed

Bressel, Tatiana A B; de Queiroz, Jana Dara Freires; Gomes Moreira, Susana Margarida; da Fonseca, Jéssyca T; Filho, Edson A; Guastaldi, Antônio Carlos; Batistuzzo de Medeiros, Silvia Regina

2017-11-28

Titanium surfaces have been modified by various approaches with the aim of improving the stimulation of osseointegration. Laser beam (Yb-YAG) treatment is a controllable and flexible approach to modifying surfaces. It creates a complex surface topography with micro and nano-scaled patterns, and an oxide layer that can improve the osseointegration of implants, increasing their usefulness as bone implant materials. Laser beam irradiation at various fluences (132, 210, or 235 J/cm 2 ) was used to treat commercially pure titanium discs to create complex surface topographies. The titanium discs were investigated by scanning electron microscopy, X-ray diffraction, and measurement of contact angles. The surface generated at a fluence of 235 J/cm 2 was used in the biological assays. The behavior of mesenchymal stem cells from an umbilical cord vein was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a mineralization assay, and an alkaline phosphatase activity assay and by carrying out a quantitative real-time polymerase chain reaction for osteogenic markers. CHO-k1 cells were also exposed to titanium discs in the MTT assay. The best titanium surface was that produced by laser beam irradiation at 235 J/cm 2 fluence. Cell proliferation analysis revealed that the CHO-k1 and mesenchymal stem cells behaved differently. The laser-processed titanium surface increased the proliferation of CHO-k1 cells, reduced the proliferation of mesenchymal stem cells, upregulated the expression of the osteogenic markers, and enhanced alkaline phosphatase activity. The laser-treated titanium surface modulated cellular behavior depending on the cell type, and stimulated osteogenic differentiation. This evidence supports the potential use of laser-processed titanium surfaces as bone implant materials, and their use in regenerative medicine could promote better outcomes.

Reversal of retinal and optic disc ischemia in a patient with sickle cell trait and glaucoma secondary to traumatic hyphema.

PubMed

Wax, M B; Ridley, M E; Magargal, L E

1982-07-01

A 14-year-old black boy with sickle cell trait, who sustained a traumatic hyphema, developed moderately elevated intraocular pressure that failed to respond to carbonic anhydrase inhibitors and osmotic agents. On the tenth postinjury day, a sudden increased cupping of the optic disc and partial central retinal artery obstruction caused painless loss of vision. Reversal of the cupping, the retinal ischemia, and the intraocular pressure was documented following anterior chamber paracentesis, and visual acuity returned to 6/6. Pathophysiology of the posterior ischemia is discussed. This case documents the potentially debilitating course of traumatic hyphema in "benign" sickle cell trait and its avoidance with proper management. The authors endorse recent suggestions for careful observation of any sickle cell patient with traumatic hyphema, and recommend anterior chamber paracentesis, supplemental oxygen, and avoidance of osmotic agents, if secondary glaucoma develops following the initial trauma.

Mechanical phenotype of cancer cells: cell softening and loss of stiffness sensing.

PubMed

Lin, Hsi-Hui; Lin, Hsiu-Kuan; Lin, I-Hsuan; Chiou, Yu-Wei; Chen, Horn-Wei; Liu, Ching-Yi; Harn, Hans I-Chen; Chiu, Wen-Tai; Wang, Yang-Kao; Shen, Meng-Ru; Tang, Ming-Jer

2015-08-28

The stiffness sensing ability is required to respond to the stiffness of the matrix. Here we determined whether normal cells and cancer cells display distinct mechanical phenotypes. Cancer cells were softer than their normal counterparts, regardless of the type of cancer (breast, bladder, cervix, pancreas, or Ha-RasV12-transformed cells). When cultured on matrices of varying stiffness, low stiffness decreased proliferation in normal cells, while cancer cells and transformed cells lost this response. Thus, cancer cells undergo a change in their mechanical phenotype that includes cell softening and loss of stiffness sensing. Caveolin-1, which is suppressed in many tumor cells and in oncogene-transformed cells, regulates the mechanical phenotype. Caveolin-1-upregulated RhoA activity and Y397FAK phosphorylation directed actin cap formation, which was positively correlated with cell elasticity and stiffness sensing in fibroblasts. Ha-RasV12-induced transformation and changes in the mechanical phenotypes were reversed by re-expression of caveolin-1 and mimicked by the suppression of caveolin-1 in normal fibroblasts. This is the first study to describe this novel role for caveolin-1, linking mechanical phenotype to cell transformation. Furthermore, mechanical characteristics may serve as biomarkers for cell transformation.

Inducible clindamycin and methicillin resistant Staphylococcus aureus in a tertiary care hospital, Kathmandu, Nepal.

PubMed

Adhikari, R P; Shrestha, S; Barakoti, A; Amatya, R

2017-07-11

Staphylococcus aureus, an important nosocomial pathogen, is frequently associated with infections in human. The management of the infections by it especially methicillin resistant ones is often difficult because methicillin resistant S. aureus is usually resistant to multiple antibiotics. Macrolide-lincosamide streptogramin B family of antibiotics is commonly used to treat such infections as an alternative to vancomycin. This study was conducted over the period of one and half year from November 2013-April 2015 in Microbiology laboratory of Nepal Medical College and Teaching Hospital, Kathmandu, Nepal to find the incidence of different phenotypes of MLS B resistance among S. aureus from clinical samples and their association with methicillin resistance. Two hundred seventy isolates of S. aureus were included in the study. Methicillin resistance was detected by cefoxitin disc diffusion method and inducible clindamycin resistance by erythromycin and clindamycin disc approximation test (D-test). Of the 270 clinical isolates of S. aureus, 25.1% (68/270) were MRSA. Erythromycin and clindamycin resistance was seen in 54.4% (147/270) and 41.8% (113/270) isolates respectively. Resistance to erythromycin and clindamycin were higher in MRSA as compared to MSSA (erythromycin-resistance: 88.2% Vs 39.1% and clindamycin-resistance: 79.4% Vs 41.8%). The overall prevalence of i MLS B and c MLS B phenotype was 11.48% (31/270) and 29.25% (79/270) respectively. Both i MLS B and c MLS B phenotypes predominated in MRSA strains. Detection rate of MRSA in our study shows the necessity to improve in healthcare practices and to formulate new policy for the control of MRSA infections. Clindamycin resistance in the form of i MLS B and c MLS B especially among MRSA emphasizes the need of D-test to be performed routinely in our set up while using clindamycin as an alternative choice to anti-staphylococcal antibiotics like vancomycin and linezolid in the treatment of staphylococcal infections.

Detection of Genetically Altered Copper Levels in Drosophila Tissues by Synchrotron X-Ray Fluorescence Microscopy

PubMed Central

Lye, Jessica C.; Hwang, Joab E. C.; Paterson, David; de Jonge, Martin D.; Howard, Daryl L.; Burke, Richard

2011-01-01

Tissue-specific manipulation of known copper transport genes in Drosophila tissues results in phenotypes that are presumably due to an alteration in copper levels in the targeted cells. However direct confirmation of this has to date been technically challenging. Measures of cellular copper content such as expression levels of copper-responsive genes or cuproenzyme activity levels, while useful, are indirect. First-generation copper-sensitive fluorophores show promise but currently lack the sensitivity required to detect subtle changes in copper levels. Moreover such techniques do not provide information regarding other relevant biometals such as zinc or iron. Traditional techniques for measuring elemental composition such as inductively coupled plasma mass spectroscopy are not sensitive enough for use with the small tissue amounts available in Drosophila research. Here we present synchrotron x-ray fluorescence microscopy analysis of two different Drosophila tissues, the larval wing imaginal disc, and sectioned adult fly heads and show that this technique can be used to detect changes in tissue copper levels caused by targeted manipulation of known copper homeostasis genes. PMID:22053217

Decreased physical function and increased pain sensitivity in mice deficient for type IX collagen.

PubMed

Allen, Kyle D; Griffin, Timothy M; Rodriguiz, Ramona M; Wetsel, William C; Kraus, Virginia B; Huebner, Janet L; Boyd, Lawrence M; Setton, Lori A

2009-09-01

In mice with Col9a1 gene inactivation (Col9a1(-/-)), osteoarthritis (OA) and intervertebral disc degeneration develop prematurely. The aim of this study was to investigate Col9a1(-/-) mice for functional and symptomatic changes that may be associated with these pathologies. Col9a1(-/-) and wild-type mice were investigated for reflexes, functional impairment (beam walking, pole climbing, wire hang, grip strength), sensorimotor skills (rotarod), mechanical sensitivity (von Frey hair), and thermal sensitivity (hot plate/tail flick). Gait was also analyzed to determine velocity, stride frequency, symmetry, percentage stance time, stride length, and step width. Postmortem, sera obtained from the mice were analyzed for hyaluronan, and their knees and spines were graded histologically for degeneration. Col9a1(-/-) mice had compensatory gait changes, increased mechanical sensitivity, and impaired physical ability. Col9a1(-/-) mice ambulated with gaits characterized by increased percentage stance times and shorter stride lengths. These mice also had heightened mechanical sensitivity and were deficient in contact righting, wire hang, rotarod, and pole climbing tasks. Male Col9a1(-/-) mice had the highest mean serum hyaluronan levels and strong histologic evidence of cartilage erosion. Intervertebral disc degeneration was also detected, with Col9a1(-/-) mice having an increased incidence of disc tears. These data describe a Col9a1(-/-) behavioral phenotype characterized by altered gait, increased mechanical sensitivity, and impaired function. These gait and functional differences suggest that Col9a1(-/-) mice select locomotive behaviors that limit joint loads. The nature and magnitude of behavioral changes were largest in male mice, which also had the greatest evidence of knee degeneration. These findings suggest that Col9a1(-/-) mice present behavioral changes consistent with anatomic signs of OA and intervertebral disc degeneration.

SAP is required for the development of innate phenotype in H2-M3-restricted CD8+ T cells1

PubMed Central

Bediako, Yaw; Bian, Yao; Zhang, Hong; Cho, Hoonsik; Stein, Paul L.; Wang, Chyung-Ru

2012-01-01

H2-M3-restricted T cells have a pre-activated surface phenotype, rapidly expand and produce cytokines upon stimulation and as such, are classified as innate T cells. Unlike most innate T cells, M3-restricted T cells also express CD8αβ co-receptors and a diverse TCR repertoire: hallmarks of conventional MHC Ia-restricted CD8+ T cells. Although iNKT cells are also innate lymphocytes, they are selected exclusively on hematopoietic cells (HC), while M3-restricted T cells can be selected on either hematopoietic or thymic epithelial cells (TEC). Moreover, their phenotypes differ depending on what cells mediate their selection. Though there is a clear correlation between selection on HC and development of innate phenotype, the underlying mechanism remains unclear. SAP is required for the development of iNKT cells and mediates signals from SLAM receptors that are exclusively expressed on HC. Based on their dual selection pathway, M3-restricted T cells present a unique model for studying the development of innate T cell phenotype. Using both polyclonal and transgenic mouse models we demonstrate that while M3-restricted T cells are capable of developing in the absence of SAP, SAP is required for HC-mediated selection, development of pre-activated phenotype and heightened effector functions of M3-restricted T cells. These findings are significant because they directly demonstrate the need for SAP in HC-mediated acquisition of innate T cell phenotype and suggest that due to their SAP-dependent HC-mediated selection, M3-restricted T cells develop a pre-activated phenotype and an intrinsic ability to proliferate faster upon stimulation, allowing for an important role in the early response to infection. PMID:23041566

Intra- and Interdimeric Caspase-8 Self-Cleavage Controls Strength and Timing of CD95-Induced Apoptosis

PubMed Central

Kallenberger, Stefan M.; Beaudouin, Joël; Claus, Juliane; Fischer, Carmen; Sorger, Peter K.; Legewie, Stefan; Eils, Roland

2014-01-01

Apoptosis in response to the ligand CD95L (also known as Fas ligand) is initiated by caspase-8, which is activated by dimerization and self-cleavage at death-inducing signaling complexes (DISCs). Previous work indicated that the degree of substrate cleavage by caspase-8 determines whether a cell dies or survives in response to a death stimulus. To determine how a death ligand stimulus is effectively translated into caspase-8 activity, we assessed this activity over time in single cells with compartmentalized probes that are cleaved by caspase-8, and used multiscale modeling to simultaneously describe single-cell and population data with an ensemble of single-cell models. We derived and experimentally validated a minimal model in which cleavage of caspase-8 in the enzymatic domain occurs in an interdimeric manner through interaction between DISCs, whereas prodomain cleavage sites are cleaved in an intradimeric manner within DISCs. Modeling indicated that sustained membrane-bound caspase-8 activity is followed by transient cytosolic activity, which can be interpreted as a molecular timer mechanism reflected by a limited lifetime of active caspase-8. The activation of caspase-8 by combined intra- and interdimeric cleavage ensures weak signaling at low concentrations of CD95L and strongly accelerated activation at higher ligand concentrations, thereby contributing to precise control of apoptosis. PMID:24619646

Cytotoxic Effects of the Radiocontrast Agent Iotrolan and Anesthetic Agents Bupivacaine and Lidocaine in Three-Dimensional Cultures of Human Intervertebral Disc Nucleus Pulposus Cells: Identification of the Apoptotic Pathways

PubMed Central

Iwasaki, Koji; Sudo, Hideki; Yamada, Katsuhisa; Ito, Manabu; Iwasaki, Norimasa

2014-01-01

Background Discography and discoblock are imaging procedures used to diagnose discogenic low back pain. Although needle puncture of the intervertebral disc (IVD) itself induces disc degeneration, the agents used in these procedures may also have harmful effects on IVD cells. The purpose of this study was to analyze whether radiocontrast agents and local anesthetic agents have detrimental effects on human nucleus pulposus (NP) cells. Methods Healthy human NP cells were cultured for 7 days in three-dimensional (3D) cell–alginate bead composites, and were then exposed to clinically relevant doses of a radiocontrast agent (iotrolan) or local anesthetic (lidocaine or bupivacaine). Cell viability and apoptosis were measured by confocal microscopy and flow cytometry. On the basis of caspase expression profiles, the apoptotic pathways activated by the agents were identified by Western blot analysis. Results The radiocontrast agent iotrolan did not affect NP cell viability or induce apoptosis. In contrast, both the anesthetic agents significantly decreased cell viability and increased the apoptotic cell number in a time- and dose-dependent manner. After 120 min, 2% lidocaine and 0.5% bupivacaine decreased percent live cells to 13% and 10%, respectively (p<0.05). The number of apoptotic cells was doubled by increasing lidocaine dosage from 1% to 2% (23% and 42%) and bupivacaine from 0.25% to 0.50% (25% and 48%) (p<0.05). Western blot analysis revealed that both anesthetic agents upregulated cleaved caspase-3 and caspase-8, whereas only bupivacaine upregulated cleaved caspase-9. Conclusions/Significance The present study demonstrates that iotrolan does not affect the viability of healthy human NP cells. In contrast, the two anesthetic agents commonly used in discography or discoblock may cause extensive damage to IVDs by inducing apoptotic cell death. PMID:24642945

Co-culture of Adult Mesenchymal Stem Cells and Nucleus Pulposus Cells in Bilaminar Pellets for Intervertebral Disc Regeneration.

PubMed

Allon, Aliza A; Schneider, Richard A; Lotz, Jeffrey C

2009-01-01

Our goal is to optimize stem cell-based tissue engineering strategies in the context of the intervertebral disc environment. We explored the benefits of co-culturing nucleus pulposus cells (NPC) and adult mesenchymal stem cells (MSC) using a novel spherical bilaminar pellet culture system where one cell type is enclosed in a sphere of the other cell type. Our 3D system provides a structure that exploits embryonic processes such as tissue induction and condensation. We observed a unique phenomenon: the budding of co-culture pellets and the formation of satellite pellets that separate from the main pellet. MSC and NPC co-culture pellets were formed with three different structural organizations. The first had random organization. The other two had bilaminar organization with either MSC inside and NPC outside or NPC inside and MSC outside. By 14 days, all co-culture pellets exhibited budding and spontaneously generated satellite pellets. The satellite pellets were composed of both cell types and, surprisingly, all had the same bilaminar organization with MSC on the inside and NPC on the outside. This organization was independent of the structure of the main pellet that the satellites stemmed from. The main pellets generated satellite pellets that spontaneously organized into a bilaminar structure. This implies that structural organization occurs naturally in this cell culture system and may be inherently favorable for cell-based tissue engineering strategies. The occurrence of budding and the organization of satellite pellets may have important implications for the use of co-culture pellets in cell-based therapies for disc regeneration. From a therapeutic point of view, the generation of satellite pellets may be a beneficial feature that would serve to spread donor cells throughout the host matrix and restore normal matrix composition in a sustainable way, ultimately renewing tissue function.

Catalytically active Yersinia outer protein P induces cleavage of RIP and caspase-8 at the level of the DISC independently of death receptors in dendritic cells.

PubMed

Gröbner, Sabine; Adkins, Irena; Schulz, Sebastian; Richter, Kathleen; Borgmann, Stefan; Wesselborg, Sebastian; Ruckdeschel, Klaus; Micheau, Olivier; Autenrieth, Ingo B

2007-10-01

Yersinia outer protein P (YopP) is injected by Y. enterocolitica into host cells thereby inducing apoptotic and necrosis-like cell death in dendritic cells (DC). Here we show the pathways involved in DC death caused by the catalytic activity of YopP. Infection with Yersinia enterocolitica, translocating catalytically active YopP into DC, triggered procaspase-8 cleavage and c-FLIPL degradation. YopP-dependent caspase-8 activation was, however, not mediated by tumor necrosis factor (TNF) receptor family members since the expression of both CD95/Fas/APO-1 and TRAIL-R2 on DC was low, and DC were resistant to apoptosis induced by agonistic anti-CD95 antibodies or TNF-related apoptosis-inducing ligand (TRAIL). Moreover, DC from TNF-Rp55-/- mice were not protected against YopP-induced cell death demonstrating that TNF-R1 is also not involved in this process. Activation of caspase-8 was further investigated by coimmunoprecitation of FADD from Yersinia-infected DC. We found that both cleaved caspase-8 and receptor interacting protein 1 (RIP1) were associated with the Fas-associated death domain (FADD) indicating the formation of an atypical death-inducing signaling complex (DISC). Furthermore, degradation of RIP mediated by the Hsp90 inhibitor geldanamycin significantly impaired YopP-induced cell death. Altogether our findings indicate that Yersinia-induced DC death is independent of death domain containing receptors, but mediated by RIP and caspase-8 at the level of DISC.

Z-disc-associated, Alternatively Spliced, PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy*

PubMed Central

Lin, Xiaoyan; Ruiz, Janelle; Bajraktari, Ilda; Ohman, Rachel; Banerjee, Soojay; Gribble, Katherine; Kaufman, Joshua D.; Wingfield, Paul T.; Griggs, Robert C.; Fischbeck, Kenneth H.; Mankodi, Ami

2014-01-01

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. PMID:24668811

Development of innovative biopolymers and related composites for bone tissue regeneration: study of their interaction with human osteoprogenitor cells.

PubMed

Basile, Maria Assunta; d'Ayala, Giovanna Gomez; Laurienzo, Paola; Malinconico, Mario; Della Ragione, Fulvio; Oliva, Adriana

2012-01-01

In the framework of a project aiming to improve the properties of poly(ε-caprolactone) (PCL)-based devices, we prepared novel composites and tested their in vitro biocompatibility and osteogenic capacity on human mesenchymal stromal cells (MSC) from bone marrow. We prepared two functionalized derivatives, PCL-g-MAGMA and PCL-g-DMAEA, by insertion of anhydride groups by radical grafting of maleic anhydride (MA) and glycidyl-methacrylate (GMA) molecules, and by insertion of N-(dimethylamino)ethylacrylate (DMAEA) of tertiary amines groups, respectively. In addition, in order to improve the osteoconductive properties of the materials, we also prepared the corresponding composites containing the mineral component of bone, namely hydroxyapatite (HA). Mesenchymal stromal cells (MSC) derived from bone marrow were prepared, plated onto a number of discs obtained from these functionalized derivatives and tested in terms of adhesion and vitality (by MTT test and SEM observation), and the expression of alkaline phosphatase, the early marker of osteoblastic phenotype. The biological in vitro assessment of the functionalized materials, PCL-g-MAGMA and PCL-g-DMAEA, appeared promising only in part, in particular the cells exhibited very poor adhesion to PCL-g-MAGMA. On the contrary, the related composites, PCL-g-MAGMA-HA and PCL-g-DMAEA-HA clearly showed that the addition of HA greatly ameliorated the cell-material interaction. In particular, a surprisingly increased response characterized PCL-g-MAGMA-HA, either in terms of adhesion and vitality or in terms of alkaline phosphatase activity. Altogether these studies showed that the addition of HA nanowhiskers resulted for all basic materials, in particular PCL-g-MAGMA, in improved cell adhesion and performance.

[Role of different scale structures of titanium implant in the biological behaviors of human umbilical vein endothelial cells].

PubMed

Liang, N W; Shi, L; Huang, Y; Deng, X L

2017-02-18

To study the role of different scale structure of Ti implants on the biological behaviors of human umbilical vein endothelial cell (HUVECs) and to reveal the role of material surface topographical features on peri-implant angiogenesis. Titanium (Ti) discs with different surface structures (Ti discs with smooth surface, Ti discs with nano scale structure, Ti discs with micro scale structure and Ti discs with micro/nano scale structure, named as SM-Ti, Nano-Ti, Micro-Ti and Micro/Nano-Ti, respectively) were prepared and their surface topographical features were confirmed via scanning electron microscopy (SEM) observation. HUVECs were cultured on these Ti discs. Biological outcomes of HUVECs on different surfaces were carried out, including cell adhesive capacity, proliferation, vascular endothelial growth factor (VEGF) production and intracellular expression of Ca(2+). The results of SEM images and immunofluorescence double staining of rhodamine-phalloidin and DAPI showed that compared with the SM-Ti and Nano-Ti group, the adhesive capacity and proliferation behavior of HUVECs on the surfaces of Micro-Ti and Micro/Nano-Ti was decreased. The results of culturing HUVECs on different groups of Ti discs after 24 hours showed that the cells number grew from (18±4) to (42±6)/ vision on SM-Ti, (28±6) to (52±10)/vision on Nano-Ti, (20±4) to (21±6)/vision on Micro-Ti and (16±4) to (18±6)/vision on Micro/Nano-Ti. Moreover, compared with the adhesion and proliferation of HUVECs on SM-Ti group and Nano-Ti, the adhesion and proliferation of HUVECs on Micro-Ti group and Micro/Nano-Ti group was significantly reduced (P<0.05).The results of enzyme-linked immunosorbent assay (ELISA) showed that the VEGF productions of SM-Ti, Nano-Ti, Micro-Ti and Micro/Nano-Ti were (690±35) ng/L, (560±20) ng/L, (474±43) ng/L and (517±29) ng/L, respectively. Moreover, compared with the VEGF production of HUVECs on SM-Ti group, the VEGF production of HUVECs on Micro-Ti group and Micro/Nano-Ti group was significantly reduced (P<0.05). The results of Ca(2+) ion detection showed that the Ca(2+) expression of HUVECs on Micro-Ti and Micro/Nano-Ti was significantly higher than that on the surface of SM-Ti and Nano-Ti. These results implied that the over expressed Ca(2+) might contributed to the impaired biological function of HUVECs on Micro-Ti and Micro/Nano-Ti. Different topographical features on titanium influenced the biological behaviors of the HUVECs, which may illustrate how topographical features of Ti implant affect peri-implant angiogenesis. These results also suggest that the biological behaviors of HUVECs might be relative to the changed expression of intracellular Ca(2+).

Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory.

PubMed

Jia, Dongya; Jolly, Mohit Kumar; Kulkarni, Prakash; Levine, Herbert

2017-06-22

Waddington's epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of "landscape" in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an "attractor" that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of "cancer attractors"-hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes "recanalization", i.e., the exit from "cancer attractors" and re-entry into "normal attractors", is more likely to succeed rather than a conventional approach that targets individual molecules/pathways.

Identification of immunophenotypic subtypes with different prognoses in extranodal natural killer/T-cell lymphoma, nasal type.

PubMed

Yu, Jian-Bo; Zuo, Zhuo; Zhang, Wen-Yan; Yang, Qun-Pei; Zhang, Ying-Chun; Tang, Yuan; Zhao, Sha; Mo, Xian-Ming; Liu, Wei-Ping

2014-11-01

To analyze the differentiation characteristics of extranodal natural killer/T-cell lymphoma, nasal type, one nude mouse model, cell lines SNK6 and SNT8, and 16 fresh human samples were analyzed by flow cytometry immunophenotyping and immunohistochemistry staining; and 115 archived cases were used for phenotypic detection and prognostic analysis. We found that CD25 was expressed by most tumor cells in all samples, and CD56(+)CD25(+) cells were the predominant population in the mouse model, the 2 cell lines, and 10 of the 16 fresh tumor samples; in the other 6 fresh tumor samples, the predominant cell population was of the CD16(+)CD25(+) phenotype, and only a minor population showed the CD56(+)CD25(+) phenotype. The phenotype detected by immunohistochemistry staining generally was consistent with the phenotype found by flow cytometry immunophenotyping. According to the expression of CD56 and CD16, 115 cases could be classified into 3 phenotypic subtypes: CD56(-)CD16(-), CD56(+)CD16(-), and CD56(dim/-)CD16(+). Patients with tumors of the CD56(dim/-)CD16(+) phenotype had a poorer prognosis than patients with tumors of the other phenotypes. Differentiation of extranodal natural killer/T-cell lymphoma, nasal type apparently resembles the normal natural killer cell developmental pattern, and these tumors can be classified into 3 phenotypic subtypes of different aggressiveness. Expression of CD56(dim/-)CD16(+) implies a poorer prognosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Patterning of wound-induced intercellular Ca2+ flashes in a developing epithelium

NASA Astrophysics Data System (ADS)

Narciso, Cody; Wu, Qinfeng; Brodskiy, Pavel; Garston, George; Baker, Ruth; Fletcher, Alexander; Zartman, Jeremiah

2015-10-01

Differential mechanical force distributions are increasingly recognized to provide important feedback into the control of an organ’s final size and shape. As a second messenger that integrates and relays mechanical information to the cell, calcium ions (Ca2+) are a prime candidate for providing important information on both the overall mechanical state of the tissue and resulting behavior at the individual-cell level during development. Still, how the spatiotemporal properties of Ca2+ transients reflect the underlying mechanical characteristics of tissues is still poorly understood. Here we use an established model system of an epithelial tissue, the Drosophila wing imaginal disc, to investigate how tissue properties impact the propagation of Ca2+ transients induced by laser ablation. The resulting intercellular Ca2+ flash is found to be mediated by inositol 1,4,5-trisphosphate and depends on gap junction communication. Further, we find that intercellular Ca2+ transients show spatially non-uniform characteristics across the proximal-distal axis of the larval wing imaginal disc, which exhibit a gradient in cell size and anisotropy. A computational model of Ca2+ transients is employed to identify the principle factors explaining the spatiotemporal patterning dynamics of intercellular Ca2+ flashes. The relative Ca2+ flash anisotropy is principally explained by local cell shape anisotropy. Further, Ca2+ velocities are relatively uniform throughout the wing disc, irrespective of cell size or anisotropy. This can be explained by the opposing effects of cell diameter and cell elongation on intercellular Ca2+ propagation. Thus, intercellular Ca2+ transients follow lines of mechanical tension at velocities that are largely independent of tissue heterogeneity and reflect the mechanical state of the underlying tissue.

Self-organisation of dodeca-dendronized fullerene into supramolecular discs and helical columns containing a nanowire-like core.

PubMed

Guerra, Sebastiano; Iehl, Julien; Holler, Michel; Peterca, Mihai; Wilson, Daniela A; Partridge, Benjamin E; Zhang, Shaodong; Deschenaux, Robert; Nierengarten, Jean-François; Percec, Virgil

2015-06-01

Twelve chiral and achiral self-assembling dendrons have been grafted onto a [60]fullerene hexa-adduct core by copper-catalyzed alkyne azide "click" cycloaddition. The structure adopted by these compounds was determined by the self-assembling peripheral dendrons. These twelve dendrons mediate the self-organisation of the dendronized [60]fullerene into a disc-shaped structure containing the [60]fullerene in the centre. The fullerene-containing discs self-organise into helical supramolecular columns with a fullerene nanowire-like core, forming a 2D columnar hexagonal periodic array. These unprecedented supramolecular structures and their assemblies are expected to provide new developments in chiral complex molecular systems and their application to organic electronics and solar cells.

DISC1, PDE4B, and NDE1 at the centrosome and synapse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradshaw, Nicholas J.; Ogawa, Fumiaki; Antolin-Fontes, Beatriz

Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at synapses in cultured neurons. NDE1 is phosphorylated by cAMP-dependantmore » Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.« less

Unsupervised automated high throughput phenotyping of RNAi time-lapse movies.

PubMed

Failmezger, Henrik; Fröhlich, Holger; Tresch, Achim

2013-10-04

Gene perturbation experiments in combination with fluorescence time-lapse cell imaging are a powerful tool in reverse genetics. High content applications require tools for the automated processing of the large amounts of data. These tools include in general several image processing steps, the extraction of morphological descriptors, and the grouping of cells into phenotype classes according to their descriptors. This phenotyping can be applied in a supervised or an unsupervised manner. Unsupervised methods are suitable for the discovery of formerly unknown phenotypes, which are expected to occur in high-throughput RNAi time-lapse screens. We developed an unsupervised phenotyping approach based on Hidden Markov Models (HMMs) with multivariate Gaussian emissions for the detection of knockdown-specific phenotypes in RNAi time-lapse movies. The automated detection of abnormal cell morphologies allows us to assign a phenotypic fingerprint to each gene knockdown. By applying our method to the Mitocheck database, we show that a phenotypic fingerprint is indicative of a gene's function. Our fully unsupervised HMM-based phenotyping is able to automatically identify cell morphologies that are specific for a certain knockdown. Beyond the identification of genes whose knockdown affects cell morphology, phenotypic fingerprints can be used to find modules of functionally related genes.

Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells.

PubMed

Yang, Min; Ma, Bo; Shao, Hanshuang; Clark, Amanda M; Wells, Alan

2016-07-07

Metastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment. Here we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages. We found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells. Our findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due to malignant metastases in breast cancer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scott, F.; Stec, B; Pop, C

The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis1, 2, 3. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation4, 5. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed andmore » isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.« less

Regeneration of spine disc and joint cartilages under temporal and space modulated laser radiation

NASA Astrophysics Data System (ADS)

Sobol, E.; Shekhter, A.; Baskov, A.; Baskov, V.; Baum, O.; Borchshenko, I.; Golubev, V.; Guller, A.; Kolyshev, I.; Omeltchenko, A.; Sviridov, A.; Zakharkina, O.

2009-02-01

The effect of laser radiation on the generation of hyaline cartilage in spine disc and joints has been demonstrated. The paper considers physical processes and mechanisms of laser regeneration, presents results of investigations aimed to optimize laser settings and to develop feedback control system for laser reconstruction of spine discs. Possible mechanisms of laser-induced regeneration include: (1) Space and temporary modulated laser beam induces nonhomogeneous and pulse repetitive thermal expansion and stress in the irradiated zone of cartilage. Mechanical effect due to controllable thermal expansion of the tissue and micro and nano gas bubbles formation in the course of the moderate (up to 45-50 oC) heating of the NP activate biological cells (chondrocytes) and promote cartilage regeneration. (2) Nondestructive laser radiation leads to the formation of nano and micro-pores in cartilage matrix. That promotes water permeability and increases the feeding of biological cells. Results provide the scientific and engineering basis for the novel low-invasive laser procedures to be used in orthopedics for the treatment cartilages of spine and joints. The technology and equipment for laser reconstruction of spine discs have been tested first on animals, and then in a clinical trial. Since 2001 the laser reconstruction of intervertebral discs have been performed for 340 patients with chronic symptoms of low back or neck pain who failed to improve with non-operative care. Substantial relief of back pain was obtained in 90% of patients treated who returned to their daily activities. The experiments on reparation of the defects in articular cartilage of the porcine joints under temporal and spase modulated laser radiation have shown promising results.

Choosing sheep (Ovis aries) as animal model for temporomandibular joint research: Morphological, histological and biomechanical characterization of the joint disc.

PubMed

Angelo, D F; Morouço, P; Alves, N; Viana, T; Santos, F; González, R; Monje, F; Macias, D; Carrapiço, B; Sousa, R; Cavaco-Gonçalves, S; Salvado, F; Peleteiro, C; Pinho, M

2016-12-01

Preclinical trials are essential to the development of scientific technologies. Remarkable molecular and cellular research has been done using small animal models. However, significant differences exist regarding the articular behavior between these models and humans. Thus, large animal models may be more appropriate to perform trials involving the temporomandibular joint (TMJ). The aim of this work was to make a morphological (anatomic dissection and white light 3D scanning system), histological (TMJ in bloc was removed for histologic analysis) and biomechanical characterization (tension and compression tests) of sheep TMJ comparing the obtained results with human data. Results showed that sheep processus condylaris and fossa mandibularis are anatomically similar to the same human structures. TMJ disc has an elliptical perimeter, thinner in the center than in periphery. Peripheral area acts as a ring structure supporting the central zone. The disc cells display both fibroblast and chondrocyte-like morphology. Marginal area is formed by loose connective tissue, with some chondrocyte-like cells and collagen fibers in diverse orientations. Discs obtained a tensile modulus of 3.97±0.73MPa and 9.39±1.67MPa, for anteroposterior and mediolateral assessment. The TMJ discs presented a compressive modulus (E) of 446.41±5.16MPa and their maximum stress value (σmax) was 18.87±1.33MPa. Obtained results suggest that these animals should be considered as a prime model for TMJ research and procedural training. Further investigations in the field of oromaxillofacial surgery involving TMJ should consider sheep as a good animal model due to its resemblance of the same joint in humans. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Lactoferricin mediates anti-inflammatory and anti-catabolic effects via inhibition of IL-1 and LPS activity in the intervertebral disc.

PubMed

Kim, Jae-Sung; Ellman, Michael B; Yan, Dongyao; An, Howard S; Kc, Ranjan; Li, Xin; Chen, Di; Xiao, Guozhi; Cs-Szabo, Gabriella; Hoskin, David W; Buechter, Doug D; Van Wijnen, Andre J; Im, Hee-Jeong

2013-09-01

The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production, and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. Copyright © 2013 Wiley Periodicals, Inc.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

PubMed

Urbanska, Marta; Winzi, Maria; Neumann, Katrin; Abuhattum, Shada; Rosendahl, Philipp; Müller, Paul; Taubenberger, Anna; Anastassiadis, Konstantinos; Guck, Jochen

2017-12-01

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells. © 2017. Published by The Company of Biologists Ltd.

In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timraz, Sara B.H., E-mail: sara.timraz@kustar.ac.ae; Farhat, Ilyas A.H., E-mail: ilyas.farhat@outlook.com; Alhussein, Ghada, E-mail: ghada.alhussein@kustar.ac.ae

In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, andmore » reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. - Highlights: • Ability of human smooth muscle cells to alter phenotype in culture is evaluated. • Examined the effect of passaging human smooth muscle cells on phenotype. • Phenotype is assessed based on morphology, proliferation, markers, and migration. • Multi-resolution assessment methodology, single-cell and cell-population. • Lower and higher passages than P7 adopted a contractile and synthetic phenotype respectively.« less

Studying Cancer Stem Cell Dynamics on PDMS Surfaces for Microfluidics Device Design

PubMed Central

Zhang, Weijia; Choi, Dong Soon; Nguyen, Yen H.; Chang, Jenny; Qin, Lidong

2013-01-01

This systematic study clarified a few interfacial aspects of cancer cell phenotypes on polydimethylsiloxane (PDMS) substrates and indicated that the cell phenotypic equilibrium greatly responds to cell-to-surface interactions. We demonstrated that coatings of fibronectin, bovine serum albumin (BSA), or collagen with or without oxygen-plasma treatments of the PDMS surfaces dramatically impacted the phenotypic equilibrium of breast cancer stem cells, while the variations of the PDMS elastic stiffness had much less such effects. Our results showed that the surface coatings of collagen and fibronectin on PDMS maintained breast cancer cell phenotypes to be nearly identical to the cultures on commercial polystyrene Petri dishes. The surface coating of BSA provided a weak cell-substrate adhesion that stimulated the increase in stem-cell-like subpopulation. Our observations may potentially guide surface modification approaches to obtain specific cell phenotypes. PMID:23900274

Different modes of APC/C activation control growth and neuron-glia interaction in the developing Drosophila eye.

PubMed

Neuert, Helen; Yuva-Aydemir, Yeliz; Silies, Marion; Klämbt, Christian

2017-12-15

The development of the nervous system requires tight control of cell division, fate specification and migration. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that affects different steps of cell cycle progression, as well as having postmitotic functions in nervous system development. It can therefore link different developmental stages in one tissue. The two adaptor proteins, Fizzy/Cdc20 and Fizzy-related/Cdh1, confer APC/C substrate specificity. Here, we show that two distinct modes of APC/C function act during Drosophila eye development. Fizzy/Cdc20 controls the early growth of the eye disc anlage and the concomitant entry of glial cells onto the disc. In contrast, fzr/cdh1 acts during neuronal patterning and photoreceptor axon growth, and subsequently affects neuron-glia interaction. To further address the postmitotic role of Fzr/Cdh1 in controlling neuron-glia interaction, we identified a series of novel APC/C candidate substrates. Four of our candidate genes are required for fzr/cdh1 -dependent neuron-glia interaction, including the dynein light chain Dlc90F Taken together, our data show how different modes of APC/C activation can couple early growth and neuron-glia interaction during eye disc development. © 2017. Published by The Company of Biologists Ltd.

Decapentaplegic and growth control in the developing Drosophila wing.

PubMed

Akiyama, Takuya; Gibson, Matthew C

2015-11-19

As a central model for morphogen action during animal development, the bone morphogenetic protein 2/4 (BMP2/4)-like ligand Decapentaplegic (Dpp) is proposed to form a long-range signalling gradient that directs both growth and pattern formation during Drosophila wing disc development. While the patterning role of Dpp secreted from a stripe of cells along the anterior-posterior compartmental boundary is well established, the mechanism by which a Dpp gradient directs uniform cell proliferation remains controversial and poorly understood. Here, to determine the precise spatiotemporal requirements for Dpp during wing disc development, we use CRISPR-Cas9-mediated genome editing to generate a flippase recognition target (FRT)-dependent conditional null allele. By genetically removing Dpp from its endogenous stripe domain, we confirm the requirement of Dpp for the activation of a downstream phospho-Mothers against dpp (p-Mad) gradient and the regulation of the patterning targets spalt (sal), optomotor blind (omb; also known as bifid) and brinker (brk). Surprisingly, however, third-instar wing blade primordia devoid of compartmental dpp expression maintain relatively normal rates of cell proliferation and exhibit only mild defects in growth. These results indicate that during the latter half of larval development, the Dpp morphogen gradient emanating from the anterior-posterior compartment boundary is not directly required for wing disc growth.

Transient inhibition of connective tissue infiltration and collagen deposition into porous poly(lactic-co-glycolic acid) discs.

PubMed

Love, Ryan J; Jones, Kim S

2013-12-01

Connective tissue rapidly proliferates on and around biomaterials implanted in vivo, which impairs the function of the engineered tissues, biosensors, and devices. Glucocorticoids can be utilized to suppress tissue ingrowth, but can only be used for a limited time because they nonselectively arrest cell proliferation in the local environment. The present study examined use of a prolyl-4-hydroxylase inhibitor, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), to suppress connective tissue ingrowth in porous PLGA discs implanted in the peritoneal cavity for 28 days. The prolyl-4-hydroxylase inhibitor was found to be effective at inhibiting collagen deposition within and on the outer surface of the disc, and also limited connective tissue ingrowth, but not to the extent of glucocorticoid inhibition. Finally, it was discovered that 1,4-DPCA suppressed Scavenger Receptor A expression on a macrophage-like cell culture, which may account for the drug's ability to limit connective tissue ingrowth in vivo. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Drosophila Growth and Development in the Absence of dMyc and dMnt

PubMed Central

Pierce, Sarah B.; Yost, Cynthia; Anderson, Sarah A. R.; Flynn, Erin M.; Delrow, Jeffrey; Eisenman, Robert N.

2008-01-01

Myc oncoproteins are essential regulators of the growth and proliferation of mammalian cells. In Drosophila the single ortholog of Myc (dMyc), encoded by the dm gene, influences organismal size and the growth of both mitotic and endoreplicating cells. A null mutation in dm results in attenuated endoreplication and growth arrest early in larval development. Drosophila also contains a single ortholog of the mammalian Mad/Mnt transcriptional repressor proteins (dMnt), which is thought to antagonize dMyc function. Here we show that animals lacking both dMyc and dMnt display increased viability and grow significantly larger and develop further than dMyc single mutants. We observe increased endoreplication and growth of larval tissues in these double mutants and disproportionate growth of the imaginal discs. Gene expression analysis indicates that loss of dMyc leads to decreased expression of genes required for ribosome biogenesis and protein synthesis. The additional loss of dMnt partially rescues expression of a small number of dMyc and dMnt genes that are primarily involved in rRNA synthesis and processing. Our results indicate that dMnt repression is normally overridden by dMyc activation during larval development. Therefore the severity of the dm null phenotype is likely due to unopposed repression by dMnt on a subset of genes critical for cell and organismal growth. Surprisingly, considerable growth and development can occur in the absence of both dMyc and dMnt. PMID:18241851

Novel isoforms of Dlg are fundamental for neuronal development in Drosophila.

PubMed

Mendoza, Carolina; Olguín, Patricio; Lafferte, Gabriela; Thomas, Ulrich; Ebitsch, Susanne; Gundelfinger, Eckart D; Kukuljan, Manuel; Sierralta, Jimena

2003-03-15

Drosophila discs-large (dlg) mutants exhibit multiple developmental abnormalities, including severe defects in neuronal differentiation and synaptic structure and function. These defects have been ascribed to the loss of a single gene product, Dlg-A, a scaffold protein thought to be expressed in many cell types. Here, we describe that additional isoforms arise as a consequence of different transcription start points and alternative splicing of dlg. At least five different dlg gene products are predicted. We identified a subset of dlg-derived cDNAs that include novel exons encoding a peptide homologous to the N terminus of the mammalian protein SAP97/hDLG (S97N). Dlg isoforms containing the S97N domain are expressed at larval neuromuscular junctions and within the CNS of both embryos and larvae but are not detectable in epithelial tissues. Strong hypomorphic dlg alleles exhibit decreased expression of S97N, which may account for neural-specific aspects of the pleiomorphic dlg mutant phenotype. Selective inhibition of the expression of S97N-containing proteins in embryos by double-strand RNA leads to severe defects in neuronal differentiation and axon guidance, without overt perturbations in epithelia. These results indicate that the differential expression of dlg products correlates with distinct functions in non-neural and neural cells. During embryonic development, proteins that include the S97N domain are essential for proper neuronal differentiation and organization, acting through mechanisms that may include the adequate localization of cell fate determinants.

Effects of the intradiscal implantation of stromal vascular fraction plus platelet rich plasma in patients with degenerative disc disease.

PubMed

Comella, Kristin; Silbert, Robert; Parlo, Michelle

2017-01-13

Stromal vascular fraction (SVF) can easily be obtained from a mini-lipoaspirate procedure of fat tissue and platelet rich plasma (PRP) can be obtained from peripheral blood. The SVF contains a mixture of cells including ADSCs and growth factors and has been depleted of the adipocyte (fat cell) population. We evaluated the safety and efficacy of administering SVF and PRP intra-discally into patients with degenerative disc disease. A total of 15 patients underwent a local tumescent liposuction procedure to remove approximately 60 ml of fat tissue. The fat was separated to isolate the SVF and the cells were delivered into the disc nucleus of patients with degenerative disc disease. The subjects were then monitored for adverse events, range of motion, visual analog scale (VAS), present pain intensity (PPI), Oswestry Disability Index (ODI), Beck Depression Inventory (BDI), Dallas Pain Questionnaire and Short Form (SF)-12 scores over a period of 6 months. Safety events were followed for 12 months. No severe adverse events (SAEs) were reported during a 12 month follow up period with no incidences of infection. Patients demonstrated statistically significant improvements in several parameters including flexion, pain ratings, VAS, PPI, and short form questionnaires. In addition, both ODI and BDI data was trending positive and a majority of patients reported improvements in their Dallas Pain Questionnaire scores. Overall, patients were pleased with the treatment results. More importantly, the procedure demonstrated a strong safety profile with no severe adverse events or complications linked to the therapy. Trial registration NCT02097862. Name of registry: www.clinicaltrials.gov . https://clinicaltrials.gov/ct2/show/NCT02097862?term=bioheart&rank=6 . Date of registration: March 25, 2014; Date of enrollment: March 2014.

Antibacterial Efficacy of Octenisept, Alexidine, Chlorhexidine, and Sodium Hypochlorite against Enterococcus faecalis Biofilms.

PubMed

Bukhary, Sundus; Balto, Hanan

2017-04-01

The purpose of this study was to evaluate the antibacterial effectiveness of Octenisept (OCT; Schülke & Mayr GmBH, Norderstedt, Germany), 1% alexidine (ALX) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), and 2% chlorhexidine (CHX) against Enterococcus faecalis biofilm using confocal laser scanning microscopy. Root dentin discs were prepared from extracted human teeth, sterilized, and inoculated with E. faecalis strain (ATCC 29212) to establish 3-week-old biofilm model. Infected dentin discs were exposed to OCT (n = 20), 1% ALX (n = 20), and 2% CHX (n = 20) for 10 minutes. Dentin discs (n = 15) exposed to 5.25% sodium hypochlorite (NaOCl) were used as a positive control, whereas specimens exposed to saline (n = 15) were used as a negative control. After exposure, the dentin discs were stained with fluorescent LIVE/DEAD BacLight dye (Invitrogen Molecular Probes, Eugene, OR) and analyzed with confocal laser scanning microscopy to determine the proportion of dead cells in the biofilm. Statistical analysis was performed using the Kruskal-Wallis and Mann-Whitney U tests (P < .05). The highest proportion of dead cells was found in the 5.25% NaOCl group (94.14%; range, 92.30%-98.20%) compared with the experimental groups (P < .05). A significantly greater proportion of dead cells was found in the OCT group (74.14%; range, 70.03%-78.96%) compared with the 1% ALX and 2% CHX groups (P < .05). The proportion of dead cells was 43.89% (range, 24.86%-55.63%) and 42.78% (range, 25.45%-55.06%) in the 1% ALX and 2% CHX groups, respectively, with no statistical significant difference between the 2 groups (P > .05). NaOCl had significantly greater antimicrobial activity against E. faecalis biofilms compared with OCT, CHX, and ALX. OCT was more effective than CHX and ALX. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Tumor Necrosis Factor-α– and Interleukin-1β–Dependent Matrix Metalloproteinase-3 Expression in Nucleus Pulposus Cells Requires Cooperative Signaling via Syndecan 4 and Mitogen-Activated Protein Kinase–NF-κB Axis

PubMed Central

Wang, Xin; Wang, Hua; Yang, Hao; Li, Jun; Cai, Qiqing; Shapiro, Irving M.; Risbud, Makarand V.

2015-01-01

Matrix metalloproteinase-3 (MMP-3) plays an important role in intervertebral disc degeneration, a ubiquitous condition closely linked to low back pain and disability. Elevated expression of syndecan 4, a cell surface heparan sulfate proteoglycan, actively controls disc matrix catabolism. However, the relationship between MMP-3 expression and syndecan 4 in the context of inflammatory disc disease has not been clearly defined. We investigated the mechanisms by which cytokines control MMP-3 expression in rat and human nucleus pulposus cells. Cytokine treatment increased MMP-3 expression and promoter activity. Stable silencing of syndecan 4 blocked cytokine-mediated MMP-3 expression; more important, syndecan 4 did not mediate its effects through NF-κB or mitogen-activated protein kinase (MAPK) pathways. However, treatment with MAPK and NF-κB inhibitors resulted in partial blocking of the inductive effect of cytokines on MMP-3 expression. Loss-of-function studies confirmed that NF-κB, p38α/β2/γ/δ, and extracellular signal–regulated kinase (ERK) 2, but not ERK1, contributed to cytokine-dependent induction of MMP3 promoter activity. Similarly, inhibitor treatments, lentiviral short hairpin-p65, and short hairpin-IκB kinase β significantly decreased cytokine-dependent up-regulation in MMP-3 expression. Finally, we show that transforming growth factor-β can block the up-regulation of MMP-3 induced by tumor necrosis factor (TNF)-α by counteracting the NF-κB pathway and syndecan 4 expression. Taken together, our results suggest that cooperative signaling through syndecan 4 and the TNF receptor 1–MAPK–NF-κB axis is required for TNF-α–dependent expression of MMP-3 in nucleus pulposus cells. Controlling these pathways may slow the progression of intervertebral disc degeneration and matrix catabolism. PMID:25063530

Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time.

PubMed

Harder, Nathalie; Mora-Bermúdez, Felipe; Godinez, William J; Wünsche, Annelie; Eils, Roland; Ellenberg, Jan; Rohr, Karl

2009-11-01

Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations.

PubMed

Grasso, Carole; Anaka, Matthew; Hofmann, Oliver; Sompallae, Ramakrishna; Broadley, Kate; Hide, Winston; Berridge, Michael V; Cebon, Jonathan; Behren, Andreas; McConnell, Melanie J

2016-09-09

The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.

Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data

PubMed Central

Kussell, Edo

2017-01-01

Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells’ response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution. PMID:28267748

Discovery of a “White-Gray-Opaque” Tristable Phenotypic Switching System in Candida albicans: Roles of Non-genetic Diversity in Host Adaptation

PubMed Central

Guan, Guobo; Dai, Yu; Nobile, Clarissa J.; Liang, Weihong; Cao, Chengjun; Zhang, Qiuyu; Zhong, Jin; Huang, Guanghua

2014-01-01

Non-genetic phenotypic variations play a critical role in the adaption to environmental changes in microbial organisms. Candida albicans, a major human fungal pathogen, can switch between several morphological phenotypes. This ability is critical for its commensal lifestyle and for its ability to cause infections. Here, we report the discovery of a novel morphological form in C. albicans, referred to as the “gray” phenotype, which forms a tristable phenotypic switching system with the previously reported white and opaque phenotypes. White, gray, and opaque cell types differ in a number of aspects including cellular and colony appearances, mating competency, secreted aspartyl proteinase (Sap) activities, and virulence. Of the three cell types, gray cells exhibit the highest Sap activity and the highest ability to cause cutaneous infections. The three phenotypes form a tristable phenotypic switching system, which is independent of the regulation of the mating type locus (MTL). Gray cells mate over 1,000 times more efficiently than do white cells, but less efficiently than do opaque cells. We further demonstrate that the master regulator of white-opaque switching, Wor1, is essential for opaque cell formation, but is not required for white-gray transitions. The Efg1 regulator is required for maintenance of the white phenotype, but is not required for gray-opaque transitions. Interestingly, the wor1/wor1 efg1/efg1 double mutant is locked in the gray phenotype, suggesting that Wor1 and Efg1 could function coordinately and play a central role in the regulation of gray cell formation. Global transcriptional analysis indicates that white, gray, and opaque cells exhibit distinct gene expression profiles, which partly explain their differences in causing infections, adaptation ability to diverse host niches, metabolic profiles, and stress responses. Therefore, the white-gray-opaque tristable phenotypic switching system in C. albicans may play a significant role in a wide range of biological aspects in this common commensal and pathogenic fungus. PMID:24691005

Discovery of a "white-gray-opaque" tristable phenotypic switching system in candida albicans: roles of non-genetic diversity in host adaptation.

PubMed

Tao, Li; Du, Han; Guan, Guobo; Dai, Yu; Nobile, Clarissa J; Liang, Weihong; Cao, Chengjun; Zhang, Qiuyu; Zhong, Jin; Huang, Guanghua

2014-04-01

Non-genetic phenotypic variations play a critical role in the adaption to environmental changes in microbial organisms. Candida albicans, a major human fungal pathogen, can switch between several morphological phenotypes. This ability is critical for its commensal lifestyle and for its ability to cause infections. Here, we report the discovery of a novel morphological form in C. albicans, referred to as the "gray" phenotype, which forms a tristable phenotypic switching system with the previously reported white and opaque phenotypes. White, gray, and opaque cell types differ in a number of aspects including cellular and colony appearances, mating competency, secreted aspartyl proteinase (Sap) activities, and virulence. Of the three cell types, gray cells exhibit the highest Sap activity and the highest ability to cause cutaneous infections. The three phenotypes form a tristable phenotypic switching system, which is independent of the regulation of the mating type locus (MTL). Gray cells mate over 1,000 times more efficiently than do white cells, but less efficiently than do opaque cells. We further demonstrate that the master regulator of white-opaque switching, Wor1, is essential for opaque cell formation, but is not required for white-gray transitions. The Efg1 regulator is required for maintenance of the white phenotype, but is not required for gray-opaque transitions. Interestingly, the wor1/wor1 efg1/efg1 double mutant is locked in the gray phenotype, suggesting that Wor1 and Efg1 could function coordinately and play a central role in the regulation of gray cell formation. Global transcriptional analysis indicates that white, gray, and opaque cells exhibit distinct gene expression profiles, which partly explain their differences in causing infections, adaptation ability to diverse host niches, metabolic profiles, and stress responses. Therefore, the white-gray-opaque tristable phenotypic switching system in C. albicans may play a significant role in a wide range of biological aspects in this common commensal and pathogenic fungus.

Meloidogyne paranaensis n. sp. (Nemata: Meloidogynidae), a Root-Knot Nematode Parasitizing Coffee in Brazil

PubMed Central

Carneiro, R. M. D. G.; Carneiro, R. G.; Abrantes, I. M. O.; Santos, M. S. N. A.; Almeida, M. R. A.

1996-01-01

A root-knot nematode parasitizing coffee in Paran  State, Brazil, is described as Meloidogyne paranaensis n. sp. The suggested common name is Paraná coffee root-knot nematode. The perineal pattern is similar to that of M. incognita; the labial disc and medial lips of the female are fused and asymmetric and rectangular; the lateral lips are small, triangular, and fused laterally with the head region. The female stylet is 15.0-17.5 μm long, with broad, distinctly set-off knobs; the distance from the dorsal esophageal gland orifice (DGO) to the stylet base is 4.2-5.5 μm. Males have a high, round head cap continuous with the body contour. The labial disc is fused with the medial lips to form an elongate lip structure. The head region is frequently marked by an incomplete annulation. The stylet is robust, 20-27 μm long, usually with round to transversely elongate knobs, sometimes with one or two projections protruding from the shaft. The stylet length of second-stage juveniles is 13-14 μm, the distance of the DGO to the stylet base is 4.0-4.5 μm, and the tail length is 48-51 μm. Biochemically, the esterase (F₁) and malate dehydrogenase (N₁) phenotypes are the most useful characters to differentiate M. paranaensis from other species. However, the esterase phenotype appears similar to that of M. konaensis. Reproduction is by mitotic parthenogenesis, 3n = 50-52. In differential host tests, tobacco, watermelon, and tomato were good hosts, whereas cotton, pepper, and peanut were nonhosts. PMID:19277133

Apparent Genetic Complexity Generated by Developmental Thresholds: The Apterous Locus in DROSOPHILA MELANOGASTER

PubMed Central

Stevens, Mary E.; Bryant, Peter J.

1985-01-01

Mutations at the apterous (ap) locus in Drosophila melanogaster give rise to three distinct phenotypes: aberrant wings, female sterility and precocious adult death. The wing phenotype includes five types of abnormality: blistering, deficiencies, duplications, high-order repetitions and transformation of structures. The mildest phenotype is seen with homozygous apblt animals which have either normal or slightly blistered wings. Most alleles produce, in the homozygote, a deficient wing in which part or all of the wing margin and wing blade is missing, but wing hinge and notum regions are normal. Animals hemizygous for each of 20 ap alleles, as well as apID/apXa heterozygotes, show duplication of parts of the notum associated with complete wing deficiency. Animals heterozygous for apc and the other tested ap alleles show repetitions of parts of the anterior wing margin, an engrailed-like transformation of posterior wing margin into anterior margin or both. Both apblt and apc show similar phenotypes in homozygotes and hemizygotes, yet both produce a less extreme phenotype than that of the other hemizygotes, suggesting that neither mutation causes loss of the entire ap+ function. The 15 alleles that cause precocious death and female sterility occur in six complementation groups based on complementation for these phenotypes. This supports the previous conclusion that the effects of apterous mutations on the wing do not correlate with their effects on viability and fertility. We propose an explanation for the effects of apterous mutations on the wing in which quantitative reductions in the activity of gene product give rise to qualitatively different phenotypes because of different threshold requirements of the ap+ function for critical events in wing disc development. PMID:3924726

Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-14-1-0115 TITLE: Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas PRINCIPAL INVESTIGATOR: Kyuson Yun...CA130273 - Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0115 5c. PROGRAM...hypothesis, we originally proposed to transform neural stem cells (NSCs) and neural progenitor cells (NPCs) in vivo by expressing an activated form

3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatta, Mitsutoki, E-mail: hatta@college.fdcnet.ac.jp; Naganuma, Kaori; Kato, Kenichi

In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histonemore » H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.« less

Image-guided genomics of phenotypically heterogeneous populations reveals vascular signalling during symbiotic collective cancer invasion

PubMed Central

Konen, J.; Summerbell, E.; Dwivedi, B.; Galior, K.; Hou, Y.; Rusnak, L.; Chen, A.; Saltz, J.; Zhou, W.; Boise, L. H.; Vertino, P.; Cooper, L.; Salaita, K.; Kowalski, J.; Marcus, A. I.

2017-01-01

Phenotypic heterogeneity is widely observed in cancer cell populations. Here, to probe this heterogeneity, we developed an image-guided genomics technique termed spatiotemporal genomic and cellular analysis (SaGA) that allows for precise selection and amplification of living and rare cells. SaGA was used on collectively invading 3D cancer cell packs to create purified leader and follower cell lines. The leader cell cultures are phenotypically stable and highly invasive in contrast to follower cultures, which show phenotypic plasticity over time and minimally invade in a sheet-like pattern. Genomic and molecular interrogation reveals an atypical VEGF-based vasculogenesis signalling that facilitates recruitment of follower cells but not for leader cell motility itself, which instead utilizes focal adhesion kinase-fibronectin signalling. While leader cells provide an escape mechanism for followers, follower cells in turn provide leaders with increased growth and survival. These data support a symbiotic model of collective invasion where phenotypically distinct cell types cooperate to promote their escape. PMID:28497793

Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory

PubMed Central

Kulkarni, Prakash; Levine, Herbert

2017-01-01

Waddington’s epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of “landscape” in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an “attractor” that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of “cancer attractors”—hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes “recanalization”, i.e., the exit from “cancer attractors” and re-entry into “normal attractors”, is more likely to succeed rather than a conventional approach that targets individual molecules/pathways. PMID:28640191

Enhancement of matrix production and cell proliferation in human annulus cells under bioreactor culture.

PubMed

Yang, Xinlin; Wang, Daidong; Hao, Jianrong; Gong, Meiqing; Arlet, Vincent; Balian, Gary; Shen, Francis H; Li, Xudong Joshua

2011-06-01

Tissue engineering is a promising approach for treatment of disc degeneration. Herein, we evaluated effects of rotating bioreactor culture on the extracellular matrix production and proliferation of human annulus fibrosus (AF) cells. AF cells were embedded into alginate beads, and then cultured up to 3 weeks in a rotating wall vessel bioreactor or a static vessel. By real-time reverse transcription-polymerase chain reaction, expression of aggrecan, collagen type I and type II, and collagen prolyl 4-hydroxylase II was remarkably elevated, whereas expression of matrix metalloproteinase 3 and a disintegrin and metalloproteinase with thrombospondin motifs 5 was significantly decreased under bioreactor. Biochemical analysis revealed that the levels of the whole cell-associated proteoglycan and collagen were approximately five- and twofolds in rotating bioreactor, respectively, compared to those in static culture. Moreover, AF cell proliferation was augmented in rotating bioreactor. DNA contents were threefolds higher in rotating bioreactor than that in static culture. Expression of the proliferating cell nuclear antigen was robustly enhanced in rotating bioreactor as early as 1 week. Our findings suggested that rotating bioreactor culture would be an effective technique for expansion of human annulus cells for tissue engineering driven treatment of disc degeneration.

Collective Motion in Bacterial Populations with Mixed Phenotypic Behaviors

NASA Astrophysics Data System (ADS)

Hoeger, Kentaro; Strickland, Ben; Shoup, Daniel; Ursell, Tristan

The motion of large, densely packed groups of organisms is often qualitatively distinct from the motion of individuals, yet hinges on individual properties and behaviors. Collective motion of bacteria depends strongly on the phenotypic behaviors of individual cells, the physical interactions between cells, and the geometry of their environment, often with multiple phenotypes coexisting in a population. Thus, to characterize how these selectively important interactions affect group traits, such as cell dispersal, spatial segregation of phenotypes, and material transport in groups, we use a library of Bacillus subtilis mutants that modulate chemotaxis, motility, and biofilm formation. By mixing phenotypes and observing bacterial behaviors and motion at single cell resolution, we probe collective motion as a function of phenotypic mixture and environmental geometry. Our work demonstrates that collective microbial motion exhibits a transition, from `turbulence' to semiballistic burrowing, as phenotypic composition varies. This work illuminates the role that individual cell behaviors play in the emergence of collective motion, and may signal qualitatively distinct regimes of material transport in bacterial populations. University of Oregon.

Expression and functional roles of estrogen receptor GPR30 in human intervertebral disc.

PubMed

Wei, Aiqun; Shen, Bojiang; Williams, Lisa A; Bhargav, Divya; Yan, Feng; Chong, Beng H; Diwan, Ashish D

2016-04-01

Estrogen withdrawal, a characteristic of female aging, is associated with age-related intervertebral disc (IVD) degeneration. The function of estrogen is mediated by two classic nuclear receptors, estrogen receptor (ER)-α and -β, and a membrane bound G-protein-coupled receptor 30 (GPR30). To date, the expression and function of GPR30 in human spine is poorly understood. This study aimed to evaluate GPR30 expression in IVD, and its role in estrogen-related regulation of proliferation and apoptosis of disc nucleus pulposus (NP) cells. GPR30 expression was examined in 30 human adult NP and 9 fetal IVD. Results showed that GPR30 was expressed in NP cells at both mRNA and protein levels. In human fetal IVD, GPR30 protein was expressed in the NP at 12-14 weeks gestation, but was undetectable at 8-11 weeks. The effect of 17β-estradiol (E2) on GPR30-mediated proliferation and interleukin-1β (IL-1β)-induced apoptosis of NP cells was investigated. Cultured NP cells were treated with or without E2, GPR30 antagonist G36, and ER antagonist ICI 182,780. NP cell viability was tested by MTS assay. Apoptosis was determined by flow cytometry using fluorescence labeled annexin-V, TUNEL assay and immumnocytochemical staining of activated caspase-3. E2 enhanced cell proliferation and prevented IL-1β-induced cell death, but the effect was partially blocked by G36 and completely abrogated by a combination of ICI 182,780 and G36. This study demonstrates that GPR30 is expressed in human IVD to transmit signals triggering E2-induced NP cell proliferation and protecting against IL-1β-induced apoptosis. The effects of E2 on NP cells require both GPR30 and classic estrogen receptors. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

[Analysis of factors related to the number of mesenchymal stem cells derived from synovial fluid of the temporomandibular joint].

PubMed

Sun, Y P; Zheng, Y H; Zhang, Z G

2017-06-09

Objective: To analyze related factors on the number of mesenchymal stem cells in the synovial fluid of the temporomandibular joint (TMJ) and provide an research basis for understanding of the source and biological role of mesenchymal stem cells derived from synovial fluid in TMJ. Methods: One hundred and twenty-two synovial fluid samples from 91 temporomandibular disorders (TMD) patients who visited in Department of TMJ Center, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University from March 2013 to December 2013 were collected in this study, and 6 TMJ synovial fluid samples from 6 normal volunteers who were studying in the North Campus of Sun Yat-sen University were also collected, so did their clinical information. Then the relation between the number of mesenchymal stem cells derived from synovial fluid and the health status of the joints, age of donor, disc perforation, condylar bony destruction, blood containing and visual analogue scale score of pain were investigated using Mann-Whitney U test and Spearman rank correlation test. Results: The number of mesenchymal stem cells derived from synovial fluid had no significant relation with visual analogue scale score of pain ( r= 0.041, P= 0.672), blood containing ( P= 0.063), condylar bony destruction ( P= 0.371). Linear correlation between the number of mesenchymal stem cells derived from synovial fluid and age of donor was very week ( r= 0.186, P= 0.043). The number of mesenchymal stem cells up-regulated when the joint was in a disease state ( P= 0.001). The disc perforation group had more mesenchymal stem cells in synovial fluid than without disc perforation group ( P= 0.042). Conclusions: The number of mesenchymal stem cells derived from synovial fluid in TMJ has no correlation with peripheral blood circulation and condylar bony destruction, while has close relation with soft tissue structure damage of the joint.

Role of vitamin D3 in treatment of lumbar disc herniation--pain and sensory aspects: study protocol for a randomized controlled trial.

PubMed

Sedighi, Mahsa; Haghnegahdar, Ali

2014-09-25

Vitamin D receptors have been identified in the spinal cord, nerve roots, dorsal root ganglia and glial cells, and its genetic polymorphism association with the development of lumbar disc degeneration and herniation has been documented. Metabolic effects of active vitamin D metabolites in the nucleus pulposus and annulus fibrosus cells have been studied. Lumbar disc herniation is a process that involves immune and inflammatory cells and processes that are targets for immune regulatory actions of vitamin D as a neurosteroid hormone. In addition to vitamin D's immune modulatory properties, its receptors have been identified in skeletal muscles. It also affects sensory neurons to modulate pain. In this study, we aim to study the role of vitamin D3 in discogenic pain and related sensory deficits. Additionally, we will address how post-treatment 25-hydroxy vitamin D3 level influences pain and sensory deficits severity. The cut-off value for serum 25-hydroxy vitamin D3 that would be efficacious in improving pain and sensory deficits in lumbar disc herniation will also be studied. We will conduct a randomized, placebo-controlled, double-blind clinical trial. Our study population will include 380 cases with one-level and unilateral lumbar disc herniation with duration of discogenic pain less than 8 weeks. Individuals who do not have any contraindications, will be divided into three groups based on serum 25-hydroxy vitamin D3 level, and each group will be randomized to receive either a single-dose 300,000-IU intramuscular injection of vitamin D3 or placebo. All patients will be under conservative treatment. Pre-treatment and post-treatment assessments will be performed with the McGill Pain Questionnaire and a visual analogue scale. For the 15-day duration of this study, questionnaires will be filled out during telephone interviews every 3 days (a total of five times). The initial and final interviews will be scheduled at our clinic. After 15 days, serum 25-hydroxy vitamin D3 levels will be measured for those who have received vitamin D3 (190 individuals). Iranian Registry for Clinical Trials ID: IRCT2014050317534N1 (trial registration: 5 June 2014).

In Vitro Characterization of a Stem-Cell-Seeded Triple-Interpenetrating-Network Hydrogel for Functional Regeneration of the Nucleus Pulposus

PubMed Central

Smith, Lachlan J.; Gorth, Deborah J.; Showalter, Brent L.; Chiaro, Joseph A.; Beattie, Elizabeth E.; Elliott, Dawn M.; Mauck, Robert L.; Chen, Weiliam

2014-01-01

Intervertebral disc degeneration is implicated as a major cause of low-back pain. There is a pressing need for new regenerative therapies for disc degeneration that restore native tissue structure and mechanical function. To that end we investigated the therapeutic potential of an injectable, triple-interpenetrating-network hydrogel comprised of dextran, chitosan, and teleostean, for functional regeneration of the nucleus pulposus (NP) of the intervertebral disc in a series of biomechanical, cytotoxicity, and tissue engineering studies. Biomechanical properties were evaluated as a function of gelation time, with the hydrogel reaching ∼90% of steady-state aggregate modulus within 10 h. Hydrogel mechanical properties evaluated in confined and unconfined compression were comparable to native human NP properties. To confirm containment within the disc under physiological loading, toluidine-blue-labeled hydrogel was injected into human cadaveric spine segments after creation of a nucleotomy defect, and the segments were subjected to 10,000 cycles of loading. Gross analysis demonstrated no implant extrusion, and further, that the hydrogel interdigitated well with native NP. Constructs were next surface-seeded with NP cells and cultured for 14 days, confirming lack of hydrogel cytotoxicity, with the hydrogel maintaining NP cell viability and promoting proliferation. Next, to evaluate the potential of the hydrogel to support cell-mediated matrix production, constructs were seeded with mesenchymal stem cells (MSCs) and cultured under prochondrogenic conditions for up to 42 days. Importantly, the hydrogel maintained MSC viability and promoted proliferation, as evidenced by increasing DNA content with culture duration. MSCs differentiated along a chondrogenic lineage, evidenced by upregulation of aggrecan and collagen II mRNA, and increased GAG and collagen content, and mechanical properties with increasing culture duration. Collectively, these results establish the therapeutic potential of this novel hydrogel for functional regeneration of the NP. Future work will confirm the ability of this hydrogel to normalize the mechanical stability of cadaveric human motion segments, and advance the material toward human translation using preclinical large-animal models. PMID:24410394

Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside.

PubMed

Bach, Frances C; Tellegen, Anna R; Beukers, Martijn; Miranda-Bedate, Alberto; Teunissen, Michelle; de Jong, Willem A M; de Vries, Stefan A H; Creemers, Laura B; Benz, Karin; Meij, Björn P; Ito, Keita; Tryfonidou, Marianna A

2018-05-29

The socioeconomic burden of chronic back pain related to intervertebral disc (IVD) disease is high and current treatments are only symptomatic. Minimally invasive strategies that promote biological IVD repair should address this unmet need. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) during IVD maturation and degeneration. The regenerative potential of NC-secreted substances on CLCs and mesenchymal stromal cells (MSCs) has already been demonstrated. However, identification of these substances remains elusive. Innovatively, this study exploits the regenerative NC potential by using healthy porcine NC-derived matrix (NCM) and employs the dog as a clinically relevant translational model. NCM increased the glycosaminoglycan and DNA content of human and canine CLC aggregates and facilitated chondrogenic differentiation of canine MSCs in vitro . Based on these results, NCM, MSCs and NCM+MSCs were injected in mildly (spontaneously) and moderately (induced) degenerated canine IVDs in vivo and, after six months of treatment, were analyzed. NCM injected in moderately (induced) degenerated canine IVDs exerted beneficial effects at the macroscopic and MRI level, induced collagen type II-rich extracellular matrix production, improved the disc height, and ameliorated local inflammation. MSCs exerted no (additive) effects. In conclusion, NCM induced in vivo regenerative effects on degenerated canine IVDs. NCM may, comparable to demineralized bone matrix in bone regeneration, serve as 'instructive matrix', by locally releasing growth factors and facilitating tissue repair. Therefore, intradiscal NCM injection could be a promising regenerative treatment for IVD disease, circumventing the cumbersome identification of bioactive NC-secreted substances.

Bilateral Non-arteritic Anterior Ischaemic Optic Neuropathy as the Presentation of Systemic Amyloidosis.

PubMed

Kanaan, M Z; Lorenzi, A R; Thampy, N; Pandit, R; Dayan, Margaret

2017-12-01

A 75-year-old hypertensive female with stable idiopathic intermediate uveitis presented with bilateral sequential optic neuropathy with optic disc swelling. The optic neuropathy in the first affected eye (right) was thought to be due to non-arteritic anterior ischaemic optic neuropathy (NAION). Asymptomatic left optic disc swelling was found at routine review 2 months later, and a diagnosis of giant cell arteritis (GCA) was sought. Temporal artery duplex ultrasound showed the "halo sign," but a subsequent temporal artery biopsy showed light-chain (AL) amyloidosis with no signs of giant cell arteritis. In this case, bilateral sequential ischaemic optic neuropathy mimicking non-arteritic anterior ischaemic optic neuropathy was the presenting sign of systemic amyloidosis involving the temporal arteries.

5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila

NASA Astrophysics Data System (ADS)

Huang, Yu Shan; Ku, Hui Yu; Tsai, Yun Chi; Chang, Chin Hao; Pao, Sih Hua; Sun, Y. Henry; Chiou, Arthur

2017-03-01

5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the eye-antenna disc primordium (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments.

Cytokeratin characterization of human prostatic carcinoma and its derived cell lines.

PubMed

Nagle, R B; Ahmann, F R; McDaniel, K M; Paquin, M L; Clark, V A; Celniker, A

1987-01-01

Two murine monoclonal anti-cytokeratin antibodies with defined specificity were shown to distinguish between basal cells and luminal cells in human prostate tissue. Forty-one biopsies or transurethral resection specimens were characterized using these two antibodies. In cases of benign prostatic hyperplasia, focal loss of the basal cell layer was noted in areas of glandular proliferation. Ten cases of adenocarcinoma of the prostate, varying in Gleason's histological grade from 2 to 4, were also studied. In each case the carcinoma was shown to represent the luminal cell phenotype with no evidence of involvement of the basal cell phenotype. An analysis of three established metastatic prostatic carcinoma cell lines (DU-145, PC-3, and LNCaP) using two-dimensional electrophoresis showed that the cytokeratin complement of each cell line was slightly different but retained the phenotype of the luminal cell. It was concluded that during both hyperplasia and neoplastic transformation of the prostate, the luminal cell phenotype is primarily involved and that the basal cell phenotype does not appear to contribute to either intraluminal proliferation or invasive cell populations.

The Time Course of Deafness and Retinal Degeneration in a Kunming Mouse Model for Usher Syndrome.

PubMed

Yao, Lu; Zhang, Lei; Qi, Lin-Song; Liu, Wei; An, Jing; Wang, Bin; Xue, Jun-Hui; Zhang, Zuo-Ming

2016-01-01

Usher syndrome is a group of autosomal recessive diseases characterized by congenital deafness and retinitis pigmentosa. In a mouse model for Usher syndrome, KMush/ush, discovered in our laboratory, we measured the phenotypes, characterized the architecture and morphology of the retina, and quantified the level of expression of pde6b and ush2a between postnatal (P) days 7, and 56. Electroretinograms and auditory brainstem response were used to measure visual and auditory phenotypes. Fundus photography and light microscopy were used to measure the architecture and morphology of the retina. Quantitative real-time PCR was used to measure the expression levels of mRNA. KMush/ush mice had low amplitudes and no obvious waveforms of Electroretinograms after P14 compared with controls. Thresholds of auditory brainstem response in our model were higher than those of controls after P14. By P21, the retinal vessels of KMush/ush mice were attenuated and their optic discs had a waxy pallor. The retinas of KMush/ush mice atrophied and the choroidal vessels were clearly visible. Notably, the architecture of each retinal layer was not different as compared with control mice at P7, while the outer nuclear layer (ONL) and other retinal layers of KMush/ush mice were attenuated significantly between P14 and P21. ONL cells were barely seen in KMush/ush mice at P56. As compared with control mice, the expression of pde6b and ush2a in KMush/ush mice declined significantly after P7. This study is a first step toward characterizing the progression of disease in our mouse model. Future studies using this model may provide insights about the etiology of the disease and the relationships between genotypes and phenotypes providing a valuable resource that could contribute to the foundation of knowledge necessary to develop therapies to prevent the retinal degeneration in patients with Usher Syndrome.

Common genetic variation drives molecular heterogeneity in human iPSCs.

PubMed

Kilpinen, Helena; Goncalves, Angela; Leha, Andreas; Afzal, Vackar; Alasoo, Kaur; Ashford, Sofie; Bala, Sendu; Bensaddek, Dalila; Casale, Francesco Paolo; Culley, Oliver J; Danecek, Petr; Faulconbridge, Adam; Harrison, Peter W; Kathuria, Annie; McCarthy, Davis; McCarthy, Shane A; Meleckyte, Ruta; Memari, Yasin; Moens, Nathalie; Soares, Filipa; Mann, Alice; Streeter, Ian; Agu, Chukwuma A; Alderton, Alex; Nelson, Rachel; Harper, Sarah; Patel, Minal; White, Alistair; Patel, Sharad R; Clarke, Laura; Halai, Reena; Kirton, Christopher M; Kolb-Kokocinski, Anja; Beales, Philip; Birney, Ewan; Danovi, Davide; Lamond, Angus I; Ouwehand, Willem H; Vallier, Ludovic; Watt, Fiona M; Durbin, Richard; Stegle, Oliver; Gaffney, Daniel J

2017-06-15

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.

Overexpression of molecular chaperons GRP78 and GRP94 in CD44(hi)/CD24(lo) breast cancer stem cells.

PubMed

Nami, Babak; Ghasemi-Dizgah, Armin; Vaseghi, Akbar

2016-01-01

Breast cancer stem cell with CD44(hi)/CD24(lo) phonotype is described having stem cell properties and represented as the main driving factor in breast cancer initiation, growth, metastasis and low response to anti-cancer agents. Glucoseregulated proteins (GRPs) are heat shock protein family chaperons that are charged with regulation of protein machinery and modulation of endoplasmic reticulum homeostasis whose important roles in stem cell development and invasion of various cancers have been demonstrated. Here, we investigated the expression levels of GRP78 and GRP94 in CD44(hi)/CD24(lo) phenotype breast cancer stem cells (BCSCs). MCF7, T-47D and MDA-MB-231 breast cancer cell lines were used. CD44(hi)/CD24(lo) phenotype cell population were analyzed and sorted by fluorescence-activated cell sorting (FACS). Transcriptional and translational expression of GRP78 and GRP94 were investigated by western blotting and quantitative real time PCR. RESULTS showed different proportion of CD44(hi)/CD24(lo) phenotype cell population in their original bulk cells. The ranking of the cell lines in terms of CD44(hi)/CD24(lo) phenotype cell population was as MCF7

Dynamic apical surface rings in superficial layer cells of koi Cyprinus carpio scale epidermis.

PubMed

DePasquale, J A

2016-09-01

This study examined the novel ring-shaped structures found in the apical surface of individual cells of the scale epidermis of koi Cyprinus carpio. These apical rings are highly dynamic structures with lifetimes ranging from a few to several minutes. While several ring forms were observed, the predominant ring morphology is circular or oval. Two distinct ring forms were identified and designated type I and type II. Type I rings have a well-defined outer border that encircles the surface microridges. Type II rings are smooth-surfaced, dinner-plate-like structures with membranous folds or compressed microridges in the centre. Type II rings appear less frequently than type I rings. Type I rings form spontaneously, arising from swollen or physically interrupted microridges but without initially perturbing the encircled microridges. After persisting for up to several minutes the ring closes in a centripetal movement to form a circular or irregular-shaped structure, the terminal disc. The terminal disc eventually disappears, leaving behind a submembranous vesicle-like structure, the terminal body. Type I rings can undergo multiple cycles of formation and closing. Recycling epidermal apical rings form through centrifugal expansion from the terminal disc followed by apparent contraction back to the disc structure, whereupon the cycle may repeat or cease. The findings demonstrate a novel skin surface structure in fishes and are discussed with respect to communication with the external aqueous environment. © 2016 The Fisheries Society of the British Isles.

The Homothorax homeoprotein activates the nuclear localization of another homeoprotein, Extradenticle, and suppresses eye development in Drosophila

PubMed Central

Pai, Chi-Yun; Kuo, Tung-Sheng; Jaw, Thomas J.; Kurant, Estee; Chen, Cheng-Tse; Bessarab, Dmitri A.; Salzberg, Adi; Sun, Y. Henry

1998-01-01

The Extradenticle (Exd) protein in Drosophila acts as a cofactor to homeotic proteins. Its nuclear localization is regulated. We report the cloning of the Drosophila homothorax (hth) gene, a homolog of the mouse Meis1 proto-oncogene that has a homeobox related to that of exd. Comparison with Meis1 finds two regions of high homology: a novel MH domain and the homeodomain. In imaginal discs, hth expression coincides with nuclear Exd. hth and exd also have virtually identical, mutant clonal phenotypes in adults. These results suggest that hth and exd function in the same pathway. We show that hth acts upstream of exd and is required and sufficient for Exd protein nuclear localization. We also show that hth and exd are both negative regulators of eye development; their mutant clones caused ectopic eye formation. Targeted expression of hth, but not of exd, in the eye disc abolished eye development completely. We suggest that hth acts with exd to delimit the eye field and prevent inappropriate eye development. PMID:9450936

High-resolution imaging of the retinal nerve fiber layer in normal eyes using adaptive optics scanning laser ophthalmoscopy.

PubMed

Takayama, Kohei; Ooto, Sotaro; Hangai, Masanori; Arakawa, Naoko; Oshima, Susumu; Shibata, Naohisa; Hanebuchi, Masaaki; Inoue, Takashi; Yoshimura, Nagahisa

2012-01-01

To conduct high-resolution imaging of the retinal nerve fiber layer (RNFL) in normal eyes using adaptive optics scanning laser ophthalmoscopy (AO-SLO). AO-SLO images were obtained in 20 normal eyes at multiple locations in the posterior polar area and a circular path with a 3-4-mm diameter around the optic disc. For each eye, images focused on the RNFL were recorded and a montage of AO-SLO images was created. AO-SLO images for all eyes showed many hyperreflective bundles in the RNFL. Hyperreflective bundles above or below the fovea were seen in an arch from the temporal periphery on either side of a horizontal dividing line to the optic disc. The dark lines among the hyperreflective bundles were narrower around the optic disc compared with those in the temporal raphe. The hyperreflective bundles corresponded with the direction of the striations on SLO red-free images. The resolution and contrast of the bundles were much higher in AO-SLO images than in red-free fundus photography or SLO red-free images. The mean hyperreflective bundle width around the optic disc had a double-humped shape; the bundles at the temporal and nasal sides of the optic disc were narrower than those above and below the optic disc (P<0.001). RNFL thickness obtained by optical coherence tomography correlated with the hyperreflective bundle widths on AO-SLO (P<0.001) AO-SLO revealed hyperreflective bundles and dark lines in the RNFL, believed to be retinal nerve fiber bundles and Müller cell septa. The widths of the nerve fiber bundles appear to be proportional to the RNFL thickness at equivalent distances from the optic disc.

Mechanical Restoration and Failure Analyses of a Hydrogel and Scaffold Composite Strategy for Annulus Fibrosus Repair

PubMed Central

Long, Rose G; Bürki, Alexander; Zysset, Philippe; Eglin, David; Grijpma, Dirk W.; Blanquer, Sebastien BG; Hecht, Andrew C; Iatridis, James C

2015-01-01

Unrepaired defects in the annulus fibrosus of intervertebral discs are associated with degeneration and persistent back pain. A clinical need exists for a disc repair strategy that can seal annular defects, be easily delivered during surgical procedures, and restore biomechanics with low risk of herniation. Multiple annulus repair strategies were developed using poly(trimethylene carbonate) scaffolds optimized for cell delivery, polyurethane membranes designed to prevent herniation, and fibrin-genipin adhesive tuned to annulus fibrosus shear properties. This three-part study evaluated repair strategies for biomechanical restoration, herniation risk and failure mode in torsion, bending and compression at physiological and hyper-physiological loads using a bovine injury model. Fibrin-genipin hydrogel restored some torsional stiffness, bending ROM and disc height loss, with negligible herniation risk and failure was observed histologically at the fibrin-genipin mid-substance following rigorous loading. Scaffold-based repairs partially restored biomechanics, but had high herniation risk even when stabilized with sutured membranes and failure was observed histologically at the interface between scaffold and fibrin-genipin adhesive. Fibrin-genipin was the simplest annulus fibrosus repair solution evaluated that involved an easily deliverable adhesive that filled irregularly-shaped annular defects and partially restored disc biomechanics with low herniation risk, suggesting further evaluation for disc repair may be warranted. PMID:26577987

A selective inhibition of c-Fos/activator protein-1 as a potential therapeutic target for intervertebral disc degeneration and associated pain.

PubMed

Makino, Hiroto; Seki, Shoji; Yahara, Yasuhito; Shiozawa, Shunichi; Aikawa, Yukihiko; Motomura, Hiraku; Nogami, Makiko; Watanabe, Kenta; Sainoh, Takeshi; Ito, Hisakatsu; Tsumaki, Noriyuki; Kawaguchi, Yoshiharu; Yamazaki, Mitsuaki; Kimura, Tomoatsu

2017-12-05

Intervertebral disc (IVD) degeneration is a major cause of low back pain. The transcription factor c-Fos/Activator Protein-1 (AP-1) controls the expression of inflammatory cytokines and matrix metalloproteinases (MMPs) that contribute to the pathogenesis IVD degeneration. We investigated the effects of inhibition of c-Fos/AP-1 on IVD degeneration and associated pain. A selective inhibitor, T-5224, significantly suppressed the interleukin-1β-induced up-regulation of Mmp-3, Mmp-13 and Adamts-5 transcription in human nucleus pulposus cells and in a mouse explant culture model of IVD degeneration. We used a tail disc percutaneous needle puncture method to further assess the effects of oral administration of T-5224 on IVD degeneration. Analysis of disc height, T2-magnetic resonance imaging (MRI) findings, and histology revealed that IVD degeneration was significantly mitigated by T-5224. Further, oral administration of T-5224 ameliorated pain as indicated by the extended tail-flick latency in response to heat stimulation of rats with needle-puncture-induced IVD degeneration. These findings suggest that the inhibition of c-Fos/AP-1 prevents disc degeneration and its associated pain and that T-5224 may serve as a drug for the prevention of IVD degeneration.

Hyaluronic Acid (HA)-Polyethylene glycol (PEG) as injectable hydrogel for intervertebral disc degeneration patients therapy

NASA Astrophysics Data System (ADS)

Putri Kwarta, Cityta; Widiyanti, Prihartini; Siswanto

2017-05-01

Chronic Low Back Pain (CLBP) is one health problem that is often encountered in a community. Inject-able hydrogels are the newest way to restore the disc thickness and hydration caused by disc degeneration by means of minimally invasive surgery. Thus, polymers can be combined to improve the characteristic properties of inject-able hydrogels, leading to use of Hyaluronic Acid (a natural polymer) and Polyethylene glycol (PEG) with Horse Radish Peroxide (HRP) cross linker enzymes. The swelling test results, which approaches were the ideal disc values, were sampled with variation of enzyme concentrations of 0.25 µmol/min/mL. The enzyme concentrations were 33.95%. The degradation test proved that the sample degradation increased along with the decrease of the HRP enzyme concentration. The results of the cytotoxicity assay with MTT assay method showed that all samples resulted in the 90% of living cells are not toxic. In vitro injection, models demonstrated that higher concentration of the enzymes was less state of gel which would rupture when released from the agarose gel. The functional group characterization shows the cross linking bonding in sample with enzyme adding. The conclusion of this study is PEG-HA-HRP enzyme are safe polymer composites which have a potential to be applied as an injectable hydrogel for intervertebral disc degeneration.

Interference of HTLV-1 Tax Protein with Cell Polarity Regulators: Defining the Subcellular Localization of the Tax-DLG1 Interaction

PubMed Central

Marziali, Federico; Bugnon Valdano, Marina; Brunet Avalos, Clarisse; Moriena, Lucía; Cavatorta, Ana Laura; Gardiol, Daniela

2017-01-01

Human T cell leukemia virus (HTLV)-1 Tax (Tax) protein is very important in viral replication and cell transformation. Tax localizes in the nucleus and cytoplasm in association with organelles. Some activities of Tax depend on interactions with PDZ (PSD-95/Discs Large/Z0-1) domain–containing proteins such as Discs large protein 1 (DLG1) which is involved in cell polarity and proliferation. The DLG1 interaction results in a cytoplasmic co-localization pattern resembling vesicular aggregates, the nature of which is still unknown. To further explore the role of PDZ proteins in HTLV-1 cell transformation, we deeply investigated the Tax-DLG1 association. By fluorescence resonance energy transfer (FRET), we detected, for the first time, the direct binding of Tax to DLG1 within the cell. We showed that the interaction specifically affects the cellular distribution of not only DLG1, but also Tax. After studying different cell structures, we demonstrated that the aggregates distribute into the Golgi apparatus in spatial association with the microtubule-organizing center (MTOC). This study contributes to understand the biological significance of Tax-PDZ interactions. PMID:29168728

Interference of HTLV-1 Tax Protein with Cell Polarity Regulators: Defining the Subcellular Localization of the Tax-DLG1 Interaction.

PubMed

Marziali, Federico; Bugnon Valdano, Marina; Brunet Avalos, Clarisse; Moriena, Lucía; Cavatorta, Ana Laura; Gardiol, Daniela

2017-11-23

Human T cell leukemia virus (HTLV)-1 Tax (Tax) protein is very important in viral replication and cell transformation. Tax localizes in the nucleus and cytoplasm in association with organelles. Some activities of Tax depend on interactions with PDZ (PSD-95/Discs Large/Z0-1) domain-containing proteins such as Discs large protein 1 (DLG1) which is involved in cell polarity and proliferation. The DLG1 interaction results in a cytoplasmic co-localization pattern resembling vesicular aggregates, the nature of which is still unknown. To further explore the role of PDZ proteins in HTLV-1 cell transformation, we deeply investigated the Tax-DLG1 association. By fluorescence resonance energy transfer (FRET), we detected, for the first time, the direct binding of Tax to DLG1 within the cell. We showed that the interaction specifically affects the cellular distribution of not only DLG1, but also Tax. After studying different cell structures, we demonstrated that the aggregates distribute into the Golgi apparatus in spatial association with the microtubule-organizing center (MTOC). This study contributes to understand the biological significance of Tax-PDZ interactions.

The Drosophila muscle LIM protein, Mlp84B, cooperates with D-titin to maintain muscle structural integrity.

PubMed

Clark, Kathleen A; Bland, Jennifer M; Beckerle, Mary C

2007-06-15

Muscle LIM protein (MLP) is a cytoskeletal LIM-only protein expressed in striated muscle. Mutations in human MLP are associated with cardiomyopathy; however, the molecular mechanism by which MLP functions is not established. A Drosophila MLP homolog, mlp84B, displays many of the same features as the vertebrate protein, illustrating the utility of the fly for the study of MLP function. Animals lacking Mlp84B develop into larvae with a morphologically intact musculature, but the mutants arrest during pupation with impaired muscle function. Mlp84B displays muscle-specific expression and is a component of the Z-disc and nucleus. Preventing nuclear retention of Mlp84B does not affect its function, indicating that Mlp84B site of action is likely to be at the Z-disc. Within the Z-disc, Mlp84B is colocalized with the N-terminus of D-titin, a protein crucial for sarcomere organization and stretch mechanics. The mlp84B mutants phenotypically resemble weak D-titin mutants. Furthermore, reducing D-titin activity in the mlp84B background leads to pronounced enhancement of the mlp84B muscle defects and loss of muscle structural integrity. The genetic interactions between mlp84B and D-titin reveal a role for Mlp84B in maintaining muscle structural integrity that was not obvious from analysis of the mlp84B mutants themselves, and suggest Mlp84B and D-titin cooperate to stabilize muscle sarcomeres.

Stochastic loss and gain of symmetric divisions in the C. elegans epidermis perturbs robustness of stem cell number

PubMed Central

Katsanos, Dimitris; Koneru, Sneha L.; Mestek Boukhibar, Lamia; Gritti, Nicola; Ghose, Ritobrata; Appleford, Peter J.; Doitsidou, Maria; Woollard, Alison; van Zon, Jeroen S.; Poole, Richard J.

2017-01-01

Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens. PMID:29108019

Analysis of cell cycle-related proteins in gastric intramucosal differentiated-type cancers based on mucin phenotypes: a novel hypothesis of early gastric carcinogenesis based on mucin phenotype

PubMed Central

2010-01-01

Background Abnormalities of cell cycle regulators are common features in human cancers, and several of these factors are associated with the early development of gastric cancers. However, recent studies have shown that gastric cancer tumorigenesis was characterized by mucin expression. Thus, expression patterns of cell cycle-related proteins were investigated in the early phase of differentiated-type gastric cancers to ascertain any mechanistic relationships with mucin phenotypes. Methods Immunostaining for Cyclins D1, A, E, and p21, p27, p53 and β-catenin was used to examine impairments of the cell cycle in 190 gastric intramucosal differentiated-type cancers. Mucin phenotypes were determined by the expressions of MUC5AC, MUC6, MUC2 and CD10. A Ki-67 positive rate (PR) was also examined. Results Overexpressions of p53, cyclin D1 and cyclin A were significantly more frequent in a gastric phenotype than an intestinal phenotype. Cyclin A was overexpressed in a mixed phenotype compared with an intestinal phenotype, while p27 overexpression was more frequent in an intestinal phenotype than in a mixed phenotype. Reduction of p21 was a common feature of the gastric intramucosal differentiated-type cancers examined. Conclusions Our results suggest that the levels of some cell cycle regulators appear to be associated with mucin phenotypes of early gastric differentiated-type cancers. PMID:20525401

SHH-dependent knockout of HIF-1 alpha accelerates the degenerative process in mouse intervertebral disc.

PubMed

Wu, W J; Zhang, X K; Zheng, X F; Yang, Y H; Jiang, S D; Jiang, L S

2013-01-01

Hypoxia-inducible factor-1alpha (HIF-1 alpha) has been reported to have an important role in the metabolism and synthesis of extracellular matrix of the nucleus pulposus cells (NPCs) and was assumed to be involved in the process of intervertebral disc degeneration. The objective of this study was to investigate the role of HIF-1alpha in disc degeneration in vivo using a conditional HIF-1alpha knockout (KO) mouse model. ShhCre transgenic mice were mated with HIF-1 alpha fl/fl mice to generate conditional HIF-1alpha KO mice (HIF-1alpha fl/fl-ShhCre+). Three mice of each genotype (Wide-type and HIF-1alpha KO) at the age of 3 days, 6, and 12 weeks were sacrificed after genotyping. Five lumbar disc samples were harvested from each mouse, with a total of 45 disc samples for each genotype. In situ hybridization and immunohistochemical analysis were used to check the efficacy of HIF-1alpha knockout. Histological grading of the disc degeneration was performed according to the classification system proposed by Boos et al. Picro-sirius red staining, Safranine O/fast green staining and immunohistochemical study were used to evaluate the expression of aggrecan, type-II collagen and vascular endothelial growth factor (VEGF). Histologic analysis revealed more NPC deaths and signs of degeneration in HIF-1alpha KO mice and the degeneration scores of HIF-1alpha KO mice were significantly higher than those of the Wide-type mice at the age of 6 weeks and 12 weeks. There were less expressions of aggrecan, type-II collagen and VEGF in the intervertebral discs of HIF1-alpha KO mice than in those of wild-type mice. Taken together, the results of our study indicated that HIF-1alpha is a pivotal contributor to NPC survival and the homeotasis of extracellular matrix through the HIF-1alpha/VEGF signaling pathway, and plays an important role in the development of disc degeneration.

In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina.

PubMed

Reyes-Aguirre, Luis I; Quintero, Heberto; Estrada-Leyva, Brenda; Lamas, Mónica

2018-01-01

microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.

Increased alternative lengthening of telomere phenotypes of telomerase-negative immortal cells upon trichostatin--a treatment.

PubMed

Jung, A Ra; Yoo, Jeong Eun; Shim, Yhong-Hee; Choi, Ye-Na; Jeung, Hei-Cheul; Chung, Hyun Cheol; Rha, Sun Young; Oh, Bong-Kyeong

2013-03-01

Human immortal cells maintain their telomeres either by telomerase or by alternative lengthening of telomeres (ALT) that is based on homologous telomeric recombination. Previous studies showed that the ALT mechanism is activated in non-ALT cells when heterochromatic features are reduced. In this study, we examined the ALT phenotypes of ALT cells after treatment with trichostatin-A (TSA), which is an inhibitor of histone deacetylases and causes global chromatin decondensation. The ALT cells remained telomerase-negative after TSA treatment. ALT-associated promyelocytic leukemia (PML) nuclear bodies and telomere sister chromatid exchanges, typical ALT phenotypes, markedly increased in the TSA-treated cells, while the telomere length remained unchanged. In addition, telomerase expression in the ALT cells suppressed TSA-mediated ALT phenotype enhancement. Our results show that certain ALT phenotypes become more pronounced when chromatin is decondensed, and also suggest that the ALT mechanism may compete with telomerase for telomere maintenance in cells that lack heterochromatin.

Transfer cell wall ingrowths and vein loading characteristics in pea leaf discs. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wimmers, L.E.; Turgeon, R.

1987-04-01

Transfer cell wall ingrowths are thought to increase transport capacity by increasing plasmalemma surface area. Leaf minor vein phloem transfer cells presumably enhance phloem loading. In Pisum sativum cv. Little marvel grown under different light regimes (150 to 1000 ..mu..mol photons m/sup -2/ sec/sup -1/) there is a positive correlation between light intensity and wall ingrowth area in phloem transfer cells. The extent of ingrowth and correlation to light intensity is greatest in minor veins, decreasing as vein size increases. Vein loading was assayed by floating abraded leaf discs on /sup 14/C-sucrose (10 mM). There is a positive correlation betweenmore » uptake and transfer cell wall area, although the latter increased more than the former. The difference in uptake is stable throughout the photoperiod, and is also stable in mature leaves for at least four days after plants are transfered to a different light intensity. Sucrose uptake is biphasic. The saturable component of uptake is sensitive to light intensity, the Km for sucrose is negatively correlated to light intensity, while V/sub max/remains unchanged.« less

Genipin cross-linked type II collagen/chondroitin sulfate composite hydrogel-like cell delivery system induces differentiation of adipose-derived stem cells and regenerates degenerated nucleus pulposus.

PubMed

Zhou, Xiaopeng; Wang, Jingkai; Fang, Weijing; Tao, Yiqing; Zhao, Tengfei; Xia, Kaishun; Liang, Chengzhen; Hua, Jianming; Li, Fangcai; Chen, Qixin

2018-04-15

Nucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration and consequent lower back pain. Although adipose-derived stem cell (ADSC)-based therapy is regarded to be promising for the treatment of degenerated NP, there is a lack of viable cell carriers to transplant ADSCs into the NP while maintaining cell function. In this study, we developed a type II collagen/chondroitin sulfate (CS) composite hydrogel-like ADSC (CCSA) delivery system with genipin as the cross-linking agent. The induction effect of the scaffold on ADSC differentiation was studied in vitro, and a rat coccygeal vertebrae degeneration model was used to investigate the regenerative effect of the CCSA system on the degenerated NP in vivo. The results showed that the CCSA delivery system cross-linked with 0.02% genipin was biocompatible and promoted the expressions of NP-specific genes. After the injection of the CCSA system, the disc height, water content, extracellular matrix synthesis, and structure of the degenerated NP were partly restored. Our CCSA delivery system uses minimally invasive approaches to promote the regeneration of degenerated NP and provides an exciting new avenue for the treatment of degenerative disc disease. Nucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration and consequent lower back pain. Stem cell-based tissue engineering is a promising method in NP regeneration, but there is a lack of viable cell carriers to transplant ADSCs into the NP while maintaining cell function. In this study, we developed a type II collagen/chondroitin sulfate (CS) composite hydrogel-like ADSC (CCSA) delivery system with genipin as the cross-linking agent. Although several research groups have studied the fabrication of injectable hydrogel with biological matrix, our study differs from other works. We chose type II collagen and CS, the two primary native components in the NP, as the main materials and combined them according to the natural ratio of collagen and sGAG in the NP. The delivery system is preloaded with ADSCs and can be injected into the NP with a needle, followed by in situ gelation. Genipin is used as a cross-linker to improve the bio-stability of the scaffold, with low cytotoxicity. We investigated the stimulatory effects of our scaffold on the differentiation of ADSCs in vitro and the regenerative effect of the CCSA delivery system on degenerated NP in vivo. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells.

PubMed Central

Shamah, S M; Stiles, C D; Guha, A

1993-01-01

Malignant astrocytoma is the most common primary human brain tumor. Most astrocytomas express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could close an autocrine loop. It is not known whether these autocrine loops contribute to the transformed phenotype of astrocytoma cells or are incidental to that phenotype. Here we show that dominant-negative mutants of the PDGF ligand break the autocrine loop and revert the phenotype of BALB/c 3T3 cells transformed by the PDGF-A or PDGF-B (c-sis) gene. Then, we show that these mutants are selective in that they do not alter the phenotype of 3T3 cells transformed by an activated Ha-ras or v-src gene or by simian virus 40. Finally, we show that these mutants revert the transformed phenotype of two independent human astrocytoma cell lines. They have no effect on the growth of human medulloblastoma, bladder carcinoma, or colon carcinoma cell lines. These observations are consistent with the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas. Images PMID:8246942

Asymmetries of Dark and Bright Negative Afterimages Are Paralleled by Subcortical ON and OFF Poststimulus Responses.

PubMed

Li, Hui; Liu, Xu; Andolina, Ian M; Li, Xiaohong; Lu, Yiliang; Spillmann, Lothar; Wang, Wei

2017-02-22

Humans are more sensitive to luminance decrements than increments, as evidenced by lower thresholds and shorter latencies for dark stimuli. This asymmetry is consistent with results of neurophysiological recordings in dorsal lateral geniculate nucleus (dLGN) and primary visual cortex (V1) of cat and monkey. Specifically, V1 population responses demonstrate that darks elicit higher levels of activation than brights, and the latency of OFF responses in dLGN and V1 is shorter than that of ON responses. The removal of a dark or bright disc often generates the perception of a negative afterimage, and here we ask whether there also exist asymmetries for negative afterimages elicited by dark and bright discs. If so, do the poststimulus responses of subcortical ON and OFF cells parallel such afterimage asymmetries? To test these hypotheses, we performed psychophysical experiments in humans and single-cell/S-potential recordings in cat dLGN. Psychophysically, we found that bright afterimages elicited by luminance decrements are stronger and last longer than dark afterimages elicited by luminance increments of equal sizes. Neurophysiologically, we found that ON cells responded to the removal of a dark disc with higher firing rates that were maintained for longer than OFF cells to the removal of a bright disc. The ON and OFF cell asymmetry was most pronounced at long stimulus durations in the dLGN. We conclude that subcortical response strength differences between ON and OFF channels parallel the asymmetries between bright and dark negative afterimages, further supporting a subcortical origin of bright and dark afterimage perception. SIGNIFICANCE STATEMENT Afterimages are physiological aftereffects following stimulation of the eye, the study of which helps us to understand how our visual brain generates visual perception in the absence of physical stimuli. We report, for the first time to our knowledge, asymmetries between bright and dark negative afterimages elicited by luminance decrements and increments, respectively. Bright afterimages are stronger and last longer than dark afterimages. Subcortical neuronal recordings of poststimulus responses of ON and OFF cells reveal similar asymmetries with respect to response strength and duration. Our results suggest that subcortical differences between ON and OFF channels help explain intensity and duration asymmetries between bright and dark afterimages, supporting the notion of a subcortical origin of bright and dark afterimages. Copyright © 2017 the authors 0270-6474/17/371984-13$15.00/0.

Generation of novel bone forming cells (monoosteophils) from the cathelicidin-derived peptide LL-37 treated monocytes.

PubMed

Zhang, Zhifang; Shively, John E

2010-11-15

Bone generation and maintenance involve osteoblasts, osteoclasts, and osteocytes which originate from unique precursors and rely on key growth factors for differentiation. However, an incomplete understanding of bone forming cells during wound healing has led to an unfilled clinical need such as nonunion of bone fractures. Since circulating monocytes are often recruited to sites of injury and may differentiate into various cell types including osteoclasts, we investigated the possibility that circulating monocytes in the context of tissue injury may also contribute to bone repair. In particular, we hypothesized that LL-37 (produced from hCAP-18, cathelicidin), which recruits circulating monocytes during injury, may play a role in bone repair. Treatment of monocytes from blood with LL-37 for 6 days resulted in their differentiation to large adherent cells. Growth of LL-37-differentiated monocytes on osteologic discs reveals bone-like nodule formation by scanning electron microscopy (SEM). In vivo transplantation studies in NOD/SCID mice show that LL-37-differentiated monocytes form bone-like structures similar to endochondral bone formation. Importantly, LL-37-differentiated monocytes are distinct from conventional monocyte-derived osteoclasts, macrophages, and dendritic cells and do not express markers of the mesenchymal stem cells (MSC) lineage, distinguishing them from the conventional precursors of osteoblasts. Furthermore, LL-37 differentiated monocytes express intracellular proteins of both the osteoblast and osteoclast lineage including osteocalcin (OC), osteonectin (ON), bone sialoprotein II (BSP II), osteopontin (OP), RANK, RANKL, MMP-9, tartrate resistant acid phosphatase (TRAP), and cathepsin K (CK). Blood derived monocytes treated with LL-37 can be differentiated into a novel bone forming cell that functions both in vitro and in vivo. We propose the name monoosteophil to indicate their monocyte derived lineage and their bone forming phenotype. These cells may have wide ranging implications in the clinic including repair of broken bones and treatment of osteoporosis.

Surface topography of hydroxyapatite promotes osteogenic differentiation of human bone marrow mesenchymal stem cells.

PubMed

Yang, Wanlei; Han, Weiqi; He, Wei; Li, Jianlei; Wang, Jirong; Feng, Haotian; Qian, Yu

2016-03-01

Effective and safe induction of osteogenic differentiation is one of the key elements of bone tissue engineering. Surface topography of scaffold materials was recently found to promote osteogenic differentiation. Utilization of this topography may be a safer approach than traditional induction by growth factors or chemicals. The aim of this study is to investigate the enhancement of osteogenic differentiation by surface topography and its mechanism of action. Hydroxyapatite (HA) discs with average roughness (Ra) of surface topography ranging from 0.2 to 1.65 μm and mean distance between peaks (RSm) ranging from 89.7 to 18.6 μm were prepared, and human bone-marrow mesenchymal stem cells (hBMSCs) were cultured on these discs. Optimal osteogenic differentiation was observed on discs with surface topography characterized by Ra ranging from 0.77 to 1.09 μm and RSm ranging from 53.9 to 39.3 μm. On this surface configuration of HA, hBMSCs showed oriented attachment, F-actin arrangement, and a peak in the expression of Yes-associated protein (YAP) and PDZ binding motif (TAZ) (YAP/TAZ). These results indicated that the surface topography of HA promoted osteogenic differentiation of hBMSCs, possibly by increasing cell attachment and promoting the YAP/TAZ signaling pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

Shh signaling from the nucleus pulposus is required for the postnatal growth and differentiation of the mouse intervertebral disc.

PubMed

Dahia, Chitra Lekha; Mahoney, Eric; Wylie, Christopher

2012-01-01

Intervertebral discs (IVD) are essential components of the vertebral column. They maintain separation, and provide shock absorbing buffers, between adjacent vertebrae, while also allowing movements between them. Each IVD consists of a central semi-liquid nucleus pulposus (NP) surrounded by a multi-layered fibrocartilagenous annulus fibrosus (AF). Although the IVDs grow and differentiate after birth along with the vertebral column, little is known about the mechanism of this. Understanding the signals that control normal IVD growth and differentiation would also provide potential therapies for degenerative disc disease, which is the major cause of lower back pain and affects a large proportion of the population. In this work, we show that during postnatal growth of the mouse, Sonic hedgehog (Shh) signaling from the NP cells controls many aspects of growth and differentiation of both the NP cells themselves and of the surrounding AF, and that it acts, at least partly, by regulating other signaling pathways in the NP and AF. Recent studies have shown that the NP cells arise from the embryonic notochord, which acts as a major signaling center in the embryo. This work shows that this notochord-derived tissue continues to carry out a major signaling function in the postnatal body and that the IVDs are signaling centers, in addition to their already known functions in the mechanics of vertebral column function.

Exosomes derived from mesenchymal non-small cell lung cancer cells promote chemoresistance.

PubMed

Lobb, Richard J; van Amerongen, Rosa; Wiegmans, Adrian; Ham, Sunyoung; Larsen, Jill E; Möller, Andreas

2017-08-01

Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes. © 2017 UICC.

Cross-regulatory protein-protein interactions between Hox and Pax transcription factors.

PubMed

Plaza, Serge; Prince, Frederic; Adachi, Yoshitsugu; Punzo, Claudio; Cribbs, David L; Gehring, Walter J

2008-09-09

Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein-protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP-EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein-DNA and protein-RNA interactions; it is also involved in protein-protein interactions.

Cross-regulatory protein–protein interactions between Hox and Pax transcription factors

PubMed Central

Plaza, Serge; Prince, Frederic; Adachi, Yoshitsugu; Punzo, Claudio; Cribbs, David L.; Gehring, Walter J.

2008-01-01

Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein–protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP–EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein–DNA and protein–RNA interactions; it is also involved in protein–protein interactions. PMID:18755899

‘Particle genetics’: treating every cell as unique

PubMed Central

Yvert, Gaël

2014-01-01

Genotype-phenotype relations are usually inferred from a deterministic point of view. For example, quantitative trait loci (QTL), which describe regions of the genome associated with a particular phenotype, are based on a mean trait difference between genotype categories. However, living systems comprise huge numbers of cells (the ‘particles’ of biology). Each cell can exhibit substantial phenotypic individuality, which can have dramatic consequences at the organismal level. Now, with technology capable of interrogating individual cells, it is time to consider how genotypes shape the probability laws of single cell traits. The possibility of mapping single cell probabilistic trait loci (PTL), which link genomic regions to probabilities of cellular traits, is a promising step in this direction. This approach requires thinking about phenotypes in probabilistic terms, a concept that statistical physicists have been applying to particles for a century. Here, I describe PTL and discuss their potential to enlarge our understanding of genotype-phenotype relations. PMID:24315431

A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

PubMed

Garzón, Ingrid; Carriel, Victor; Marín-Fernández, Ana Belén; Oliveira, Ana Celeste; Garrido-Gómez, Juan; Campos, Antonio; Sánchez-Quevedo, María Del Carmen; Alaminos, Miguel

2012-01-01

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas

DTIC Science & Technology

2015-07-01

AWARD NUMBER: W81XWH-14-1-0115 TITLE: Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas PRINCIPAL INVESTIGATOR: Kyuson Yun...YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE July 2015 2. REPORT TYPE Annual 3. DATES COVERED 1 Jul 2014 - 30 Jun 2015 4. TITLE AND SUBTITLE Cell ...of Origin and Cancer Stem Cell Phenotype in Medulloblastomas 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0115 5c. PROGRAM ELEMENT NUMBER 6

Total analysis systems with Thermochromic Etching Discs technology.

PubMed

Avella-Oliver, Miquel; Morais, Sergi; Carrascosa, Javier; Puchades, Rosa; Maquieira, Ángel

2014-12-16

A new analytical system based on Thermochromic Etching Discs (TED) technology is presented. TED comprises a number of attractive features such as track independency, selective irradiation, a high power laser, and the capability to create useful assay platforms. The analytical versatility of this tool opens up a wide range of possibilities to design new compact disc-based total analysis systems applicable in chemistry and life sciences. In this paper, TED analytical implementation is described and discussed, and their analytical potential is supported by several applications. Microarray immunoassay, immunofiltration assay, solution measurement, and cell culture approaches are herein addressed in order to demonstrate the practical capacity of this system. The analytical usefulness of TED technology is herein demonstrated, describing how to exploit this tool for developing truly integrated analytical systems that provide solutions within the point of care framework.

Interleukin-like EMT inducer regulates partial phenotype switching in MITF-low melanoma cell lines

PubMed Central

Noguchi, Ken; Dalton, Annamarie C.; Howley, Breege V.; McCall, Buckley J.; Yoshida, Akihiro; Diehl, J. Alan

2017-01-01

ILEI (FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell biological process that confers metastatic properties to a tumor cell. Initially, we found that ILEI mRNA is highly expressed in melanoma metastases but not in primary tumors, suggesting that ILEI contributes to the malignant properties of melanoma. While melanoma is not an epithelial cell-derived tumor and does not undergo a traditional EMT, melanoma undergoes a similar process known as phenotype switching in which high (micropthalmia-related transcription factor) MITF expressing (MITF-high) proliferative cells switch to a low expressing (MITF-low) invasive state. We observed that MITF-high proliferative cells express low levels of ILEI (ILEI-low) and MITF-low invasive cells express high levels of ILEI (ILEI-high). We found that inducing phenotype switching towards the MITF-low invasive state increases ILEI mRNA expression, whereas phenotype switching towards the MITF-high proliferative state decreases ILEI mRNA expression. Next, we used in vitro assays to show that knockdown of ILEI attenuates invasive potential but not MITF expression or chemoresistance. Finally, we used gene expression analysis to show that ILEI regulates several genes involved in the MITF-low invasive phenotype including JARID1B, HIF-2α, and BDNF. Gene set enrichment analysis suggested that ILEI-regulated genes are enriched for JUN signaling, a known regulator of the MITF-low invasive phenotype. In conclusion, we demonstrate that phenotype switching regulates ILEI expression, and that ILEI regulates partial phenotype switching in MITF-low melanoma cell lines. PMID:28545079

Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc.

PubMed

Bhunia, Bibhas K; Kaplan, David L; Mandal, Biman B

2018-01-16

Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ∼2.2-fold and ∼1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ∼11-, ∼5.1-, and ∼6.7-fold increases in col I α 1 , sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.

Phenotype and function of CD209+ bovine blood dendritic cells, monocyte-derived-dendritic cells and monocyte-derived macrophages

USDA-ARS?s Scientific Manuscript database

Phylogenic comparisons of the mononuclear phagocyte system (MPS) of humans and mice demonstrate phenotypic divergence of dendritic cell (DC) subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny a...

Ultrastructural changes in the receptor parts of retinal rods in experimental alloxan-induced diabetes in rabbits.

PubMed

Zarebska, A; Łańcut, M; Bakiera, K; Matejko, E; Czerny, K; Kiś, G; Wójtowicz, Z

2001-01-01

Mature rabbits were administered a single dose of alloxan at the dose 100 mg/kg b.m. After 3 and 6 weeks and after 3 and 6 months, the samples of the retina were taken from the areas immediate to the papilla of the optic nerve. Ultrathin sections were dyed according to the Reynold's method, and the receptive parts of the rods were examined under electron microscope BS-500 Tesla. After 6 weeks following alloxan administration, distinct morphological changes in the form of enlargement of certain discs in the receptive parts of rod cells were observed. After 3 months the majority of the discs was damaged, and after 6 months only single, quite well preserved rod cells were found to be present in the retina.

Phenotypic states become increasingly sensitive to perturbations near a bifurcation in a synthetic gene network.

PubMed

Axelrod, Kevin; Sanchez, Alvaro; Gore, Jeff

2015-08-24

Microorganisms often exhibit a history-dependent phenotypic response after exposure to a stimulus which can be imperative for proper function. However, cells frequently experience unexpected environmental perturbations that might induce phenotypic switching. How cells maintain phenotypic states in the face of environmental fluctuations remains an open question. Here, we use environmental perturbations to characterize the resilience of phenotypic states in a synthetic gene network near a critical transition. We find that far from the critical transition an environmental perturbation may induce little to no phenotypic switching, whereas close to the critical transition the same perturbation can cause many cells to switch phenotypic states. This loss of resilience was observed for perturbations that interact directly with the gene circuit as well as for a variety of generic perturbations-such as salt, ethanol, or temperature shocks-that alter the state of the cell more broadly. We obtain qualitatively similar findings in natural gene circuits, such as the yeast GAL network. Our findings illustrate how phenotypic memory can become destabilized by environmental variability near a critical transition.

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

PubMed

Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

2015-12-01

The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Interaction of micron and nano-sized particles with cells of the dura mater.

PubMed

Papageorgiou, Iraklis; Marsh, Rainy; Tipper, Joanne L; Hall, Richard M; Fisher, John; Ingham, Eileen

2014-10-01

Intervertebral total disc replacements (TDR) are used in the treatment of degenerative spinal disc disease. There are, however, concerns that they may be subject to long-term failure due to wear. The adverse effects of TDR wear have the potential to manifest in the dura mater and surrounding tissues. The aim of this study was to investigate the physiological structure of the dura mater, isolate the resident dural epithelial and stromal cells and analyse the capacity of these cells to internalise model polymer particles. The porcine dura mater was a collagen-rich structure encompassing regularly arranged fibroblastic cells within an outermost epithelial cell layer. The isolated dural epithelial cells had endothelial cell characteristics (positive for von Willebrand factor, CD31, E-cadherin and desmoplakin) and barrier functionality whereas the fibroblastic cells were positive for collagen I and III, tenascin and actin. The capacity of the dural cells to take up model particles was dependent on particle size. Nanometer sized particles readily penetrated both types of cells. However, dural fibroblasts engulfed micron-sized particles at a much higher rate than dural epithelial cells. The study suggested that dural epithelial cells may offer some barrier to the penetration of micron-sized particles but not nanometer sized particles. © 2014 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc.

Ganglion cell distribution and retinal resolution in the Florida manatee, Trichechus manatus latirostris.

PubMed

Mass, Alla M; Ketten, Darlene R; Odell, Daniel K; Supin, Alexander Ya

2012-01-01

The topographic organization of retinal ganglion cells was examined in the Florida manatee (Trichechus manatus latirostris) to assess ganglion cell size and distribution and to estimate retinal resolution. The ganglion cell layer of the manatee's retina was comprised primarily of large neurons with broad intercellular spaces. Cell sizes varied from 10 to 60 μm in diameter (mean 24.3 μm). The retinal wholemounts from adult animals measured 446-501 mm(2) in area with total ganglion cell counts of 62,000-81,800 (mean 70,200). The cell density changed across the retina, with the maximum in the area below the optic disc and decreasing toward the retinal edges and in the immediate vicinity of the optic disc. The maximum cell density ranged from 235 to 337 cells per millimeter square in the adult retinae. Two wholemounts obtained from juvenile animals were 271 and 282 mm(2) in area with total cell numbers of 70,900 and 68,700, respectively (mean 69,800), that is, nearly equivalent to those of adults, but juvenile retinae consequently had maximum cell densities that were higher than those of adults: 478 and 491 cells per millimeter square. Calculations indicate a retinal resolution of ∼19' (1.6 cycles per degree) in both adult and juvenile retinae. Copyright © 2011 Wiley Periodicals, Inc.

Hoxb4 overexpression in CD4 memory phenotype T cells increases the central memory population upon homeostatic proliferation.

PubMed

Frison, Héloïse; Giono, Gloria; Thébault, Paméla; Fournier, Marilaine; Labrecque, Nathalie; Bijl, Janet J

2013-01-01

Memory T cell populations allow a rapid immune response to pathogens that have been previously encountered and thus form the basis of success in vaccinations. However, the molecular pathways underlying the development and maintenance of these cells are only starting to be unveiled. Memory T cells have the capacity to self renew as do hematopoietic stem cells, and overlapping gene expression profiles suggested that these cells might use the same self-renewal pathways. The transcription factor Hoxb4 has been shown to promote self-renewal divisions of hematopoietic stem cells resulting in an expansion of these cells. In this study we investigated whether overexpression of Hoxb4 could provide an advantage to CD4 memory phenotype T cells in engrafting the niche of T cell deficient mice following adoptive transfer. Competitive transplantation experiments demonstrated that CD4 memory phenotype T cells derived from mice transgenic for Hoxb4 contributed overall less to the repopulation of the lymphoid organs than wild type CD4 memory phenotype T cells after two months. These proportions were relatively maintained following serial transplantation in secondary and tertiary mice. Interestingly, a significantly higher percentage of the Hoxb4 CD4 memory phenotype T cell population expressed the CD62L and Ly6C surface markers, characteristic for central memory T cells, after homeostatic proliferation. Thus Hoxb4 favours the maintenance and increase of the CD4 central memory phenotype T cell population. These cells are more stem cell like and might eventually lead to an advantage of Hoxb4 T cells after subjecting the cells to additional rounds of proliferation.

Protein SUMOylation is Involved in Cell-cycle Progression and Cell Morphology in Giardia lamblia.

PubMed

Di Genova, Bruno M; da Silva, Richard C; da Cunha, Júlia P C; Gargantini, Pablo R; Mortara, Renato A; Tonelli, Renata R

2017-07-01

The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α-tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

Functional involvement of human discs large tumor suppressor in cytokinesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Unno, Kenji; Hanada, Toshihiko; Chishti, Athar H.

2008-10-15

Cytokinesis is the final step of cell division that completes the separation of two daughter cells. We found that the human discs large (hDlg) tumor suppressor homologue is functionally involved in cytokinesis. The guanylate kinase (GUK) domain of hDlg mediates the localization of hDlg to the midbody during cytokinesis, and over-expression of the GUK domain in U2OS and HeLa cells impaired cytokinesis. Mouse embryonic fibroblasts (MEFs) derived from dlg mutant mice contained an increased number of multinucleated cells and showed reduced proliferation in culture. A kinesin-like motor protein, GAKIN, which binds directly to the GUK domain of hDlg, exhibited amore » similar intracellular distribution pattern with hDlg throughout mitosis and localized to the midbody during cytokinesis. However, the targeting of hDlg and GAKIN to the midbody appeared to be independent of each other. The midbody localization of GAKIN required its functional kinesin-motor domain. Treatment of cells with the siRNA specific for hDlg and GAKIN caused formation of multinucleated cells and delayed cytokinesis. Together, these results suggest that hDlg and GAKIN play functional roles in the maintenance of midbody architecture during cytokinesis.« less

Platform for combined analysis of functional and biomolecular phenotypes of the same cell.

PubMed

Kelbauskas, L; Ashili, S; Zeng, J; Rezaie, A; Lee, K; Derkach, D; Ueberroth, B; Gao, W; Paulson, T; Wang, H; Tian, Y; Smith, D; Reid, B; Meldrum, Deirdre R

2017-03-16

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression.

Patterns of magnetic resonance imaging abnormalities in symptomatic patients with Krabbe disease correspond to phenotype.

PubMed

Abdelhalim, Ahmed N; Alberico, Ronald A; Barczykowski, Amy L; Duffner, Patricia K

2014-02-01

Initial magnetic resonance imaging studies of individuals with Krabbe disease were analyzed to determine whether the pattern of abnormalities corresponded to the phenotype. This was a retrospective, nonblinded study. Families/patients diagnosed with Krabbe disease submitted medical records and magnetic resonance imaging discs for central review. Institutional review board approval/informed consents were obtained. Sixty-four magnetic resonance imaging scans were reviewed by two neuroradiologists and a child neurologist according to phenotype: early infantile (onset 0-6 months) = 39 patients; late infantile (onset 7-12 months) = 10 patients; later onset (onset 13 months-10 years) = 11 patients; adolescent (onset 11-20 years) = one patient; and adult (21 years or greater) = three patients. Local interpretations were compared with central review. Magnetic resonance imaging abnormalities differed among phenotypes. Early infantile patients had a predominance of increased intensity in the dentate/cerebellar white matter as well as changes in the deep cerebral white matter. Later onset patients did not demonstrate involvement in the dentate/cerebellar white matter but had extensive involvement of the deep cerebral white matter, parieto-occipital region, and posterior corpus callosum. Late infantile patients exhibited a mixed pattern; 40% had dentate/cerebellar white matter involvement while all had involvement of the deep cerebral white matter. Adolescent/adult patients demonstrated isolated corticospinal tract involvement. Local and central reviews primarily differed in interpretation of the early infantile phenotype. Analysis of magnetic resonance imaging in a large cohort of symptomatic patients with Krabbe disease demonstrated imaging abnormalities correspond to specific phenotypes. Knowledge of these patterns along with typical clinical signs/symptoms should promote earlier diagnosis and facilitate treatment. Copyright © 2014 Elsevier Inc. All rights reserved.

Bar represses dPax2 and decapentaplegic to regulate cell fate and morphogenetic cell death in Drosophila eye.

PubMed

Kang, Jongkyun; Yeom, Eunbyul; Lim, Janghoo; Choi, Kwang-Wook

2014-01-01

The coordinated regulation of cell fate and cell survival is crucial for normal pattern formation in developing organisms. In Drosophila compound eye development, crystalline arrays of hexagonal ommatidia are established by precise assembly of diverse cell types, including the photoreceptor cells, cone cells and interommatidial (IOM) pigment cells. The molecular basis for controlling the number of cone and IOM pigment cells during ommatidial pattern formation is not well understood. Here we present evidence that BarH1 and BarH2 homeobox genes are essential for eye patterning by inhibiting excess cone cell differentiation and promoting programmed death of IOM cells. Specifically, we show that loss of Bar from the undifferentiated retinal precursor cells leads to ectopic expression of Prospero and dPax2, two transcription factors essential for cone cell specification, resulting in excess cone cell differentiation. We also show that loss of Bar causes ectopic expression of the TGFβ homolog Decapentaplegic (Dpp) posterior to the morphogenetic furrow in the larval eye imaginal disc. The ectopic Dpp expression is not responsible for the formation of excess cone cells in Bar loss-of-function mutant eyes. Instead, it causes reduction in IOM cell death in the pupal stage by antagonizing the function of pro-apoptotic gene reaper. Taken together, this study suggests a novel regulatory mechanism in the control of developmental cell death in which the repression of Dpp by Bar in larval eye disc is essential for IOM cell death in pupal retina.

Competition among gene regulatory networks imposes order within the eye-antennal disc of Drosophila

PubMed Central

Weasner, Bonnie M.; Kumar, Justin P.

2013-01-01

The eye-antennal disc of Drosophila gives rise to numerous adult tissues, including the compound eyes, ocelli, antennae, maxillary palps and surrounding head capsule. The fate of each tissue is governed by the activity of unique gene regulatory networks (GRNs). The fate of the eye, for example, is controlled by a set of fourteen interlocking genes called the retinal determination (RD) network. Mutations within network members lead to replacement of the eyes with head capsule. Several studies have suggested that in these instances all retinal progenitor and precursor cells are eliminated via apoptosis and as a result the surrounding head capsule proliferates to compensate for retinal tissue loss. This model implies that the sole responsibility of the RD network is to promote the fate of the eye. We have re-analyzed eyes absent mutant discs and propose an alternative model. Our data suggests that in addition to promoting an eye fate the RD network simultaneously functions to actively repress GRNs that are responsible for directing antennal and head capsule fates. Compromising the RD network leads to the inappropriate expression of several head capsule selector genes such as cut, Lim1 and wingless. Instead of undergoing apoptosis, a population of mutant retinal progenitors and precursor cells adopt a head capsule fate. This transformation is accompanied by an adjustment of cell proliferation rates such that just enough head capsule is generated to produce an intact adult head. We propose that GRNs simultaneously promote primary fates, inhibit alternative fates and establish cell proliferation states. PMID:23222441

Evaluation of Enterococcus faecalis clinical isolates with 'penicillin-resistant, ampicillin-susceptible' phenotype as reported by Vitek-2 Compact system.

PubMed

Tan, Yen Ee; Ng, Lily S Y; Tan, Thean Yen

2014-10-01

It has been recently reported that ampicillin susceptibility cannot accurately predict piperacillin and imipenem susceptibilities in penicillin-resistant, ampicillin-susceptible (Pen-R, Amp-S) Enterococcus faecalis isolates, contrary to the current Clinical and Laboratory Standards Institute (CLSI) recommendations. This has important therapeutic implications. Such isolates were noted after the use of Vitek-2 Compact system AST-GP67 susceptibility cards in a Singapore general hospital and they were increasing in numbers. The primary aim of this study was to evaluate these clinical isolates against microbroth dilution (MBD) technique and other commonly used antimicrobial susceptibility test (AST) methods for penicillin and ampicillin. The secondary aim was to evaluate whether ampicillin susceptibility could indeed be a reliable surrogate marker for piperacillin and imipenem susceptibilities in E. faecalis isolates that were confirmed Pen-R, Amp-S.From 2009 to 2013, a total of 49 isolates (5%) of 983 non-duplicate E. faecalis tested by Vitek-2 displayed the 'Pen-R, Amp-S' phenotype in a general hospital in Singapore. These were tested against MBD which was the reference method, Etest and disc diffusion for penicillin and ampicillin. Susceptibilities to piperacillin and imipenem were also tested using MBD. In addition, β-lactamase production test was performed. Forty E. faecalis isolates with penicillin-susceptible, ampicillin-susceptible (Pen-S, Amp-S) phenotype were included for comparative purposes.The categorical agreement rate was 100% for all AST methods in ampicillin reporting for the 'Pen-R, Amp-S' group of E. faecalis isolates. However, a large number of isolates (46 isolates, 93.9%) fell into the major error category for penicillin testing by the Vitek-2 system. Penicillin minimum inhibitory concentrations (MICs) generated by the Vitek-2 system for the majority of these isolates were two doubling dilutions higher compared to those obtained by the reference test. The Etest method correlated well with the MBD method. Thirty-two isolates (65.3%) were in categorical agreement with the MBD method when tested by the disc diffusion method for penicillin. Only three E. faecalis isolates (6.1%) were confirmed to have the uncommon penicillin resistance phenotype, with two of them showing resistance to piperacillin and intermediate to imipenem. β-lactamase production test was negative for all isolates. Among the Pen-S, Amp-S E. faecalis isolates, the categorical agreement was 100% for penicillin and ampicillin in all the tested methods.Enterococcus faecalis with 'Pen-R, Amp-S' phenotype reported by the Vitek-2 system using AST-GP67 susceptibility cards must be confirmed with a reference test, the Etest method being a good alternative. The Vitek-2 system generated higher penicillin MIC readings compared to MBD in this study. The actual prevalence of this uncommon penicillin resistance phenotype in E. faecalis was found to be low in this institution. More studies are required to confirm the reliability of ampicillin as a surrogate marker for piperacillin and imipenem susceptibilities in these isolates.

Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution

PubMed Central

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D.; Rainey, Paul B.; de Visser, J. Arjan G. M.; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology. PMID:27077662

Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution.

PubMed

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D; Rainey, Paul B; de Visser, J Arjan G M; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes-via growth-over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

Lasp1 misexpression influences chondrocyte differentiation in the vertebral column.

PubMed

Hermann-Kleiter, Natascha; Ghaffari-Tabrizi, Nassim; Blumer, Michael J F; Schwarzer, Christoph; Mazur, Magdalena A; Artner, Isabella

2009-01-01

The mouse mutant wavy tail Tg(Col1a1-lacZ)304ng was created through transgene insertion and exhibits defects of the vertebral column. Homozygous mutant animals have compressed tail vertebrae and wedge-shaped intervertebral discs, resulting in a meandering tail. Delayed closure of lumbar neural arches and lack of processus spinosi have been observed; these defects become most prominent during the transition from cartilage to bone. The spina bifida was resistant to folic acid treatment, while retinoic acid administration caused severe skeletal defects in the mutant, but none in wild type control animals. The transgene integrated at chromosome 11 band D, in an area of high gene density. The insertion site was located between the transcription start sites of the Rpl23 and Lasp1 genes. LASP1 (an actin binding protein involved in cell migration and survival) was found to be produced in resting and hypertrophic chondrocytes in the vertebrae. In mutant vertebrae, temporal and spatial misexpression of Lasp1 was observed, indicating that alterations in Lasp1 transcription are most likely responsible for the observed phenotype. These data reveal a yet unappreciated role of Lasp1 in chondrocyte differentiation during cartilage to bone transition.

Implications of the Hybrid Epithelial/Mesenchymal Phenotype in Metastasis

PubMed Central

Jolly, Mohit Kumar; Boareto, Marcelo; Huang, Bin; Jia, Dongya; Lu, Mingyang; Ben-Jacob, Eshel; Onuchic, José N.; Levine, Herbert

2015-01-01

Transitions between epithelial and mesenchymal phenotypes – the epithelial to ­mesenchymal transition (EMT) and its reverse the mesenchymal to epithelial transition (MET) – are hallmarks of cancer metastasis. While transitioning between the epithelial and mesenchymal phenotypes, cells can also attain a hybrid epithelial/mesenchymal (E/M) (i.e., partial or intermediate EMT) phenotype. Cells in this phenotype have mixed epithelial (e.g., adhesion) and mesenchymal (e.g., migration) properties, thereby allowing them to move collectively as clusters. If these clusters reach the bloodstream intact, they can give rise to clusters of circulating tumor cells (CTCs), as have often been seen experimentally. Here, we review the operating principles of the core regulatory network for EMT/MET that acts as a “three-way” switch giving rise to three distinct phenotypes – E, M and hybrid E/M – and present a theoretical framework that can elucidate the role of many other players in regulating epithelial plasticity. Furthermore, we highlight recent studies on partial EMT and its association with drug resistance and tumor-initiating potential; and discuss how cell–cell communication between cells in a partial EMT phenotype can enable the formation of clusters of CTCs. These clusters can be more apoptosis-resistant and have more tumor-initiating potential than singly moving CTCs with a wholly mesenchymal (complete EMT) phenotype. Also, more such clusters can be formed under inflammatory conditions that are often generated by various therapies. Finally, we discuss the multiple advantages that the partial EMT or hybrid E/M phenotype have as compared to a complete EMT phenotype and argue that these collectively migrating cells are the primary “bad actors” of metastasis. PMID:26258068

Extracellular matrix components and culture regimen selectively regulate cartilage formation by self-assembling human mesenchymal stem cells in vitro and in vivo.

PubMed

Ng, Johnathan; Wei, Yiyong; Zhou, Bin; Burapachaisri, Aonnicha; Guo, Edward; Vunjak-Novakovic, Gordana

2016-12-09

Cartilage formation from self-assembling mesenchymal stem cells (MSCs) in vitro recapitulate important cellular events during mesenchymal condensation that precedes native cartilage development. The goal of this study was to investigate the effects of cartilaginous extracellular matrix (ECM) components and culture regimen on cartilage formation by self-assembling human MSCs in vitro and in vivo. Human bone marrow-derived MSCs (hMSCs) were seeded and compacted in 6.5-mm-diameter transwell inserts with coated (type I, type II collagen) or uncoated (vehicle) membranes, at different densities (0.5 × 10 6 , 1.0 × 10 6 , 1.5 × 10 6 per insert). Pellets were formed by aggregating hMSCs (0.25 × 10 6 ) in round-bottomed wells. All tissues were cultured for up to 6 weeks for in vitro analyses. Discs (cultured for 6, 8 or 10 weeks) and pellets (cultured for 10 weeks) were implanted subcutaneously in immunocompromised mice to evaluate the cartilage stability in vivo. Type I and type II collagen coatings enabled cartilage disc formation from self-assembling hMSCs. Without ECM coating, hMSCs formed dome-shaped tissues resembling the pellets. Type I collagen, expressed in the prechondrogenic mesenchyme, improved early chondrogenesis versus type II collagen. High seeding density improved cartilage tissue properties but resulted in a lower yield of disc formation. Discs and pellets exhibited compositional and organizational differences in vitro and in vivo. Prolonged chondrogenic induction of the discs in vitro expedited endochondral ossification in vivo. The outcomes of cartilage tissues formed from self-assembling MSCs in vitro and in vivo can be modulated by the control of culture parameters. These insights could motivate new directions for engineering cartilage and bone via a cartilage template from self-assembling MSCs.

Genetic Variants Associated with Optic Nerve Vertical Cup-to-Disc Ratio Are Risk Factors for Primary Open Angle Glaucoma in a US Caucasian Population

PubMed Central

Fan, Bao Jian; Wang, Dan Yi; Pasquale, Louis R.; Haines, Jonathan L.

2011-01-01

Purpose. Genetically complex disorders, such as primary open angle glaucoma (POAG), may include highly heritable quantitative traits as part of the overall phenotype, and mapping genes influencing the related quantitative traits may effectively identify genetic risk factors predisposing to the complex disease. Recent studies have identified SNPs associated with optic nerve area and vertical cup-to-disc ratio (VCDR). The purpose of this study was to evaluate the association between these SNPs and POAG in a US Caucasian case-control sample. Methods. Five SNPs previously associated with optic disc area, or VCDR, were genotyped in 539 POAG cases and 336 controls. Genotype data were analyzed for single SNP associations and SNP interactions with VCDR and POAG. Results. SNPs associated with VCDR rs1063192 (CDKN2B) and rs10483727 (SIX1/SIX6) were also associated with POAG (P = 0.0006 and P = 0.0043 for rs1063192 and rs10483727, respectively). rs1063192, associated with smaller VCDR, had a protective effect (odds ratio [OR] = 0.73; 95% confidence interval [CI], 0.58–0.90), whereas rs10483727, associated with larger VCDR, increased POAG risk (OR = 1.33; 95% CI, 1.08–1.65). POAG risk associated with increased VCDR was significantly influenced by the C allele of rs1900004 (ATOH7), associated with increased optic nerve area (P-interaction = 0.025; OR = 1.89; 95% CI, 1.22–2.94). Conclusions. Genetic variants influencing VCDR are associated with POAG in a US Caucasian population. Variants associated with optic nerve area are not independently associated with disease but can influence the effects of VCDR variants suggesting that increased optic disc area can significantly contribute to POAG risk when coupled with risk factors controlling VCDR. PMID:21398277

Biofilm Formation and β-Lactamase Production in Burn Isolates of Pseudomonas aeruginosa.

PubMed

Heydari, Samira; Eftekhar, Fereshteh

2015-03-01

Pseudomonas aeruginosa is an important nosocomial pathogen characterized by its innate resistance to multiple antimicrobial agents. Plasmid-mediated drug resistance also occurs by the production of extended-spectrum β-lactamases (ESBL), metallo β-lactamases (MBL), and AmpC β-lactamases. Another important factor for establishment of chronic infections by P. aeruginosa is biofilm formation mediated by the psl gene cluster. The aim of this study was to evaluate biofilm formation and presence of the pslA gene in burn isolates of P. aeruginosa as well as the association of antibiotic resistance, MBL, ESBL and AmpC β-lactamase production with biofilm formation among the isolates. Sixty-two burn isolates of P. aeruginosa were obtained from Shahid Motahari Hospital in Tehran from August to October 2011. Antibiotic susceptibility was determined by the disc diffusion assay. MBL, AmpC and ESBL production were screened using the double disc synergy test, AmpC disc test and combined disc diffusion assay, respectively. The potential to form biofilm was measured using the microtiter plate assay and pslA gene was detected using specific primers and PCR. Biofilm formation was observed in 43.5% of the isolates, of which 66.7% produced strong and 33.3% formed weak biofilms. All biofilm-positive and 14.2% of biofilm-negative isolates harbored the pslA gene. MBL, AmpC and ESBL production were significantly higher in the biofilm-positive isolates (70.3%, 62.9% and 33.3%, respectively) compared to the biofilm-negative strains (31.4%, 34.2% and 20%, respectively). Overall, 19 isolates (30.6%) co-produced MBL and AmpC, among which the majority were biofilm-positive (63.1%). Finally, four isolates (6.4%) had all three enzymes, of which 3 (75%) produced biofilm. Biofilm formation (both strong and weak) strongly correlated with pslA gene carriage. Biofilm formation also correlated with MBL and AmpC β-lactamase production. More importantly, multiple-β-lactamase phenotype was associated with formation of strong biofilms.

Coriandrum sativum Suppresses Aβ42-Induced ROS Increases, Glial Cell Proliferation, and ERK Activation.

PubMed

Liu, Quan Feng; Jeong, Haemin; Lee, Jang Ho; Hong, Yoon Ki; Oh, Youngje; Kim, Young-Mi; Suh, Yoon Seok; Bang, Semin; Yun, Hye Sup; Lee, Kyungho; Cho, Sung Man; Lee, Sung Bae; Jeon, Songhee; Chin, Young-Won; Koo, Byung-Soo; Cho, Kyoung Sang

2016-01-01

Alzheimer's disease (AD), the most common neurodegenerative disease, has a complex and widespread pathology that is characterized by the accumulation of amyloid [Formula: see text]-peptide (A[Formula: see text]) in the brain and various cellular abnormalities, including increased oxidative damage, an amplified inflammatory response, and altered mitogen-activated protein kinase signaling. Based on the complex etiology of AD, traditional medicinal plants with multiple effective components are alternative treatments for patients with AD. In the present study, we investigated the neuroprotective effects of an ethanol extract of Coriandrum sativum (C. sativum) leaves on A[Formula: see text] cytotoxicity and examined the molecular mechanisms underlying the beneficial effects. Although recent studies have shown the benefits of the inhalation of C. sativum oil in an animal model of AD, the detailed molecular mechanisms by which C. sativum exerts its neuroprotective effects are unclear. Here, we found that treatment with C. sativum extract increased the survival of both A[Formula: see text]-treated mammalian cells and [Formula: see text]42-expressing flies. Moreover, C. sativum extract intake suppressed [Formula: see text]-induced cell death in the larval imaginal disc and brain without affecting A[Formula: see text]42 expression and accumulation. Interestingly, the increases in reactive oxygen species levels and glial cell number in AD model flies were reduced by C. sativum extract intake. Additionally, C. sativum extract inhibited the epidermal growth factor receptor- and A[Formula: see text]-induced phosphorylation of extracellular signal-regulated kinase (ERK). The constitutively active form of ERK abolished the protective function of C. sativum extract against the [Formula: see text]-induced eye defect phenotype in Drosophila. Taken together, these results suggest that C. sativum leaves have antioxidant, anti-inflammatory, and ERK signaling inhibitory properties that are beneficial for patients with AD.

Automated assembly of microfluidic "lab-on-a-disc"

NASA Astrophysics Data System (ADS)

Berger, M.; Müller, T.; Voebel, T.; Baum, C.; Glennon, T.; Mishra, R.; Kinahan, D.; King, D.; Ducrée, J.; Brecher, C.

2018-02-01

Point-of-care (POC) testing attracts more and more attention in the medical health sector because of their specific property to perform the diagnostic close to the patient. The fast diagnosis right at the hospital or the doctor's office improves the medical reaction time and the chances for a successful healing process. One of this POC test systems is a "Lab-on-a-Disc" (LoaD) which looks like a compact disc crisscrossed with microfluidic tubes and cavities. The fluid to be analysed is placed in the LoaD and an external device then rotates the LoaD. The cavities inside the LoaD and the centrifugal force ensure a clearly defined sequence of the analysis. Furthermore, we aim for an inexpensive manufacture of the medical product without neglecting its quality and functionality. Therefore, the Fraunhofer IPT works on an assembly cell to implement dissoluble films concisely into the disc. This dissoluble film demonstrates its successful usage as a gate for the fluid, which opens after a predefined moment in the cycle. Furthermore, we investigate to integrate a laser welding process into our gantry system and demonstrate its efficiency with the welding of polymer discs. This procedure is clinically safe because no further laser absorption material is needed in the sealing process, which might pollute the LoaD. Moreover, this process allows the alignment of several discs before the welding and therefore leads to precisely manufactured LoaDs in large quantities. All these methods together enable a fast, costefficient and reliable mass production to bring POC testing among the people.

Prevalence of ESBL-producing Pseudomonas aeruginosa isolates in Warsaw, Poland, detected by various phenotypic and genotypic methods

PubMed Central

Róg, Patrycja; Smolińska-Król, Katarzyna; Ćmiel, Milena; Słoczyńska, Alicja; Patzer, Jan; Dzierżanowska, Danuta; Wolinowska, Renata; Starościak, Bohdan; Tyski, Stefan

2017-01-01

Knowledge of the prevalence of ESBL enzymes among P. aeruginosa strains compared to the Enterobacteraiceae family is limited. The phenotypic tests recommended by EUCAST for the detection of ESBL-producing Enterobacteriaceae are not always suited for P. aeruginosa strains. This is mainly due to the presence of other families of ESBLs in P. aeruginosa isolates more often than in Enterobacteriaceae, production of natural AmpC cephalosporinase and its overexpression, and co-production of metallo-β-lactamases. The aim of this study was to determine the occurrence of ESBLs in P. aeruginosa isolated from patients from hospitals in Warsaw, to evaluate the ESBL production of these isolates using currently available phenotypic tests, their modifications, multiplex PCR and molecular typing of ESBL-positive isolates by PFGE. Clinical isolates of P. aeruginosa were collected in 2000-2014 from four Warsaw hospitals. Based on the data obtained in this study, we suggest using three DDST methods with inhibitors, such as clavulanic acid, sulbactam and imipenem, to detect ESBL-producing P. aeruginosa strains. Depending on the appearance of the plates, we suggest a reduction in the distance between discs with antibiotics to 15 mm and the addition of boronic acid at 0.4 mg per disc. The analysed isolates carried genes encoding ESBL from the families VEB (69 isolates with VEB-9), GES (6 with GES-1, 1 GES-5, 5 GES-13 and 2 with GES-15), OXA-2 (12 with OXA-15, 1 OXA-141, 1 OXA-210, 1 OXA-543 and 1 with OXA-544) and OXA-10 (5 isolates with OXA-74 and one with OXA-142). The most important result of this study was the discovery of three new genes, blaGES-15, blaOXA-141 and blaOXA-142; their nucleotide sequences have been submitted to the NCBI GenBank. It is also very important to note that this is the first report on the epidemiological problem of VEB-9-producing bacterial strains, not only in Poland but also worldwide. PMID:28658322

Preparation of adult Drosophila eyes for thin sectioning and microscopic analysis.

PubMed

Jenny, Andreas

2011-08-27

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes. The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g.). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g.). The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined.

The NHERF1 PDZ2 Domain Regulates PKA–RhoA–p38-mediated NHE1 Activation and Invasion in Breast Tumor Cells

PubMed Central

Cardone, Rosa A.; Bellizzi, Antonia; Busco, Giovanni; Weinman, Edward J.; Dell'Aquila, Maria E.; Casavola, Valeria; Azzariti, Amalia; Mangia, Anita; Paradiso, Angelo

2007-01-01

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na+/H+ exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1α expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na+/H+ exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling. PMID:17332506

Synthesis, characterization and cell behavior of fluoridated hydroxyapatite

NASA Astrophysics Data System (ADS)

Qu, Haibo

Fluorine-containing hydroxyapatite (Ca5(PO4) 3(OH)1-xFx FHA), where F- partially replaces OH- in hydroxyapatite (HA), is recognized as a possible biomaterial for bone and tooth implants and gaining attention in the last several years as a possible alternative to HA. In this study, FHA powders were synthesized through a pH-cycling method. It was discovered that fluorine incorporation increased with the fluorine content in the initial solution and the number of pH cycles employed. A relatively low fluorine incorporation efficiency, ˜60%, was attained for most of the FHA samples. The short time of stay at each pH cycle and the limited number of cycles used are believed to be the main reasons of the low fluorine incorporation into the apatite structure. It was also revealed that the FHA particles produced by the pH-cycling method were inhomogeneous. They were a mixture of hydroxyapatite and F-rich apatite (or FA) particles. The mechanisms of incorporation of fluorine ions into hydroxyapatite by a pH cyclicing method were studied using TEM, XRD and fluorine measurement. Instead of forming laminated structures as reported by other research groups, a mixture of nano-sized F-rich apatite (FHA) and hydroxyapatite (HA) particles were obtained using the pH-cyclicing method. After calcination, these FHA particles were homogenized and became single phased FHA. The effect of fluorine content, preparing method, and sintering temperature on both the bulk density and biaxial flexural strength of sintered FHA was studied. Both uniaxially pressed un-milled (UPU) and cold isostatically pressed milled (IPM) FHA discs were sintered at temperatures between 1200˜400°C at an interval of 100°C. It was found that the fluorine content had a significant impact on the sintering behavior, densification, and mechanical properties of FHA discs. A close correlation between the sintered density and biaxial flexural strength of the specimens was revealed, where the biaxial flexural strength increased exponentially with the sintered density. FHA discs with various fluorine contents have been used to investigate the effect of fluorine content on osteoblastic cell behaviors. Rat osteosarcoma (ROS 17/28) cells were cultured on FHA discs for appropriate times. The osteoblastic cell behaviors were examined in terms of cell attachment, proliferation, morphology and differentiation. The fluorine content in FHA strongly affected the cell activities. More cell attachment and proliferation were observed on the fluorine-containing FHA than pure HA. Fluorine content also affected the differentiation behaviors of osteoblastic cells. Cells on fluorine-containing FHA had higher alkaline phosphatase (ALP) activity than pure HA in 2 weeks. The morphology of the cells showed that it took less time for cells to cover the surface of fluorine-containing samples than that of pure HA. These results suggested that fluorine ions had a significant impact on osteoblastic cell behaviors.

Dysregulated miR-127-5p contributes to type II collagen degradation by targeting matrix metalloproteinase-13 in human intervertebral disc degeneration.

PubMed

Hua, Wen-Bin; Wu, Xing-Huo; Zhang, Yu-Kun; Song, Yu; Tu, Ji; Kang, Liang; Zhao, Kang-Cheng; Li, Shuai; Wang, Kun; Liu, Wei; Shao, Zeng-Wu; Yang, Shu-Hua; Yang, Cao

2017-08-01

Intervertebral disc degeneration (IDD) is a chronic disease associated with the degradation of extracellular matrix (ECM). Matrix metalloproteinase (MMP)-13 is a major enzyme that mediates the degradation of ECM components. MMP-13 has been predicted to be a potential target of miR-127-5p. However, the exact function of miR-127-5p in IDD is still unclear. We designed this study to evaluate the correlation between miR-127-5p level and the degeneration of human intervertebral discs and explore the potential mechanisms. miR-127-5p levels and MMP-13 mRNA levels were detected by quantitative real-time polymerase chain reaction (qPCR). To determine whether MMP-13 is a target of miR-127-5p, dual luciferase reporter assays were performed. miR-127-5p mimic and miR-127-5p inhibitor were used to overexpress or downregulate miR-127-5p expression in human NP cells, respectively. Small interfering RNA (siRNA) was used to knock down MMP-13 expression in human NP cells. Type II collagen expression in human NP cells was detected by qPCR, western blotting, and immunofluorescence staining. We confirmed that miR-127-5p was significantly downregulated in nucleus pulposus (NP) tissue of degenerative discs and its expression was inversely correlated with MMP-13 mRNA levels. We reveal that MMP-13 may act as a target of miR-127-5p. Expression of miR-127-5p was inversely correlated with type II collagen expression in human NP cells. Moreover, suppression of MMP-13 expression by siRNA blocked downstream signaling and increased type II collagen expression. Dysregulated miR-127-5p contributed to the degradation of type II collagen by targeting MMP-13 in human IDD. Our findings highlight that miR-127-5p may serve as a new therapeutic target in IDD. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Nasal lavage, blood or sputum: Which is best for phenotyping asthma?

PubMed

de Farias, Camyla F; Amorim, Maria M F; Dracoulakis, Michel; Caetano, Lilian B; Santoro, Ilka L; Fernandes, Ana L G

2017-05-01

Determination of asthma phenotypes, particularly inflammatory phenotypes, helps guide treatment and management of this heterogeneous disease. Induced sputum cytology has been the gold standard for determination of inflammatory phenotypes, but sputum induction is fairly invasive and technically challenging. Blood and nasal lavage cytology have been suggested as substitutes, but have not been fully verified. The aim of this study is to determine the accuracy of blood and nasal lavage cytometry as indicators of inflammatory phenotypes in asthma. Clinical evaluation, Asthma Control Questionnaire (ACQ) and spirometry were performed for 121 adult asthma patients, and blood, nasal lavage and induced sputum samples were taken. Eosinophils and neutrophils were counted in three samples from each subject. Inflammatory phenotypes (eosinophilic, neutrophilic, mixed and paucicellular) and cells counts were analysed using Venn diagram and receiver operating characteristic (ROC) curve, respectively. ACQ score, spirometry and bronchodilator response did not differ among subjects with different inflammatory phenotypes. Inflammatory phenotypes defined by nasal lavage cytometry were in better concordance than those defined by blood cell counts with phenotypes determined by sputum cytology, and were significantly correlated with sputum phenotypes. For eosinophilia, nasal lavage cytology showed better accuracy than blood cytology (area under the curve (AUC): 0.89 vs 0.65). For all phenotypes, sensitivity and positive and negative predictive power were higher for nasal lavage cytometry than for blood. Blood cell counts gave a high level of false positives for all inflammatory phenotypes. We recommend nasal lavage cytology over blood cell count as a substitute for sputum cytology to identify inflammatory phenotypes in asthma. © 2016 Asian Pacific Society of Respirology.

Tumor necrosis factor-α-dependent infiltration of macrophages into the dorsal root ganglion in a rat disc herniation model.

PubMed

You, Changcheng; Zhu, Kai; Liu, Xiaoqi; Xi, Chunyang; Zhang, Zhipeng; Xu, Gongping; Yan, Jinglong

2013-11-01

A prospective molecular mechanism of macrophages infiltration in experimental disc herniation. To investigate the mechanisms of macrophages infiltration into the dorsal root ganglion (DRG) in a rat model of disc herniation. Macrophages infiltrate the DRG after application of nucleus pulposus (NP) on the DRG, and may play an important role in radiculopathy. However, the mechanisms of macrophages infiltration after NP application remain poorly understood. After experimental disc herniation in this study, we investigated changes in the expression of ED1 (a marker of macrophages) and vascular cell adhesion molecule-1 (VCAM-1) in DRG using immunofluorescence. We also investigated the expression of ED1 and VCAM-1 in DRG by treatment with tumor necrosis factor-α (TNF-α) inhibitor at the time of surgery. We found a massive ED1-positive macrophages infiltrated the DRG, and VCAM-1-like immunoreactivity vessels became evident after NP application. Furthermore, both macrophage infiltration and VCAM-1 expression were prevented by treatment with TNF-α inhibitor at the time of surgery. These findings indicated that macrophages infiltration into the DRG was TNF-α-dependent, and might be partly mediated by VCAM-1 in the early stage of experimental lumbar disc herination. Taken together, this study provides important preliminary data suggesting that TNF-α plays an important role in the macrophage infiltration. N/A.

Photoreceptor disc shedding in the living human eye

PubMed Central

Kocaoglu, Omer P.; Liu, Zhuolin; Zhang, Furu; Kurokawa, Kazuhiro; Jonnal, Ravi S.; Miller, Donald T.

2016-01-01

Cone photoreceptors undergo a daily cycle of renewal and shedding of membranous discs in their outer segments (OS), the portion responsible for light capture. These physiological processes are fundamental to maintaining photoreceptor health, and their dysfunction is associated with numerous retinal diseases. While both processes have been extensively studied in animal models and postmortem eyes, little is known about them in the living eye, in particular human. In this study, we report discovery of the optical signature associated with disc shedding using a method based on adaptive optics optical coherence tomography (AO-OCT) in conjunction with post-processing methods to track and monitor individual cone cells in 4D. The optical signature of disc shedding is characterized by an abrupt transient loss in the cone outer segment tip (COST) reflection followed by its return that is axially displaced anteriorly. Using this signature, we measured the temporal and spatial properties of shedding events in three normal subjects. Average duration of the shedding event was 8.8 ± 13.4 minutes, and average length loss of the OS was 2.1 μm (7.0% of OS length). Prevalence of cone shedding was highest in the morning (14.3%) followed by the afternoon (5.7%) and evening (4.0%), with load distributed across the imaged patch. To the best of our knowledge these are the first images of photoreceptor disc shedding in the living retina. PMID:27895995

Reduced sympathetic innervation after alteration of target cell neurotransmitter phenotype in transgenic mice.

PubMed Central

Cho, S; Son, J H; Park, D H; Aoki, C; Song, X; Smith, G P; Joh, T H

1996-01-01

Neurotransmitters play a variety of important roles during nervous system development. In the present study, we hypothesized that neurotransmitter phenotype of both projecting and target cells is an important factor for the final synaptic linkage and its specificity. To test this hypothesis, we used transgenic techniques to convert serotonin/melatonin-producing cells of the pineal gland into cells that also produce dopamine and investigated the innervation of the phenotypically altered target cells. This phenotypic alteration markedly reduced the noradrenergic innervation originating from the superior cervical ganglia. Although the mechanism by which the reduction occurs is presently unknown, quantitative enzyme-linked immunoassay showed the presence of the equivalent amounts of nerve growth factor (NGF) in the control and transgenic pineal glands, suggesting that it occurred in a NGF-independent manner. The results suggest that target neurotransmitter phenotype influences the formation of afferent connections during development. Images Fig. 3 Fig. 4 PMID:8610132

Platform for combined analysis of functional and biomolecular phenotypes of the same cell

PubMed Central

Kelbauskas, L.; Ashili, S.; Zeng, J.; Rezaie, A.; Lee, K.; Derkach, D.; Ueberroth, B.; Gao, W.; Paulson, T.; Wang, H.; Tian, Y.; Smith, D.; Reid, B.; Meldrum, Deirdre R.

2017-01-01

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression. PMID:28300162

Hepatic Oval Cells Have the Side Population Phenotype Defined by Expression of ATP-Binding Cassette Transporter ABCG2/BCRP1

PubMed Central

Shimano, Koichi; Satake, Makoto; Okaya, Atsuhito; Kitanaka, Junichi; Kitanaka, Nobue; Takemura, Motohiko; Sakagami, Masafumi; Terada, Nobuyuki; Tsujimura, Tohru

2003-01-01

Organ-specific stem cells can be identified by the side population (SP) phenotype, which is defined by the property to effectively exclude the Hoechst 33342 dye. The ATP-binding cassette transporter ABCG2/BCRP1 mediates the SP phenotype. Because hepatic oval cells possess several characteristics of stem cells, we examined whether they have the SP phenotype using the 2-acetylaminofluorene/partial hepatectomy (PH) model. Fluorescence-activated cell sorting analysis showed that a population of non-parenchymal cells containing oval cells, prepared on day 7 after PH, carried a significant number of SP cells, whereas that of non-parenchymal cells without oval cells, prepared on day 0 after PH, did not. Northern blot analysis using total liver RNA obtained on various days after PH showed that the expression of ABCG2/BCRP1 mRNA increased after PH, reaching the highest level on day 7, and then gradually decreased. This pattern of changes in the ABCG2/BCRP1 mRNA level was well correlated to that in the number of oval cells. Furthermore, in situ hybridization revealed that oval cells were the sites of expression of ABCG2/BCRP1 mRNA. These results indicate that oval cells have the SP phenotype defined by expression of ABCG2/BCRP1, suggesting that oval cells may represent stem cells in the liver. PMID:12819005

Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

PubMed Central

Bao, Bin; Wang, Zhiwei; Ali, Shadan; Kong, Dejuan; Banerjee, Sanjeev; Ahmad, Aamir; Li, Yiwei; Azmi, Asfar S.; Miele, Lucio; Sarkar, Fazlul H.

2011-01-01

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of Epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein. PMID:21503965

Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2β1 Signaling

PubMed Central

Murphy, Kaitlin C.; Hoch, Allison I.; Harvestine, Jenna N.; Zhou, Dejie

2016-01-01

The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone-forming potential. MSCs rapidly lose their induced phenotype upon removal of the soluble stimuli; however, their bone-forming potential can be sustained when provided with continued instruction via extracellular matrix (ECM) cues. In comparison with dissociated cells, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining their osteogenic potential. We hypothesized that entrapment of MSC spheroids formed from osteogenically induced cells would exhibit better preservation of their bone-forming potential than would dissociated cells from monolayer culture. Spheroids exhibited comparable osteogenic potential and increased proangiogenic potential with or without osteogenic preconditioning versus monolayer-cultured MSCs. Spheroids were then entrapped in collagen hydrogels, and the osteogenic stimulus was removed. In comparison with entrapped dissociated MSCs, spheroids exhibited significantly increased markers of osteogenic differentiation. The capacity of MSC spheroids to retain their osteogenic phenotype upon withdrawal of inductive cues was mediated by α2β1 integrin binding to cell-secreted ECM. These results demonstrate the capacity of spheroidal culture to sustain the mineral-producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair. Significance Despite the promise of mesenchymal stem cells (MSCs) for cell-based therapies for tissue repair and regeneration, there is little evidence that transplanted MSCs directly contribute to new bone formation, suggesting that induced cells rapidly lose their osteogenic phenotype or undergo apoptosis. In comparison with dissociated cells, MSC spheroids exhibit increased trophic factor secretion and improved cell survival. The loss of phenotype represents a significant clinical challenge for cell therapies, yet there is no evidence for whether MSC spheroids retain their osteogenic phenotype upon entrapment in a clinically relevant biomaterial. These findings demonstrate that MSC spheroids retain their osteogenic phenotype better than do dissociated MSCs, and this is due to integrin engagement with the cell-secreted extracellular matrix. These data provide evidence for a novel approach for potentiating the use of MSCs in bone repair. PMID:27365484

Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R; Weissman, Amir; Binah, Ofer

2016-12-01

Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Phenotypic Description of the Spanish Multicentre Genetic Glaucoma Group Cohort

PubMed Central

Gamundi, Maria José; Duch, Susana; Rios, Jose; Carballo, Miguel; Study Group, EMEIGG

2017-01-01

Introduction The aim of the study was to make a phenotypic description of the Spanish multicentre glaucoma group cohort of patients. Design Retrospective, observational, multicentre, cohort study. Material and Methods The clinical charts of 152 patients with hereditary glaucoma from18 Spanish eye centres were reviewed in order to make an epidemiologic description of the type of glaucoma and associated factors. True hereditary cases were compared with familiar cases according to the Gong et al. criteria. Results 61% were true hereditary cases and 39% familiar cases. Ocular comorbidity, optic disc damage, and visual field mean defect were significantly more severe in hereditary patients, who required significantly more first-line hypotensive drugs and surgical interventions to control intraocular pressure than familiar patients. Conclusions The strength of the hereditary component of glaucoma seems to worsen the clinical course, causing more structural and functional damage and requiring more intense glaucoma treatment. The family history of glaucoma should be carefully investigated and taken into consideration when making treatment decisions or intensifying previous treatment. PMID:29082038

Functional and phenotypic characterization of CD8+CD28+ and CD28- T cells in atopic individuals sensitized to Dermatophagoides pteronyssinus.

PubMed

Lourenço, O; Fonseca, A M; Paiva, A; Arosa, F A; Taborda-Barata, L

2006-01-01

CD8+ T suppressor cells may play a role in immunoregulation. Recent studies have characterized this population by the lack of the CD28 molecule. These CD8+CD28 T cells differ phenotypically and functionally from CD8 + CD28 + T cells. Little is known about CD8 + CD28 cells in atopy. Our aim was to analyze the phenotype and functional properties of CD8 + CD28T cells in atopic and non-atopic individuals. Peripheral blood mononuclear cells (PBMC) were obtained after density gradient centrifugation. CD8 + CD28 and CD8 + CD28 + T cells were isolated using immunomagnetic beads. Relative percentages of these cells and expression of several phenotypic markers were analyzed by flow cytometry. Proliferation was assessed by thymidine incorporation in isolated populations and in co-cultures with PBMC using Dermatophagoides pteronyssinus as stimulus. Cytokine synthesis was evaluated in culture supernatants by cytometric bead array. The relative percentages of CD8+CD28 T cells and their phenotypic expression in atopic and non-atopic volunteers were not significantly different. However, CD8 + CD28 T cells showed greater proliferation than did CD8+CD28+ T cells when stimulated with D. pteronyssinus, although cytokine synthesis patterns were similar. CD8+CD28 co-cultures with PBMC showed greater proliferation than CD8+CD28+ T cell co-cultures, but cytokine synthesis patterns were not different. Our data confirm phenotypic and functional differences between CD28+ and CD28 T cells, irrespective of atopic status. Purified human CD8+CD28 T cells, freshly isolated from peripheral blood, do not have suppressor properties on allergen-specific proliferation or on cytokine synthesis in PBMC.

Effect of Apoptotic Cell Recognition on Macrophage Polarization and Mycobacterial Persistence

PubMed Central

de Oliveira Fulco, Tatiana; Andrade, Priscila Ribeiro; de Mattos Barbosa, Mayara Garcia; Pinto, Thiago Gomes Toledo; Ferreira, Paula Fernandez; Ferreira, Helen; da Costa Nery, José Augusto; Real, Suzana Côrte; Borges, Valéria Matos; Moraes, Milton Ozório; Sarno, Euzenir Nunes; Sampaio, Elizabeth Pereira

2014-01-01

Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-β) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-β. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection. PMID:25024361

Radiofrequency treatment alters cancer cell phenotype

NASA Astrophysics Data System (ADS)

Ware, Matthew J.; Tinger, Sophia; Colbert, Kevin L.; Corr, Stuart J.; Rees, Paul; Koshkina, Nadezhda; Curley, Steven; Summers, H. D.; Godin, Biana

2015-07-01

The importance of evaluating physical cues in cancer research is gradually being realized. Assessment of cancer cell physical appearance, or phenotype, may provide information on changes in cellular behavior, including migratory or communicative changes. These characteristics are intrinsically different between malignant and non-malignant cells and change in response to therapy or in the progression of the disease. Here, we report that pancreatic cancer cell phenotype was altered in response to a physical method for cancer therapy, a non-invasive radiofrequency (RF) treatment, which is currently being developed for human trials. We provide a battery of tests to explore these phenotype characteristics. Our data show that cell topography, morphology, motility, adhesion and division change as a result of the treatment. These may have consequences for tissue architecture, for diffusion of anti-cancer therapeutics and cancer cell susceptibility within the tumor. Clear phenotypical differences were observed between cancerous and normal cells in both their untreated states and in their response to RF therapy. We also report, for the first time, a transfer of microsized particles through tunneling nanotubes, which were produced by cancer cells in response to RF therapy. Additionally, we provide evidence that various sub-populations of cancer cells heterogeneously respond to RF treatment.

21 CFR 172.886 - Petroleum wax.

Code of Federal Regulations, 2012 CFR

2012-04-01

... disc approximately 3/16-inch thick with a hole bored in the center to closely fit the stem of the... joints will prevent freezing. Do not use grease on stopcocks or joints. Spectrophotometric cells. Fused quartz cells, optical path length in the range of 5.000 centimeters ±0.005 centimeter; also for checking...

21 CFR 172.886 - Petroleum wax.

Code of Federal Regulations, 2013 CFR

2013-04-01

... disc approximately 3/16-inch thick with a hole bored in the center to closely fit the stem of the... joints will prevent freezing. Do not use grease on stopcocks or joints. Spectrophotometric cells. Fused quartz cells, optical path length in the range of 5.000 centimeters ±0.005 centimeter; also for checking...