Development of Methods for Carrier-Mediated Targeted Delivery of Antiviral Compounds Using Monoclonal Antibodies

DTIC Science & Technology

1987-04-01

2. Comparison of Immunofluorescent Staining in Formaldehyde-Fixed Pichlnde Virus-Infected Cells That Had Been either Dried prior to Reaction with...was undertaken. 37 &aa&&3&M ^.{m^mmsmmmmmmmmmiä B. Experimental Methods General Procedures and Instrumentation. When required, reactions and...period, the reaction mixture was red and efficient stirring became very difficult. After the addition was complete, the reaction mixture was allowed

Intracellular pathways following uptake of bevacizumab in RPE cells.

PubMed

Aboul Naga, Shereen Hassan; Dithmer, Michaela; Chitadze, Guranda; Kabelitz, Dieter; Lucius, Ralph; Roider, Johann; Klettner, Alexa

2015-02-01

The anti-VEGF antibody bevacizumab is widely used off-label for the treatment of various ocular diseases, most commonly in age-related macular degeneration and diabetic macular edema. Bevacizumab is able to penetrate the retina and is found in the choroid after intravitreal injection in a time dependent manner. It has previously been shown to be taken up by the retinal pigment epithelium (RPE). In this study, we have investigated the intracellular pathway following uptake of bevacizumab in RPE cells, tested both in primary porcine RPE cells and in the human cell line ARPE19. Bevacizumab displays a characteristic, time-dependent pattern of intracellular distribution, as detected by immunofluorescence and pulse chase experiments. In both primary cells and the cell line, intracellular bevacizumab can be found after seven days, as detected by immunofluorescence and Western blotting. Immediately after application, bevacizumab partially colocalizes with Rab5, indicating some uptake in early endosomes. Intracellularly, bevacizumab is detected in the cytoskeletal fraction, aligning with actin filaments, as revealed by subcellular fractioning and immunofluorescence. Bevacizumab seems to travel along actin filaments by myosin7a, as determined by triple staining immunofluorescence. Interestingly, over a period of seven days, bevacizumab seems to accumulate in certain storage areas, as observed by immunofluorescence. Furthermore, results obtained with immunocytochemistry, Western blotting and flow cytometry indicate that bevacizumab may be released from the RPE cells via exosomes. In conclusion, bevacizumab is taken up by and transported in the retinal pigment epithelial cells in a characteristic, time-dependent manner, where it seems to move along actin filaments by myosin7a and seem to be partially released from the cells via exosomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ovarian phagocyte subsets and their distinct tissue distribution patterns.

PubMed

Carlock, Colin; Wu, Jean; Zhou, Cindy; Ross, April; Adams, Henry; Lou, Yahuan

2013-01-01

Ovarian macrophages, which play critical roles in various ovarian events, are probably derived from multiple lineages. Thus, a systemic classification of their subsets is a necessary first step for determination of their functions. Utilizing antibodies to five phagocyte markers, i.e. IA/IE (major histocompatibility complex class II), F4/80, CD11b (Mac-1), CD11c, and CD68, this study investigated subsets of ovarian phagocytes in mice. Three-color immunofluorescence and flow cytometry, together with morphological observation on isolated ovarian cells, demonstrated complicated phenotypes of ovarian phagocytes. Four macrophage and one dendritic cell subset, in addition to many minor phagocyte subsets, were identified. A dendritic cell-like population with a unique phenotype of CD11c(high)IA/IE⁻F4/80⁻ was also frequently observed. A preliminary age-dependent study showed dramatic increases in IA/IE⁺ macrophages and IA/IE⁺ dendritic cells after puberty. Furthermore, immunofluorescences on ovarian sections showed that each subset displayed a distinct tissue distribution pattern. The pattern for each subset may hint to their role in an ovarian function. In addition, partial isolation of ovarian macrophage subset using CD11b antibodies was attempted. Establishment of this isolation method may have provided us a tool for more precise investigation of each subset's functions at the cellular and molecular levels.

Immunofluorescence findings in rapid whitening of scalp hair.

PubMed

Guin, J D; Kumar, V; Petersen, B H

1981-09-01

Rapid whitening of scalp hair developed during a three-month period along with a diffuse, subtotal alopecia in a patient. Immunofluorescence microscopy of biopsy material showed prominent deposits of IgG and IgM in a granular pattern in the epithelium of the lower portions of hair follicles. Some return of the color and amount of scalp hair occurred within a year, but occasional bouts of hair loss continued to occur. It is theorized that the rapid graying was caused by a selective loss of pigmented hair, which was perhaps caused by an immunologic mechanism. Some of the findings suggest that the cause of this patient's loss of hair color may be different from those of patients who have been previously described as having rapid whitening of scalp hair because of alopecia areata or vitiligo.

[Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope].

PubMed

Gao, Yi-ning; Wang, Dan-ying; Pan, Zong-fu; Mei, Yu-qin; Wang, Zhi-qiang; Zhu, Dan-yan; Lou, Yi-jia

2012-07-01

To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope. Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes. The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons. Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.

Clinico-laboratory aspects of anti-nuclear and anti-native DNA antibody tests.

PubMed

Webb, J

1978-01-01

Available techniques for detection of anti-nuclear antibodies are here briefly reviewed. The relatively insensitive LE cell test has been largely supplanted by the indirect immunofluorescent ANA test which should be reported in terms of titre and pattern. Specific measurement of nDNA antibodies is now a regular technique in SLE diagnosis and management.

Biological characterization of metanephric mesenchymal stem cells from the Beijing duck.

PubMed

Chen, Jia; Pu, Yabin; Sun, Yujiao; Zhang, Ping; Li, Qian; Wang, Kunfu; Wang, Wenjie; Ma, Yuehui; Guan, Weijun

2016-02-01

Mesenchymal stem cells (MSCs) possess self-proliferation and multi-directional differentiation abilities. Previous studies on MSCs have mostly focused on the bone marrow, lungs, pancreas and umbilical cord blood, with few studies on metanephric tissues in ducks. For the present study, the Beijing duck was selected as an experimental animal. Duck embryo metanephric mesenchymal stem cells (MMSCs) were studied. MMSC isolation culture, analysis of biological characteristics, induced differentiation and identification were performed in preliminary experiments. In the current study, surface antigens and gene expression patterns were detected using immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. The induced cells, adipocytes, hepatocytes, epithelial cells and islet cells were identified by oil red O staining, periodic acid-Schiff staining, immunofluorescence and dithizone staining, respectively. RT-PCR was performed for detection of specific marker genes. The results suggested that the biological characteristics of MMSCs were similar to those of the MSCs previously analyzed. Primary MMSCs were sub-cultured to passage 21. The induced cells exhibit typical staining and immunofluorescence indicating the expression of specific genes. This demonstrates that MMSCs may be a novel alternative source of MSCs for experimental and clinical applications.

The synaptic ribbon is critical for sound encoding at high rates and with temporal precision

PubMed Central

Chakrabarti, Rituparna; Picher, Maria Magdalena; Neef, Jakob; Jung, SangYong; Gültas, Mehmet; Maxeiner, Stephan

2018-01-01

We studied the role of the synaptic ribbon for sound encoding at the synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in mice lacking RIBEYE (RBEKO/KO). Electron and immunofluorescence microscopy revealed a lack of synaptic ribbons and an assembly of several small active zones (AZs) at each synaptic contact. Spontaneous and sound-evoked firing rates of SGNs and their compound action potential were reduced, indicating impaired transmission at ribbonless IHC-SGN synapses. The temporal precision of sound encoding was impaired and the recovery of SGN-firing from adaptation indicated slowed synaptic vesicle (SV) replenishment. Activation of Ca2+-channels was shifted to more depolarized potentials and exocytosis was reduced for weak depolarizations. Presynaptic Ca2+-signals showed a broader spread, compatible with the altered Ca2+-channel clustering observed by super-resolution immunofluorescence microscopy. We postulate that RIBEYE disruption is partially compensated by multi-AZ organization. The remaining synaptic deficit indicates ribbon function in SV-replenishment and Ca2+-channel regulation. PMID:29328020

An immunohistochemical study of APG-2 protein in the rat hippocampus after transient forebrain ischemia.

PubMed

Lee, Mun-Yong; Choi, Yun-Sik; Choi, Jeong-Sun; Min, Do Sik; Chun, Myung-Hoon; Kim, Ok Nyu; Lee, Sang Bok; Kim, Seong Yun

2002-01-11

The cellular localization and spatiotemporal expression pattern of APG-2 protein, a member of the heat shock protein 110 family, were investigated in the rat hippocampus after transient forebrain ischemia. The spatiotemporal patterns of immunoreactivity of both APG-2 and glial fibrillary acidic protein were very similar, indicating that reactive astrocytes express APG-2, which was confirmed by double immunofluorescence histochemistry. Colocalization of APG-2 and a neuronal marker NeuN in the neurons of the CA2 and CA3 subfields was also confirmed.

Pointwise mutual information quantifies intratumor heterogeneity in tissue sections labeled with multiple fluorescent biomarkers.

PubMed

Spagnolo, Daniel M; Gyanchandani, Rekha; Al-Kofahi, Yousef; Stern, Andrew M; Lezon, Timothy R; Gough, Albert; Meyer, Dan E; Ginty, Fiona; Sarachan, Brion; Fine, Jeffrey; Lee, Adrian V; Taylor, D Lansing; Chennubhotla, S Chakra

2016-01-01

Measures of spatial intratumor heterogeneity are potentially important diagnostic biomarkers for cancer progression, proliferation, and response to therapy. Spatial relationships among cells including cancer and stromal cells in the tumor microenvironment (TME) are key contributors to heterogeneity. We demonstrate how to quantify spatial heterogeneity from immunofluorescence pathology samples, using a set of 3 basic breast cancer biomarkers as a test case. We learn a set of dominant biomarker intensity patterns and map the spatial distribution of the biomarker patterns with a network. We then describe the pairwise association statistics for each pattern within the network using pointwise mutual information (PMI) and visually represent heterogeneity with a two-dimensional map. We found a salient set of 8 biomarker patterns to describe cellular phenotypes from a tissue microarray cohort containing 4 different breast cancer subtypes. After computing PMI for each pair of biomarker patterns in each patient and tumor replicate, we visualize the interactions that contribute to the resulting association statistics. Then, we demonstrate the potential for using PMI as a diagnostic biomarker, by comparing PMI maps and heterogeneity scores from patients across the 4 different cancer subtypes. Estrogen receptor positive invasive lobular carcinoma patient, AL13-6, exhibited the highest heterogeneity score among those tested, while estrogen receptor negative invasive ductal carcinoma patient, AL13-14, exhibited the lowest heterogeneity score. This paper presents an approach for describing intratumor heterogeneity, in a quantitative fashion (via PMI), which departs from the purely qualitative approaches currently used in the clinic. PMI is generalizable to highly multiplexed/hyperplexed immunofluorescence images, as well as spatial data from complementary in situ methods including FISSEQ and CyTOF, sampling many different components within the TME. We hypothesize that PMI will uncover key spatial interactions in the TME that contribute to disease proliferation and progression.

Pattern of glomerular diseases in Oman: a study based on light microscopy and immunofluorescence.

PubMed

Alwahaibi, Nasar Yousuf; Alhabsi, Taiseer Ahmed; Alrawahi, Samira Abdullah

2013-03-01

Light microscopy and immunofluorescence play an important part in the final diagnosis of renal biopsy. The aim of this study was to analyze the pattern of various glomerular diseases in Oman. A total of 424 renal biopsies were retrospectively analyzed at the Sultan Qaboos University Hospital between 1999 and 2010. Focal and segmental glomerulosclerosis (FSGS), minimal change disease (MCD), membranous glomerulopathy (MGN) and IgA nephropathy were the most common primary glomerular diseases encountered, accounting for 21.2%, 17%, 12.3% and 8.3%, respectively, of all cases. Lupus nephritis was the most common secondary glomerular disease and was the most prevalent among all biopsies, accounting for 30.4% of all biopsies. Amyloidosis was seen in only two cases. The presence of fluorescein isothiocyanatefibrin in all renal cases was low when compared with IgG, IgA, IgM, C3 and C1q markers. In conclusion, based on the findings of this study, lupus nephritis was the most common of all glomerular diseases and FSGS was the most common primary glomerular disease. The importance of fluorescein isothiocyanate-fibrin in the diagnosis of renal biopsy needs to be further investigated.

A novel method for detecting IgA endomysial antibodies by using human umbilical vein endothelial cells.

PubMed

Castellino, F; Scaglione, N; Grosso, S B; Sategna-Guidetti, C

2000-01-01

Although tissue transglutaminase was recently identified as the main autoantigen recognized by endomysial antibodies in coeliac patients, anti-endomysium antibody detection still persists as the gold standard for coeliac disease screening and diagnosis. (1) To evaluate human umbilical vein cells (HUVEC) as an alternative source of endomysial antigen and to assess their suitability in the diagnosis of coeliac disease. (2) To verify whether tissue transglutaminase is one target antigen eliciting the endomysial antibody fraction of coeliac serum IgA. University teaching hospital. Sera from 123 untreated adults with biopsy-proven coeliac disease and 84 controls (40 healthy and 44 diseased) were assessed by indirect immunofluorescence, using HUVEC on glass slides prepared by cytocentrifugation and permeabilized by using Triton X (0.5%). Indirect immunofluorescence was performed: (1) using coeliac disease serum samples on HUVEC with or without prior incubation with tissue transglutaminase; and (2) incubating both HUVEC and monkey oesophagus with goat anti-guinea pig tissue transglutaminase antibody. All the coeliac patients, who were also positive on monkey oesophagus, showed the typical fluorescent homogeneous cytoplasmic stain on HUVEC. All control sera were negative both on HUVEC and on monkey oesophagus. IgA antibodies did not react with non-permeabilized cells, with intact membrane. Preincubation of coeliac sera with tissue transglutaminase abolished the typical fluorescent pattern. The incubation of anti-tissue transglutaminase antibody with monkey oesophagus and HUVEC resulted in an immunofluorescence staining pattern identical to that obtained with positive coeliac sera. (1) As a substrate for anti-endomysial antibody, HUVEC may provide the same diagnostic accuracy as monkey oesophagus, thus bypassing economical and ethical problems. The HUVEC antigen reacting with IgA from coeliac disease sera is an intracellular rather than a cell-surface antigen, as IgA antibodies reacted only with permeabilized cells. (2) Pretreatment of untreated coeliac sera with tissue transglutaminase abolished almost completely the specific staining; incubation with anti-tissue transglutaminase antibody elicited the characteristic fluorescent pattern, thus confirming that tissue transglutaminase represents the prominent autoantigen in coeliac disease.

Antibodies to Ro/La, Cenp-B, and snRNPs antigens in autoimmune hepatitis of North America versus Asia: patterns of immunofluorescence, ELISA reactivities, and HLA association.

PubMed

Parveen, S; Morshed, S A; Arima, K; Nishioka, M; Czaja, A J; Chow, W C; Ng, H S

1998-06-01

To assess whether demography is one of the important factors determining antibody response to nuclear antigens [ANA: SSA-Ro (52K and 60K), SSB-La, snRNPs (A, 70K, B'/B), and Cenp-B], we investigated 95 and 47 sera of autoimmune hepatitis (AIH) from North America and Asia, respectively, by immunofluorescent (IF) and recombinant ELISA. Correlations among nuclear IF patterns, ELISA, and disease indices were analyzed. The frequency and titer of individual antibodies differed significantly between the groups. Patients with speckled patterns were younger in both regions and had higher aspartate aminotransferase levels only in North America. HLA-A1, B8, DQ2, and DR4 or DR3 or both in North America, and A2, B61, DQ7, and DR4 in Asia were predominant. In Asia, B61 correlated with anti-70K, and DQ7 correlated with antibodies to 52K, Cenp-B, and B'/B. In North America, A1, B8, DR3 haplotype, and DQ2 correlated with antibodies to A and 70K. Anti-B'/B and DR4 in North America, and A2 in Asia, were associated with concurrent immunologic disorder. Individual ANA clusters correlated with individual HLA in the demography, and different HLA alleles might determine disease expression as well as different ANA being produced in AIH.

Isolation and Total Synthesis of Stolonines A–C, Unique Taurine Amides from the Australian Marine Tunicate Cnemidocarpa stolonifera

PubMed Central

Tran, Trong D.; Pham, Ngoc B.; Ekins, Merrick; Hooper, John N. A.; Quinn, Ronald J.

2015-01-01

Cnemidocarpa stolonifera is an underexplored marine tunicate that only occurs on the tropical to subtropical East Coast of Australia, with only two pyridoacridine compounds reported previously. Qualitative analysis of the lead-like enhanced fractions of C. stolonifera by LC-MS dual electrospray ionization coupled with PDA and ELSD detectors led to the identification of three new natural products, stolonines A–C (1–3), belonging to the taurine amide structure class. Structures of the new compounds were determined by NMR and MS analyses and later verified by total synthesis. This is the first time that the conjugates of taurine with 3-indoleglyoxylic acid, quinoline-2-carboxylic acid and β-carboline-3-carboxylic acid present in stolonines A–C (1–3), respectively, have been reported. An immunofluorescence assay on PC3 cells indicated that compounds 1 and 3 increased cell size, induced mitochondrial texture elongation, and caused apoptosis in PC3 cells. PMID:26204949

A Cell-Based Screen Reveals that the Albendazole Metabolite, Albendazole Sulfone, Targets Wolbachia

PubMed Central

Bray, Walter M.; White, Pamela M.; Ruybal, Jordan; Lokey, R. Scott; Debec, Alain; Sullivan, William

2012-01-01

Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts. PMID:23028321

Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks.

PubMed

Mah, Li-Jeen; Orlowski, Christian; Ververis, Katherine; Vasireddy, Raja S; El-Osta, Assam; Karagiannis, Tom C

2011-01-25

Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds.

Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks

PubMed Central

2011-01-01

Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds. PMID:21261999

Anti-ENA antibody profiles in patients with hepatitis C virus infection.

PubMed

Litwin, Christine M; Rourk, Angela R

2018-03-01

The presence of antinuclear antibodies (ANA) has been described following hepatitis C virus (HCV). Very few studies have investigated the presence of anti-extractable nuclear antigens (ENA) in HCV infection. The aim of this study was to assess the prevalence of ENA antibodies in 100 patients with HCV infection compared to the prevalence of ENA antibodies in 100 healthy control patients. Sera from patients were tested for ENA using a multiplex microbead immunoassay. Sera positive for ENA were subsequently tested for ANA using an indirect immunofluorescence assay and titered if positive. Fourteen (14%) of the 100 patients with HCV infection had anti-ENA antibodies: four each showed anti-SSA antibodies and anti-dsDNA antibodies, three each had RNP antibodies and Scl-70 antibodies, and one each had anti-SSB, centromere B, Sm, and Sm/RNP antibodies. Ten of the 14 patients positive for anti-ENA were positive by indirect immunofluorescence staining (IFA) with titers ranging from 1:40 to 1:160. Five had antinuclear patterns, one had combined antinuclear and cytoplasmic patterns, and four only had a cytoplasmic pattern. Three of the 100 healthy control patients had ANA positive titers (1:80 and 1:320) and anti-ENA antibodies: one anti-Scl-70 and two anti-RNP. The prevalence of anti-ENA antibodies was significantly higher in the patients with HCV infections than in the healthy controls. Other studies of anti-ENA profiles in patients with HCV infection have identified similar patterns of positivity for anti-SSA, anti-SSB, anti-dsDNA, anti-RNP, anti- Sm/RNP, Scl-70, centromere B, and anti-Sm. © 2017 Wiley Periodicals, Inc.

Automated indirect immunofluorescence evaluation of antinuclear autoantibodies on HEp-2 cells.

PubMed

Voigt, Jörn; Krause, Christopher; Rohwäder, Edda; Saschenbrecker, Sandra; Hahn, Melanie; Danckwardt, Maick; Feirer, Christian; Ens, Konstantin; Fechner, Kai; Barth, Erhardt; Martinetz, Thomas; Stöcker, Winfried

2012-01-01

Indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells is considered as the gold standard screening method for the detection of antinuclear autoantibodies (ANA). However, in terms of automation and standardization, it has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Although there are already some automation solutions for IIF incubation in the market, the automation of result evaluation is still in its infancy. Therefore, the EUROPattern Suite has been developed as a comprehensive automated processing and interpretation system for standardized and efficient ANA detection by HEp-2 cell-based IIF. In this study, the automated pattern recognition was compared to conventional visual interpretation in a total of 351 sera. In the discrimination of positive from negative samples, concordant results between visual and automated evaluation were obtained for 349 sera (99.4%, kappa = 0.984). The system missed out none of the 272 antibody-positive samples and identified 77 out of 79 visually negative samples (analytical sensitivity/specificity: 100%/97.5%). Moreover, 94.0% of all main antibody patterns were recognized correctly by the software. Owing to its performance characteristics, EUROPattern enables fast, objective, and economic IIF ANA analysis and has the potential to reduce intra- and interlaboratory variability.

Automated Indirect Immunofluorescence Evaluation of Antinuclear Autoantibodies on HEp-2 Cells

PubMed Central

Voigt, Jörn; Krause, Christopher; Rohwäder, Edda; Saschenbrecker, Sandra; Hahn, Melanie; Danckwardt, Maick; Feirer, Christian; Ens, Konstantin; Fechner, Kai; Barth, Erhardt; Martinetz, Thomas; Stöcker, Winfried

2012-01-01

Indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells is considered as the gold standard screening method for the detection of antinuclear autoantibodies (ANA). However, in terms of automation and standardization, it has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Although there are already some automation solutions for IIF incubation in the market, the automation of result evaluation is still in its infancy. Therefore, the EUROPattern Suite has been developed as a comprehensive automated processing and interpretation system for standardized and efficient ANA detection by HEp-2 cell-based IIF. In this study, the automated pattern recognition was compared to conventional visual interpretation in a total of 351 sera. In the discrimination of positive from negative samples, concordant results between visual and automated evaluation were obtained for 349 sera (99.4%, kappa = 0.984). The system missed out none of the 272 antibody-positive samples and identified 77 out of 79 visually negative samples (analytical sensitivity/specificity: 100%/97.5%). Moreover, 94.0% of all main antibody patterns were recognized correctly by the software. Owing to its performance characteristics, EUROPattern enables fast, objective, and economic IIF ANA analysis and has the potential to reduce intra- and interlaboratory variability. PMID:23251220

Ciprofloxacin Derivatives Affect Parasite Cell Division and Increase the Survival of Mice Infected with Toxoplasma gondii

PubMed Central

Martins-Duarte, Erica S.; Dubar, Faustine; Lawton, Philippe; França da Silva, Cristiane; C. Soeiro, Maria de Nazaré; de Souza, Wanderley; Biot, Christophe; Vommaro, Rossiane C.

2015-01-01

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against T. gondii in vitro, compared with the original compound. Cipro derivatives are expected to target the parasite’s DNA gyrase complex in the apicoplast. The activity of these compounds in vivo, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives in vivo, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects T. gondii tachyzoites in vitro, by immunofluorescence and transmission electron microscopy (TEM). When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13–25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment). Light microscopy examination early (6 and 24h) post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition - with the appearance of ‘tethered’ parasites – malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against C. parvum, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in T. gondii tachyzoites, supporting that these compounds targets the apicoplast. Our results show that Cipro derivatives improved the survival of mice acutely infected with T. gondii and inhibited parasite replication early in the first cycle of infection in vitro, highlighting their therapeutic potential for the treatment of toxoplasmosis. PMID:25950173

Autofluorescence and non-specific immunofluorescent labeling in frozen bovine intestinal tissue sections: Solutions for multi-color immunofluorescence experiments

USDA-ARS?s Scientific Manuscript database

Autofluorescence and non-specific immunofluorescent labeling are common challenges associated with immunofluorescence experiments. Autofluorescence typically demonstrates a broad emission spectrum, increasing the potential for overlap with experiments that utilize multiple fluorophores. During immun...

ANAlyte: A modular image analysis tool for ANA testing with indirect immunofluorescence.

PubMed

Di Cataldo, Santa; Tonti, Simone; Bottino, Andrea; Ficarra, Elisa

2016-05-01

The automated analysis of indirect immunofluorescence images for Anti-Nuclear Autoantibody (ANA) testing is a fairly recent field that is receiving ever-growing interest from the research community. ANA testing leverages on the categorization of intensity level and fluorescent pattern of IIF images of HEp-2 cells to perform a differential diagnosis of important autoimmune diseases. Nevertheless, it suffers from tremendous lack of repeatability due to subjectivity in the visual interpretation of the images. The automatization of the analysis is seen as the only valid solution to this problem. Several works in literature address individual steps of the work-flow, nonetheless integrating such steps and assessing their effectiveness as a whole is still an open challenge. We present a modular tool, ANAlyte, able to characterize a IIF image in terms of fluorescent intensity level and fluorescent pattern without any user-interactions. For this purpose, ANAlyte integrates the following: (i) Intensity Classifier module, that categorizes the intensity level of the input slide based on multi-scale contrast assessment; (ii) Cell Segmenter module, that splits the input slide into individual HEp-2 cells; (iii) Pattern Classifier module, that determines the fluorescent pattern of the slide based on the pattern of the individual cells. To demonstrate the accuracy and robustness of our tool, we experimentally validated ANAlyte on two different public benchmarks of IIF HEp-2 images with rigorous leave-one-out cross-validation strategy. We obtained overall accuracy of fluorescent intensity and pattern classification respectively around 85% and above 90%. We assessed all results by comparisons with some of the most representative state of the art works. Unlike most of the other works in the recent literature, ANAlyte aims at the automatization of all the major steps of ANA image analysis. Results on public benchmarks demonstrate that the tool can characterize HEp-2 slides in terms of intensity and fluorescent pattern with accuracy better or comparable with the state of the art techniques, even when such techniques are run on manually segmented cells. Hence, ANAlyte can be proposed as a valid solution to the problem of ANA testing automatization. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Impact of immunofluorescence on the histological pattern of pediatric kidney biopsies from northern Pakistan.

PubMed

Ali, Akhtar; Ali, Mohammad Usman; Ali, Mahrukh Ayesha

2011-01-01

Kidney biopsy is an investigation for diagnosis and prognosis of a variety of nephritides. It also influences therapeutic options. Immunofluorescence (IMF) greatly adds in identifying the pathologies which may not be obvious on light microscopy (L/M), such as IgM, IgA nephropathy, pauci-immune glomerulonephritis, and anti-glomerular basement membrane disease. We present here data of 170 pediatric kidney biopsies from July 2005 to December 2009 from Department of Nephrology and Hypertension, Lady Reading Hospital, Peshawar, Pakistan. The study was undertaken to see whether IMF would alter the histological pattern of pediatric kidney biopsies and to compare these data with an earlier data from our department of 415 pediatric kidney biopsies done over 7-year period from 1998 to 2005, which were analyzed with L/M alone. Out of 170 kidney biopsies using L/M and IMF, IgM turns out to be most common pattern (20%), followed by minimal change disease (MCD) (17.05%), focal and segmental glomerulosclerosis (FSGS) (15.88%), chronic sclerosing glomerulonephritis (Chr. sclerosing GN) (12.35%), mesangio proliferative glomerulonephritis (MPGN) (7.65%), mesangio capillary glomerulonephritis (MCGN) (6.47%), membranous glomerulonephritis (Mem. GN) (5.29%), IgA nephropathy (5.29%), cresentic glomerulonephritis (Cres. GN) (3.53%), lupus nephritis (2.96%), and others (3.53%). Comparing these results of 170 cases with 415 renal biopsies without IMF, IgM dominated the histological pattern in IMF group whereas MCD followed by FSGS and MPGN were prominent in group without IMF. Therefore, variation in the overall histological pattern with IMF technique proved statistically significant (p < 0.0001). Addition of IMF has altered the frequency of MCD, a change from 24% (100/415) to 17% (29/170), FSGS from 18.3% (76/415) to 15.88% (27/170), and MPGN from 17.35% (72/415) to 7.65% (13/170).

Pointwise mutual information quantifies intratumor heterogeneity in tissue sections labeled with multiple fluorescent biomarkers

PubMed Central

Spagnolo, Daniel M.; Gyanchandani, Rekha; Al-Kofahi, Yousef; Stern, Andrew M.; Lezon, Timothy R.; Gough, Albert; Meyer, Dan E.; Ginty, Fiona; Sarachan, Brion; Fine, Jeffrey; Lee, Adrian V.; Taylor, D. Lansing; Chennubhotla, S. Chakra

2016-01-01

Background: Measures of spatial intratumor heterogeneity are potentially important diagnostic biomarkers for cancer progression, proliferation, and response to therapy. Spatial relationships among cells including cancer and stromal cells in the tumor microenvironment (TME) are key contributors to heterogeneity. Methods: We demonstrate how to quantify spatial heterogeneity from immunofluorescence pathology samples, using a set of 3 basic breast cancer biomarkers as a test case. We learn a set of dominant biomarker intensity patterns and map the spatial distribution of the biomarker patterns with a network. We then describe the pairwise association statistics for each pattern within the network using pointwise mutual information (PMI) and visually represent heterogeneity with a two-dimensional map. Results: We found a salient set of 8 biomarker patterns to describe cellular phenotypes from a tissue microarray cohort containing 4 different breast cancer subtypes. After computing PMI for each pair of biomarker patterns in each patient and tumor replicate, we visualize the interactions that contribute to the resulting association statistics. Then, we demonstrate the potential for using PMI as a diagnostic biomarker, by comparing PMI maps and heterogeneity scores from patients across the 4 different cancer subtypes. Estrogen receptor positive invasive lobular carcinoma patient, AL13-6, exhibited the highest heterogeneity score among those tested, while estrogen receptor negative invasive ductal carcinoma patient, AL13-14, exhibited the lowest heterogeneity score. Conclusions: This paper presents an approach for describing intratumor heterogeneity, in a quantitative fashion (via PMI), which departs from the purely qualitative approaches currently used in the clinic. PMI is generalizable to highly multiplexed/hyperplexed immunofluorescence images, as well as spatial data from complementary in situ methods including FISSEQ and CyTOF, sampling many different components within the TME. We hypothesize that PMI will uncover key spatial interactions in the TME that contribute to disease proliferation and progression. PMID:27994939

A Nanoscale Interface Promoting Molecular and Functional Differentiation of Neural Cells

NASA Astrophysics Data System (ADS)

Posati, Tamara; Pistone, Assunta; Saracino, Emanuela; Formaggio, Francesco; Mola, Maria Grazia; Troni, Elisabetta; Sagnella, Anna; Nocchetti, Morena; Barbalinardo, Marianna; Valle, Francesco; Bonetti, Simone; Caprini, Marco; Nicchia, Grazia Paola; Zamboni, Roberto; Muccini, Michele; Benfenati, Valentina

2016-08-01

Potassium channels and aquaporins expressed by astrocytes are key players in the maintenance of cerebral homeostasis and in brain pathophysiologies. One major challenge in the study of astrocyte membrane channels in vitro, is that their expression pattern does not resemble the one observed in vivo. Nanostructured interfaces represent a significant resource to control the cellular behaviour and functionalities at micro and nanoscale as well as to generate novel and more reliable models to study astrocytes in vitro. However, the potential of nanotechnologies in the manipulation of astrocytes ion channels and aquaporins has never been previously reported. Hydrotalcite-like compounds (HTlc) are layered materials with increasing potential as biocompatible nanoscale interface. Here, we evaluate the effect of the interaction of HTlc nanoparticles films with primary rat neocortical astrocytes. We show that HTlc films are biocompatible and do not promote gliotic reaction, while favouring astrocytes differentiation by induction of F-actin fibre alignment and vinculin polarization. Western Blot, Immunofluorescence and patch-clamp revealed that differentiation was accompanied by molecular and functional up-regulation of both inward rectifying potassium channel Kir 4.1 and aquaporin 4, AQP4. The reported results pave the way to engineering novel in vitro models to study astrocytes in a in vivo like condition.

Cardiac toxicity of 5-ring polycyclic aromatic hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during zebrafish development

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

Incardona, John P., E-mail: john.incardona@noaa.gov; Linbo, Tiffany L.; Scholz, Nathaniel L.

Petroleum-derived compounds, including polycyclic aromatic hydrocarbons (PAHs), commonly occur as complex mixtures in the environment. Recent studies using the zebrafish experimental model have shown that PAHs are toxic to the embryonic cardiovascular system, and that the severity and nature of this developmental cardiotoxicity varies by individual PAH. In the present study we characterize the toxicity of the relatively higher molecular weight 5-ring PAHs benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), and benzo[k]fluoranthene (BkF). While all three compounds target the cardiovascular system, the underlying role of the ligand-activated aryl hydrocarbon receptor (AHR2) and the tissue-specific induction of the cytochrome p450 metabolic pathway (CYP1A) weremore » distinct for each. BaP exposure (40 {mu}M) produced AHR2-dependent bradycardia, pericardial edema, and myocardial CYP1A immunofluorescence. By contrast, BkF exposure (4-40 {mu}M) caused more severe pericardial edema, looping defects, and erythrocyte regurgitation through the atrioventricular valve that were AHR2-independent (i.e., absent myocardial or endocardial CYP1A induction). Lastly, exposure to BeP (40 {mu}M) yielded a low level of CYP1A+ signal in the vascular endothelium of the head and trunk, without evident toxic effects on cardiac function or morphogenesis. Combined with earlier work on 3- and 4-ring PAHs, our findings provide a more complete picture of how individual PAHs may drive the cardiotoxicity of mixtures in which they predominate. This will improve toxic injury assessments and risk assessments for wild fish populations that spawn in habitats altered by overlapping petroleum-related human impacts such as oil spills, urban stormwater runoff, or sediments contaminated by legacy industrial activities. -- Highlights: Black-Right-Pointing-Pointer PAH compounds with 5 rings in different arrangements caused differential tissue-specific patterns of CYP1A induction in zebrafish embryos. Black-Right-Pointing-Pointer These compounds produced differential cardiac developmental toxicity that did not strictly correlate with associated CYP1A induction. Black-Right-Pointing-Pointer Cardiotoxicity of benzo(a)pyrene was partially dependent on the AHR2 isoform, while benzo(k)fluoranthene cardiotoxicity was not. Black-Right-Pointing-Pointer Individual PAH compounds have distinct toxicokinetic pathways in fish embryos, and act through different toxic mechanisms.« less

AMPK modulatory activity of olive-tree leaves phenolic compounds: Bioassay-guided isolation on adipocyte model and in silico approach.

PubMed

Jiménez-Sánchez, Cecilia; Olivares-Vicente, Mariló; Rodríguez-Pérez, Celia; Herranz-López, María; Lozano-Sánchez, Jesús; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto; Encinar, José Antonio; Micol, Vicente

2017-01-01

Olive-tree polyphenols have demonstrated potential for the management of obesity-related pathologies. We aimed to explore the capacity of Olive-tree leaves extract to modulate triglyceride accumulation and AMP-activated protein kinase activity (AMPK) on a hypertrophic adipocyte model. Intracellular triglycerides and AMPK activity were measured on the hypertrophic 3T3-L1 adipocyte model by AdipoRed and immunofluorescence microscopy, respectively. Reverse phase high performance liquid chromatography coupled to time-of-flight mass detection with electrospray ionization (RP-HPLC-ESI-TOF/MS) was used for the fractionation of the extract and the identification of the compounds. In-silico molecular docking of the AMPK alpha-2, beta and gamma subunits with the identified compounds was performed. Olive-tree leaves extract decreased the intracellular lipid accumulation through AMPK-dependent mechanisms in hypertrophic adipocytes. Secoiridoids, cinnamic acids, phenylethanoids and phenylpropanoids, flavonoids and lignans were the candidates predicted to account for this effect. Molecular docking revealed that some compounds may be AMPK-gamma modulators. The modulatory effects of compounds over the alpha and beta AMPK subunits appear to be less probable. Olive-tree leaves polyphenols modulate AMPK activity, which may become a therapeutic aid in the management of obesity-associated disturbances. The natural occurrence of these compounds may have important nutritional implications for the design of functional ingredients.

A flavonoid compound library screen revealed potent antiviral activity of plant-derived flavonoids on human enterovirus A71 replication.

PubMed

Min, Nyo; Leong, Pok Thim; Lee, Regina Ching Hua; Khuan, Jeffery Seng Eng; Chu, Justin Jang Hann

2018-02-01

Hand Foot Mouth Disease (HFMD), resulting from human enterovirus A71 (HEVA71) infection can cause severe neurological complications leading to fatality in young children. Currently, there is no approved antiviral for therapeutic treatment against HEVA71 infection. In this study, a 500-compound flavonoid library was screened to identify potential inhibitors of HEVA71 using high-throughput immunofluorescence-based phenotypic screening method. Two lead flavonoid compounds, ST077124 and ST024734 at the non-cytotoxic concentration of 50 μM were found to be effective antivirals that inhibited replication of HEVA71, reducing infectious viral titers by 3.5 log 10  PFU/ml and 2.5 log 10  PFU/ml respectively. Our study revealed that ST077124 is a specific antiviral compound that inhibits human enteroviruses while ST024734 exhibited antiviral activity against human enteroviruses as well as dengue virus type-2. We also identified that both compounds affected the viral RNA transcription and translation machinery of HEVA71 but did not interfere with the viral internal ribosomal entry site (IRES) activity. Hence, our findings strongly suggest that ST077124 and ST024734 are effective antiviral compounds of minimal cytotoxicity and could serve as promising therapeutic agents against HEVA71 infection. Copyright © 2017 Elsevier B.V. All rights reserved.

AMPK modulatory activity of olive–tree leaves phenolic compounds: Bioassay-guided isolation on adipocyte model and in silico approach

PubMed Central

Jiménez-Sánchez, Cecilia; Olivares-Vicente, Mariló; Rodríguez-Pérez, Celia; Herranz-López, María; Lozano-Sánchez, Jesús; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto; Encinar, José Antonio; Micol, Vicente

2017-01-01

Scope Olive-tree polyphenols have demonstrated potential for the management of obesity-related pathologies. We aimed to explore the capacity of Olive-tree leaves extract to modulate triglyceride accumulation and AMP-activated protein kinase activity (AMPK) on a hypertrophic adipocyte model. Methods Intracellular triglycerides and AMPK activity were measured on the hypertrophic 3T3-L1 adipocyte model by AdipoRed and immunofluorescence microscopy, respectively. Reverse phase high performance liquid chromatography coupled to time-of-flight mass detection with electrospray ionization (RP-HPLC-ESI-TOF/MS) was used for the fractionation of the extract and the identification of the compounds. In-silico molecular docking of the AMPK alpha-2, beta and gamma subunits with the identified compounds was performed. Results Olive-tree leaves extract decreased the intracellular lipid accumulation through AMPK-dependent mechanisms in hypertrophic adipocytes. Secoiridoids, cinnamic acids, phenylethanoids and phenylpropanoids, flavonoids and lignans were the candidates predicted to account for this effect. Molecular docking revealed that some compounds may be AMPK-gamma modulators. The modulatory effects of compounds over the alpha and beta AMPK subunits appear to be less probable. Conclusions Olive-tree leaves polyphenols modulate AMPK activity, which may become a therapeutic aid in the management of obesity-associated disturbances. The natural occurrence of these compounds may have important nutritional implications for the design of functional ingredients. PMID:28278224

Design, Synthesis and Antitumor Activity of Novel link-bridge and B-Ring Modified Combretastatin A-4 (CA-4) Analogues as Potent Antitubulin Agents

PubMed Central

Duan, Yong-Tao; Man, Ruo-Jun; Tang, Dan-Jie; Yao, Yong-Fang; Tao, Xiang-Xiang; Yu, Chen; Liang, Xin-Yi; Makawana, Jigar A.; Zou, Mei-Juan; Wang, Zhong-Chang; Zhu, Hai-Liang

2016-01-01

A series of 12 novel acylhydrazone, chalcone and amide–bridged analogues of combretastatin A-4 were designed and synthesized toward tubulin. All these compounds were determined by elemental analysis, 1H NMR, and MS. Among them, compound 7 with acylhydrazone-bridge, bearing a benzyl at the indole-N position, was identified as a potent antiproliferative agent against a panel of cancer cell lines with IC50 values ranging from 0.08 to 35.6 μM. In contrast, its cytotoxic effects on three normal human cells were minimal. Cellular studies have revealed that the induction of apoptosis by compound 7 was associated with a collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, alterations in the expression of some cell cycle-related proteins (Cyclin B1, Cdc25c, Cdc2, P21) and some apoptosis-related proteins (Bax, PARP, Bcl-2, Caspase3). The docking mode showed the binding posture of CA-4 and compound 7 are similar in the colchicine-binding pocket of tubulin, as confirmed by colchicine-tubulin competitive binding assay, tubulin polymerization inhibitory activity, extracellular protein expression determination assay and confocal immunofluorescence microscopy. In vivo study, compound 7 effectively inhibited A549 xenograft tumor growth without causing significant loss of body weight suggesting that compound 7 is a promising new antimitotic agent with clinical potential. PMID:27138035

Compounds from Cynomorium songaricum with Estrogenic and Androgenic Activities Suppress the Oestrogen/Androgen-Induced BPH Process.

PubMed

Wang, Xueni; Tao, Rui; Yang, Jing; Miao, Lin; Wang, Yu; Munyangaju, Jose Edouard; Wichai, Nuttapong; Wang, Hong; Zhu, Yan; Liu, Erwei; Chang, Yanxu; Gao, Xiumei

2017-01-01

To investigate the phytoestrogenic and phytoandrogenic activities of compounds isolated from CS and uncover the role of CS in prevention of oestrogen/androgen-induced BPH. Cells were treated with CS compounds, and immunofluorescence assay was performed to detect the nuclear translocation of ER α or AR in MCF-7 or LNCaP cells; luciferase reporter assay was performed to detect ERs or AR transcriptional activity in HeLa or AD293 cells; MTT assay was performed to detect the cell proliferation of MCF-7 or LNCaP cells. Oestrogen/androgen-induced BPH model was established in rat and the anti-BPH, anti-estrogenic, and anti-androgenic activities of CS in vivo were further investigated. The nuclear translocation of ER α was stimulated by nine CS compounds, three of which also stimulated AR translocation. The transcriptional activities of ER α and ER β were induced by five compounds, within which only ECG induced AR transcriptional activity as well. Besides, ECG stimulated the proliferation of both MCF-7 cells and LNCaP cells. CS extract suppressed oestrogen/androgen-induced BPH progress in vivo by downregulation of E2 and T level in serum and alteration of the expressions of ER α , ER β , and AR in the prostate. Our data demonstrates that compounds from CS exhibit phytoestrogenic and phytoandrogenic activities, which may contribute to inhibiting the oestrogen/androgen-induced BPH development.

On Variations in the Level of PER in Glial Clocks of Drosophila Optic Lobe and Its Negative Regulation by PDF Signaling.

PubMed

Górska-Andrzejak, Jolanta; Chwastek, Elżbieta M; Walkowicz, Lucyna; Witek, Kacper

2018-01-01

We show that the level of the core protein of the circadian clock Period (PER) expressed by glial peripheral oscillators depends on their location in the Drosophila optic lobe. It appears to be controlled by the ventral lateral neurons (LNvs) that release the circadian neurotransmitter Pigment Dispersing Factor (PDF). We demonstrate that glial cells of the distal medulla neuropil (dMnGl) that lie in the vicinity of the PDF-releasing terminals of the LNvs possess receptors for PDF (PDFRs) and express PER at significantly higher level than other types of glia. Surprisingly, the amplitude of PER molecular oscillations in dMnGl is increased twofold in PDF-free environment, that is in Pdf 0 mutants. The Pdf 0 mutants also reveal an increased level of glia-specific protein REPO in dMnGl. The photoreceptors of the compound eye (R-cells) of the PDF-null flies, on the other hand, exhibit de-synchrony of PER molecular oscillations, which manifests itself as increased variability of PER-specific immunofluorescence among the R-cells. Moreover, the daily pattern of expression of the presynaptic protein Bruchpilot (BRP) in the lamina terminals of the R-cells is changed in Pdf 0 mutant. Considering that PDFRs are also expressed by the marginal glia of the lamina that surround the R-cell terminals, the LNv pacemakers appear to be the likely modulators of molecular cycling in the peripheral clocks of both the glial cells and the photoreceptors of the compound eye. Consequently, some form of PDF-based coupling of the glial clocks and the photoreceptors of the eye with the central LNv pacemakers must be operational.

On Variations in the Level of PER in Glial Clocks of Drosophila Optic Lobe and Its Negative Regulation by PDF Signaling

PubMed Central

Górska-Andrzejak, Jolanta; Chwastek, Elżbieta M.; Walkowicz, Lucyna; Witek, Kacper

2018-01-01

We show that the level of the core protein of the circadian clock Period (PER) expressed by glial peripheral oscillators depends on their location in the Drosophila optic lobe. It appears to be controlled by the ventral lateral neurons (LNvs) that release the circadian neurotransmitter Pigment Dispersing Factor (PDF). We demonstrate that glial cells of the distal medulla neuropil (dMnGl) that lie in the vicinity of the PDF-releasing terminals of the LNvs possess receptors for PDF (PDFRs) and express PER at significantly higher level than other types of glia. Surprisingly, the amplitude of PER molecular oscillations in dMnGl is increased twofold in PDF-free environment, that is in Pdf0 mutants. The Pdf0 mutants also reveal an increased level of glia-specific protein REPO in dMnGl. The photoreceptors of the compound eye (R-cells) of the PDF-null flies, on the other hand, exhibit de-synchrony of PER molecular oscillations, which manifests itself as increased variability of PER-specific immunofluorescence among the R-cells. Moreover, the daily pattern of expression of the presynaptic protein Bruchpilot (BRP) in the lamina terminals of the R-cells is changed in Pdf0 mutant. Considering that PDFRs are also expressed by the marginal glia of the lamina that surround the R-cell terminals, the LNv pacemakers appear to be the likely modulators of molecular cycling in the peripheral clocks of both the glial cells and the photoreceptors of the compound eye. Consequently, some form of PDF-based coupling of the glial clocks and the photoreceptors of the eye with the central LNv pacemakers must be operational. PMID:29615925

Cell/surface interactions on laser micro-textured titanium-coated silicon surfaces.

PubMed

Mwenifumbo, Steven; Li, Mingwei; Chen, Jianbo; Beye, Aboubaker; Soboyejo, Wolé

2007-01-01

This paper examines the effects of nano-scale titanium coatings, and micro-groove/micro-grid patterns on cell/surface interactions on silicon surfaces. The nature of the cellular attachment and adhesion to the coated/uncoated micro-textured surfaces was elucidated by the visualization of the cells and relevant cytoskeletal & focal adhesion proteins through scanning electron microscopy and immunofluorescence staining. Increased cell spreading and proliferation rates are observed on surfaces with 50 nm thick Ti coatings. The micro-groove geometries have been shown to promote contact guidance, which leads to reduced scar tissue formation. In contrast, smooth surfaces result in random cell orientations and the increased possibility of scar tissue formation. Immunofluorescence cell staining experiments also reveal that the actin stress fibers are aligned along the groove dimensions, with discrete focal adhesions occurring along the ridges, within the grooves and at the ends of the cell extensions. The implications of the observed cell/surface interactions are discussed for possible applications of silicon in implantable biomedical systems.

Immunological cross-reactivity of cultured rat hippocampal neurons with goldfish brain proteins synthesized during memory consolidation.

PubMed

Schmidt, R; Löffler, F; Müller, H W; Seifert, W

1986-10-29

Ependymins are goldfish brain glycoproteins exhibiting a specifically enhanced rate of synthesis when the animals adopt a new pattern of swimming behavior. With specific antisera against ependymins it has become possible to look for ependymin-like immunoreactivity in other animal species, both qualitatively by immunofluorescence staining and quantitatively by radioimmunoassay. Ependymin-like immunoreactivity was detected not only in other fish but also in rat brain. In the rat radioimmunoassay measurements were highest for the hippocampal formation and for cultured neurons derived from the embryonic hippocampus. Immunofluorescence staining was performed on various cell culture systems derived from rat brain, in order to establish which cell type contains the antigen. Only neuronal cell populations reacted with the anti-ependymin antisera. Cells derived from embryonic rat brain hippocampus which resembled pyramidal neurons stained particularly bright for ependymin-like immunoreactivity. The antigenic material was distributed throughout the cytoplasm including the neuronal extensions. Various neuron-specific antisera have been used to counterstain the cells containing ependymin-like immunoreactivity.

Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by Widefield Epifluorescence, super-resolution light, and immunoelectron microscopy.

PubMed

Killingsworth, Murray C; Lai, Ken; Wu, Xiaojuan; Yong, Jim L C; Lee, C Soon

2012-11-01

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy.

Quantum Dot Immunocytochemical Localization of Somatostatin in Somatostatinoma by Widefield Epifluorescence, Super-resolution Light, and Immunoelectron Microscopy

PubMed Central

Lai, Ken; Wu, Xiaojuan; Yong, Jim L. C.; Lee, C. Soon

2012-01-01

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy. PMID:22899862

Comparison of desmoglein ELISA and indirect immunofluorescence using two substrates (monkey oesophagus and normal human skin) in the diagnosis of pemphigus.

PubMed

Ng, Patricia P L; Thng, Steven T G; Mohamed, Khatija; Tan, Suat Hoon

2005-11-01

A prospective study was performed to assess the usefulness of desmoglein enzyme-linked immunosorbent assay testing compared with indirect immunofluorescence in the diagnosis of new cases of pemphigus, as well as to compare the relative sensitivities of monkey oesophagus and normal human skin as substrates for indirect immunofluorescence. These tests were performed on the sera of 29 consecutive new cases of pemphigus diagnosed over a 2-year period based on clinical, histological and direct immunofluorescence findings. Desmoglein enzyme-linked immunosorbent assay was positive in all patients whereas indirect immunofluorescence was positive in only 25 of 29 patients. All four patients with negative indirect immunofluorescence had positive antinuclear antibodies or cytoplasmic fluorescence that could have masked the anti-intercellular antibodies. Desmoglein enzyme-linked immunosorbent assay appeared to reflect the disease activity better than indirect immunofluorescence in a few patients who had active disease of recent onset. Monkey oesophagus was found to be superior or equal to human skin as a substrate for indirect immunofluorescence in both pemphigus vulgaris and foliaceus.

Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells.

PubMed

Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M; Jans, David A; Tamir, Sharon; Kehn-Hall, Kylene

2016-11-01

The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

The spindle kinesin-like protein HsEg5 is an autoantigen in systemic lupus erythematosus.

PubMed

Whitehead, C M; Winkfein, R J; Fritzler, M J; Rattner, J B

1996-10-01

Autoantibodies directed against the mitotic spindle apparatus (MSA) have been shown to target an antigen referred to as NuMA (nuclear mitotic apparatus). In this study, we identified a second MSA antigen as the spindle kinesin-like protein HsEg5. We studied the frequency of antibodies to HsEg5 in human sera that demonstrate the MSA pattern of staining, the frequency of autoantibodies to HsEg5 in patients with systemic lupus erythematosus (SLE), and the clinical features of patients with antibodies to HsEg5. A prototype serum from an SLE patient was used to isolate a 4.8-kilobase complementary DNA (cDNA) from a HeLa cDNA library. Western blot, immunoprecipitation, and sequence analysis revealed that the antigen was an approximately 130-kd protein, HsEg5. The frequency of autoantibodies to recombinant HsEg5 in 51 sera that demonstrated an MSA pattern of staining on HEp-2 and HeLa cells was detected by immunoblotting 2 constructs of the cDNA. The clinical features of patients with antibodies directed against HsEg5 was obtained by retrospective chart review. The antigen responsible for the MSA-35 pattern was identified as the human kinesin-like protein HsEg5. Seven of 51 sera (14%) that demonstrated an MSA pattern of staining reacted with recombinant HsEg5. Six of 7 of the HsEg5-positive patients (86%) had SLE, and 1 had Sjögren's syndrome. The indirect immunofluorescent staining pattern of sera that reacted with HsEg5 could be distinguished from the other sera that reacted with NuMA. In an unselected cohort of 52 SLE patients, 3 (6%) had autoantibodies reactive with the recombinant HsEg5. Autoantibodies to MSA fall into 2 major classes: those reactive with NuMA and those reactive with HsEg5. Autoantibodies to HsEg5 are found in a lower frequency than NuMA in sera that demonstrate the MSA pattern of staining and appear to be specifically associated with SLE. HsEg5 can be distinguished from NuMA by indirect immunofluorescence and Western blotting.

Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing.

PubMed

Ricchiuti, Vincent; Adams, Joseph; Hardy, Donna J; Katayev, Alexander; Fleming, James K

2018-01-01

Indirect immunofluorescence (IIF) is considered by the American College of Rheumatology (ACR) and the international consensus on ANA patterns (ICAP) the gold standard for the screening of anti-nuclear antibodies (ANA). As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells) were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6%) than when compared to the current method (89.4%). Moreover, the software was consistent with technologist reading in 80.6-97.5% of patterns and 71.0-93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity.

Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing

PubMed Central

Ricchiuti, Vincent; Adams, Joseph; Hardy, Donna J.; Katayev, Alexander; Fleming, James K.

2018-01-01

Indirect immunofluorescence (IIF) is considered by the American College of Rheumatology (ACR) and the international consensus on ANA patterns (ICAP) the gold standard for the screening of anti-nuclear antibodies (ANA). As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells) were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6%) than when compared to the current method (89.4%). Moreover, the software was consistent with technologist reading in 80.6–97.5% of patterns and 71.0–93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity. PMID:29780386

Use of HCA in Subproteome-immunization and Screening of Hybridoma Supernatants to Define Distinct Antibody Binding Patterns

PubMed Central

Szafran, Adam T.; Mancini, Maureen G.; Nickerson, Jeffrey A.; Edwards, Dean P.; Mancini, Michael A.

2016-01-01

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. PMID:26521976

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns.

PubMed

Szafran, Adam T; Mancini, Maureen G; Nickerson, Jeffrey A; Edwards, Dean P; Mancini, Michael A

2016-03-01

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. Copyright © 2015 Elsevier Inc. All rights reserved.

Reversal of multidrug resistance by morning glory resin glycosides in human breast cancer cells.

PubMed

Figueroa-González, Gabriela; Jacobo-Herrera, Nadia; Zentella-Dehesa, Alejandro; Pereda-Miranda, Rogelio

2012-01-27

Reversal of multidrug resistance (MDR) by thirty resin glycosides from the morning glory family (Convolvulaceae) was evaluated in vinblastine-resistant human breast carcinoma cells (MCF-7/Vin). The effects of these amphipathic compounds on the cytotoxicity and P-glycoprotein (P-gp)-mediated MDR were estimated with the sulforhodamine B colorimetric assay. Active noncytotoxic compounds exerted a potentiation effect of vinblastine susceptibility by 1- to over 1906-fold at tested concentrations of 5 and 25 μg/mL. Murucoidin V (1) enhanced vinblastine activity 255-fold when incorporated at 25 μg/mL and also, based on flow cytometry, significantly increased the intracellular accumulation of rhodamine 123 with the use of reserpine as a positive control for a MDR reversal agent. Incubation of MCF-7/Vin cells with 1 caused an increase in uptake and notably lowered the efflux rate of rhodamine 123. Decreased expression of P-glycoprotein by compound 1 was detected by immunofluorescence flow cytometry after incubation with an anti-P-gp monoclonal antibody. These results suggest that resin glycosides represent potential efflux pump inhibitors for overcoming MDR in cancer therapy.

[Expression and localization of transmembrane protein CMTM2 in human testis and sperm].

PubMed

Zhang, X W; Lan, K; Yang, W B; Li, Q; Zhao, Y P; Yin, H Q; Kite, B; Bai, W J; Xu, T

2017-08-18

To study the expression of transmembrane protein CMTM2 in the testis and sperm of adult males and to approach the potential function of the protein in the male reproductive system. The expression of CMTM2 in human testis and sperm was confirmed by Western blot. Immunohistochemical staining was used for detecting CMTM2 localization in the testis tissue, TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction, that is, immunofluorescent staining was used for detecting CMTM2 localization in both the testis and sperm before and after the acrosome reaction. CMTM2 was presented in both human testis and sperm. In the testis, CMTM2 immunoreactive particles were observed mainly in the membrane of the different stages of spermatogenic cells. In the human sperm, its immunoreactivity was restrictively localized to the posterior head where sperm-egg fusion occurred, and the CMTM2 localization was not affected by sperm acrosome reaction. CMTM2 was widely expressed in seminiferous tubules of the human testis, mainly in the cell membranes of spermatogenic cells, which was consistent with the previous reports. The immunofluorescence performed on frozen human testis slides showed similar findings with immunohistochemistry, which gave weight to the localization of CMTM2 in the cell membranes of spermatogenic cells at different stages. TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction. CMTM2 was localized at the posterior head of sperm before and after acrosome reaction. The localization and expression of CMTM2 were not affected by sperm acrosome reaction. Expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. The expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. However, it still remains to be further elucidated about the definite role of CMTM2 in male reproductive system and the process of spermatogenesis. And in vitro fertilization experiments are needed to confirm the role of CMTM2 in fertilization in future.

Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014–2015

PubMed Central

Chan, Edward K. L.; Damoiseaux, Jan; Carballo, Orlando Gabriel; Conrad, Karsten; de Melo Cruvinel, Wilson; Francescantonio, Paulo Luiz Carvalho; Fritzler, Marvin J.; Garcia-De La Torre, Ignacio; Herold, Manfred; Mimori, Tsuneyo; Satoh, Minoru; von Mühlen, Carlos A.; Andrade, Luis E. C.

2015-01-01

During the 12th International Workshop on Autoantibodies and Autoimmunity held in Sao Paulo, Brazil, on August 28, 2014, a full day session was devoted to establishing a consensus on the nomenclature of staining patterns observed in the antinuclear antibody (ANA) indirect immunofluorescence test on HEp-2 cells. The current report summarizes the collective agreements with input from the host Brazilian and international communities that represented research, clinical, and diagnostic service laboratories. Patterns are categorized in three major groups (nuclear, cytoplasmic, and mitotic patterns) and each pattern has been defined and described in detail. The consensus nomenclature and representative patterns are made available online at the international consensus on antinuclear antibody pattern (ICAP) website (www.ANApatterns.org). To facilitate continuous improvement and input, specific comments on ICAP are encouraged and these will be discussed in subsequent ICAP meetings. The ultimate goal with the establishment of the ICAP is to promote harmonization and understanding of autoantibody test nomenclature, as well as interpretation guidelines for ANA testing, thereby optimizing usage in patient care. PMID:26347739

Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting - strengths and weaknesses.

PubMed

Hormann, Wymke; Hahn, Melanie; Gerlach, Stefan; Hochstrate, Nicola; Affeldt, Kai; Giesen, Joyce; Fechner, Kai; Damoiseaux, Jan G M C

2017-11-27

Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis. We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months. Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation. Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

How should a district general hospital immunology service screen for anti-nuclear antibodies? An ‘in-the-field’ audit

PubMed Central

Hira-Kazal, R; Shea-Simonds, P; Peacock, J L; Maher, J

2015-01-01

Anti-nuclear antibody (ANA) testing assists in the diagnosis of several immune-mediated disorders. The gold standard method for detection of these antibodies is by indirect immunofluorescence testing on human epidermoid laryngeal carcinoma (HEp-2) cells. However, many laboratories test for these antibodies using solid-phase assays such as enzyme-linked immunosorbent assay (ELISA), which allows for higher throughput testing at reduced cost. In this study, we have audited the performance of a previously established ELISA assay to screen for ANA, making comparison with the gold standard HEp-2 immunofluorescence test. A prospective and unselected sample of 89 consecutive ANA test requests by consultant rheumatologists were evaluated in parallel over a period of 10 months using both tests. ELISA and HEp-2 screening assays yielded 40 (45%) and 72 (81%) positive test results, respectively, demonstrating lack of concordance between test methods. Using standard and clinical samples, it was demonstrated that the ELISA method did not detect several ANA with nucleolar, homogeneous and speckled immunofluorescence patterns. None of these ELISANEG HEp-2POS ANA were reactive with a panel of six extractable nuclear antigens or with double-stranded DNA. Nonetheless, 13 of these samples (15%) originated from patients with recognized ANA-associated disease (n = 7) or Raynaud's phenomenon (n = 6). We conclude that ELISA screening may fail to detect clinically relevant ANA that lack defined specificity for antigen. PMID:25412573

An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells.

PubMed

Koch, Peter; Herzig, Stefan; Matthes, Jan

Pore-forming subunits of voltage gated calcium channels (VGCC) are large membrane proteins (260kDa) containing 24 transmembrane domains. Despite transfection with viral promoter driven vectors, biochemical analysis of VGCC is often hampered by rather low expression levels in heterologous systems rendering VGCC challenging targets. Especially in immunofluorescent detection, calcium channels are demanding proteins. We provide an expert step-by-step protocol with adapted conditions for handling procedures (tsA-201 cell culture, transient transfection, incubation time and temperature at 28°C or 37°C and immunostaining) to address the L-type calcium-channel pore Ca v 1.2 in an immunofluorescent approach. We performed immunocytochemical analysis of Ca v 1.2 expression at single-cell level in combination with detection of different markers for cellular organelles. We show confluency levels and shapes of tsA-201 cells at different time points during an experiment. Our experiments reveal sufficient levels of Ca v 1.2 protein and a correct Ca v 1.2 expression pattern in polygonal shaped cells already 12h after transfection. A sequence of elaborated protocol modifications allows subcellular localization analysis of Ca v 1.2 in an immunocytochemical approach. We provide a protocol that may be used to achieve insights into physiological and pathophysiological processes involving voltage gated calcium channels. Our protocol may be used for expression analysis of other challenging proteins and efficient overexpression may be exploited in related biochemical techniques requiring immunolabels. Copyright © 2016 Elsevier Inc. All rights reserved.

Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells

PubMed Central

Jin, Rong; Xia, Yiqun; Chen, Qiuxiang; Li, Wulan; Chen, Dahui; Ye, Hui; Zhao, Chengguang; Du, Xiaojing; Shi, Dengjian; Wu, Jianzhang; Liang, Guang

2016-01-01

Background The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells. Methods The expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay. Results High levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation and invasion, arrested the cell cycle, and induced apoptosis in vitro. Conclusion The asymmetric mono-carbonyl analog of curcumin Da0324 exhibited significantly improved antigastric cancer activity. Da0324 may be a promising NF-κB inhibitor for the selective targeting of cancer cells. However, further studies are needed in animals to validate these findings for the therapeutic use of Da0324. PMID:27042000

A step towards standardization: A method for end-point titer determination by fluorescence index of an automated microscope. End-point titer determination by fluorescence index.

PubMed

Carbone, Teresa; Gilio, Michele; Padula, Maria Carmela; Tramontano, Giuseppina; D'Angelo, Salvatore; Pafundi, Vito

2018-05-01

Indirect Immunofluorescence (IIF) is widely considered the Gold Standard for Antinuclear Antibody (ANA) screening. However, the high inter-reader variability remains the major disadvantage associated with ANA testing and the main reason for the increasing demand of the computer-aided immunofluorescence microscope. Previous studies proposed the quantification of the fluorescence intensity as an alternative for the classical end-point titer evaluation. However, the different distribution of bright/dark light linked to the nature of the self-antigen and its location in the cells result in different mean fluorescence intensities. The aim of the present study was to correlate Fluorescence Index (F.I.) with end-point titers for each well-defined ANA pattern. Routine serum samples were screened for ANA testing on HEp-2000 cells using Immuno Concepts Image Navigator System, and positive samples were serially diluted to assign the end-point titer. A comparison between F.I. and end-point titers related to 10 different staining patterns was made. According to our analysis, good technical performance of F.I. (97% sensitivity and 94% specificity) was found. A significant correlation between quantitative reading of F.I. and end-point titer groups was observed using Spearman's test and regression analysis. A conversion scale of F.I. in end-point titers for each recognized ANA-pattern was obtained. The Image Navigator offers the opportunity to improve worldwide harmonization of ANA test results. In particular, digital F.I. allows quantifying ANA titers by using just one sample dilution. It could represent a valuable support for the routine laboratory and an effective tool to reduce inter- and intra-laboratory variability. Copyright © 2018. Published by Elsevier B.V.

Microtubules in root hairs.

PubMed

Traas, J A; Braat, P; Emons, A M; Meekes, H; Derksen, J

1985-06-01

The microtubules of root hairs of Raphanus sativus, Lepidium sativum, Equisetum hyemale, Limnobium stoloniferum, Ceratopteris thalictroides, Allium sativum and Urtica dioica were investigated using immunofluorescence and electron microscopy. Arrays of cortical microtubules were observed in all hairs. The microtubules in the hairs show net axial orientations, but in Allium and Urtica helical microtubule patterns are also present. Numerical parameters of microtubules in Raphanus, Equisetum and Limnobium were determined from dry-cleave preparations. The results are discussed with respect to cell wall deposition and cell morphogenesis.

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

Kim, Woo Taek; Franceschi, V.R.; Okita, T.W.

The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.

A high throughput drug screening assay to identify compounds that promote oligodendrocyte differentiation using acutely dissociated and purified oligodendrocyte precursor cells.

PubMed

Lariosa-Willingham, Karen D; Rosler, Elen S; Tung, Jay S; Dugas, Jason C; Collins, Tassie L; Leonoudakis, Dmitri

2016-09-05

Multiple sclerosis is caused by an autoimmune response resulting in demyelination and neural degeneration. The adult central nervous system has the capacity to remyelinate axons in part through the generation of new oligodendrocytes (OLs). To identify clinical candidate compounds that may promote remyelination, we have developed a high throughput screening (HTS) assay to identify compounds that promote the differentiation of oligodendrocyte precursor cells (OPCs) into OLs. Using acutely dissociated and purified rat OPCs coupled with immunofluorescent image quantification, we have developed an OL differentiation assay. We have validated this assay with a known promoter of differentiation, thyroid hormone, and subsequently used the assay to screen the NIH clinical collection library. We have identified twenty-seven hit compounds which were validated by dose response analysis and the generation of half maximal effective concentration (EC50) values allowed for the ranking of efficacy. The assay identified novel promoters of OL differentiation which we attribute to (1) the incorporation of an OL toxicity pre-screen to allow lowering the concentrations of toxic compounds and (2) the utilization of freshly purified, non-passaged OPCs. These features set our assay apart from other OL differentiation assays used for drug discovery efforts. This acute primary OL-based differentiation assay should be of use to those interested in screening large compound libraries for the identification of drugs for the treatment of MS and other demyelinating diseases.

Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

PubMed Central

Bezek, D M; Baker, J C; Kaneene, J B

1988-01-01

A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood samples collected using ethylenediaminetetraacetic acid as an anticoagulant. Bovine viral diarrhea virus antigen was detected by immunofluorescence between 3 and 11 days postinfection in lymphocyte smears and 3 to 12 days postinfection in buffy coat smears. Isolation of virus from both lymphocytes and buffy coat preparations correlated with detection of immunofluorescence. Serum neutralizing antibody to bovine virus diarrhea virus was detected on day 10 postinfection. Buffy coat smears were as sensitive as lymphocyte smears for the detection of antigen by immunofluorescence. It appeared that immunofluorescent staining of white blood cells was an effective method of detecting bovine virus diarrhea viral antigen. PMID:2836047

Sudan Black B masks Mycobacterium avium subspecies paratuberculosis immunofluorescent antibody labeling

USDA-ARS?s Scientific Manuscript database

Autofluorescence and non-specific immunofluorescent labeling are common challenges associated with immunofluorescence experiments. Autofluorescence typically demonstrates a broad emission spectrum, increasing the potential for overlap with experiments that utilize multiple fluorophores. During immun...

Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells

PubMed Central

Fe Lanfranco, Maria; Loane, David J.; Mocchetti, Italo; Burns, Mark P.; Villapol, Sonia

2017-01-01

Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope® fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques to visualize the mRNA expression pattern of pro- and anti-inflammatory cytokines in microglia/macrophages cells. Using these methods, we can associate one mRNA to specific cell types when combining with different cellular markers by immunofluorescence. Results from RNAscope® probes IL-1β, TNFα, TGFβ, IL-10 or Arg1, showed colocalization with antibodies for microglia/macrophage cells. These target probes showed adequate sensitivity and specificity to detect mRNA expression. New FISH detection techniques combined with immunohistochemical techniques will help to jointly determine the protein and mRNA localization, as well as provide reliable quantification of the mRNA expression levels. PMID:29238736

Localization of the human 64kD autoantigen D1 to myofibrils in a subset of extraocular muscle fibers

NASA Technical Reports Server (NTRS)

Conley, C. A.; Fowler, V. M.

1999-01-01

PURPOSE. To evaluate the tissue-specific expression pattern of the 64kD human autoantigen D1, a tropomodulin-related protein that may be involved in thyroid-associated ophthalmopathy. METHODS. Recombinant 64kD human autoantigen D1 was generated in a bacterial expression system and used to immunize rabbits. Specific antibodies were affinity-purified and used for Western blots on normal and hyperthyroid rat and rabbit tissue, and immunofluorescence localization on cryosections of rat tissue. RESULTS. Anti-64kD human autoantigen D1 antibodies recognize specifically a approximately 70kD polypeptide in western blots of extraocular muscle, sternothyroid muscle, and smooth muscle. Immunofluorescence staining demonstrates that the 64kD human autoantigen D1 localizes to myofibrils in slow fibers from rat extraocular and sternothyroid muscle. The level of this protein is not altered in extraocular muscles from hyperthyroid rabbits. CONCLUSIONS. The 64kD human autoantigen D1 is expressed in slow fibers of extraocular and sternothyroid muscles as a component of myofibrils, and is not upregulated in conditions of hyperthyroidism.

Canine melanoma diagnosis: RACK1 as a potential biological marker.

PubMed

Campagne, C; Julé, S; Alleaume, C; Bernex, F; Ezagal, J; Château-Joubert, S; Estrada, M; Aubin-Houzelstein, G; Panthier, J-J; Egidy, G

2013-11-01

Melanoma diagnosis in dogs can be challenging due to the variety of histological appearances of canine melanocytic neoplasms. Markers of malignancy are needed. Receptor for activated C-kinase 1 (RACK1) was found to characterize melanomas in other mammals. We investigated the value of RACK1 detection in the classification of 19 cutaneous and 5 mucosal melanocytic neoplasms in dogs. These tumors were categorized as melanocytomas or benign and melanomas or malignant after evaluation of their morphology, mitotic index, and Ki-67 growth fraction. Using immunofluorescence, we confirmed microphthalmia-associated transcription factor (MITF) as a marker of normal and transformed melanocytic cells in dog tissues. All control (n = 10) and tumoral (n = 24) samples stained positively for MITF (34/34, 100%). Whereas RACK1 was not detected in healthy skin melanocytes, melanocytic lesions were all positive for RACK1 signal (24/24, 100%). RACK1 cytoplasmic staining appeared with 2 distinct distribution patterns: strong, diffuse, and homogeneous or granular and heterogeneous. All melanoma samples (13/13, 100%) stained homogeneously for RACK1. All melanocytomas (11/11, 100%) stained heterogeneously for RACK1. Immunohistochemistry was less consistent than immunofluorescence for all labelings in melanocytic lesions, which were often very pigmented. Thus, the fluorescent RACK1-MITF labeling pattern helped to distinguish melanomas from melanocytomas. Furthermore, RACK1 labeling correlated with 2 of 11 morphological features linked to malignancy: cell and nuclear size. These results suggest that RACK1 may be used as a marker in dog melanomas.

Dense Deposit Disease Mimicking a Renal Small Vessel Vasculitis

PubMed Central

Singh, Lavleen; Bhardwaj, Swati; Sinha, Aditi; Bagga, Arvind; Dinda, Amit

2016-01-01

Dense deposit disease is caused by fluid-phase dysregulation of the alternative complement pathway and frequently deviates from the classic membranoproliferative pattern of injury on light microscopy. Other patterns of injury described for dense deposit disease include mesangioproliferative, acute proliferative/exudative, and crescentic GN. Regardless of the histologic pattern, C3 glomerulopathy, which includes dense deposit disease and C3 GN, is defined by immunofluorescence intensity of C3c two or more orders of magnitude greater than any other immune reactant (on a 0–3 scale). Ultrastructural appearances distinguish dense deposit disease and C3 GN. Focal and segmental necrotizing glomerular lesions with crescents, mimicking a small vessel vasculitis such as ANCA-associated GN, are a very rare manifestation of dense deposit disease. We describe our experience with this unusual histologic presentation and distinct clinical course of dense deposit disease, discuss the pitfalls in diagnosis, examine differential diagnoses, and review the relevant literature. PMID:26361799

Tissue-Specific, Development-Dependent Phenolic Compounds Accumulation Profile and Gene Expression Pattern in Tea Plant [Camellia sinensis

PubMed Central

Li, Weiwei; Zhao, Lei; Meng, Fei; Wang, Yunsheng; Tan, Huarong; Yang, Hua; Wei, Chaoling; Wan, Xiaochun; Gao, Liping; Xia, Tao

2013-01-01

Phenolic compounds in tea plant [Camellia sinensis (L.)] play a crucial role in dominating tea flavor and possess a number of key pharmacological benefits on human health. The present research aimed to study the profile of tissue-specific, development-dependent accumulation pattern of phenolic compounds in tea plant. A total of 50 phenolic compounds were identified qualitatively using liquid chromatography in tandem mass spectrometry technology. Of which 29 phenolic compounds were quantified based on their fragmentation behaviors. Most of the phenolic compounds were higher in the younger leaves than that in the stem and root, whereas the total amount of proanthocyanidins were unexpectedly higher in the root. The expression patterns of 63 structural and regulator genes involved in the shikimic acid, phenylpropanoid, and flavonoid pathways were analyzed by quantitative real-time polymerase chain reaction and cluster analysis. Based on the similarity of their expression patterns, the genes were classified into two main groups: C1 and C2; and the genes in group C1 had high relative expression level in the root or low in the bud and leaves. The expression patterns of genes in C2-2-1 and C2-2-2-1 groups were probably responsible for the development-dependent accumulation of phenolic compounds in the leaves. Enzymatic analysis suggested that the accumulation of catechins was influenced simultaneously by catabolism and anabolism. Further research is recommended to know the expression patterns of various genes and the reason for the variation in contents of different compounds in different growth stages and also in different organs. PMID:23646127

The Sound Pattern of Japanese Surnames

ERIC Educational Resources Information Center

Tanaka, Yu

2017-01-01

Compound surnames in Japanese show complex phonological patterns, which pose challenges to current theories of phonology. This dissertation proposes an account of the segmental and prosodic issues in Japanese surnames and discusses their theoretical implications. Like regular compound words, compound surnames may undergo a sound alternation known…

Transitional epithelial lesions of the ureter in renal transplant rejection.

PubMed

Katz, J P; Greenstein, S M; Hakki, A; Miller, A; Katz, S M; Simonian, S

1988-04-01

The spectrum of ureteric lesions of human renal allografts, long attributed exclusively to postsurgical complications such as ischemia, has recently been shown to include the types of rejection seen in the kidney. Since the rejected ureter also exhibits transitional epithelial lesions that may impact on renal and ureteral function, we studied, by light, immunohistochemical, immunofluorescent, and electron microscopic techniques, ureters of 65 irreversibly rejected kidneys. Seven unused cadaver kidneys served as controls. Urothelial lesions, noticed in 57 of 65 ureters (88%), ranged from minimal basal vacuolization to complete sloughing with or without necrosis of the epithelial lining. Epithelial exfoliation was noticed in 31 cases (54.4%), and basal vacuolization, severe enough to produce cleavage of the epithelial junctions and thus create bullae, was noticed in 21 cases (36.8%). Immunofluorescent and immunoperoxidase stains, performed in 16 cases, were all positive for immunoglobulins but yielded varied results ranging from granular to linear staining, particularly in the region of the basal cells and the basement membrane. Electron microscopic findings confirmed the light microscopic alterations. By contrast, control ureters showed no lesions. Urothelial ureteric lesions might impede ureteral functions and result in obstruction or infection, thus compounding the consequences of renal allograft rejection. Moreover, elucidation of the pathophysiology of the process will advance the understanding of various cutaneous and transitional epithelial autoimmune conditions.

VPS53 mutations cause progressive cerebello-cerebral atrophy type 2 (PCCA2).

PubMed

Feinstein, Miora; Flusser, Hagit; Lerman-Sagie, Tally; Ben-Zeev, Bruria; Lev, Dorit; Agamy, Orly; Cohen, Idan; Kadir, Rotem; Sivan, Sara; Leshinsky-Silver, Esther; Markus, Barak; Birk, Ohad S

2014-05-01

Progressive cerebello-cerebral atrophy (PCCA) leading to profound mental retardation, progressive microcephaly, spasticity and early onset epilepsy, was diagnosed in four non-consanguineous apparently unrelated families of Jewish Moroccan ancestry. Common founder mutation(s) were assumed. Genome-wide linkage analysis and whole exome sequencing were done, followed by realtime PCR and immunofluorescent microscopy. Genome-wide linkage analysis mapped the disease-associated gene to 0.5 Mb on chromosome 17p13.3. Whole exome sequencing identified only two mutations within this locus, which were common to the affected individuals: compound heterozygous mutations in VPS53, segregating as expected for autosomal recessive heredity within all four families, and common in Moroccan Jews (∼1:37 carrier rate). The Golgi-associated retrograde protein (GARP) complex is involved in the retrograde pathway recycling endocytic vesicles to Golgi; c.2084A>G and c.1556+5G>A VPS53 founder mutations are predicted to affect the C-terminal domain of VPS53, known to be critical to its role as part of this complex. Immunofluorescent microscopy demonstrated swollen and abnormally numerous CD63 positive vesicular bodies, likely intermediate recycling/late endosomes, in fibroblasts of affected individuals. Autosomal recessive PCCA type 2 is caused by VPS53 mutations.

Learning language from the input: why innate constraints can't explain noun compounding.

PubMed

Ramscar, Michael; Dye, Melody

2011-02-01

Do the production and interpretation of patterns of plural forms in noun-noun compounds reveal the workings of innate constraints that govern morphological processing? The results of previous studies on compounding have been taken to support a number of important theoretical claims: first, that there are fundamental differences in the way that children and adults learn and process regular and irregular plurals, second, that these differences reflect formal constraints that govern the way the way regular and irregular plurals are processed in language, and third, that these constraints are unlikely to be the product of learning. In a series of seven experiments, we critically assess the evidence that is cited in support of these arguments. The results of our experiments provide little support for the idea that substantively different factors govern the patterns of acquisition, production and interpretation patterns of regular and irregular plural forms in compounds. Once frequency differences between regular and irregular plurals are accounted for, we find no evidence of any qualitative difference in the patterns of interpretation and production of regular and irregular plural nouns in compounds, in either adults or children. Accordingly, we suggest that the pattern of acquisition of both regular and irregular plurals in compounds is consistent with a simple account, in which children learn the conventions that govern plural compounding using evidence that is readily available in the distribution patterns of adult speech. Copyright © 2010 Elsevier Inc. All rights reserved.

The pattern of tyrosine phosphorylation in human sperm in response to binding to zona pellucida or hyaluronic acid.

PubMed

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny; Huszar, Gabor

2014-05-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.

The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

PubMed Central

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny

2014-01-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP. PMID:24077441

The Association of Palmitoylethanolamide with Luteolin Decreases Autophagy in Spinal Cord Injury.

PubMed

Siracusa, Rosalba; Paterniti, Irene; Bruschetta, Giuseppe; Cordaro, Marika; Impellizzeri, Daniela; Crupi, Rosalia; Cuzzocrea, Salvatore; Esposito, Emanuela

2016-08-01

Spinal cord injury (SCI) is a devastating condition of the central nervous system (CNS) often resulting in severe functional impairment and for which there are not yet restorative therapies. In the present study, we performed a widely used model of SCI to determine the neuroprotective propriety of palmitoylethanolamide (PEA) and the antioxidant effect of a flavonoid luteolin (Lut), given as a co-ultramicronized compound co-ultraPEALut. In particular, by western blot analysis and immunofluorescence staining, we investigated whether this compound (at the dose of 1 mg/kg) was able to modulate autophagy. Our results showed that treatment with co-ultraPEALut after SCI reduced the expression of proteins promoter of autophagy such as Beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (MAP-LC3). In contrast, this compound decreased the levels of mammalian target of rapamycin (mTOR), p-Akt, and p-70S6K which are proteins that inhibit autophagy. These data confirmed that the protective role of co-ultraPEALut is associated with inhibition of excessive autophagy and regulation of protein degradation. Therefore, treatment with co-ultraPEALut could be considered as a possible therapeutic approach in an acute traumatic lesion like SCI.

Variations of attractors and wavelet spectra of the immunofluorescence distributions for women in the pregnant period

NASA Astrophysics Data System (ADS)

Galich, Nikolay E.

2008-07-01

Communication contains the description of the immunology data treatment. New nonlinear methods of immunofluorescence statistical analysis of peripheral blood neutrophils have been developed. We used technology of respiratory burst reaction of DNA fluorescence in the neutrophils cells nuclei due to oxidative activity. The histograms of photon count statistics the radiant neutrophils populations' in flow cytometry experiments are considered. Distributions of the fluorescence flashes frequency as functions of the fluorescence intensity are analyzed. Statistic peculiarities of histograms set for women in the pregnant period allow dividing all histograms on the three classes. The classification is based on three different types of smoothing and long-range scale averaged immunofluorescence distributions, their bifurcation and wavelet spectra. Heterogeneity peculiarities of long-range scale immunofluorescence distributions and peculiarities of wavelet spectra allow dividing all histograms on three groups. First histograms group belongs to healthy donors. Two other groups belong to donors with autoimmune and inflammatory diseases. Some of the illnesses are not diagnosed by standards biochemical methods. Medical standards and statistical data of the immunofluorescence histograms for identifications of health and illnesses are interconnected. Peculiarities of immunofluorescence for women in pregnant period are classified. Health or illness criteria are connected with statistics features of immunofluorescence histograms. Neutrophils populations' fluorescence presents the sensitive clear indicator of health status.

Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function.

PubMed

Ferro, Austin; Carbone, Emily; Marzouk, Evan; Siegel, Asher; Nguyen, Donna; Polley, Kailen; Hartman, Jessilyn; Frederick, Kimberley; Ives, Stephen; Lagalwar, Sarita

2017-01-22

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ataxias and other movement disorders marked by progressive degeneration of the cerebellum. The goal of this protocol is to assess mitochondrial dysfunction in Spinocerebellar ataxia type 1 (SCA1) and assess the efficacy of pharmacological targeting of metabolic respiration via the water-soluble compound succinic acid to slow disease progression. This approach is applicable to other cerebellar diseases and can be adapted to a host of water-soluble therapies. Ex vivo analysis of mitochondrial respiration is used to detect and quantify disease-related changes in mitochondrial function. With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic booster succinic acid by dissolving this compound directly into the home cage drinking water. The ability of the drug to pass the blood brain barrier can be deduced using high performance liquid chromatography (HPLC). The efficacy of these compounds can then be tested using multiple behavioral paradigms including the accelerating rotarod, balance beam test and footprint analysis. Cytoarchitectural integrity of the cerebellum can be assessed using immunofluorescence assays that detect Purkinje cell nuclei and Purkinje cell dendrites and soma. These methods are robust techniques for determining mitochondrial dysfunction and the efficacy of treatment with water-soluble compounds in cerebellar neurodegenerative disease.

Accuracy of Immunofluorescence in the Diagnosis of Primary Ciliary Dyskinesia

PubMed Central

Frost, Emily; Dixon, Mellisa; Ollosson, Sarah; Kilpin, Kate; Patel, Mitali; Scully, Juliet; Rogers, Andrew V.; Mitchison, Hannah M.; Bush, Andrew; Hogg, Claire

2017-01-01

Rationale: The standard approach to diagnosis of primary ciliary dyskinesia (PCD) in the United Kingdom consists of assessing ciliary function by high-speed microscopy and ultrastructure by election microscopy, but equipment and expertise is not widely available internationally. The identification of biallelic disease-causing mutations is also diagnostic, but many disease-causing genes are unknown, and testing is not widely available outside the United States. Fluorescent antibodies to ciliary proteins are used to validate research genetic studies, but diagnostic utility in this disease has not been systematically evaluated. Objectives: To determine utility of a panel of six fluorescent labeled antibodies as a diagnostic tool for PCD. Methods: The study used immunofluorescent labeling of nasal brushings from a discovery cohort of 35 patients diagnosed with PCD by ciliary ultrastructure, and a diagnostic accuracy cohort of 386 patients referred with symptoms suggestive of disease. The results were compared with diagnostic outcome. Measurements and Main Results: Immunofluorescence correctly identified mislocalized or absent staining in 100% of the discovery cohort. In the diagnostic cohort immunofluorescence successfully identified 22 of 25 patients with PCD and normal staining in all 252 in whom PCD was considered highly unlikely. In addition, immunofluorescence provided a result in 55% (39) of cases that were previously inconclusive. Immunofluorescence results were available within 14 days, costing $187 per sample compared with electron microscopy (27 days; cost $1,452). Conclusions: Immunofluorescence is a highly specific diagnostic test for PCD, and it improves the speed and availability of diagnostic testing. However, sensitivity is limited and immunofluorescence is not suitable as a stand-alone test. PMID:28199173

Delay, change and bifurcation of the immunofluorescence distribution attractors in health statuses diagnostics and in medical treatment

NASA Astrophysics Data System (ADS)

Galich, Nikolay E.; Filatov, Michael V.

2008-07-01

Communication contains the description of the immunology experiments and the experimental data treatment. New nonlinear methods of immunofluorescence statistical analysis of peripheral blood neutrophils have been developed. We used technology of respiratory burst reaction of DNA fluorescence in the neutrophils cells nuclei due to oxidative activity. The histograms of photon count statistics the radiant neutrophils populations' in flow cytometry experiments are considered. Distributions of the fluorescence flashes frequency as functions of the fluorescence intensity are analyzed. Statistic peculiarities of histograms set for healthy and unhealthy donors allow dividing all histograms on the three classes. The classification is based on three different types of smoothing and long-range scale averaged immunofluorescence distributions and their bifurcation. Heterogeneity peculiarities of long-range scale immunofluorescence distributions allow dividing all histograms on three groups. First histograms group belongs to healthy donors. Two other groups belong to donors with autoimmune and inflammatory diseases. Some of the illnesses are not diagnosed by standards biochemical methods. Medical standards and statistical data of the immunofluorescence histograms for identifications of health and illnesses are interconnected. Possibilities and alterations of immunofluorescence statistics in registration, diagnostics and monitoring of different diseases in various medical treatments have been demonstrated. Health or illness criteria are connected with statistics features of immunofluorescence histograms. Neutrophils populations' fluorescence presents the sensitive clear indicator of health status.

Expression of agrin, dystroglycan, and utrophin in normal renal tissue and in experimental glomerulopathies.

PubMed

Raats, C J; van den Born, J; Bakker, M A; Oppers-Walgreen, B; Pisa, B J; Dijkman, H B; Assmann, K J; Berden, J H

2000-05-01

The dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane.

Expression of Agrin, Dystroglycan, and Utrophin in Normal Renal Tissue and in Experimental Glomerulopathies

PubMed Central

Raats, C. J. Ilse; van den Born, Jacob; Bakker, Marinka A. H.; Oppers-Walgreen, Birgitte; Pisa, Brenda J. M.; Dijkman, Henry B. P. M.; Assmann, Karel J. M.; Berden, Jo H. M.

2000-01-01

The dystrophin-glycoprotein complex, which comprises α- and β-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against α- and β-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane. PMID:10793086

Novel HLA-G-Binding Leukocyte Immunoglobulin-Like Receptor (LILR) Expression Patterns in Human Placentas and Umbilical Cords

PubMed Central

McIntire, Ramsey H.; Sifers, Travis; Platt, J. Sue; Ganacias, Karen G.; Langat, Daudi K.; Hunt, Joan S.

2008-01-01

Human placentas are sources of cytokines, hormones and other substances that program receptive cells. One of these substances is HLA-G, which influences the functioning of both leukocytes and endothelial cells. In this study we investigated the possibility that these and/or other types of cells in extraembryonic fetal tissues might respond to HLA-G by interacting with one or another of the leukocyte immunoglobulin-like receptors (LILR). LILRB1 is expressed by most leukocytes and LILRB2 is expressed primarily by monocytes, macrophages and dendritic cells. Analysis of term placentas by immunohistochemistry and Real Time PCR demonstrated that LILRB1 and LILRB2 protein and specific messages are produced in the mesenchyme of term villous placenta but are differently localized. LILRB1 was abundant in stromal cells and LILRB2 was prominent perivascularly. Neither receptor was identified in trophoblast. Further investigation using double label immunofluorescence indicated that placental vascular smooth muscle but not endothelia exhibit LILRB2. Term umbilical cord exhibited the same LILRB2 patterns as term placenta. Samples obtained by laser capture dissection of vascular smooth muscle in umbilical cords demonstrated LILRB2 mRNA, and double labeling immunofluorescence showed that cord vascular smooth muscle but not endothelium exhibited LILRB2 protein. The presence of LILRB1 in placental stromal cells and LILRB2 in vascular smooth muscle strongly suggest that HLA-G has novel functions in these tissues that could include regulation of placental immunity as well as development and function of the extraembryonic vasculature. PMID:18538388

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro.

PubMed

Tansriratanawong, Kallapat; Ishikawa, Hiroshi; Toyomura, Junko; Sato, Soh

2017-10-01

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial-endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

Immunofluorescence reveals unusual patterns of labelling for connexin43 localized to calbindin-D28K-positive interstitial cells in the pineal gland.

PubMed

Tsao, D D; Wang, S G; Lynn, B D; Nagy, J I

2017-06-01

Gap junctions between cells in the pineal gland have been described ultrastructurally, but their connexin constituents have not been fully characterized. We used immunofluorescence in combination with markers of pineal cells to document the cellular localization of connexin43 (Cx43). Immunofluorescence labelling of Cx43 with several different antibodies was widely distributed throughout the pineal, whereas another connexin examined, connexin26, was not found in pineal but only in surrounding leptomeninges. Labelling apparently associated with plasma membranes was visualized either as fine Cx43-puncta (1-2 μm) or as unusually large pools of Cx43 ranging up to 4-7 μm in diameter or length. These puncta and pools were highly concentrated in perivascular spaces, where they were associated with numerous cells devoid of labelling for markers of pinealocytes (e.g. tryptophan hydroxylase and serotonin), and where they were minimally associated with blood vessels and lacked association with resident macrophages. Astrocytes labelled for glial fibrillary acidic protein were largely restricted to the anterior pole of the pineal gland, where they displayed only fine and sparse Cx43-puncta along their processes. Labelling for Cx43 was localized largely though not exclusively to the somata and long processes of a subpopulation of perivascular interstitial cells that were immunopositive for calbindin-D28K. These cells were often located among dense bundles or termination areas of sympathetic fibres labelled for tyrosine hydroxylase or serotonin. The results indicate that interstitial cells form abundant gap junctions composed of Cx43, and suggest that gap junction-mediated intracellular communication by these cells supports the activities of pinealocytes. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

A paperless autoimmunity laboratory: myth or reality?

PubMed

Lutteri, Laurence; Dierge, Laurine; Pesser, Martine; Watrin, Pascale; Cavalier, Etienne

2016-08-01

Testing for antinuclear antibodies is the most frequently prescribed analysis for the diagnosis of rheumatic diseases. Indirect immunofluorescence remains the gold standard method for their detection despite the increasing use of alternative techniques. In order to standardize the manual microscopy reading, automated acquisition and interpretation systems have emerged. This publication enables us to present our method of interpretation and characterization of antinuclear antibodies based on a cascade of analyses and to share our everyday experience of the G Sight from Menarini. The positive/negative discrimination on Hep cells 2000 is correct in 85% of the cases. In most of the false negative results, it is a question of aspecific or low titers patterns, but a few cases of SSA speckled patterns of low titers demonstrated a probability index below 8. Regarding the pattern recognition, some types and mixed patterns are not properly recognized. Concerning the probability index correlated in some studies to final titer, the weak fluorescence of certain patterns and the random presence of artifacts that distort the index don't lead us to continue it in our daily practice. In conclusion, automated reading systems facilitate the reporting of results and traceability of patterns but still require the expertise of a laboratory technologist for positive/negative discrimination and for pattern recognition.

OUR EXPERIENCE WITH IMMUNOFLUORESCENCE (RELATING TO 250 SERA STUDIED AT THE NANCY DERMATOLOGICAL CLINIC)

DTIC Science & Technology

The principle of immunofluorescence, discovered by Coons in 1942, was applied to the diagnosis of syphilis by Deacon, Falcon and Harris in 1957...technique and to determine its value (Dauget, Fribourg-Blanc, Thivolet). The research reported here, relating to 250 sera, seems to confirm the great importance of the immunofluorescence test (IF) in the diagnosis of syphilis .

[Evaluation of a Computer-Aided Microscope System and Its Anti-Nuclear Antibody Test Kit for Indirect Immunofluorescence Assay].

PubMed

Hayashi, Nobuhide; Saegusa, Jun; Uto, Kenichi; Oyabu, Chinami; Saito, Toshiharu; Sato, Itsuko; Kawano, Seiji; Kumagai, Shunichi

2016-02-01

Antinuclear antibody (ANA) testing is indispensable for diagnosing and understanding clinical conditions of autoimmune diseases. The indirect immunofluorescence assay (IFA) is the gold standard for ANA screening, and it can detect more than 100 different antibodies, such as anti-PCNA as well as anti-cytoplasmic antibodies. However, complicated procedures of conventional IFA and visual interpretation require highly skilled laboratory staff. This study evaluates the capability, characteristics, and applicability of the recently developed ANA detection system (EUROPattern Cosmic IFA System, EPA) using HEp20-10 cells and the automated pattern recognition microscope. Findings using EPA and conventional methods were compared in 282 sera obtained from connective tissue disease patients and 250 sera from healthy individuals. The concordance of the positivity rate, antibody titer (within +/- 1 tube difference), and the accurate recognition rate of ANA patterns between the automated EPA method and the microscopic judgement of the EPA image by eye was 98.9, 97.4, and 55.3%, respectively. The EPA method showed concordance of the positivity rate as high as 93.3% and concordance of the antibody titer as high as 94.0% (within +/- 1 titer) compared with the conventional method. Regarding the four typical patterns of ANA (homogeneous, speckled, nucleolar, and centromere), large differences between the EPA and conventional methods were not observed, and the rate of concordance between the final EPA result and the conventional method was from 94.1 to 100%. The positivity rate of ANA using the EPA and conventional methods showed marked agreement among the six connective tissue diseases (SLE, MCTD, SSc, PM/DM, and SS) and healthy individuals. Although the EPA system is not considered a complete system and laboratory staff should verify the results, it is a useful system for routine ANA analysis because it contributes to ANA standardization and an efficient workflow.

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays

PubMed Central

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-01-01

Aims: To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). Methods: The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques—an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. Results: All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (κ > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (κ = 0.63). Conclusion: The assay kit marketed as Varelisa allows accurate detection of LKM1. PMID:12461054

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays.

PubMed

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-12-01

To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). The assay kit marketed as Varelisa allows accurate detection of LKM1.

X inactivation in a mammal species with three sex chromosomes.

PubMed

Veyrunes, Frédéric; Perez, Julie

2018-06-01

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.

Development of a central nervous system axonal myelination assay for high throughput screening.

PubMed

Lariosa-Willingham, Karen D; Rosler, Elen S; Tung, Jay S; Dugas, Jason C; Collins, Tassie L; Leonoudakis, Dmitri

2016-04-22

Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.

Anti-adenovirus activities of shikonin, a component of Chinese herbal medicine in vitro.

PubMed

Gao, Hong; Liu, Lei; Qu, Zhang-Yi; Wei, Feng-Xiang; Wang, Shu-Qiu; Chen, Guang; Qin, Le; Jiang, Fu-Yang; Wang, Ying-Chen; Shang, Lei; Gao, Chun-Yan

2011-01-01

Radix Lithosperm eyrthrorhizon is a common prescription compound in traditional Chinese medicine. Shikonin is a major component of Radix Lithospermi and has various biological activities. We have investigated the inhibitory effect of shikonin on the growth of adenovirus type 3 (AdV3) in vitro. The antiviral function of shikonin against AdV3 and its virus inhibition ratio were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method (MTT). The expression of hexon protein in AdV3 was determined by immunofluorescence assay using laser scanning confocal microscopy (LSCM) and Western blot analysis. In addition, the rate of apoptosis in cells infected by AdV3 was determined by flow cytometry. Shikonin (0.0156-1 µM) inhibited growth of AdV3 in a concentration-dependent manner with a virus inhibition rate of 23.8-69.1%. Expression of hexon protein in AdV3 was higher in the virus control group than in the shikonin-treated groups as determined by immunofluorescence assay and Western blotting (p<0.05). The rate of shikonin-treated HeLa cell apoptosis had a statistically significant decrease with increasing concentration of drug (p<0.05). Our data demonstrate that shikonin possesses anti-AdV3 capabilities and that the potential antiviral mechanism might involve inhibiting the degree of apoptosis and hexon protein expression of AdV.

Automated Quantification of DNA Demethylation Effects in Cells via 3D Mapping of Nuclear Signatures and Population Homogeneity Assessment1

PubMed Central

Gertych, Arkadiusz; Wawrowsky, Kolja A.; Lindsley, Erik; Vishnevsky, Eugene; Farkas, Daniel L.; Tajbakhsh, Jian

2009-01-01

Background Today’s advanced microscopic imaging applies to the preclinical stages of drug discovery that employ high-throughput and high-content three-dimensional (3D) analysis of cells to more efficiently screen candidate compounds. Drug efficacy can be assessed by measuring response homogeneity to treatment within a cell population. In this study topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti-cancer agents: 5-azacytidine (5-AZA) and octreotide (OCT). Methods Mouse pituitary folliculostellate TtT-GF cells treated with 5-AZA and OCT for 48 hours, and untreated populations, were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei (n=163). Cell images were processed utilizing an automated 3D analysis software that we developed by combining seeded watershed segmentation to extract nuclear shells with measurements of Kullback-Leibler’s (K-L) divergence to analyze cell population homogeneity in the relative nuclear distribution patterns of MeC versus DAPI stained sites. Each cell was assigned to one of the four classes: similar, likely similar, unlikely similar and dissimilar. Results Evaluation of the different cell groups revealed a significantly higher number of cells with similar or likely similar MeC/DAPI patterns among untreated cells (~100%), 5-AZA-treated cells (90%), and a lower degree of same type of cells (64%) in the OCT-treated population. The latter group contained (28%) of unlikely similar or dissimilar (7%) cells. Conclusion Our approach was successful in the assessment of cellular behavior relevant to the biological impact of the applied drugs, i.e. the reorganization of MeC/DAPI distribution by demethylation. In a comparison with other metrics, K-L divergence has proven to be a more valuable and robust tool for categorization of individual cells within a population, with potential applications in epigenetic drug screening. PMID:19459215

Flow cytometric immunofluorescence of rat anterior pituitary cells

NASA Technical Reports Server (NTRS)

Hatfield, J. Michael; Hymer, W. C.

1985-01-01

A flow cytometric immunofluorescence technique was developed for the quantification of growth hormone, prolactin, and luteinizing hormone producing cells. The procedure is based on indirect-immunofluorescence of intracellular hormone using an EPICS V cell sorter and can objectively count 50,000 cells in about 3 minutes. It can be used to study the dynamics of pituitary cell populations under various physiological and pharmacological conditions.

Rapid and simple method of photobleaching to reduce background autofluorescence in lung tissue sections.

PubMed

Kumar, B Santhosh; Sandhyamani, S; Nazeer, Shaiju S; Jayasree, R S

2015-02-01

Autofluorescence exhibited by tissues often interferes with immunofluorescence. Using imaging and spectral analysis, we observed remarkable reduction of autofluorescence of formalin fixed paraffin embedded tissues irradiated with light prior to incubation with immunofluorescent dyes. The technique of photobleaching offers significant improvement in the quality and specificity of immunofluorescence. This has the potential for better techniques for disease diagnosis.

Synthesis and antitumor activity of some substituted indazole derivatives.

PubMed

Abbassi, Najat; Rakib, El Mostapha; Chicha, Hakima; Bouissane, Latifa; Hannioui, Abdellah; Aiello, Cinzia; Gangemi, Rosaria; Castagnola, Patrizio; Rosano, Camillo; Viale, Maurizio

2014-06-01

Some new N-[6-indazolyl]arylsulfonamides and N-[alkoxy-6-indazolyl]arylsulfonamides were prepared by the reduction of 2-alkyl-6-nitroindazoles with SnCl2 in different alcohols, followed by coupling the corresponding amine with arylsulfonyl chlorides in pyridine. The newly synthesized compounds were evaluated for their antiproliferative and apoptotic activities against two human tumor cell lines: A2780 (ovarian carcinoma) and A549 (lung adenocarcinoma). Preliminary in vitro pharmacological studies revealed that N-(2-allyl-2H-indazol-6-yl)-4-methoxybenzenesulfonamide 4 and N-[7-ethoxy-2-(4-methyl-benzyl)-2H-indazol-6-yl]-4-methyl-benzenesulfonamide 9 exhibited significant antiproliferative activity against the A2780 and A549 cell lines with IC50 values in the range from 4.21 to 18.6 µM, and also that they trigger apoptosis in a dose-dependent manner. Furthermore, both active compounds were able to cause an arrest of cells in the G2/M phase of the cell cycle, typical but not exclusive of tubulin interacting agents, although only infrequent interactions with the microtubule network were observed by immunofluorescence microscopy, while docking analysis showed a possible different behavior between the two active compounds. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of criteria for the postmortem diagnosis of mycoplasmal pneumonia of swine.

PubMed Central

Armstrong, C H; Scheidt, A B; Thacker, H L; Runnels, L J; Freeman, M J

1984-01-01

Ten swine from each of five herds believed to be affected with mycoplasmal pneumonia of swine and ten swine from each of five herds believed to be mycoplasmal pneumonia-free were selected for postmortem study. Lungs from the 100 swine were examined; grossly and microscopically for lesions typical of mycoplasmal pneumonia of swine and culturally and by an indirect immunofluorescent procedure for the presence of Mycoplasma hyopneumoniae. Nineteen of the lungs had both gross and microscopic lesions typical of mycoplasmal pneumonia of swine and 13 (68%) of these were infected, i.e. were culturally and/or indirect immunofluorescent positive. Absence of gross lesions did not prove freedom from mycoplasmal pneumonia, 14 of 73 (19%) grossly normal lungs were found to be infected with M. hyopneumoniae. Comparison of the indirect immunofluorescent and cultural examination, as methods of diagnosing mycoplasma pneumonia, revealed that neither procedure alone was reliable in the case of negative results. Ten lungs were indirect immunofluorescent negative and culturally positive and seven were culturally negative and indirect immunofluorescent positive (11 lungs were positive by both procedures). It was concluded that a definitive diagnosis of mycoplasmal pneumonia of swine requires that M. hyopneumoniae be visualized in indirect immunofluorescent stained lung sections or that it be recovered culturally. PMID:6434167

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

PubMed

Son, Kyung-No; Liang, Zhiguo; Lipton, Howard L

2015-09-01

Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Thiosemicarbazones and Phthalyl-Thiazoles compounds exert antiviral activity against yellow fever virus and Saint Louis encephalitis virus.

PubMed

Pacca, Carolina Colombelli; Marques, Rafael Elias; Espindola, José Wanderlan P; Filho, Gevânio B O Oliveira; Leite, Ana Cristina Lima; Teixeira, Mauro Martins; Nogueira, Mauricio L

2017-03-01

Arboviruses, arthropod-borneviruses, are frequency associated to human outbreak and represent a serious health problem. The genus Flavivirus, such as Yellow Fever Virus (YFV) and Saint Louis Encephalitis Virus (SLEV), are important pathogens with high morbidity and mortality worldwide. In Brazil, YFV is maintained in sylvatic cycle, but many cases are notified annually, despite the efficiency of vaccine. SLEV causes an acute encephalitis and is widely distributed in the Americas. There is no specific antiviral drugs for these viruses, only supporting treatment that can alleviate symptoms and prevent complications. Here, we evaluated the potential anti-YFV and SLEV activity of a series of thiosemicarbazones and phthalyl-thiazoles. Plaque reduction assay, flow cytometry, immunofluorescence and cellular viability were used to test the compounds in vitro. Treated cells showed efficient inhibition of the viral replication at concentrations that presented minimal toxicity to cells. The assays showed that phthalyl-thiazole and phenoxymethyl-thiosemicarbazone reduced 60% of YFV replication and 75% of SLEV replication. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Targeting of Arenavirus RNA Synthesis by a Carboxamide-Derivatized Aromatic Disulfide with Virucidal Activity

PubMed Central

Sepúlveda, Claudia S.; García, Cybele C.; Levingston Macleod, Jesica M.

2013-01-01

Several arenaviruses can cause severe hemorrhagic fever (HF) in humans, representing a public health threat in endemic areas of Africa and South America. The present study characterizes the potent virucidal activity of the carboxamide-derivatized aromatic disulfide NSC4492, an antiretroviral zinc finger-reactive compound, against Junín virus (JUNV), the causative agent of Argentine HF. The compound was able to inactivate JUNV in a time and temperature-dependent manner, producing more than 99 % reduction in virus titer upon incubation with virions at 37°C for 90 min. The ability of NSC4492-treated JUNV to go through different steps of the multiplication cycle was then evaluated. Inactivated virions were able to bind and enter into the host cell with similar efficiency as control infectious particles. In contrast, treatment with NSC4492 impaired the capacity of JUNV to drive viral RNA synthesis, as measured by quantitative RT-PCR, and blocked viral protein expression, as determined by indirect immunofluorescence. These results suggest that the disulfide NSC4492 targets on the arenavirus replication complex leading to impairment in viral RNA synthesis. Additionally, analysis of VLP produced in NSC4492-treated cells expressing JUNV matrix Z protein revealed that the compound may interact with Z resulting in an altered aggregation behavior of this protein, but without affecting its intrinsic self-budding properties. The potential perspectives of NSC4492 as an inactivating vaccinal compound for pathogenic arenaviruses are discussed. PMID:24278404

Cancer associated fibroblasts (CAFs) are activated in cutaneous basal cell carcinoma and in the peritumoural skin.

PubMed

Omland, Silje Haukali; Wettergren, Erika Elgstrand; Mollerup, Sarah; Asplund, Maria; Mourier, Tobias; Hansen, Anders Johannes; Gniadecki, Robert

2017-10-07

Cutaneous basal cell carcinoma (BCC) is the commonest cancer worldwide. BCC is locally invasive and the surrounding stromal microenvironment is pivotal for tumourigenesis. Cancer associated fibroblasts (CAFs) in the microenvironment are essential for tumour growth in a variety of neoplasms but their role in BCC is poorly understood. Material included facial BCC and control skin from the peritumoural area and from the buttocks. With next-generation sequencing (NGS) we compared mRNA expression between BCC and peritumoural skin. qRT-PCR, immunohistochemical and immunofluorescent staining were performed to validate the NGS results and to investigate CAF-related cyto-and chemokines. NGS revealed upregulation of 65 genes in BCC coding for extracellular matrix components pointing at CAF-related matrix remodeling. qRT-PCR showed increased mRNA expression of CAF markers FAP-α, PDGFR-β and prolyl-4-hydroxylase in BCC. Peritumoural skin (but not buttock skin) also exhibited high expression of PDGFR-β and prolyl-4-hydroxylase but not FAP-α. We found a similar pattern for the CAF-associated chemokines CCL17, CCL18, CCL22, CCL25, CXCL12 and IL6 with high expression in BCC and peritumoural skin but absence in buttock skin. Immunofluorescence revealed correlation between FAP-α and PDGFR-β and CXCL12 and CCL17. Matrix remodeling is the most prominent molecular feature of BCC. CAFs are present within BCC stroma and associated with increased expression of chemokines involved in tumour progression and immunosuppression (CXCL12, CCL17). Fibroblasts from chronically sun-exposed skin near tumours show gene expression patterns resembling that of CAFs, indicating that stromal fibroblasts in cancer-free surgical BCC margins exhibit a tumour promoting phenotype.

Uniformity under in vitro conditions: Changes in the phenotype of cancer cell lines derived from different medulloblastoma subgroups.

PubMed

Chlapek, Petr; Zitterbart, Karel; Kren, Leos; Filipova, Lenka; Sterba, Jaroslav; Veselska, Renata

2017-01-01

Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.

Up-regulated ephrinB3/EphB3 expression in intractable temporal lobe epilepsy patients and pilocarpine induced experimental epilepsy rat model.

PubMed

Huang, Hao; Li, Ruohan; Yuan, Jinxian; Zhou, Xin; Liu, Xi; Ou, Shu; Xu, Tao; Chen, Yangmei

2016-05-15

EphB family receptor tyrosine kinases, in cooperation with cell surface-bound ephrinB ligands, play a critical role in maintenance of dendritic spine morphogenesis, axons guidance, synaptogenesis, synaptic reorganization and plasticity in the central nervous system (CNS). However, the expression pattern of ephrinB/EphB in intractable temporal lobe epilepsy (TLE) and the underlying molecular mechanisms during epileptogenesis remain poorly understood. Here we investigated the expression pattern and cellular distribution of ephrinB/EphB in intractable TLE patients and lithium chloride-pilocarpine induced TLE rats using real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry, double-labeled immunofluorescence and Western blot analysis. Compared to control groups, ephrinB3 and EphB3 mRNA expression were significantly up-regulated in intractable TLE patients and TLE rats, while the mRNA expression trend of ephrinB1/2 and EphB1/2/4/6 in intractable TLE patients and TLE rats were inconsistent. Western blot analysis and semi-quantitative immunohistochemistry confirmed that ephrinB3 and EphB3 protein level were up-regulated in intractable TLE patients and TLE rats. At the same time, double-labeled immunofluorescence indicate that ephrinB3 was expressed mainly in the cytoplasm and protrusions of glia and neurons, while EphB3 was expressed mainly in the cytoplasm of neurons. Taken together, up-regulated expression of ephrinB3/EphB3 in intractable TLE patients and experimental TLE rats suggested that ephrinB3/EphB3 might be involved in the pathogenesis of TLE. Copyright © 2016 Elsevier B.V. All rights reserved.

Finding Patterns

ERIC Educational Resources Information Center

Mulvey, Bridget

2016-01-01

Students best learn science through a combination of science inquiry and language learning. This article presents a series of chemistry lessons on the naming of compounds. The weeklong unit focuses on patterns across compound names and chemical formulas and addresses several of the "Next Generation Science Standards" (NGSS Lead States…

NASA's Software Bank (NETS)

NASA Technical Reports Server (NTRS)

1992-01-01

NETS (A Neural Network Development Tool) is a software system for mimicking the human brain. It is used in a University of Arkansas project in pattern matching of chemical systems. If successful, chemists would be able to identify mixtures of compounds without long and costly separation procedures. Using NETS, the group has trained the computer to recognize pattern relationships in a known compound and associate the results to an unknown compound. The research appears to be promising.

Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue

PubMed Central

Robertson, David; Savage, Kay; Reis-Filho, Jorge S; Isacke, Clare M

2008-01-01

Background Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues. Results We present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4°C or in the longer term at -20°C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously. Conclusion This method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples. PMID:18366689

Neural activity in the hippocampus during conflict resolution.

PubMed

Sakimoto, Yuya; Okada, Kana; Hattori, Minoru; Takeda, Kozue; Sakata, Shogo

2013-01-15

This study examined configural association theory and conflict resolution models in relation to hippocampal neural activity during positive patterning tasks. According to configural association theory, the hippocampus is important for responses to compound stimuli in positive patterning tasks. In contrast, according to the conflict resolution model, the hippocampus is important for responses to single stimuli in positive patterning tasks. We hypothesized that if configural association theory is applicable, and not the conflict resolution model, the hippocampal theta power should be increased when compound stimuli are presented. If, on the other hand, the conflict resolution model is applicable, but not configural association theory, then the hippocampal theta power should be increased when single stimuli are presented. If both models are valid and applicable in the positive patterning task, we predict that the hippocampal theta power should be increased by presentation of both compound and single stimuli during the positive patterning task. To examine our hypotheses, we measured hippocampal theta power in rats during a positive patterning task. The results showed that hippocampal theta power increased during the presentation of a single stimulus, but did not increase during the presentation of a compound stimulus. This finding suggests that the conflict resolution model is more applicable than the configural association theory for describing neural activity during positive patterning tasks. Copyright © 2012 Elsevier B.V. All rights reserved.

Concentration evolution of pharmaceutically active compounds in raw urban and industrial wastewater.

PubMed

Camacho-Muñoz, Dolores; Martín, Julia; Santos, Juan Luis; Aparicio, Irene; Alonso, Esteban

2014-09-01

The distribution of pharmaceutically active compounds in the environment has been reported in several works in which wastewater treatment plants have been identified as the main source of these compounds to the environment. The concentrations of these compounds in influent wastewater can vary widely not only during the day but also along the year, because of the seasonal-consumption patterns of some pharmaceuticals. However, only few studies have attempted to assess the hourly variability of the concentrations of pharmaceutically active compounds in wastewater. In this work, the distribution and seasonal and hourly variability of twenty-one pharmaceuticals, belonging to seven therapeutic groups, have been investigated in urban and industrial wastewater. The highest concentrations of pharmaceutically active compounds, except salicylic acid, were found in urban wastewater, especially in the case of anti-inflammatory drugs and caffeine. The highest concentrations of salicylic acid were measured in industrial wastewater, reaching concentration levels up to 3295μgL(-)(1). The studied pharmaceutically active compounds showed different distribution patterns during winter and summer periods. Temporal variability of pharmaceutically active compounds during a 24-h period showed a distribution in concordance with their consumption and excretion patterns, in the case of urban wastewater, and with the schedule of industrial activities, in the case of industrial wastewater. Copyright © 2014 Elsevier Ltd. All rights reserved.

Immunofluorescence Staining Protocols for Major Autophagy Proteins Including LC3, P62, and ULK1 in Mammalian Cells in Response to Normoxia and Hypoxia.

PubMed

Li, Wen; Li, Shupeng; Li, Yifang; Lin, Xiaoying; Hu, Yongquan; Meng, Tian; Wu, Baojin; He, Rongrong; Feng, Du

2018-03-27

Immunofluorescence is an invaluable technique widely used in cell biology. This technique allows visualization of the subcellular distribution of different target proteins or organelles, by specific recognition of the antibody to the endogenous protein itself or to its antigen via the epitope. This technique can be used on tissue sections, cultured cells, or individual cells. Meanwhile, immunofluorescence can also be used in combination with non-antibody fluorescent staining, such as DAPI or fluorescent fusion proteins, e.g., GFP or YFP, etc.Autophagy is a catabolic pathway in which dysfunctional organelles and cellular components are degraded via lysosomes. During this process, cytoplasmic LC3 translocates to autophagosomal membranes. Therefore, cells undergoing autophagy can be identified by visualizing fluorescently labeled LC3 or other autophagy markers. Immunofluorescence is an important part of autophagy detection methods even if observation of the formation of autophagosome by transmission electron microscopy has become a gold standard for characterizing autophagy.By observing the immunofluorescence staining of some key autophagy proteins, we can intuitively evaluate the levels of autophagy in samples. Herein, this protocol describes the predominant method used for the research of autophagy, which mainly focuses on the immunofluorescence staining of cellular LC3, P62, and ULK1 in response to normoxia and hypoxia, by presenting the detailed materials required and methodology.

Neuroprotective Role of Novel Triazine Derivatives by Activating Wnt/β Catenin Signaling Pathway in Rodent Models of Alzheimer's Disease.

PubMed

Sinha, Anshuman; Tamboli, Riyaj S; Seth, Brashket; Kanhed, Ashish M; Tiwari, Shashi Kant; Agarwal, Swati; Nair, Saumya; Giridhar, Rajani; Chaturvedi, Rajnish Kumar; Yadav, Mange Ram

2015-08-01

It has been reported in the literature that cholinesterase inhibitors provide protection in Alzheimer's disease (AD). Recent reports have implicated triazine derivatives as cholinesterase inhibitors. These findings led us to investigate anti-cholinestrase property of some novel triazine derivatives synthesized in this laboratory. In vitro cholinesterase inhibition assay was performed using Ellman method. The potent compounds screened out from in vitro assay were further evaluated using scopolamine-induced amnesic mice model. Further, in vitro reactive oxygen species (ROS) scavenging and anti-apoptotic property of the potent compounds were demonstrated against Aβ1-42-induced neurotoxicity in rat hippocampal cells. Their neuroprotective role was assessed using Aβ1-42-induced Alzheimer's-like phenotype in rats. Further, the role of compounds on the activation of the Wnt/β-catenin pathway was studied. The results showed that the chosen compounds are having protective effect in Alzheimer's-like condition; the ex vivo results advocated their anti-cholinestrase and anti-oxidant activities. Treatment with TRZ-15 and TRZ-20 showed neuroprotective ability of the compounds as evidenced from the improved cognitive ability in the animals, and decrease in Aβ1-42 burden and cytochrome c and cleaved caspase-3 levels in the brain. This study also demonstrates positive involvement of the novel triazine derivatives in the Wnt/β-catenin pathway. Immunoblot and immunofluorescence data suggested that ratio of pGSK3/GSK3 and β-catenin got dramatically improved after treatment with TRZ-15 and TRZ-20. TRZ-15 and TRZ-20 showed neuroprotection in scopolamine-induced amnesic mice and Aβ1-42-induced Alzheimer's rat model and also activate the Wnt/β-catenin signaling pathway. These findings conclude that TRZ-15 and TRZ-20 could be a therapeutic approach to treat AD.

Immunofluorescent localization of Staphylococcos aureus antigen in acute bacterial endocarditis nephritis.

PubMed

Yum, M; Wheat, L J; Maxwell, D; Edwards, J L

1978-11-01

A 75-year-old man with Staphylococcus aureus endocarditis in whom acute diffuse proliferative glomerulonephritis developed is described. The light- and electron-microscopic changes of the glomeruli in this case were identical to those of acute poststreptococcal glomerulonephritis. Immunofluorescence revealed deposition of immunoglobulins and complement in the glomeruli. In addition, bacterial antigenic material was demonstrated in the glomeruli by indirect immunofluorescence. These observations further support the hypothesis of an immune-complex pathogenesis in this form of glomerulonephritis.

β-cell serotonin production is associated with female sex, old age, and diabetes-free condition.

PubMed

Kim, Yeong Gi; Moon, Joon Ho; Kim, Kyuho; Kim, Hyeongseok; Kim, Juok; Jeong, Ji-Seon; Lee, Junguee; Kang, Shinae; Park, Joon Seong; Kim, Hail

2017-11-25

Serotonin is known to be present in pancreatic β-cells and to play several physiological roles, including insulin secretion, β-cell proliferation, and paracrine inhibition of α-cells. However, the serotonin production of different cell lines and islets has not been compared based on age, sex, and diabetes related conditions. Here, we directly compared the serotonin concentrations in βTC and MIN6 cell lines, as well as in islets from mice using ultra-performance liquid chromatography tandem mass spectrometry. The average serotonin concentration was 5-10 ng/mg protein in the islets of male and non-pregnant female mice. The serotonin level was higher in females than males at 8 weeks, although there was no difference at 1 year. Furthermore, we observed serotonin by immunofluorescence staining in the pancreatic tissues of mice and human. Serotonin was detected by immunofluorescence staining in a portion of β-cells from islets of old female mice, but not of male or young female mice. A similar pattern was observed in human pancreas as well. In humans, serotonin production in β-cells was associated with a diabetes-free condition. Thus, serotonin production in β-cells was associated with old age, female sex, and diabetes-free condition. Copyright © 2017 Elsevier Inc. All rights reserved.

Anti-actin IgA antibodies in severe coeliac disease

PubMed Central

Granito, A; Muratori, P; Cassani, F; Pappas, G; Muratori, L; Agostinelli, D; Veronesi, L; Bortolotti, R; Petrolini, N; Bianchi, F B; Volta, U

2004-01-01

Anti-actin IgA antibodies have been found in sera of coeliacs. Our aim was to define the prevalence and clinical significance of anti-actin IgA in coeliacs before and after gluten withdrawal. One hundred and two biopsy-proven coeliacs, 95 disease controls and 50 blood donors were studied. Anti-actin IgA were evaluated by different methods: (a) antimicrofilament positivity on HEp-2 cells and on cultured fibroblasts by immunofluorescence; (b) anti-actin positivity by enzyme-linked immuosorbent assay (ELISA); and (c) presence of the tubular/glomerular pattern of anti-smooth muscle antibodies on rat kidney sections by immunofluorescence. Antimicrofilament IgA were present in 27% of coeliacs and in none of the controls. Antimicrofilament antibodies were found in 25 of 54 (46%) coeliacs with severe villous atrophy and in three of 48 (6%) with mild damage (P < 0·0001). In the 20 patients tested, antimicrofilaments IgA disappeared after gluten withdrawal in accordance with histological recovery. Our study shows a significant correlation between antimicrofilament IgA and the severity of intestinal damage in untreated coeliacs. The disappearance of antimicrofilament IgA after gluten withdrawal predicts the normalization of intestinal mucosa and could be considered a useful tool in the follow-up of severe coeliac disease. PMID:15270857

Anti-brain autoantibodies in the serum of schizophrenic patients: a case-control study.

PubMed

Margari, Francesco; Petruzzelli, Maria Giuseppina; Mianulli, Rossana; Toto, Maddalena; Pastore, Adriana; Bizzaro, Nicola; Tampoia, Marilina

2013-12-30

Schizophrenia is considered a neurodevelopmental disorder with a multifactorial pathogenesis where autoimmune factors may play a significant role. The aim of this study was to verify the presence of anti-brain autoantibodies in the serum of schizophrenic patients compared to healthy controls. Autoantibodies against brain were detected by the immunofluorescence method, utilizing sections of rat hippocampus and hypothalamus and of monkey cerebellum. Three different fluorescence patterns were observed, staining the nucleus-cytoplasm of neurons, the neuroendothelial of blood vessel and the neurofilaments. Search for other organ-specific and non organ-specific autoantibodies was performed in all sera by indirect immunofluorescence method, enzyme linked immunosorbent assay and chemiluminescence immunoassay. Results showed a significant association between schizophrenia and anti-brain autoantibodies against the neuroendothelium of blood vessel in hypothalamus, hippocampus and cerebellum; a significant nuclear and cytoplasmic staining of neurons was assessed only for the hippocampus. No other significant association was found, except between schizophrenia and anti-nuclear autoantibodies on HEp-2 cells. In conclusion, these results support the hypothesis of a significant association between schizophrenia and circulating anti-brain autoantibodies, suggesting a diffuse reactivity against the neuroendothelium of blood vessel and highlighting a nuclear and cytoplasmic staining of the neurons of hippocampus. © 2013 Published by Elsevier Ireland Ltd.

Altered expression of zonula occludens-2 precedes increased blood-brain barrier permeability in a murine model of fulminant hepatic failure.

PubMed

Shimojima, Naoki; Eckman, Christopher B; McKinney, Michael; Sevlever, Daniel; Yamamoto, Satoshi; Lin, Wenlang; Dickson, Dennis W; Nguyen, Justin H

2008-01-01

Brain edema secondary to increased blood-brain barrier (BBB) permeability is a lethal complication in fulminant hepatic failure (FHF). Intact tight junctions (TJ) between brain capillary endothelial cells are critical for normal BBB function. However, the role of TJ in FHF has not been explored. We hypothesized that alterations in the composition of TJ proteins would result in increased BBB permeability in FHF. In this study, FHF was induced in C57BL/6J mice by using azoxymethane. BBB permeability was assessed with sodium fluorescein. Expression of TJ proteins was determined by Western blot, and their cellular distribution was examined using immunofluorescent microscopy. Comatose FHF mice had significant cerebral sodium fluorescein extravasation compared with control and precoma FHF mice, indicating increased BBB permeability. Western blot analysis showed a significant decrease in zonula occludens (ZO)-2 expression starting in the precoma stage. Immunofluorescent microscopy showed a significantly altered distribution pattern of ZO-2 in isolated microvessels from precoma FHF mice. These changes were more prominent in comatose FHF animals. Significant alterations in ZO-2 expression and distribution in the tight junctions preceded the increased BBB permeability in FHF mice. These results suggest that ZO-2 may play an important role in the pathogenesis of brain edema in FHF.

Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

PubMed Central

Martin, Teresa A.; Herman, Christine T.; Limpoco, Francis T.; Michael, Madeline C.; Potts, Gregory K.; Bailey, Ryan C.

2014-01-01

Methods for the generation of substrates presenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retention of function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor-ligand interactions. This molecularly general photochemical patterning method is an enabling tool that will allow the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces. PMID:21793535

Striational antibodies in myasthenia gravis: reactivity and possible clinical significance.

PubMed

Romi, Fredrik; Skeie, Geir Olve; Gilhus, Nils Erik; Aarli, Johan Arild

2005-03-01

Myasthenia gravis is an autoimmune disease caused, in most cases, by antibodies attaching to the acetylcholine receptor. Some myasthenia gravis patients have antibodies that bind in a cross-striational pattern to skeletal and heart muscle tissue sections (striational antibodies). These antibodies react with epitopes on the muscle proteins titin and ryanodine receptor, are found mainly in sera of patients with thymoma and late-onset myasthenia gravis, and may correlate with myasthenia gravis severity. Their presence may predict an unsatisfactory outcome after thymectomy. The detection of titin and ryanodine receptor antibodies provides more specific clinical information than the immunofluorescent demonstration of striational antibodies.

Sequential Immunofluorescent Light Microscopy and Electron Microscopy of Recombination Nodules During Meiotic Prophase I.

PubMed

Anderson, Lorinda K

2017-01-01

Immunolocalization using either fluorescence for light microscopy (LM) or gold particles for electron microscopy (EM) has become a common tool to pinpoint proteins involved in recombination during meiotic prophase. Each method has its advantages and disadvantages. For example, LM immunofluorescence is comparatively easier and higher throughput compared to immunogold EM localization. In addition, immunofluorescence has the advantages that a faint signal can often be enhanced by longer exposure times and colocalization using two (or more) probes with different absorbance and emission spectra is straightforward. However, immunofluorescence is not useful if the object of interest does not label with an antibody probe and is below the resolution of the LM. In comparison, immunogold EM localization is higher resolution than immunofluorescent LM localization, and individual nuclear structures, such as recombination nodules, can be identified by EM regardless of whether they are labeled or not. However, immunogold localization has other disadvantages including comparatively low signal-to-noise ratios, more difficult colocalization using gold particles of different sizes, and the inability to evaluate labeling efficiency before examining the sample using EM (a more expensive and time-consuming technique than LM). Here we describe a method that takes advantage of the good points of both immunofluorescent LM and EM to analyze two classes of late recombination nodules (RNs), only one of which labels with antibodies to MLH1 protein, a marker of crossovers. The method can be used readily with other antibodies to analyze early recombination nodules or other prophase I structures.

Application of DNA Chip Scanning Technology for Automatic Detection of Chlamydia trachomatis and Chlamydia pneumoniae Inclusions

PubMed Central

Bogdanov, Anita; Endrész, Valeria; Urbán, Szabolcs; Lantos, Ildikó; Deák, Judit; Burián, Katalin; Önder, Kamil; Ayaydin, Ferhan; Balázs, Péter

2014-01-01

Chlamydiae are obligate intracellular bacteria that propagate in the inclusion, a specific niche inside the host cell. The standard method for counting chlamydiae is immunofluorescent staining and manual counting of chlamydial inclusions. High- or medium-throughput estimation of the reduction in chlamydial inclusions should be the basis of testing antichlamydial compounds and other drugs that positively or negatively influence chlamydial growth, yet low-throughput manual counting is the common approach. To overcome the time-consuming and subjective manual counting, we developed an automatic inclusion-counting system based on a commercially available DNA chip scanner. Fluorescently labeled inclusions are detected by the scanner, and the image is processed by ChlamyCount, a custom plug-in of the ImageJ software environment. ChlamyCount was able to measure the inclusion counts over a 1-log-unit dynamic range with a high correlation to the theoretical counts. ChlamyCount was capable of accurately determining the MICs of the novel antimicrobial compound PCC00213 and the already known antichlamydial antibiotics moxifloxacin and tetracycline. ChlamyCount was also able to measure the chlamydial growth-altering effect of drugs that influence host-bacterium interaction, such as gamma interferon, DEAE-dextran, and cycloheximide. ChlamyCount is an easily adaptable system for testing antichlamydial antimicrobials and other compounds that influence Chlamydia-host interactions. PMID:24189259

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

PubMed Central

Malyavantham, Kishore S.; Suresh, Lakshmanan

2018-01-01

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern. PMID:29364249

Early interstitial lung disease in microscopic polyangiitis: Case report and literature review.

PubMed

García-Nava, Marcos; Mateos-Toledo, Heidegger; Guevara-Canseco, Ana Patricia Georgina; Infante-González, Cesar Eduardo; Reyes-Nava, Diego Alberto; Estrada-Castro, Emilio

Microscopic polyangiitis (MPA) is a systemic disease included in the Chapel Hill 2012 Classification as necrotizing vasculitis affecting capillaries, venules and arterioles. It usually expresses antineutrophil cytoplasmic antibodies (ANCA) and has a perinuclear immunofluorescence pattern and correlation with anti-myeloperoxidase (MPO) antibodies. Capillaritis with alveolar hemorrhage is the most common manifestation of lung disease. Interstitial lung disease (ILD) is uncommon, with usual interstitial pneumonia being the predominant pattern. However, other patterns such as organizing pneumonia have been described. No guidelines exist for treating patients with ILD and, currently, ANCA-associated vasculitis (AAV) is managed along the lines of small vessel vasculitis. The prognosis with this association is uncertain, with possibilities of relapse and a fatal outcome. We present a case in which ILD was the first manifestation of MPA, without alveolar hemorrhage, with subsequent renal involvement and, in which, the established treatment produced a significant clinical improvement. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

The structural and properties of magnesium-phosphorus compounds under pressure.

PubMed

Liu, Yunxian; Wang, Chao; Lv, Pin; Sun, Hairui; Duan, Defang

2018-06-01

Inspired by the emerging of compounds with novel structures and unique properties (i.e., superconductivity and hardness) under high pressure, we systematically explored a binary Mg-P system under pressure combining first-principles calculation with structure prediction. Several stoichiometries (Mg3P, Mg2P, MgP, MgP2, and MgP3) were predicted stable under pressure. Especially, the P-P bonding patterns are different in the P-rich compounds and the Mg-rich compounds: in the former, the P-P bonding patterns form P2, P3, quadrilateral units, P-P***P chains or disordered "graphene-like" sublattice, while in the latter, the P-P bonding patterns eventually isolated P ions. The analysis of integrated crystal orbital Hamilton populations reveals that the P-P interactions are mainly responsible for the structural stability. The P-rich compounds with stoichiometries of MgP, MgP2 and MgP3 exhibit superconductive behaviors, and these phases show Tc in the range of 4.3-20 K. Our study provides useful information for understanding the Mg-P binary compounds at high pressure. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

PubMed

Margaryan, Hasmik; Dorosh, Andriy; Capkova, Jana; Manaskova-Postlerova, Pavla; Philimonenko, Anatoly; Hozak, Pavel; Peknicova, Jana

2015-03-08

Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.

Glial activation in the collagenase model of nociception associated with osteoarthritis.

PubMed

Adães, Sara; Almeida, Lígia; Potes, Catarina S; Ferreira, Ana Rita; Castro-Lopes, José M; Ferreira-Gomes, Joana; Neto, Fani L

2017-01-01

Background Experimental osteoarthritis entails neuropathic-like changes in dorsal root ganglia (DRG) neurons. Since glial activation has emerged as a key player in nociception, being reported in numerous models of neuropathic pain, we aimed at evaluating if glial cell activation may also occur in the DRG and spinal cord of rats with osteoarthritis induced by intra-articular injection of collagenase. Methods Osteoarthritis was induced by two injections, separated by three days, of 500 U of type II collagenase into the knee joint of rats. Movement-induced nociception was evaluated by the Knee-Bend and CatWalk tests during the following six weeks. Glial fibrillary acidic protein (GFAP) expression in satellite glial cells of the DRG was assessed by immunofluorescence and Western Blot analysis; the pattern of GFAP and activating transcription factor-3 (ATF-3) expression was also compared through double immunofluorescence analysis. GFAP expression in astrocytes and IBA-1 expression in microglia of the L3-L5 spinal cord segments was assessed by immunohistochemistry and Western Blot analysis. The effect of the intrathecal administration of fluorocitrate, an inhibitor of glial activation, on movement-induced nociception was evaluated six weeks after the first collagenase injection. Results GFAP expression in satellite glial cells of collagenase-injected animals was significantly increased six weeks after osteoarthritis induction. Double immunofluorescence showed GFAP upregulation in satellite glial cells surrounding ATF-3-positive neurons. In the spinal cord of collagenase-injected animals, an ipsilateral upregulation of GFAP and IBA-1 was also observed. The inhibition of glial activation with fluorocitrate decreased movement- and loading-induced nociception. Conclusion Collagenase-induced knee osteoarthritis leads to the development of nociception associated with movement of the affected joint and to the activation of glial cells in both the DRG and the spinal cord. Inhibition of glial cell activation by fluorocitrate decreases these osteoarthritis-associated nociceptive behaviours. These results suggest that glial cell activation may play a role in the development of chronic pain in this experimental model of osteoarthritis.

Alzheimer's-associated A{beta} oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal

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

Pitt, Jason; Roth, William; Lacor, Pascale

2009-10-15

It now appears likely that soluble oligomers of amyloid-{beta}{sub 1-42} peptide, rather than insoluble fibrils, act as the primary neurotoxin in Alzheimer's disease (AD). Consequently, compounds capable of altering the assembly state of these oligomers (referred to as ADDLs) may have potential for AD therapeutics. Phenolic compounds are of particular interest for their ability to disrupt A{beta} oligomerization and reduce pathogenicity. This study has focused on oleocanthal (OC), a naturally-occurring phenolic compound found in extra-virgin olive oil. OC increased the immunoreactivity of soluble A{beta} species, when assayed with both sequence- and conformation-specific A{beta} antibodies, indicating changes in oligomer structure. Analysismore » of oligomers in the presence of OC showed an upward shift in MW and a ladder-like distribution of SDS-stable ADDL subspecies. In comparison with control ADDLs, oligomers formed in the presence of OC (A{beta}-OC) showed equivalent colocalization at synapses but exhibited greater immunofluorescence as a result of increased antibody recognition. The enhanced signal at synapses was not due to increased synaptic binding, as direct detection of fluorescently-labeled ADDLs showed an overall reduction in ADDL signal in the presence of OC. Decreased binding to synapses was accompanied by significantly less synaptic deterioration assayed by drebrin loss. Additionally, treatment with OC improved antibody clearance of ADDLs. These results indicate oleocanthal is capable of altering the oligomerization state of ADDLs while protecting neurons from the synaptopathological effects of ADDLs and suggest OC as a lead compound for development in AD therapeutics.« less

Chlorpyrifos- and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2

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

Sindi, Ramya A., E-mail: ramya.sindi2010@my.ntu.ac

Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20 h before exposure to CPF or CPO for 2–8 h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length ofmore » axon-like processes compared to the control, respectively, retraction of neurites being observed within 2 h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2 h exposure and reduced levels of reactivity of the same antibody following 8 h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2 h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8 h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation. - Highlights: • Chlorpyrifos and chlorpyrifos oxon induced rapid neurite retraction in N2a cells. • This occurred following transient hyperphosphorylation of ERK 1/2. • It was concomitant with hyperphosphorylation of neurofilament heavy chain (NFH). • Subsequent NFH dephosphorylation was associated with increased phosphatase activity. • Thus, the above changes reflect key events in chlorpyrifos induced neurodegeneration.« less

Choline acetyltransferase-like immunofluorescence in epidermis of human skin.

PubMed

Johansson, O; Wang, L

1993-01-01

Using the indirect immunofluorescence approach the occurrence of choline acetyltransferase-like immunoreactivity in epidermis, except stratum basale, of human skin is described. Immunoreactive cells were also found in hair follicles, sweat gland ducts and sebaceous glands.

Immunofluorescence detection of pea protein in meat products.

PubMed

Petrášová, Michaela; Pospiech, Matej; Tremlová, Bohuslava; Javůrková, Zdeňka

2016-08-01

In this study we developed an immunofluorescence method to detect pea protein in meat products. Pea protein has a high nutritional value but in sensitive individuals it may be responsible for causing allergic reactions. We produced model meat products with various additions of pea protein and flour; the detection limit (LOD) of the method for pea flour was 0.5% addition, and for pea protein it was 0.001% addition. The repeatabilities and reproducibilities for samples both positive and negative for pea protein were all 100%. In a blind test with model products and commercial samples, there was no statistically significant difference (p > 0.05) between the declared concentrations of pea protein and flour and the immunofluorescence method results. Sensitivity was 1.06 and specificity was 1.00. These results show that the immunofluorescence method is suitable for the detection of pea protein in meat products.

Microwave-assisted extraction of polycyclic aromatic compounds from coal.

PubMed

Kerst, M; Andersson, J T

2001-08-01

Microwave-assisted extraction (MAE) of polycyclic aromatic compounds (PACs) from coal is shown to give the same pattern of compounds as Soxhlet extraction. MAE requires only 10 mL solvent and 10 min extraction time whereas Soxhlet uses 200 mL and takes 24 h. Although the yields were lower, dichloromethane (DCM) was preferred to pyridine, N-methyl-2-pyrrolidone (NMP), and NMP with CS2 because the pattern of the PACs is shown to be independent of solvent and DCM is a much more convenient solvent to work with.

Method and apparatus for coating a patterned thin film on a substrate from a fluid source with continuous feed capability

DOEpatents

Burrows, Paul E [Kennewick, WA; Sapochak, Linda S [Kennewick, WA

2009-09-22

A method and apparatus for forming patterned coatings of thin film, non-polymerizable compounds on a substrate. A mixture of the non-polymerizable compound and a liquid carrier is pumped into the interior of a heated evaporation box having an internal temperature sufficient to convert substantially all of the non-polymerizable compound and liquid carrier to a gaseous form. The non-polymerizable compound and liquid carrier are then removed from the evaporation box via exit slit in the evaporation box. Adjacent to the exit slit, and maintained in a vacuum, is a first substrate upon which the non-polymerizable compound condenses. The first substrate is in motion, for example on a web roller, thereby allowing a continuous coating of the non-polymerizable compound to be applied to the first substrate. Once the non-polymerizable compound is applied to one side of the first substrate, an energy source is then directed toward the opposite side of the first substrate. In this manner, a portion of the non-polymerizable compound is removed from the first substrate. A second substrate is then provided adjacent to the first substrate, and the non-polymerizable compound is thereby transferred from the first substrate onto the second substrate. By repeatedly transferring portions of the non-polymerizable material from the first substrate to the second substrate in this manner, the thin film, non-polymerizable materials can be formed onto the second substrate in a predetermined pattern, and in a continuous and highly efficient process.

Characterization of anticancer agents by their growth inhibitory activity and relationships to mechanism of action and structure.

PubMed

Keskin, O; Bahar, I; Jernigan, R L; Beutler, J A; Shoemaker, R H; Sausville, E A; Covell, D G

2000-04-01

An analysis of the growth inhibitory potency of 122 anticancer agents available from the National Cancer Institute anticancer drug screen is presented. Methods of singular value decomposition (SVD) were applied to determine the matrix of distances between all compounds. These SVD-derived dissimilarity distances were used to cluster compounds that exhibit similar tumor growth inhibitory activity patterns against 60 human cancer cell lines. Cluster analysis divides the 122 standard agents into 25 statistically distinct groups. The first eight groups include structurally diverse compounds with reactive functionalities that act as DNA-damaging agents while the remaining 17 groups include compounds that inhibit nucleic acid biosynthesis and mitosis. Examination of the average activity patterns across the 60 tumor cell lines reveals unique 'fingerprints' associated with each group. A diverse set of structural features are observed for compounds within these groups, with frequent occurrences of strong within-group structural similarities. Clustering of cell types by their response to the 122 anticancer agents divides the 60 cell types into 21 groups. The strongest within-panel groupings were found for the renal, leukemia and ovarian cell panels. These results contribute to the basis for comparisons between log(GI(50)) screening patterns of the 122 anticancer agents and additional tested compounds.

Hair and Scalp Changes in Cutaneous and Systemic Lupus Erythematosus.

PubMed

Udompanich, Siriorn; Chanprapaph, Kumutnart; Suchonwanit, Poonkiat

2018-06-09

Cutaneous and systemic lupus erythematosus (SLE) commonly involves the hair and scalp. Alopecia can result from direct activity of disease on the scalp or from the state of physical stress in the form of telogen effluvium. Discoid lupus erythematosus and lupus panniculitis/profundus are known to cause scarring alopecia, while accumulation of recent studies has shown that non-scarring alopecia in SLE may have different subtypes, comprising lupus erythematosus-specific and lupus erythematosus-nonspecific changes on histology. This review aims to summarize the clinical pattern, trichoscopic, histopathological, and direct immunofluorescence features of different types of alopecia in cutaneous and systemic lupus erythematosus, as well as exploring their relationship with SLE disease activity.

[Possibilities of differentiation of antinuclear antibodies].

PubMed

Müller, W; Rosenthal, M; Stojan, B

1975-10-15

Antinuclear antibodies can give diagnostic informations according to their titre values, the belonging to different classes of immune globulins and on the basis of different patterns of immunofluorescence connection. The determination of granulocyte-specific antibodies which frequently appear in progressive chronic polyarthritis further contributes to the differential-diagnostic classification of diseases of the connective tissue. An antibody against extractable nuclear antigen is specific for the so-called mixed connective tissue disease, an antimitochondrial antibody for the pseudo-LE-syndrome. Moreover, the own examinations resulted in a particularly high and frequent ability of complement fixation of the antinuclear factors in systematic lupus erythematosus and sclerodermy. In contrast to this in the progressive chronic polyarthritis the complement fixation was clearly more insignificant.

Serum antinuclear antibody in adult Thais.

PubMed

Prapinjumrune, Chanwit; Prucktrakul, Chalakorn; Sooktonglarng, Trakarn; Thongprasom, Kobkan

2017-03-01

This study investigated the presence of antinuclear antibody (ANA) in older Thais compared with middle-age and younger participants. Antinuclear antibody represents the first step in the diagnostic testing for lupus erythematosus (LE) and other autoimmune diseases. Due to the lack of reference ANA levels in older, middle-age and younger Thais healthy participants, this study will be useful for determining the proper diagnostic and treatment criteria. There were 28 older (60-76 years), 17 middle-age (41-59 years) and 13 younger (24-40 years) participants in this study. Immunofluorescence was performed to analyse the ANA staining pattern and titre levels in the participants' blood samples. The presence of serum ANA was found in 18 of 28 cases (64.3%), four of 17 (23.5%) and one of 13 cases (7.7%) of the older, middle-age and younger participants, respectively. The difference in the number of serum ANA-positive participants between the older, middle-age and younger groups was statistically significant (p < 0.05). Interestingly, the ANA positive in older participants presented more than one staining pattern. The speckled pattern was the most commonly detected ANA staining pattern in the older group, being found in 12 cases followed by cytoplasmic pattern (10 cases), homogeneous pattern (nine cases) and nucleolar pattern (five cases). In the middle-age group, the speckled pattern was found in four cases, whereas one younger participant presented a nucleolar pattern. Serum ANA positive was significantly higher in the older group compared with the middle-age and younger groups. There were variations of the serum ANA staining patterns in the older group. © 2016 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd.

FACTORS CONTROLLING THE EMISSIONS OF MONOTERPENES AND OTHER VOLATILE ORGANIC COMPOUNDS

EPA Science Inventory

Plants contain a number of volatile organic compounds, including isoprene, mono- and sesquiterpenes, alcohols, aldehydes, ketones, and esters. ndividual plant species have unique combinations of these compounds; consequently, the emission pattern for each species is also specific...

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulose hydrolysates

PubMed Central

2014-01-01

We investigated the severity of the inhibitory effects of 13 phenolic compounds usually found in spruce hydrolysates (4-hydroxy-3-methoxycinnamaldehyde, homovanilyl alcohol, vanillin, syringic acid, vanillic acid, gallic acid, dihydroferulic acid, p-coumaric acid, hydroquinone, ferulic acid, homovanillic acid, 4-hydroxybenzoic acid and vanillylidenacetone). The effects of the selected compounds on cell growth, biomass yield and ethanol yield were studied and the toxic concentration threshold was defined for each compound. Using Ethanol Red, the popular industrial strain of Saccharomyces cerevisiae, we found the most toxic compound to be 4-hydroxy-3-methoxycinnamaldehyde which inhibited growth at a concentration of 1.8 mM. We also observed that toxicity did not generally follow a trend based on the aldehyde, acid, ketone or alcohol classification of phenolic compounds, but rather that other structural properties such as additional functional groups attached to the compound may determine its toxicity. Three distinctive growth patterns that effectively clustered all the compounds involved in the screening into three categories. We suggest that the compounds have different cellular targets, and that. We suggest that the compounds have different cellular targets and inhibitory mechanisms in the cells, also compounds who share similar pattern on cell growth may have similar inhibitory effect and mechanisms of inhibition. PMID:24949277

Self-organization of human embryonic stem cells on micropatterns

PubMed Central

Deglincerti, Alessia; Etoc, Fred; Guerra, M. Cecilia; Martyn, Iain; Metzger, Jakob; Ruzo, Albert; Simunovic, Mijo; Yoney, Anna; Brivanlou, Ali H.; Siggia, Eric; Warmflash, Aryeh

2018-01-01

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate to specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. While embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, these methods do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach where human embryonic stem cells are confined to disk-shaped, sub-millimeter colonies. After 42 hours of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. Our protocol takes 3 days; it uses commercial microfabricated slides (CYTOO), human laminin-521 (LN-521) as extra-cellular matrix coating, and either conditioned or chemically-defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. PMID:27735934

Cellular organization of pre-mRNA splicing factors in several tissues. Changes in the uterus by hormone action.

PubMed

George-Téllez, R; Segura-Valdez, M L; González-Santos, L; Jiménez-García, L F

2002-05-01

In the mammalian cell nucleus, splicing factors are distributed in nuclear domains known as speckles or splicing factor compartments (SFCs). In cultured cells, these domains are dynamic and reflect transcriptional and splicing activities. We used immunofluorescence and confocal microscopy to monitor whether splicing factors in differentiated cells display similar features. Speckled patterns are observed in rat hepatocytes, beta-cells, bronchial and intestine epithelia and also in three cell types of the uterus. Moreover, the number, distribution and sizes of the speckles vary among them. In addition, we studied variations in the circular form (shape) of speckles in uterine cells that are transcriptionally modified by a hormone action. During proestrus of the estral cycle, speckles are irregular in shape while in diestrus I they are circular. Experimentally, in castrated rats luminal epithelial cells show a pattern where speckles are dramatically rounded, but they recover their irregular shape rapidly after an injection of estradiol. The same results were observed in muscle and gland epithelial cells of the uterus. We concluded that different speckled patterns are present in various cells types in differentiated tissues and that these patterns change in the uterus depending upon the presence or absence of hormones such as estradiol.

Computer-Assisted Classification Patterns in Autoimmune Diagnostics: The AIDA Project

PubMed Central

Benammar Elgaaied, Amel; Cascio, Donato; Bruno, Salvatore; Ciaccio, Maria Cristina; Cipolla, Marco; Fauci, Alessandro; Morgante, Rossella; Taormina, Vincenzo; Gorgi, Yousr; Marrakchi Triki, Raja; Ben Ahmed, Melika; Louzir, Hechmi; Yalaoui, Sadok; Imene, Sfar; Issaoui, Yassine; Abidi, Ahmed; Ammar, Myriam; Bedhiafi, Walid; Ben Fraj, Oussama; Bouhaha, Rym; Hamdi, Khouloud; Soumaya, Koudhi; Neili, Bilel; Asma, Gati; Lucchese, Mariano; Catanzaro, Maria; Barbara, Vincenza; Brusca, Ignazio; Fregapane, Maria; Amato, Gaetano; Friscia, Giuseppe; Neila, Trai; Turkia, Souayeh; Youssra, Haouami; Rekik, Raja; Bouokez, Hayet; Vasile Simone, Maria; Fauci, Francesco; Raso, Giuseppe

2016-01-01

Antinuclear antibodies (ANAs) are significant biomarkers in the diagnosis of autoimmune diseases in humans, done by mean of Indirect ImmunoFluorescence (IIF) method, and performed by analyzing patterns and fluorescence intensity. This paper introduces the AIDA Project (autoimmunity: diagnosis assisted by computer) developed in the framework of an Italy-Tunisia cross-border cooperation and its preliminary results. A database of interpreted IIF images is being collected through the exchange of images and double reporting and a Gold Standard database, containing around 1000 double reported images, has been settled. The Gold Standard database is used for optimization of a CAD (Computer Aided Detection) solution and for the assessment of its added value, in order to be applied along with an Immunologist as a second Reader in detection of autoantibodies. This CAD system is able to identify on IIF images the fluorescence intensity and the fluorescence pattern. Preliminary results show that CAD, used as second Reader, appeared to perform better than Junior Immunologists and hence may significantly improve their efficacy; compared with two Junior Immunologists, the CAD system showed higher Intensity Accuracy (85,5% versus 66,0% and 66,0%), higher Patterns Accuracy (79,3% versus 48,0% and 66,2%), and higher Mean Class Accuracy (79,4% versus 56,7% and 64.2%). PMID:27042658

Patterning of novel breast implant surfaces by enhancing silicone biocompatibility, using biomimetic topographies.

PubMed

Barr, S; Hill, E; Bayat, A

2010-04-26

Silicone biocompatibility is dictated by cell-surface interaction and its understanding is important in the field of implantation. The role of surface topography and its associated cellular morphology needs investigation to identify qualities that enhance silicone surface biocompatability. This study aims to create well-defined silicone topographies and examine how breast tissue-derived fibroblasts react and align to these surfaces. Photolithographic microelectronic techniques were modified to produce naturally inspired topographies in silicone, which were cultured with breast tissue-derived human fibroblasts. Using light, immunofluorescent and atomic force microscopy, the cytoskeletal reaction of fibroblasts to these silicone surfaces was investigated. Numerous, well-defined micron-sized pillars, pores, grooves, and ridges were manufactured and characterized in medical grade silicone. Inimitable immunofluorescent microscopy represented in our high magnification images of vinculin, vimentin, and the actin cytoskeleton highlights the differences in fibroblast adhesion between fabricated silicone surfaces. These unique figures illustrate that fibroblast adhesion and the reactions these cells have to silicone can be manipulated to enhance biointegration between the implant and the breast tissue. An alteration of fibroblast phenotype was also observed, exhibiting the propensity of these surfaces to induce categorical remodeling of fibroblasts. This unique study shows that fibroblast reactions to silicone topographies can be tailored to induce physiological changes in cells. This paves the way for further research necessary to develop more biocompatible constructs capable of eliminating capsular contracture by subverting the foreign body response.

Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

PubMed

Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

2014-09-01

We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment. Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

[Standardized indirect immunofluorescence. Differentiation of mitochondrial, microsomal and ribosomal antibodies].

PubMed

Storch, W

1977-02-15

By an extensive standardisation of the indirect immunofluorescence for the demonstration espeially of mitochondrial antibodies we succeeded in recognizing atypical fluorescence patterns and in describing their exact localisation. On the basis of absorption studies with mitochondrias, microsomas and ribosomas by comparative observation of sections of liver, stomach and kidneys of rats the preferred sort of reaction and the intensity of fluorescence of antibodies against mitochondria, microsomas and ribosomas were empirically established. Antimitochondrial antibodies react above all with the parietal cells of the stomach and the distal epithelia of the tubulus of the kidney. Antibodies against microsomas of liver and kidney are characterized by a brilliant diffuse cytoplasmatic fluorescence of the hepatocytes and by a comparatively weaker fluorescence of exclusively proximal tubuli of the kidneys of rats. Antibodies against ribosomas lead to a fluorescence especially of the main cells of the stomach. The differentiation of several cytoplasmatic antibodies is among others of interest for the diagnosis of certain autoimmune diseases. Although there are numerous still unclear findings and "overlap" phenomena the existence of high titre antibodies against mitochondrias speaks for a primarily biliary cirrhosis or a pseudo-LE-syndrome, the existence of antibodies against microsomas of kidney and liver of rats for a special form of a chronically active hepatitis and the existence of the very rare antibodies against ribosomas for an active lupus erythematodes disseminatus.

Correlation between serological and immunofluorescence results in the investigation of brucellosis in veterinary surgeons.

PubMed Central

Henderson, R J; Hill, D M; Vickers, A A; Edwards, J M; Tillett, H

1976-01-01

Four serological tests and three immunofluorescence tests for IgG, IgM, and IgA were compared for value in the investigation of brucellosis in veterinary surgeons. No one serological test stood out over the others, and the immunofluorescence tests did not appear to have advantages over the serological tests. If a laboratory is limited in time and resources then the saline agglutination or the complement fixation test would be reasonably satisfactory. The 2-mercaptoethanol test and the antihuman globulin (Coombs' test) have no advantages over the other two and could be dropped. Immunofluorescence tests are not recommended for routine testing of brucellosis sera. The results and these recommendations apply to the 'vet' sera tested; it is reasonable to suppose that what applies to 'vet' sera will also apply to sera of those who work with or are in repeated contact with cattle and who will have had previous experience of brucella antigen, that is, dairy farmers, herdsmen, or slaughter house employees. PMID:765361

Expression of Eag1 K+ channel and ErbBs in human pituitary adenomas: cytoskeleton arrangement patterns in cultured cells.

PubMed

del Pliego, Margarita González; Aguirre-Benítez, Elsa; Paisano-Cerón, Karina; Valdovinos-Ramírez, Irene; Rangel-Morales, Carlos; Rodríguez-Mata, Verónica; Solano-Agama, Carmen; Martín-Tapia, Dolores; de la Vega, María Teresa; Saldoval-Balanzario, Miguel; Camacho, Javier; Mendoza-Garrido, María Eugenia

2013-01-01

Pituitary adenomas can invade surrounded tissue, but the mechanism remains elusive. Ether à go-go-1 (Eag1) potassium channel and epidermal growth factor receptors (ErbB1 and ErbB2) have been associated to invasive phenotypes or poor prognosis in cancer patients. However, cells arrange their cytoskeleton in order to acquire a successful migration pattern. We have studied ErbBs and Eag1 expression, and cytoskeleton arrangements in 11 human pituitary adenomas. Eag1, ErbB1 and ErbB2 expression were studied by immunochemistry in tissue and cultured cells. The cytoskeleton arrangement was analyzed in cultured cells by immunofluorescence. Normal pituitary tissue showed ErbB2 expression and Eag1 only in few cells. However, Eag1 and ErbB2 were expressed in all the tumors analyzed. ErbB1 expression was observed variable and did not show specificity for a tumor characteristic. Cultured cells from micro- and macro-adenomas clinically functional organize their cytoskeleton suggesting a mesenchymal pattern, and a round leucocyte/amoeboid pattern from invasive clinically silent adenoma. Pituitary tumors over-express EGF receptors and the ErbB2 repeated expression suggests is a characteristic of adenomas. Eag 1 was express, in different extent, and could be a therapeutic target. The cytoskeleton arrangements observed suggest that pituitary tumor cells acquire different patterns: mesenchymal, and leucocyte/amoeboid, the last observed in the invasive adenomas. Amoeboid migration pattern has been associated with high invasion capacity.

A monoclonal IgM smooth muscle antibody reactive with fibroblast stress fibres produced by immunization with Treponema pallidum.

PubMed Central

Strugnell, R A; Underwood, J R; Clarke, F M; Pedersen, J S; Chalmers, P J; Faine, S; Toh, B H

1983-01-01

A monoclonal IgM smooth muscle antibody secreted by a hybrid (MMI-1) of mouse plasmacytoma NS-1 with spleen cells from mouse immunized with Treponema pallidum was detected by indirect immunofluorescence tests on frozen tissue sections and on acetone fixed monolayers of rat and human fibroblasts. The antibody did not react with acetone fixed smears of T. pallidum but reacted with smooth muscle fibres and with striations of skeletal and cardiac muscle. In non-muscle cells, the antibody stained liver in a 'polygonal' pattern, thymus with accentuated staining of the thymic medulla, renal glomeruli and the brush border and peritubular fibrils of renal tubules. In fibroblast monolayers, the antibody stained stress fibres in an interrupted pattern. Immunoblotting with muscle proteins and the antibody showed labelling of a 100K molecule. The cellular distribution of the mouse monoclonal antibody is similar to that obtained with anti-actin antibody suggesting that the corresponding antigen may be an actin binding protein. Images Fig. 3 PMID:6347470

Three-dimensional flow structure and patterns of bed shear stress in an evolving compound meander bend

USGS Publications Warehouse

Engel, Frank; Rhoads, Bruce L.

2016-01-01

Compound meander bends with multiple lobes of maximum curvature are common in actively evolving lowland rivers. Interaction among spatial patterns of mean flow, turbulence, bed morphology, bank failures and channel migration in compound bends is poorly understood. In this paper, acoustic Doppler current profiler (ADCP) measurements of the three-dimensional (3D) flow velocities in a compound bend are examined to evaluate the influence of channel curvature and hydrologic variability on the structure of flow within the bend. Flow structure at various flow stages is related to changes in bed morphology over the study timeframe. Increases in local curvature within the upstream lobe of the bend reduce outer bank velocities at morphologically significant flows, creating a region that protects the bank from high momentum flow and high bed shear stresses. The dimensionless radius of curvature in the upstream lobe is one-third less than that of the downstream lobe, with average bank erosion rates less than half of the erosion rates for the downstream lobe. Higher bank erosion rates within the downstream lobe correspond to the shift in a core of high velocity and bed shear stresses toward the outer bank as flow moves through the two lobes. These erosion patterns provide a mechanism for continued migration of the downstream lobe in the near future. Bed material size distributions within the bend correspond to spatial patterns of bed shear stress magnitudes, indicating that bed material sorting within the bend is governed by bed shear stress. Results suggest that patterns of flow, sediment entrainment, and planform evolution in compound meander bends are more complex than in simple meander bends. Moreover, interactions among local influences on the flow, such as woody debris, local topographic steering, and locally high curvature, tend to cause compound bends to evolve toward increasing planform complexity over time rather than stable configurations.

Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

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

Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn

Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified amore » novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC{sub 50} values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets.« less

Tandem application of ligand-based virtual screening and G4-OAS assay to identify novel G-quadruplex-targeting chemotypes.

PubMed

Musumeci, Domenica; Amato, Jussara; Zizza, Pasquale; Platella, Chiara; Cosconati, Sandro; Cingolani, Chiara; Biroccio, Annamaria; Novellino, Ettore; Randazzo, Antonio; Giancola, Concetta; Pagano, Bruno; Montesarchio, Daniela

2017-05-01

G-quadruplex (G4) structures are key elements in the regulation of cancer cell proliferation and their targeting is deemed to be a promising strategy in anticancer therapy. A tandem application of ligand-based virtual screening (VS) calculations together with the experimental G-quadruplex on Oligo Affinity Support (G4-OAS) assay was employed to discover novel G4-targeting compounds. The interaction of the selected compounds with the investigated G4 in solution was analysed through a series of biophysical techniques and their biological activity investigated by immunofluorescence and MTT assays. A focused library of 60 small molecules, designed as putative G4 groove binders, was identified through the VS. The G4-OAS experimental screening led to the selection of 7 ligands effectively interacting with the G4-forming human telomeric DNA. Evaluation of the biological activity of the selected compounds showed that 3 ligands of this sub-library induced a marked telomere-localized DNA damage response in human tumour cells. The combined application of virtual and experimental screening tools proved to be a successful strategy to identify new bioactive chemotypes able to target the telomeric G4 DNA. These compounds may represent useful leads for the development of more potent and selective G4 ligands. Expanding the repertoire of the available G4-targeting chemotypes with improved physico-chemical features, in particular aiming at the discovery of novel, selective G4 telomeric ligands, can help in developing effective anti-cancer drugs with fewer side effects. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2017 Elsevier B.V. All rights reserved.

Processing Rhythmic Pattern during Chinese Sentence Reading: An Eye Movement Study

PubMed Central

Luo, Yingyi; Duan, Yunyan; Zhou, Xiaolin

2015-01-01

Prosodic constraints play a fundamental role during both spoken sentence comprehension and silent reading. In Chinese, the rhythmic pattern of the verb-object (V-O) combination has been found to rapidly affect the semantic access/integration process during sentence reading (Luo and Zhou, 2010). Rhythmic pattern refers to the combination of words with different syllabic lengths, with certain combinations disallowed (e.g., [2 + 1]; numbers standing for the number of syllables of the verb and the noun respectively) and certain combinations preferred (e.g., [1 + 1] or [2 + 2]). This constraint extends to the situation in which the combination is used to modify other words. A V-O phrase could modify a noun by simply preceding it, forming a V-O-N compound; when the verb is disyllabic, however, the word order has to be O-V-N and the object is preferred to be disyllabic. In this study, we investigated how the reader processes the rhythmic pattern and word order information by recording the reader's eye-movements. We created four types of sentences by crossing rhythmic pattern and word order in compounding. The compound, embedding a disyllabic verb, could be in the correct O-V-N or the incorrect V-O-N order; the object could be disyllabic or monosyllabic. We found that the reader spent more time and made more regressions on and after the compounds when either type of anomaly was detected during the first pass reading. However, during re-reading (after all the words in the sentence have been viewed), less regressive eye movements were found for the anomalous rhythmic pattern, relative to the correct pattern; moreover, only the abnormal rhythmic pattern, not the violated word order, influenced the regressive eye movements. These results suggest that while the processing of rhythmic pattern and word order information occurs rapidly during the initial reading of the sentence, the process of recovering from the rhythmic pattern anomaly may ease the reanalysis processing at the later stage of sentence integration. Thus, rhythmic pattern in Chinese can dynamically affect both local phrase analysis and global sentence integration during silent reading. PMID:26696942

Processing Rhythmic Pattern during Chinese Sentence Reading: An Eye Movement Study.

PubMed

Luo, Yingyi; Duan, Yunyan; Zhou, Xiaolin

2015-01-01

Prosodic constraints play a fundamental role during both spoken sentence comprehension and silent reading. In Chinese, the rhythmic pattern of the verb-object (V-O) combination has been found to rapidly affect the semantic access/integration process during sentence reading (Luo and Zhou, 2010). Rhythmic pattern refers to the combination of words with different syllabic lengths, with certain combinations disallowed (e.g., [2 + 1]; numbers standing for the number of syllables of the verb and the noun respectively) and certain combinations preferred (e.g., [1 + 1] or [2 + 2]). This constraint extends to the situation in which the combination is used to modify other words. A V-O phrase could modify a noun by simply preceding it, forming a V-O-N compound; when the verb is disyllabic, however, the word order has to be O-V-N and the object is preferred to be disyllabic. In this study, we investigated how the reader processes the rhythmic pattern and word order information by recording the reader's eye-movements. We created four types of sentences by crossing rhythmic pattern and word order in compounding. The compound, embedding a disyllabic verb, could be in the correct O-V-N or the incorrect V-O-N order; the object could be disyllabic or monosyllabic. We found that the reader spent more time and made more regressions on and after the compounds when either type of anomaly was detected during the first pass reading. However, during re-reading (after all the words in the sentence have been viewed), less regressive eye movements were found for the anomalous rhythmic pattern, relative to the correct pattern; moreover, only the abnormal rhythmic pattern, not the violated word order, influenced the regressive eye movements. These results suggest that while the processing of rhythmic pattern and word order information occurs rapidly during the initial reading of the sentence, the process of recovering from the rhythmic pattern anomaly may ease the reanalysis processing at the later stage of sentence integration. Thus, rhythmic pattern in Chinese can dynamically affect both local phrase analysis and global sentence integration during silent reading.

BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

EPA Science Inventory

An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

Immunofluorescence Staining — EDRN Public Portal

Cancer.gov

Direct immunofluorescence method is used to detect the deposit of immunoglobulins, complement components, fibrinogen, etc. in tissues. This technique is usually performed on frozen sections. The primary antibody is conjugated to fluorescein binds directly with the antigen and can be detected by the fluorescent tag using a fluorescent microscope.

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing.

PubMed

Erlandsen, S L; Sherlock, L A; Bemrick, W J

1990-04-01

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.

Distribution of the muscarinic K+ channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart.

PubMed

Dobrzynski, H; Marples, D D; Musa, H; Yamanushi, T T; Henderson, Z; Takagishi, Y; Honjo, H; Kodama, I; Boyett, M R

2001-10-01

The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.

Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin.

PubMed

Schmucker, B; Ballhausen, W G; Kressel, M

1997-01-01

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.

Immunohistochemical expression and colocalization of somatostatin, carboxypeptidase-E and prohormone convertases 1 and 2 in rat brain.

PubMed

Billova, S; Galanopoulou, A S; Seidah, N G; Qiu, X; Kumar, U

2007-06-29

The processing of many peptides for their maturation in target tissue depends upon the presence of sorting receptor. Several previous studies have predicted that carboxypeptidase-E (CPE), prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2) may function as sorting elements for somatostatin (SST) for its maturation and processing to appropriate targets. However, nothing is currently known about whether brain, neuronal culture or even endocrine cells express SST, CPE, PC1 and PC2 and exhibit colocalization. Accordingly, in the present study using peroxidase immunohistochemistry, double-labeled indirect immunofluorescence immunohistochemistry and Western blot analysis, we mapped the distributional pattern of SST, CPE, PC1 and PC2 in different rat brain regions. Additionally, we also determined the colocalization of SST with CPE, PC1 and PC2 as well as colocalization of CPE with PC1 and PC2. The localization of SST, CPE, PC1 and PC2 reveals a distinct and region specific distribution pattern in the rat brain. Using an indirect double-label immunofluorescence method we observed selective neuron specific colocalization in a region specific manner in cortex, striatum and hippocampus. These studies provide the first evidence for colocalization between SST, CPE, PC1 and PC2 as well as CPE with PC1 and PC2. SST in cerebral cortex colocalized in pyramidal and non-pyramidal neurons with CPE, PC1 and PC2. Most importantly, in striatum and hippocampus colocalization was mostly observed selectively and preferentially in interneurons. CPE is also colocalized with PC1 and PC2 in a region specific manner. The data presented here provide a new insight into the distribution and colocalization of SST, CPE, PC1 and PC2 in rat brain. Taken together, our data anticipate the possibility that CPE, PC1 and PC2 might be potential target for the maturation of SST.

Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones.

PubMed Central

Tamarappoo, B K; Verkman, A S

1998-01-01

Five single-point aquaporin-2 (AQP2) mutations that cause non-X-linked nephrogenic diabetes insipidus (NDI) were characterized to establish the cellular defect and to develop therapeutic strategies. In Xenopus oocytes expressing AQP2 cRNAs, single-channel water permeabilities of mutants L22V, T126M, and A147T were similar to that of wild-type AQP2, whereas R187C and C181W were nonfunctional. In [35S]methionine pulse-chase experiments in transiently transfected CHO cells, half-times for AQP2 degradation were approximately 4 h for wild-type AQP2 and L22V, and mildly decreased for T126M (2.7 h), C181W (2.4 h), R187C (2.0 h), and A147T (1.8 h). Immunofluorescence showed three distinct AQP2-staining patterns: plasma membrane and endosomal staining (wild-type, L22V), endoplasmic reticulum (ER) staining (T126M > A147T approximately R187C), or a mixed pattern of reticular and perinuclear vesicular staining. Immunoblot of fractionated vesicles confirmed primary ER localization of T126M, R187C, and A147T. To determine if the AQP2-trafficking defect is correctable, cells were incubated with the "chemical chaperone" glycerol for 48 h. Immunoblot showed that glycerol produced a nearly complete redistribution of AQP2 (T126M, A147T, and R187C) from ER to membrane/endosome fractions. Immunofluorescence confirmed the cellular redistribution. Redistribution of AQP2 mutants was also demonstrated in transfected MDCK cells, and using the chaperones TMAO and DMSO in place of glycerol in CHO cells. Water permeability measurements indicated that functional correction was achieved. These results indicate defective mammalian cell processing of mutant AQP2 water channels in NDI, and provide evidence for pharmacological correction of the processing defect by chemical chaperones. PMID:9593782

Dynamic change of SGK expression and its role in neuron apoptosis after traumatic brain injury.

PubMed

Wu, Xinmin; Mao, Hui; Liu, Jiao; Xu, Jian; Cao, Jianhua; Gu, Xingxing; Cui, Gang

2013-01-01

Activation of specific signaling pathways in response to mechanical trauma causes delayed neuronal apoptosis; GSK-3β/β-catenin signaling plays a critical role in the apoptosis of neurons in CNS diseases, SGK was discovered as a regulator of GSK-3β/β-catenin pathway, The goal of this study was to determine if the mechanism of cell death or survival mediated by the SGK/GSK-3β/β-catenin pathway is involved in a rat model of TBI. Here, an acute traumatic brain injury model was applied to investigate the expression change and possible roles of SGK, Expression of SGK, and total-GSK-3β, phospho-GSK3β on ser-9, beta-catenin, and caspase-3 were examined by immunohistochemistry and Western blot analysis. Double immunofluorescent staining was used to observe the SGK localizations. Si-RNA was performed to identify whether SGK regulates neuron apoptosis via GSK-3β/β-catenin pathway, ultimately inhibit caspase-3 activation. Temporally, SGK expression showed an increase pattern after TBI and reached a peak at day 3. Spatially, SGK was widely expressed in the neuron, rarely in astrocytes and oligodendrocytes; in addition, the expression patterns of active caspase-3 and phospho-GSK3β were parallel with that of SGK, at the same time, the expression of β-catenin shows similarity with SGK. In vitro, to further investigate the function of SGK, a neuronal cell line PC12 was employed to establish an apoptosis model. We analyzed the association of SGK with apoptosis on PC12 cells by western blot, immunofluorescent labeling and siRNA. the results implied that SGK plays an important role in neuron apoptosis via the regulation of GSK3β/β-catenin signaling pathway; ultimately inhibit caspase-3 activation. Taken together, we inferred traumatic brain injury induced an upregulation of SGK in the central nervous system, which show a protective role in neuron apoptosis.

Involvement of p63 in the herpes simplex virus-1-induced demise of corneal cells.

PubMed

Orosz, László; Gallyas, Eva; Kemény, Lajos; Mándi, Yvette; Facskó, Andrea; Megyeri, Klára

2010-06-07

The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues, including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1, we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line (SIRC). SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of p63 and Bax proteins. Indirect immunofluorescence assays and Western blot analyses demonstrated the presence of HSV-1 glycoprotein D (gD) in the infected SIRC cell line, and the pattern of gD expression was consistent with efficient viral replication. The results of MTT and ELISA assays showed that HSV-1 elicited a strong cytopathic effect, and apoptosis played an important role in the demise of the infected cells. Mock-infected SIRC cells displayed the constitutive expression of DeltaNp63alpha. The expressions of the Bax-beta and TAp63gamma isoforms were considerably increased, whereas the level of DeltaNp63alpha was decreased in the HSV-1-infected SIRC cells. Experiments involving the use of acyclovir showed that viral DNA replication was necessary for the accumulation of TAp63gamma. These data suggest that a direct, virus-mediated cytopathic effect may play an important role in the pathogenic mechanism of herpetic keratitis. By disturbing the delicate balance between the pro-survival DeltaN and the pro-apoptotic TA isoforms, HSV-1 may cause profound alterations in the viability of the ocular cells and in the tissue homeostasis of the ocular surface.

New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis.

PubMed

Sugimoto, K; Williamson, R E; Wasteneys, G O

2000-12-01

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.

Morphologic and molecular evaluation of Chlamydia trachomatis growth in human endocervix reveals distinct growth patterns

PubMed Central

Lewis, Maria E.; Belland, Robert J.; AbdelRahman, Yasser M.; Beatty, Wandy L.; Aiyar, Ashok A.; Zea, Arnold H.; Greene, Sheila J.; Marrero, Luis; Buckner, Lyndsey R.; Tate, David J.; McGowin, Chris L.; Kozlowski, Pamela A.; O'Brien, Michelle; Lillis, Rebecca A.; Martin, David H.; Quayle, Alison J.

2014-01-01

In vitro models of Chlamydia trachomatis growth have long been studied to predict growth in vivo. Alternative or persistent growth modes in vitro have been shown to occur under the influence of numerous stressors but have not been studied in vivo. Here, we report the development of methods for sampling human infections from the endocervix in a manner that permits a multifaceted analysis of the bacteria, host and the endocervical environment. Our approach permits evaluating total bacterial load, transcriptional patterns, morphology by immunofluorescence and electron microscopy, and levels of cytokines and nutrients in the infection microenvironment. By applying this approach to two pilot patients with disparate infections, we have determined that their contrasting growth patterns correlate with strikingly distinct transcriptional biomarkers, and are associated with differences in local levels of IFNγ. Our multifaceted approach will be useful to dissect infections in the human host and be useful in identifying patients at risk for chronic disease. Importantly, the molecular and morphological analyses described here indicate that persistent growth forms can be isolated from the human endocervix when the infection microenvironment resembles the in vitro model of IFNγ-induced persistence. PMID:24959423

Ketamine induces a robust whole-brain connectivity pattern that can be differentially modulated by drugs of different mechanism and clinical profile.

PubMed

Joules, R; Doyle, O M; Schwarz, A J; O'Daly, O G; Brammer, M; Williams, S C; Mehta, M A

2015-11-01

Ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist, has been studied in relation to the glutamate hypothesis of schizophrenia and increases dissociation, positive and negative symptom ratings. Ketamine effects brain function through changes in brain activity; these activity patterns can be modulated by pre-treatment of compounds known to attenuate the effects of ketamine on glutamate release. Ketamine also has marked effects on brain connectivity; we predicted that these changes would also be modulated by compounds known to attenuate glutamate release. Here, we perform task-free pharmacological magnetic resonance imaging (phMRI) to investigate the functional connectivity effects of ketamine in the brain and the potential modulation of these effects by pre-treatment of the compounds lamotrigine and risperidone, compounds hypothesised to differentially modulate glutamate release. Connectivity patterns were assessed by combining windowing, graph theory and multivariate Gaussian process classification. We demonstrate that ketamine has a robust effect on the functional connectivity of the human brain compared to saline (87.5 % accuracy). Ketamine produced a shift from a cortically centred, to a subcortically centred pattern of connections. This effect is strongly modulated by pre-treatment with risperidone (81.25 %) but not lamotrigine (43.75 %). Based on the differential effect of these compounds on ketamine response, we suggest the observed connectivity effects are primarily due to NMDAR blockade rather than downstream glutamatergic effects. The connectivity changes contrast with amplitude of response for which no differential effect between pre-treatments was detected, highlighting the necessity of these techniques in forming an informed view of the mechanistic effects of pharmacological compounds in the human brain.

Familial primary cutaneous amyloidosis. Clinical, genetic, and immunofluorescent studies.

PubMed

Vasily, D B; Bhatia, S G; Uhlin, S R

1978-08-01

Familial primary cutaneous amyloidosis, a rare, autosomal dominant genodermatosis, affected 16 of 46 family members of German descent. Previous case reports involved families of Russian, Spanish, or Chinese descent. The finding of IgG, IgM, C3 in the amyloid deposits confirms recent reports of immunofluorescent dermal amyloid deposits.

Chemical Space Mapping and Structure-Activity Analysis of the ChEMBL Antiviral Compound Set.

PubMed

Klimenko, Kyrylo; Marcou, Gilles; Horvath, Dragos; Varnek, Alexandre

2016-08-22

Curation, standardization and data fusion of the antiviral information present in the ChEMBL public database led to the definition of a robust data set, providing an association of antiviral compounds to seven broadly defined antiviral activity classes. Generative topographic mapping (GTM) subjected to evolutionary tuning was then used to produce maps of the antiviral chemical space, providing an optimal separation of compound families associated with the different antiviral classes. The ability to pinpoint the specific spots occupied (responsibility patterns) on a map by various classes of antiviral compounds opened the way for a GTM-supported search for privileged structural motifs, typical for each antiviral class. The privileged locations of antiviral classes were analyzed in order to highlight underlying privileged common structural motifs. Unlike in classical medicinal chemistry, where privileged structures are, almost always, predefined scaffolds, privileged structural motif detection based on GTM responsibility patterns has the decisive advantage of being able to automatically capture the nature ("resolution detail"-scaffold, detailed substructure, pharmacophore pattern, etc.) of the relevant structural motifs. Responsibility patterns were found to represent underlying structural motifs of various natures-from very fuzzy (groups of various "interchangeable" similar scaffolds), to the classical scenario in medicinal chemistry (underlying motif actually being the scaffold), to very precisely defined motifs (specifically substituted scaffolds).

Identity of SMCT1 (SLC5A8) as a neuron-specific Na+-coupled transporter for active uptake of L-lactate and ketone bodies in the brain.

PubMed

Martin, Pamela M; Gopal, Elangovan; Ananth, Sudha; Zhuang, Lina; Itagaki, Shiro; Prasad, Balakrishna M; Smith, Sylvia B; Prasad, Puttur D; Ganapathy, Vadivel

2006-07-01

SMCT1 is a sodium-coupled (Na(+)-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na(+)-coupled and electrogenic. The Michaelis constant is 1.4 +/- 0.1 mm for beta-d-hydroxybutyrate, 0.21 +/- 0.04 mm for acetoacetate and 0.21 +/- 0.03 mm for alpha-ketoisocaproate. The Na(+) : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na(+)-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na(+)-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na(+) gradient and hence, contribute to the maintenance of the energy status and function of neurons.

Low work function, stable compound clusters and generation process

DOEpatents

Dinh, Long N.; Balooch, Mehdi; Schildbach, Marcus A.; Hamza, Alex V.; McLean, II, William

2000-01-01

Low work function, stable compound clusters are generated by co-evaporation of a solid semiconductor (i.e., Si) and alkali metal (i.e., Cs) elements in an oxygen environment. The compound clusters are easily patterned during deposition on substrate surfaces using a conventional photo-resist technique. The cluster size distribution is narrow, with a peak range of angstroms to nanometers depending on the oxygen pressure and the Si source temperature. Tests have shown that compound clusters when deposited on a carbon substrate contain the desired low work function property and are stable up to 600.degree. C. Using the patterned cluster containing plate as a cathode baseplate and a faceplate covered with phosphor as an anode, one can apply a positive bias to the faceplate to easily extract electrons and obtain illumination.

SAR matrices: automated extraction of information-rich SAR tables from large compound data sets.

PubMed

Wassermann, Anne Mai; Haebel, Peter; Weskamp, Nils; Bajorath, Jürgen

2012-07-23

We introduce the SAR matrix data structure that is designed to elucidate SAR patterns produced by groups of structurally related active compounds, which are extracted from large data sets. SAR matrices are systematically generated and sorted on the basis of SAR information content. Matrix generation is computationally efficient and enables processing of large compound sets. The matrix format is reminiscent of SAR tables, and SAR patterns revealed by different categories of matrices are easily interpretable. The structural organization underlying matrix formation is more flexible than standard R-group decomposition schemes. Hence, the resulting matrices capture SAR information in a comprehensive manner.

Semantically Transparent and Opaque Compounds in German Noun-Phrase Production: Evidence for Morphemes in Speaking.

PubMed

Lorenz, Antje; Zwitserlood, Pienie

2016-01-01

This study examines the lexical representation and processing of noun-noun compounds and their grammatical gender during speech production in German, a language that codes for grammatical gender (masculine, feminine, and neuter). Using a picture-word interference paradigm, participants produced determiner-compound noun phrases in response to pictures, while ignoring written distractor words. Compound targets were either semantically transparent (e.g., birdhouse) or opaque (e.g., hotdog), and their constituent nouns either had the same or a different gender (internal gender match). Effects of gender-congruent but otherwise unrelated distractor nouns, and of two morphologically related distractors corresponding to the first or second constituent were assessed relative to a completely unrelated, gender-incongruent distractor baseline. Both constituent distractors strongly facilitated compound naming, and these effects were independent of the targets' semantic transparency. This supports retrieval of constituent morphemes for semantically transparent and opaque compounds during speech production. Furthermore, gender congruency between compounds and distractors did not speed up naming in general, but interacted with gender match of the compounds' constituent nouns, and their semantic transparency. A significant gender-congruency effect was obtained with semantically transparent compounds, consisting of two constituent nouns of the same gender, only. In principle, this pattern is compatible with a multiple lemma representation account for semantically transparent, but not for opaque compounds. The data also fit with a more parsimonious, holistic representation for all compounds at the lemma level, when differences in co-activation patterns for semantically transparent and opaque compounds are considered.

Anti-Neutrophil Cytoplasmic Antibody in Behçet’s Disease

PubMed Central

Duzgun, Nursen; Sahin, Mehmet; Ayaslioglu, Ergin

2006-01-01

Anti neutrophil cytoplasmic antibody (ANCA) is strongly associated with some vasculitic disorders. Behçet’s disease (BD) is a systemic vasculitis of unknown etiology. In this study, ANCA was found to be positive in 8 out of 66 patients (10.2%) with BD by combination testing consisting of immunofluorescence and ELISA [one patient showed an atypical pattern by indirect immunofloresence techique, 6 patients were reactive to bacterial-permeability increasing protein (BPI) and one patient was reactive to Cathepsin G in ELISA]. There were no vascular manifestations such as veneous or arterial thrombosis and arterial aneurysms in ANCA-positive patients with BD. The results suggest that ANCA may be found in a minority of BD as those in previous published studies. PMID:23674967

[Transient expression and characterization of intracellular single chain Fv against the nucleocapsid protein of Hantavirus].

PubMed

Bai, Wen-tao; Xu, Zhi-kai; Zhang, Fang-lin; Luo, Wen; Liu, Yong; Wu, Xing-an; Yan, Yan

2004-11-01

To transiently express an intracellular single chain Fv of monoclonal antibody 1A8 against nucleocapsid protein of Hantavirus and characterize the immunological activities of the expressed products. COS-7 cells were transfected with mammalian expression vector 1A8-scFv-Ckappa/pCI-neo via lipofectin. The expressed product was identified by indirect immunofluorescence and immunoprecipitation. A diffuse pattern fluorescence was observed in less than 1% cytoplasm of transfected COS-7 cells. The binding of intracellular antibody fragments to NP antigen was confirmed by immunoprecipitation analysis. Transiently expressed single chain intrabodies can effectively target NP antigen in the cytoplasm. The present study may provide a new approach for treatment of Hantavirus.

Magneto immunofluorescence assay for diagnosis of celiac disease.

PubMed

Kergaravat, Silvina V; Beltramino, Luis; Garnero, Nidia; Trotta, Liliana; Wagener, Marta; Fabiano, Silvia N; Pividori, Maria Isabel; Hernandez, Silvia R

2013-10-10

A magneto immunofluorescence assay for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The ATG2 were recognized by transglutaminase enzyme immobilized on the magnetic beads and then the immunological reaction was revealed by antibodies labeled with peroxidase. The fluorescent response of the enzymatic reaction with o-phenylenediamine and H2O2 as substrates was correlated with anti-transglutaminase titer, showing EC50 and LOD values of 1:11,600 and 1:74,500 of antibody titers, respectively. A total number of 29 sera samples from clinically confirmed cases of celiac disease and 19 negative control samples were tested by the novel magneto immunofluorescence assay. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 8.1 U was the most effective cut-off value to discriminate correctly between celiac and non-celiac patients. The immunofluorescence assay exhibited a sensitivity of 96.6%, a specificity of 89.5% and an efficiency 93.8% compared with the commercial optical ELISA kit. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparison of three methods for the detection of Trichinella spiralis infections in pigs by five European laboratories*

PubMed Central

Kohler, G.; Ruitenberg, E. J.

1974-01-01

Three methods employed in the diagnosis of trichinosis (trichinoscopy, digestion method, and immunofluorescence technique) were compared by laboratories in 5 countries of the European economic community. For this purpose, material from 32 pigs infected with 50, 150, 500, and 1 500 T. spiralis larvae was examined. With none of the three methods was it possible to detect with sufficient reliability a T. spiralis infection in pigs infected with 50 larvae. The digestion method and the immunofluorescence technique yielded more reliable results when the infection dose was 150 larvae or more. With trichinoscopy, reliable results were obtained in pigs infected with 500 and 1 500 larvae. With the digestion method and trichinoscopy, the onset of infections was detectable from 3 weeks post infection, the digestion method being more reliable; the immunofluorescence technique yielded positive results from approximately 4-6 weeks post infection. The immunofluorescence technique is applicable for epidemiological surveys. As a routine diagnostic procedure in the slaughterhouse, trichinoscopy and the digestion method are possible alternatives, the latter being more sensitive. PMID:4616776

Autoantibodies against Leydig cells in patients after spermatic cord torsion.

PubMed Central

Zanchetta, R; Mastrogiacomo, I; Graziotti, P; Foresta, C; Betterle, C

1984-01-01

This study is aimed at searching for the presence of circulating antibodies against frozen sections of human testis, ovary and trophoblast in patients that had spermatic cord torsion. Sixty-eight sera samples were studied. Nine patients (13.2%) were positive for organ specific anti-testis autoantibodies. Six patients were positive for antibodies against Leydig cells: five were positive only with the indirect immunofluorescence technique of complement fixing (ITT/CF), the sixth patient was positive only with the indirect immunofluorescence technique (ITT). The other three patients were positive for antibodies against germ line cells: two patients were positive with both techniques, the third was positive only with indirect immunofluorescence technique. Eight of these patients were negative for antibodies against adrenal cortex while only one case was positive with indirect immunofluorescence technique both on adrenal cortex and Leydig cells. Human lyophilized testis absorbed the reactive antibodies against Leydig cells and germ line cells, while adrenal cortex and lyophilized testosterone were ineffective. This study shows the identification of a specific antibody against Leydig cells and germ line cells in patients after spermatic cord torsion. PMID:6362937

Ligand-based virtual screening and inductive learning for identification of SIRT1 inhibitors in natural products.

PubMed

Sun, Yunan; Zhou, Hui; Zhu, Hongmei; Leung, Siu-wai

2016-01-25

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase, and its dysregulation can lead to ageing, diabetes, and cancer. From 346 experimentally confirmed SIRT1 inhibitors, an inhibitor structure pattern was generated by inductive logic programming (ILP) with DMax Chemistry Assistant software. The pattern contained amide, amine, and hetero-aromatic five-membered rings, each of which had a hetero-atom and an unsubstituted atom at a distance of 2. According to this pattern, a ligand-based virtual screening of 1 444 880 active compounds from Chinese herbs identified 12 compounds as inhibitors of SIRT1. Three compounds (ZINC08790006, ZINC08792229, and ZINC08792355) had high affinity (-7.3, -7.8, and -8.6 kcal/mol, respectively) for SIRT1 as estimated by molecular docking software AutoDock Vina. This study demonstrated a use of ILP and background knowledge in machine learning to facilitate virtual screening.

Ligand-based virtual screening and inductive learning for identification of SIRT1 inhibitors in natural products

NASA Astrophysics Data System (ADS)

Sun, Yunan; Zhou, Hui; Zhu, Hongmei; Leung, Siu-Wai

2016-01-01

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase, and its dysregulation can lead to ageing, diabetes, and cancer. From 346 experimentally confirmed SIRT1 inhibitors, an inhibitor structure pattern was generated by inductive logic programming (ILP) with DMax Chemistry Assistant software. The pattern contained amide, amine, and hetero-aromatic five-membered rings, each of which had a hetero-atom and an unsubstituted atom at a distance of 2. According to this pattern, a ligand-based virtual screening of 1 444 880 active compounds from Chinese herbs identified 12 compounds as inhibitors of SIRT1. Three compounds (ZINC08790006, ZINC08792229, and ZINC08792355) had high affinity (-7.3, -7.8, and -8.6 kcal/mol, respectively) for SIRT1 as estimated by molecular docking software AutoDock Vina. This study demonstrated a use of ILP and background knowledge in machine learning to facilitate virtual screening.

A Model-Based Analysis of Chemical and Temporal Patterns of Cuticular Hydrocarbons in Male Drosophila melanogaster

PubMed Central

Kent, Clement; Azanchi, Reza; Smith, Ben; Chu, Adrienne; Levine, Joel

2007-01-01

Drosophila Cuticular Hydrocarbons (CH) influence courtship behaviour, mating, aggregation, oviposition, and resistance to desiccation. We measured levels of 24 different CH compounds of individual male D. melanogaster hourly under a variety of environmental (LD/DD) conditions. Using a model-based analysis of CH variation, we developed an improved normalization method for CH data, and show that CH compounds have reproducible cyclic within-day temporal patterns of expression which differ between LD and DD conditions. Multivariate clustering of expression patterns identified 5 clusters of co-expressed compounds with common chemical characteristics. Turnover rate estimates suggest CH production may be a significant metabolic cost. Male cuticular hydrocarbon expression is a dynamic trait influenced by light and time of day; since abundant hydrocarbons affect male sexual behavior, males may present different pheromonal profiles at different times and under different conditions. PMID:17896002

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

PubMed

Shackney, Stanley; Emlet, David R; Pollice, Agnese; Smith, Charles; Brown, Kathryn; Kociban, Deborah

2006-01-01

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. Copyright (c) 2005 Wiley-Liss, Inc.

Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool.

PubMed

Mesquita, Flávio da Silva; Oliveira, Danielle Bruna Leal de; Crema, Daniela; Pinez, Célia Miranda Nunes; Colmanetti, Thaís Cristina; Thomazelli, Luciano Matsumia; Gilio, Alfredo Elias; Vieira, Sandra Elisabeth; Martinez, Marina Baquerizo; Botosso, Viviane Fongaro; Durigon, Edison Luiz

The aim of this study was to evaluate the QuickVue ® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue ® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue ® RSV Test and viral load or specific strain. The QuickVue ® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. This study demonstrated that the QuickVue ® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

When experience meets language statistics: Individual variability in processing English compound words.

PubMed

Falkauskas, Kaitlin; Kuperman, Victor

2015-11-01

Statistical patterns of language use demonstrably affect language comprehension and language production. This study set out to determine whether the variable amount of exposure to such patterns leads to individual differences in reading behavior as measured via eye-movements. Previous studies have demonstrated that more proficient readers are less influenced by distributional biases in language (e.g., frequency, predictability, transitional probability) than poor readers. We hypothesized that a probabilistic bias that is characteristic of written but not spoken language would preferentially affect readers with greater exposure to printed materials in general and to the specific pattern engendering the bias. Readers of varying reading experience were presented with sentences including English compound words that can occur in 2 spelling formats with differing probabilities: concatenated (windowsill, used 40% of the time) or spaced (window sill, 60%). Linear mixed effects multiple regression models fitted to the eye-movement measures showed that the probabilistic bias toward the presented spelling had a stronger facilitatory effect on compounds that occurred more frequently (in any spelling) or belonged to larger morphological families, and on readers with higher scores on a test of exposure-to-print. Thus, the amount of support toward the compound's spelling is effectively exploited when reading, but only when the spelling patterns are entrenched in an individual's mental lexicon via overall exposure to print and to compounds with alternating spelling. We argue that research on the interplay of language use and structure is incomplete without proper characterization of how particular individuals, with varying levels of experience and skill, learn these language structures. (c) 2015 APA, all rights reserved).

Diagnosing gastrointestinal illnesses using fecal headspace volatile organic compounds

PubMed Central

Chan, Daniel K; Leggett, Cadman L; Wang, Kenneth K

2016-01-01

Volatile organic compounds (VOCs) emitted from stool are the components of the smell of stool representing the end products of microbial activity and metabolism that can be used to diagnose disease. Despite the abundance of hydrogen, carbon dioxide, and methane that have already been identified in human flatus, the small portion of trace gases making up the VOCs emitted from stool include organic acids, alcohols, esters, heterocyclic compounds, aldehydes, ketones, and alkanes, among others. These are the gases that vary among individuals in sickness and in health, in dietary changes, and in gut microbial activity. Electronic nose devices are analytical and pattern recognition platforms that can utilize mass spectrometry or electrochemical sensors to detect these VOCs in gas samples. When paired with machine-learning and pattern recognition algorithms, this can identify patterns of VOCs, and thus patterns of smell, that can be used to identify disease states. In this review, we provide a clinical background of VOC identification, electronic nose development, and review gastroenterology applications toward diagnosing disease by the volatile headspace analysis of stool. PMID:26819529

Characteristics, chemical compositions and biological activities of propolis from Al-Bahah, Saudi Arabia

PubMed Central

Elnakady, Yasser A.; Rushdi, Ahmed I.; Franke, Raimo; Abutaha, Nael; Ebaid, Hossam; Baabbad, Mohannad; Omar, Mohamed O. M.; Al Ghamdi, Ahmad A.

2017-01-01

Propolis has been used to treat several diseases since ancient times, and is an important source of bioactive natural compounds and drug derivatives. These properties have kept the interest of investigators around the world, leading to the investigation of the chemical and biological properties and application of propolis. In this report, the chemical constituents that are responsible for the anticancer activities of propolis were analyzed. The propolis was sourced from Al-Baha in the southern part of the Kingdom of Saudi Arabia. Standard protocols for chemical fractionation and bioactivity-guided chemical analysis were used to identify the bio-active ethyl acetate fraction. The extraction was performed in methanol and then analyzed by gas chromatography-mass spectrometry (GC-MS). The major compounds are triterpenoids, with a relative concentration of 74.0%; steroids, with a relative concentration of 9.8%; and diterpenoids, with a relative concentration of 7.9%. The biological activity was characterized using different approaches and cell-based assays. Propolis was found to inhibit the proliferation of cancer cells in a concentration-dependent manner through apoptosis. Immunofluorescence staining with anti-α-tubulin antibodies and cell cycle analysis indicated that tubulin and/or microtubules are the cellular targets of the L-acetate fraction. This study demonstrates the importance of Saudi propolis as anti-cancer drug candidates. PMID:28165013

Characteristics, chemical compositions and biological activities of propolis from Al-Bahah, Saudi Arabia

NASA Astrophysics Data System (ADS)

Elnakady, Yasser A.; Rushdi, Ahmed I.; Franke, Raimo; Abutaha, Nael; Ebaid, Hossam; Baabbad, Mohannad; Omar, Mohamed O. M.; Al Ghamdi, Ahmad A.

2017-02-01

Propolis has been used to treat several diseases since ancient times, and is an important source of bioactive natural compounds and drug derivatives. These properties have kept the interest of investigators around the world, leading to the investigation of the chemical and biological properties and application of propolis. In this report, the chemical constituents that are responsible for the anticancer activities of propolis were analyzed. The propolis was sourced from Al-Baha in the southern part of the Kingdom of Saudi Arabia. Standard protocols for chemical fractionation and bioactivity-guided chemical analysis were used to identify the bio-active ethyl acetate fraction. The extraction was performed in methanol and then analyzed by gas chromatography-mass spectrometry (GC-MS). The major compounds are triterpenoids, with a relative concentration of 74.0%; steroids, with a relative concentration of 9.8%; and diterpenoids, with a relative concentration of 7.9%. The biological activity was characterized using different approaches and cell-based assays. Propolis was found to inhibit the proliferation of cancer cells in a concentration-dependent manner through apoptosis. Immunofluorescence staining with anti-α-tubulin antibodies and cell cycle analysis indicated that tubulin and/or microtubules are the cellular targets of the L-acetate fraction. This study demonstrates the importance of Saudi propolis as anti-cancer drug candidates.

BCM-95 and (2-hydroxypropyl)-β-cyclodextrin reverse autophagy dysfunction and deplete stored lipids in Sap C-deficient fibroblasts.

PubMed

Tatti, Massimo; Motta, Marialetizia; Scarpa, Susanna; Di Bartolomeo, Sabrina; Cianfanelli, Valentina; Tartaglia, Marco; Salvioli, Rosa

2015-08-01

Saposin (Sap) C deficiency is a rare variant form of Gaucher disease caused by impaired Sap C expression or accelerated degradation, and associated with accumulation of glucosylceramide and other lipids in the endo/lysosomal compartment. No effective therapies are currently available for the treatment of Sap C deficiency. We previously reported that a reduced amount and enzymatic activity of cathepsin (Cath) B and Cath D, and defective autophagy occur in Sap C-deficient fibroblasts. Here, we explored the use of two compounds, BCM-95, a curcumin derivative, and (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), to improve lysosomal function of Sap C-deficient fibroblasts. Immunofluorescence and biochemical studies documented that each compound promotes an increase of the expression levels and activities of Cath B and Cath D, and efficient clearance of cholesterol (Chol) and ceramide (Cer) in lysosomes. We provide evidence that BCM-95 and HP-β-CD enhance lysosomal function promoting autophagic clearance capacity and lysosome reformation. Our findings suggest a novel pharmacological approach to Sap C deficiency directed to treat major secondary pathological aspects in this disorder. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fig volatile compounds--a first comparative study.

PubMed

Grison-Pigé, Laure; Hossaert-McKey, Martine; Greeff, Jaco M; Bessière, Jean-Marie

2002-09-01

We analysed the compounds of volatile blends released by receptive figs of twenty Ficus species to attract their specific pollinating wasps. In all, 99 different compounds were identified. The compounds are mainly terpenoids, aliphatic compounds and products from the shikimic acid pathway. In each species blend, there are few major compounds, which are generally common among floral fragrances. Most species blends also include rare compounds, but generally their proportion in the blend is low. A possible basis for species-specificity of Ficus-wasp interactions is discussed in relation to the patterns of volatiles found in this interspecies comparison. Copyright 2002 Elsevier Science Ltd.

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution

PubMed Central

Humphries, Adam; Cereser, Biancastella; Gay, Laura J.; Miller, Daniel S. J.; Das, Bibek; Gutteridge, Alice; Elia, George; Nye, Emma; Jeffery, Rosemary; Poulsom, Richard; Novelli, Marco R.; Rodriguez-Justo, Manuel; McDonald, Stuart A. C.; Wright, Nicholas A.; Graham, Trevor A.

2013-01-01

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO−) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis. PMID:23766371

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution.

PubMed

Humphries, Adam; Cereser, Biancastella; Gay, Laura J; Miller, Daniel S J; Das, Bibek; Gutteridge, Alice; Elia, George; Nye, Emma; Jeffery, Rosemary; Poulsom, Richard; Novelli, Marco R; Rodriguez-Justo, Manuel; McDonald, Stuart A C; Wright, Nicholas A; Graham, Trevor A

2013-07-02

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO(-)) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis.

Expression and distribution of voltage-gated ion channels in ferret sinoatrial node.

PubMed

Brahmajothi, Mulugu V; Morales, Michael J; Campbell, Donald L; Steenbergen, Charles; Strauss, Harold C

2010-10-01

Spontaneous diastolic depolarization in the sinoatrial (SA) node enables it to serve as pacemaker of the heart. The variable cell morphology within the SA node predicts that ion channel expression would be heterogeneous and different from that in the atrium. To evaluate ion channel heterogeneity within the SA node, we used fluorescent in situ hybridization to examine ion channel expression in the ferret SA node region and atrial appendage. SA nodal cells were distinguished from surrounding cardiac myocytes by expression of the slow (SA node) and cardiac (surrounding tissue) forms of troponin I. Nerve cells in the sections were identified by detection of GAP-43 and cytoskeletal middle neurofilament. Transcript expression was characterized for the 4 hyperpolarization-activated cation channels, 6 voltage-gated Na(+) channels, 3 voltage-gated Ca(2+) channels, 24 voltage-gated K(+) channel α-subunits, and 3 ancillary subunits. To ensure that transcript expression was representative of protein expression, immunofluorescence was used to verify localization patterns of voltage-dependent K(+) channels. Colocalizations were performed to observe any preferential patterns. Some overlapping and nonoverlapping binding patterns were observed. Measurement of different cation channel transcripts showed heterogeneous expression with many different patterns of expression, attesting to the complexity of electrical activity in the SA node. This study provides insight into the possible role ion channel heterogeneity plays in SA node pacemaker activity.

The nature of compounds: a psychocentric perspective.

PubMed

Libben, Gary

2014-01-01

Although compound words often seem to be words that themselves contain words, this paper argues that this is not the case for the vast majority of lexicalized compounds. Rather, it is claimed that as a result of acts of lexical processing, the constituents of compound words develop into new lexical representations. These representations are bound to specific morphological roles and positions (e.g., head, modifier) within a compound word. The development of these positionally bound compound constituents creates a rich network of lexical knowledge that facilitates compound processing and also creates some of the well-documented patterns in the psycholinguistic and neurolinguistic study of compounding.

Patterning of Novel Breast Implant Surfaces by Enhancing Silicone Biocompatibility, Using Biomimetic Topographies

PubMed Central

Barr, S.; Hill, E.; Bayat, A.

2010-01-01

Introduction and Aims: Silicone biocompatibility is dictated by cell-surface interaction and its understanding is important in the field of implantation. The role of surface topography and its associated cellular morphology needs investigation to identify qualities that enhance silicone surface biocompatability. This study aims to create well-defined silicone topographies and examine how breast tissue–derived fibroblasts react and align to these surfaces. Methods: Photolithographic microelectronic techniques were modified to produce naturally inspired topographies in silicone, which were cultured with breast tissue–derived human fibroblasts. Using light, immunofluorescent and atomic force microscopy, the cytoskeletal reaction of fibroblasts to these silicone surfaces was investigated. Results: Numerous, well-defined micron-sized pillars, pores, grooves, and ridges were manufactured and characterized in medical grade silicone. Inimitable immunofluorescent microscopy represented in our high magnification images of vinculin, vimentin, and the actin cytoskeleton highlights the differences in fibroblast adhesion between fabricated silicone surfaces. These unique figures illustrate that fibroblast adhesion and the reactions these cells have to silicone can be manipulated to enhance biointegration between the implant and the breast tissue. An alteration of fibroblast phenotype was also observed, exhibiting the propensity of these surfaces to induce categorical remodeling of fibroblasts. Conclusions: This unique study shows that fibroblast reactions to silicone topographies can be tailored to induce physiological changes in cells. This paves the way for further research necessary to develop more biocompatible constructs capable of eliminating capsular contracture by subverting the foreign body response. PMID:20458346

Immunofluorescence Technique Using HeLa Cells Expressing Recombinant Nucleoprotein for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus

PubMed Central

Saijo, Masayuki; Qing, Tang; Niikura, Masahiro; Maeda, Akihiko; Ikegami, Tetsuro; Sakai, Koji; Prehaud, Christophe; Kurane, Ichiro; Morikawa, Shigeru

2002-01-01

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections. PMID:11825944

Balantioides coli: morphological and ultrastructural characteristics of pig and non-human primate isolates.

PubMed

Barbosa, Alynne da Silva; Barbosa, Helene Santos; Souza, Sandra Maria de Oliveira; Dib, Laís Verdan; Uchôa, Claudia Maria Antunes; Bastos, Otilio Machado Pereira; Amendoeira, Maria Regina Reis

2018-06-26

Balantioides coli is a ciliated protozoon that inhabits the intestine of pigs, non-human primates and humans. Light microscopy studies have described over 50 species of the genus Balantioides but their validity is in doubt. Due to the limited information about this genus, this study is aimed to identify morphological characteristics of Balantioides coli isolated using fluorescence microscopy and both scanning (SEM) and transmission electron microscopy (TEM). Trophozoites isolated from the feces of pig and macaque were washed and subjected to centrifugation. These cells were fixed with paraformaldehyde for immunofluorescence. Other aliquots of these trophozoites were fixed with glutaraldehyde, post fixed with osmium tetroxide and processed for SEM and TEM. Immunofluorescence studies revealed microtubules with a longitudinal distribution to the main axis of the parasite and in the constitution of cilia. SEM demonstrated a high concentration of cilia covering the oral apparatus and a poor presence of such structures in cytopyge. TEM revealed in the plasma membrane, several associated structures were observed to delineate the cellular cortex and mucocysts. The cytoskeleton of the oral region was observed in detail and had an organization pattern consisting of microtubules, which formed files and nematodesmal networks. Organelles such as hydrogenosomes like and peroxisomes were observed close to the cortex. Macronuclei were observed, but structures that were consistent with micronuclei were not identified. Ultrastructural morphological analysis of isolates confirms its similarity to Balantioides coli. In this study were identified structures that had not yet been described, such as hydrogenosomes like and cytoskeletal structures.

Neonatal-Onset Chronic Diarrhea Caused by Homozygous Nonsense WNT2B Mutations.

PubMed

O'Connell, Amy E; Zhou, Fanny; Shah, Manasvi S; Murphy, Quinn; Rickner, Hannah; Kelsen, Judith; Boyle, John; Doyle, Jefferson J; Gangwani, Bharti; Thiagarajah, Jay R; Kamin, Daniel S; Goldsmith, Jeffrey D; Richmond, Camilla; Breault, David T; Agrawal, Pankaj B

2018-06-04

Homozygous nonsense mutations in WNT2B were identified in three individuals from two unrelated families with severe, neonatal-onset osmotic diarrhea after whole-exome sequencing was performed on trios from the two families. Intestinal biopsy samples from affected individuals were used for histology and immunofluorescence and to generate enteroids ex vivo. Histopathologic evaluation demonstrated chronic inflammatory changes in the stomach, duodenum, and colon. Immunofluorescence demonstrated diminished staining for OLFM4, a marker for intestinal stem cells (ISCs). The enteroids generated from WNT2B-deficient intestinal epithelium could not be expanded and did not survive passage. Addition of CHIR-99021 (a GSK3A and GSK3B inhibitor and activator of canonical WNT/β-CATENIN signaling) could not rescue WNT2B-deficient enteroids. Addition of supplemental recombinant murine WNT2B was able to perpetuate small enteroids for multiple passages but failed to expand their number. Enteroids showed a 10-fold increase in the expression of LEF1 mRNA and a 100-fold reduction in TLR4 expression, compared with controls by quantitative RT-PCR, indicating alterations in canonical WNT and microbial pattern-recognition signaling. In summary, individuals with homozygous nonsense mutations in WNT2B demonstrate severe intestinal dysregulation associated with decreased ISC number and function, likely explaining their diarrheal phenotype. WNT2B deficiency should be considered for individuals with neonatal-onset diarrhea. Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Effect of propofol on androgen receptor activity in prostate cancer cells.

PubMed

Tatsumi, Kenichiro; Hirotsu, Akiko; Daijo, Hiroki; Matsuyama, Tomonori; Terada, Naoki; Tanaka, Tomoharu

2017-08-15

Androgen receptor is a nuclear receptor and transcription factor activated by androgenic hormones. Androgen receptor activity plays a pivotal role in the development and progression of prostate cancer. Although accumulating evidence suggests that general anesthetics, including opioids, affect cancer cell growth and impact patient prognosis, the effect of those drugs on androgen receptor in prostate cancer is not clear. The purpose of this study was to investigate the effect of the general anesthetic propofol on androgen receptor activity in prostate cancer cells. An androgen-dependent human prostate cancer cell line (LNCaP) was stimulated with dihydrotestosterone (DHT) and exposed to propofol. The induction of androgen receptor target genes was investigated using real-time reverse transcription polymerase chain reaction, and androgen receptor protein levels and localization patterns were analyzed using immunoblotting and immunofluorescence assays. The effect of propofol on the proliferation of LNCaP cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Propofol significantly inhibited DHT-induced expression of androgen receptor target genes in a dose- and time-dependent manner, and immunoblotting and immunofluorescence assays indicated that propofol suppressed nuclear levels of androgen receptor proteins. Exposure to propofol for 24h suppressed the proliferation of LNCaP cells, whereas 4h of exposure did not exert significant effects. Together, our results indicate that propofol suppresses nuclear androgen receptor protein levels, and inhibits androgen receptor transcriptional activity and proliferation in LNCaP cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models.

PubMed

Zhang, Yanke; Gao, Baobing; Xiong, Yan; Zheng, Fangshuo; Xu, Xin; Yang, Yong; Hu, Yida; Wang, Xuefeng

2017-07-01

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.

The Progression of Haze Formation in Rabbit Corneas Following Phototherapeutic Keratectomy

PubMed Central

Gibson, Daniel J.; Tuli, Sonal S.; Schultz, Gregory S.

2013-01-01

Purpose. To determine the topographical location and time course of development of corneal haze in a phototherapeutic keratectomy model using slit lamp examination, macrophotography, quantitative image analysis, and immunofluorescence staining of corneal sections. Methods. Rabbit corneas were ablated with an excimer laser and were observed and graded for haze via slit lamp, imaged, and graded by macrophotography. Corneal sections were stained for α-smooth muscle actin (α-SMA) and tenascin-C (TNC). The distribution of haze imaged in the macrophotographs and density of α-SMA and TNC staining were compared. A daily image time course of haze formation was generated using macrophotography. Results. The first signs of corneal haze were apparent shortly after reepithelialization. The haze was distributed as a ring at the wound margin in all cases, while nearly all corneas also had some central islands of haze initiation. With time, the haze spread within the ablated zone and intensified. The pattern of immunofluorescent staining for α-SMA and TNC at the wound margin mirrored the haze distribution, spread, and intensification with time. Conclusions. The initiation and spread of subepithelial haze begins shortly after reepithelialization. The haze then spreads from the loci of initiation and becomes more dense with time, maturing as early as 14 days after wounding. The improved temporal and spatial resolution provided by these data improve the current model of light-scattering haze formation in wounded corneas, which will improve the design of studies aimed at maintaining corneal clarity following acute injury or surgery. PMID:23800768

Biostatistical analysis of quantitative immunofluorescence microscopy images.

PubMed

Giles, C; Albrecht, M A; Lam, V; Takechi, R; Mamo, J C

2016-12-01

Semiquantitative immunofluorescence microscopy has become a key methodology in biomedical research. Typical statistical workflows are considered in the context of avoiding pseudo-replication and marginalising experimental error. However, immunofluorescence microscopy naturally generates hierarchically structured data that can be leveraged to improve statistical power and enrich biological interpretation. Herein, we describe a robust distribution fitting procedure and compare several statistical tests, outlining their potential advantages/disadvantages in the context of biological interpretation. Further, we describe tractable procedures for power analysis that incorporates the underlying distribution, sample size and number of images captured per sample. The procedures outlined have significant potential for increasing understanding of biological processes and decreasing both ethical and financial burden through experimental optimization. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed Central

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results. PMID:3032390

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results.

Effects of compound Shenhua tablet on renal tubular Na+-K+-ATPase in rats with acute ischemic reperfusion injury.

PubMed

Yang, Yue; Wei, Ri-bao; Zheng, Xiao-yong; Qiu, Qiang; Cui, Shao-yuan; Yin, Zhong; Shi, Suo-zhu; Chen, Xiang-mei

2014-03-01

To observe the effect of Compound Shenhua Tablet (, SHT) on the sodium-potassium- exchanging adenosinetriphosphatase (Na(+)-K(+)-ATPase) in the renal tubular epithelial cells of rats with acute ischemic reperfusion and to investigate the mechanisms underlying the effects of SHT on renal ischemic reperfusion injury (RIRI). Fifty male Wistar rats were randomly divided into the sham surgery group, model group, astragaloside group [150 mg/(kg·d)], SHT low-dose group [1.5 g/(kg·d)] and SHT high-dose group [3.0 g/(kg·d)], with 10 rats in each group. After 1 week of continuous intragastric drug administration, surgery was performed to establish the model. At either 24 or 72 h after the surgery, 5 rats in each group were sacrificed, blood biochemistry, renal pathology, immunoblot and immunohistochemical examinations were performed, and double immunofluorescence staining was observed under a laser confocal microscope. Compared with the sham surgery group, the serum creatinine (SCr) and blood urea nitrogen (BUN) levels were significantly increased, Na(+)-K(+)-ATPase protein level was decreased, and kidney injury molecule-1 (KIM-1) protein level was increased in the model group after the surgery (P<0.01 or P<0.05). Compared with the model group, the SCr, BUN, pathological scores, Na(+)-K(+)-ATPase, and the KIM-1 protein level of the three treatment groups were significantly improved at 72 h after the surgery (P<0.05 or P<0.01). And the SCr, BUN of the SHT low- and high-dose groups, and the pathological scores of the SHT high-dose group were significantly lower than those of the astragaloside group (P<0.05). The localizations of Na(+)-K(+)-ATPase and megalin of the model group were disrupted, with the distribution areas overlapping with each other and alternately arranged. The severity of the disruption was slightly milder in three treatment groups compared with that of the model group. The results of immunofluorescence staining showed that the SHT high-dose group had a superior effect as compared with the astragaloside group and the SHT low-dose group. The SHT effectively alleviated RIRI caused by ischemic reperfusion, promoted the recovery of the polarity of renal tubular epithelial cells, and protected the renal tubules. The therapeutic effects of SHT were superior to those of astragaloside as a single agent.

Long-term hydrocephalus alters the cytoarchitecture of the adult subventricular zone

PubMed Central

Campos-Ordoñez, Tania; Herranz-Pérez, Vicente; Chaichana, Kaisorn L.; Rincon-Torroella, Jordina; Rigamonti, Daniele; García-Verdugo, Jose M.; Quiñones-Hinojosa, Alfredo; Gonzalez-Perez, Oscar

2014-01-01

Hydrocephalus can develop secondarily to a disturbance in production, flow and/or absorption of cerebrospinal fluid. Experimental models of hydrocephalus, especially subacute and chronic hydrocephalus, are few and limited, and the effects of hydrocephalus on the subventricular zone are unclear. The aim of this study was to analyze the effects of long-term obstructive hydrocephalus on the subventricular zone, which is the neurogenic niche lining the lateral ventricles. We developed a new method to induce hydrocephalus by obstructing the aqueduct of Sylvius in the mouse brain, thus simulating aqueductal stenosis in humans. In 120-day-old rodents (n = 18 per group), the degree of ventricular dilatation and cellular composition of the subventricular zone were studied by immunofluorescence and transmission electron microscopy. In adult patients (age > 18 years), the sizes of the subventricular zone, corpus callosum, and internal capsule were analyzed by magnetic resonance images obtained from patients with and without aqueductal stenosis (n=25 per group). Mice with 60-day hydrocephalus had a reduced number of Ki67+ and doublecortin+ cells on immunofluorescence, as well as decreased number of neural progenitors and neuroblasts in the subventricular zone on electron microscopy analysis as compared to non-hydrocephalic mice. Remarkably, a number of extracellular matrix structures (fractones) contacting the ventricular lumen and blood vessels were also observed around the subventricular zone in mice with hydrocephalus. In humans, the widths of the subventricular zone, corpus callosum, and internal capsule in patients with aqueductal stenosis were significantly smaller than age and gender-matched patients without aqueductal stenosis. In summary, supratentorial hydrocephalus reduces the proliferation rate of neural progenitors and modifies the cytoarchitecture and extracellular matrix compounds of the subventricular zone. In humans, this similar process reduces the subventricular niche as well as the width of corpus callosum and internal capsule. PMID:24858805

The lysosomotropic drug LeuLeu-OMe induces lysosome disruption and autophagy-independent cell death in Trypanosoma brucei

PubMed Central

Koh, Hazel X.; Aye, Htay M.; Tan, Kevin S. W.; He, Cynthia Y.

2015-01-01

Background: Trypanosoma brucei is a blood-borne, protozoan parasite that causes African sleeping sickness in humans and nagana in animals. The current chemotherapy relies on only a handful of drugs that display undesirable toxicity, poor efficacy and drug-resistance. In this study, we explored the use of lysosomotropic drugs to induce bloodstream form T. brucei cell death via lysosome destabilization. Methods: We measured drug concentrations that inhibit cell proliferation by 50% (IC_{50) for several compounds, chosen based on their lysosomotropic effects previously reported in Plasmodium falciparum. The lysosomal effects and cell death induced by L-leucyl-L-leucyl methyl ester (LeuLeu-OMe) were further analyzed by flow cytometry and immunofluorescence analyses of different lysosomal markers. The effect of autophagy in LeuLeu-OMe-induced lysosome destabilization and cytotoxicity was also investigated in control and autophagy-deficient cells. Results: LeuLeu-OMe was selected for detailed analyses due to its strong inhibitory profile against T. brucei with minimal toxicity to human cell lines in vitro. Time-dependent immunofluorescence studies confirmed an effect of LeuLeu-OMe on the lysosome. LeuLeu-OMe-induced cytotoxicity was also found to be dependent on the acidic pH of the lysosome. Although an increase in autophagosomes was observed upon LeuLeu-OMe treatment, autophagy was not required for the cell death induced by LeuLeu-OMe. Necrosis appeared to be the main cause of cell death upon LeuLeu-OMe treatment. Conclusions: LeuLeu-OMe is a lysosomotropic agent capable of destabilizing lysosomes and causing necrotic cell death in bloodstream form of T. brucei. PMID:28357304}

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage "salchichón".

PubMed

Andrade, M A Jesús; Córdoba, Juan José; Casado, Eva M A; Córdoba, María G; Rodríguez, Mar

2010-06-01

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products. Copyright 2010 Elsevier Ltd. All rights reserved.

Aberrant distribution patterns of corneodesmosomal components of tape-stripped corneocytes in atopic dermatitis and related skin conditions (ichthyosis vulgaris, Netherton syndrome and peeling skin syndrome type B).

PubMed

Igawa, Satomi; Kishibe, Mari; Honma, Masaru; Murakami, Masamoto; Mizuno, Yuki; Suga, Yasushi; Seishima, Mariko; Ohguchi, Yuka; Akiyama, Masashi; Hirose, Kenji; Ishida-Yamamoto, Akemi; Iizuka, Hajime

2013-10-01

Atopic dermatitis (AD), Netherton syndrome (NS) and peeling skin syndrome type B (PSS) may show some clinical phenotypic overlap. Corneodesmosomes are crucial for maintaining stratum corneum integrity and the components' localization can be visualized by immunostaining tape-stripped corneocytes. In normal skin, they are detected at the cell periphery. To determine whether AD, NS, PSS and ichthyosis vulgaris (IV) have differences in the corneodesmosomal components' distribution and corneocytes surface areas. Corneocytes were tape-stripped from a control group (n=12) and a disease group (37 AD cases, 3 IV cases, 4 NS cases, and 3 PSS cases), and analyzed with immunofluorescent microscopy. The distribution patterns of corneodesmosomal components: desmoglein 1, corneodesmosin, and desmocollin 1 were classified into four types: peripheral, sparse diffuse, dense diffuse and partial diffuse. Corneocyte surface areas were also measured. The corneodesmosome staining patterns were abnormal in the disease group. Other than in the 3 PSS cases, all three components showed similar patterns in each category. In lesional AD skin, the dense diffuse pattern was prominent. A high rate of the partial diffuse pattern, loss of linear cell-cell contacts, and irregular stripping manners were unique to NS. Only in PSS was corneodesmosin staining virtually absent. The corneocyte surface areas correlated significantly with the rate of combined sparse and dense diffuse patterns of desmoglein 1. This method may be used to assess abnormally differentiated corneocytes in AD and other diseases tested. In PSS samples, tape stripping analysis may serve as a non-invasive diagnostic test. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Role of endothelial permeability hotspots and endothelial mitosis in determining age-related patterns of macromolecule uptake by the rabbit aortic wall near branch points.

PubMed

Chooi, K Yean; Comerford, Andrew; Cremers, Stephanie J; Weinberg, Peter D

2016-07-01

Transport of macromolecules between plasma and the arterial wall plays a key role in atherogenesis. Scattered hotspots of elevated endothelial permeability to macromolecules occur in the aorta; a fraction of them are associated with dividing cells. Hotspots occur particularly frequently downstream of branch points, where lesions develop in young rabbits and children. However, the pattern of lesions varies with age, and can be explained by similar variation in the pattern of macromolecule uptake. We investigated whether patterns of hotspots and mitosis also change with age. Evans' Blue dye-labeled albumin was injected intravenously into immature or mature rabbits and its subsequent distribution in the aortic wall around intercostal branch ostia examined by confocal microscopy and automated image analysis. Mitosis was detected by immunofluorescence after adding 5-bromo-2-deoxiuridine to drinking water. Hotspots were most frequent downstream of branches in immature rabbits, but a novel distribution was observed in mature rabbits. Neither pattern was explained by mitosis. Hotspot uptake correlated spatially with the much greater non-hotspot uptake (p < 0.05), and the same pattern was seen when only the largest hotspots were considered. The pattern of hotspots changes with age. The data are consistent with there being a continuum of local permeabilities rather than two distinct mechanisms. The distribution of the dye, which binds to elastin and collagen, was similar to that of non-binding tracers and to lesions apart from a paucity at the lateral margins of branches that can be explained by lower levels of fibrous proteins in those regions. Copyright © 2016. Published by Elsevier Ireland Ltd.

Effects of low dose treatment of tributyltin on the regulation of estrogen receptor functions in MCF-7 cells.

PubMed

Sharan, Shruti; Nikhil, Kumar; Roy, Partha

2013-06-01

Endocrine disrupting chemicals are the natural/synthetic compounds which mimic or inhibit the actions of endogenous hormones. Organotin compounds, such as tributyltin (TBT) are typical environmental contaminants and suspected endocrine-disrupting chemical. The present study evaluates the estrogenic potential of this compound in vitro in ER (+) breast adenocarcinoma, MCF-7 cell line. Our data showed that tributyltin chloride (TBTCl) had agonistic activities for estrogen receptor-α (ER-α). Its estrogenic potential was checked using cell proliferation assay, aromatase assay, transactivation assay, and protein expression analysis. Low dose treatment of TBTCl had a proliferative effect on MCF-7 cells and resulted in up-regulation of aromatase enzyme activity and enhanced estradiol production in MCF-7 cells. Immunofluorescence staining showed translocation of ER-α from cytoplasm to nucleus and increased expression of ER-α, 3β-HSD and aromatase on treatment with increasing doses of TBTCl. Further, to decipher the probable signaling pathways involved in its action, the MCF-7 cells were transfected with different pathway dependent luciferase reporter plasmids (CRE, SRE, NF-κB and AP1). A significant increase in CRE and SRE and decrease in NF-κB regulated pathway were observed (p<0.05). Our results thus showed that the activation of SRE by TBTCl may be due to ligand dependent ER-α activation of the MAPK pathway and increased phosphorylation of ERK. In summary, the present data suggests that low dose of tributyltin genomically and non-genomically augmented estrogen dependent signaling by targeting various pathways. Copyright © 2013 Elsevier Inc. All rights reserved.

Automated antinuclear immunofluorescence antibody screening: a comparative study of six computer-aided diagnostic systems.

PubMed

Bizzaro, Nicola; Antico, Antonio; Platzgummer, Stefan; Tonutti, Elio; Bassetti, Danila; Pesente, Fiorenza; Tozzoli, Renato; Tampoia, Marilina; Villalta, Danilo

2014-03-01

Indirect immunofluorescence (IIF) plays an important role in immunological assays for detecting and measuring autoantibodies. However, the method is burdened by some unfavorable features: the need for expert morphologists, the subjectivity of interpretation, and a low degree of standardization and automation. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of anti-nuclear antibodies (ANA), the biomedical industry has developed technological solutions which might significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading. We collected 104 ANA-positive sera from patients with a confirmed clinical diagnosis of autoimmune disease and 40 ANA-negative sera from healthy blood donors. One aliquot of each serum, without information about pattern and titer, was sent to six laboratories of our group, where the sera were tested with the IIF manual method provided by each of the six manufacturers of automatic systems. Assignment of result (pos/neg), of pattern and titer was made by consensus at a meeting attended by all members of the research team. Result was assigned if consensus for pos/neg was reached by at least four of six certifiers, while for the pattern and for the titer, the value observed with higher frequency (mode) was adopted. Seventeen ANA-positive sera and six ANA-negative sera were excluded. Therefore, the study with the following automatic instrumentation was conducted on 92 ANA-positive sera and on 34 ANA-negative sera: Aklides, EUROPattern, G-Sight (I-Sight-IFA), Helios, Image Navigator, and Nova View. Analytical imprecision was measured in five aliquots of the same serum, randomly added to the sample series. Overall sensitivity of the six automated systems was 96.7% and overall specificity was 89.2%. Most false negatives were recorded for cytoplasmic patterns, whereas among nuclear patterns those with a low level of fluorescence (i.e., multiple nuclear dots, midbody, nuclear rim) were sometimes missed. The intensity values of the light signal of various instruments showed a good correlation with the titer obtained by manual reading (Spearman's rho between 0.672 and 0.839; P<0.0001 for all the systems). Imprecision ranged from 1.99% to 25.2% and, for all the systems, it was lower than that obtained by the manual IIF test (39.1%). The accuracy of pattern recognition, which is for now restricted to the most typical patterns (homogeneous, speckled, nucleolar, centromere, multiple nuclear dots and cytoplasmic) was limited, ranging from 52% to 79%. This study, which is the first to compare the diagnostic accuracy of six systems for automated ANA-IIF reading on the same series of sera, showed that all systems are able to perform very well the task for which they were created. Indeed, cumulative automatic discrimination between positive and negative samples had 95% accuracy. All the manufacturers are actively continuing the development of new and more sophisticated software for a better definition in automatic recognition of patterns and light signal conversion in end-point titer. In the future, this may avert the need for serum dilution for titration, which will be a great advantage in economic terms and time-saving. Copyright © 2013 Elsevier B.V. All rights reserved.

Two novel bursting patterns in the Duffing system with multiple-frequency slow parametric excitations

NASA Astrophysics Data System (ADS)

Han, Xiujing; Zhang, Yi; Bi, Qinsheng; Kurths, Jürgen

2018-04-01

This paper aims to report two novel bursting patterns, the turnover-of-pitchfork-hysteresis-induced bursting and the compound pitchfork-hysteresis bursting, demonstrated for the Duffing system with multiple-frequency parametric excitations. Typically, a hysteresis behavior between the origin and non-zero equilibria of the fast subsystem can be observed due to delayed pitchfork bifurcation. Based on numerical analysis, we show that the stable equilibrium branches, related to the non-zero equilibria resulted from the pitchfork bifurcation, may become the ones with twists and turns. Then, the novel bursting pattern turnover-of-pitchfork-hysteresis-induced bursting is revealed accordingly. In particular, we show that additional pitchfork bifurcation points may appear in the fast subsystem under certain parameter conditions. This creates multiple delay-induced hysteresis behavior and helps us to reveal the other novel bursting pattern, the compound pitchfork-hysteresis bursting. Besides, effects of parameters on the bursting patterns are studied to explore the relation of these two novel bursting patterns.

Perception and Bias in the Processing of Compound versus Phrasal Stress: Evidence from Event-Related Brain Potentials

ERIC Educational Resources Information Center

McCauley, Stewart M; Hestvik, Arild; Vogel, Irene

2013-01-01

Previous research using picture/word matching tasks has demonstrated a tendency to incorrectly interpret phrasally stressed strings as compounds. Using event-related potentials, we sought to determine whether this pattern stems from poor perceptual sensitivity to the compound/phrasal stress distinction, or from a post-perceptual bias in behavioral…

Number of Sense Effects of Chinese Disyllabic Compounds in the Two Hemispheres

ERIC Educational Resources Information Center

Huang, Chih-Ying; Lee, Chia-Ying; Huang, Hsu-Wen; Chou, Chia-Ju

2011-01-01

The current study manipulated the visual field and the number of senses of the first character in Chinese disyllabic compounds to investigate how the related senses (polysemy) of the constituted character in the compounds were represented and processed in the two hemispheres. The ERP results in experiment 1 revealed crossover patterns in the left…

Weathering Patterns of Forensic Biomarker Compounds and PAHs in Coastal Marsh Sediment Samples since the 2010 Deepwater Horizon Oil Spill

NASA Astrophysics Data System (ADS)

Overton, E. B.; Meyer, B.; Miles, S.; Olson, G.; Adhikari, P. L.

2016-02-01

It has been well established that the composition of oil, when spilled into the marine environment, undergoes substantial changes caused by weathering. The general sequence of this compositional change begins with straight chain alkanes (the fastest to degrade), followed by low molecular weight branched and cyclic alkanes and, finally the aromatics. Most resistant to weathering are the higher molecular weight cyclic and branched alkanes (i.e., the "forensic biomarker compounds" such as the hopanes and steranes) and tri-aromatic ringed steroids. The composition of these biomarker compounds is particularly resistant to change because they are not affected by evaporative weathering, are not water soluble, and are not readily degraded by microbial and/or photo-oxidation. However, after extensive time in the environment, being subjected to numerous weathering factors, biomarker compositional patterns are beginning to exhibit significant changes. This presentation will describe the general weathering patterns of petroleum residues in sediment samples collected from marsh areas of coastal Louisiana over a five year period. Particular attention will focus on compositional changes that have been observed in the steranes and diasteranes compounds that traditionally have been considered the most resistant to compositional changes due to weathering.

In situ detection of GM1 and GM2 gangliosides using immunohistochemical and immunofluorescent techniques for auxiliary diagnosis of canine and feline gangliosidoses.

PubMed

Kohyama, Moeko; Yabuki, Akira; Ochiai, Kenji; Nakamoto, Yuya; Uchida, Kazuyuki; Hasegawa, Daisuke; Takahashi, Kimimasa; Kawaguchi, Hiroaki; Tsuboi, Masaya; Yamato, Osamu

2016-03-31

GM1 and GM2 gangliosidoses are progressive neurodegenerative lysosomal storage diseases resulting from the excessive accumulation of GM1 and GM2 gangliosides in the lysosomes, respectively. The diagnosis of gangliosidosis is carried out based on comprehensive findings using various types of specimens for histological, ultrastructural, biochemical and genetic analyses. Therefore, the partial absence or lack of specimens might have resulted in many undiagnosed cases. The aim of the present study was to establish immunohistochemical and immunofluorescent techniques for the auxiliary diagnosis of canine and feline gangliosidoses, using paraffin-embedded brain specimens stored for a long period. Using hematoxylin and eosin staining, cytoplasmic accumulation of pale to eosinophilic granular materials in swollen neurons was observed in animals previously diagnosed with GM1 or GM2 gangliosidosis. The immunohistochemical and immunofluorescent techniques developed in this study clearly demonstrated the accumulated material to be either GM1 or GM2 ganglioside. Immunohistochemical and immunofluorescent techniques using stored paraffin-embedded brain specimens are useful for the retrospective diagnosis of GM1 and GM2 gangliosidoses in dogs and cats.

Increased serum anti-N-methyl-D-aspartate receptor antibody immunofluorescence in psychiatric patients with past catatonia

PubMed Central

Lin, Chin-Chuen; Hung, Yi-Yung; Tsai, Meng-Chang

2017-01-01

Objective Anti-N-methyl-D-aspartate receptor (NMDAR) antibody was thought to be the cause of anti-NMDAR encephalitis, with manifestations similar to catatonia and schizophrenia. Anti-NMDAR antibody in neuropsychiatric patients who had catatonia before were investigated in a follow-up evaluation. The intensity of antibody immunofluorescence was quantified and compared with healthy controls. Method Nineteen patients (eight males and eleven females) agreed to be followed-up. Thirteen had the diagnosis of schizophrenia, two had the diagnosis of major depressive disorder, two had bipolar disorder, one had postpartum depression, and one had herpes simplex encephalitis. No patient had catatonia during the follow-up. Nineteen sex-matched healthy controls were recruited. Results Using Mann-Whitney U test, patients had greater intensity of anti-NMDAR antibody immunofluorescence than the healthy controls (121,979 ± 86,526 vs. 47,692 ± 26,102, p = 0.003). No correlation was found between immunofluorescence intensity and catatonia scales or symptom severity scores. Neuropsychiatric patients with past catatonia showed greater anti-NMDAR antibody response than the healthy controls. Conclusion NMDAR dysfunction might play a role in the mechanism underlying catatonia. Further studies are needed to confirm this finding. PMID:29073246

Increased serum anti-N-methyl-D-aspartate receptor antibody immunofluorescence in psychiatric patients with past catatonia.

PubMed

Lin, Chin-Chuen; Hung, Yi-Yung; Tsai, Meng-Chang; Huang, Tiao-Lai

2017-01-01

Anti-N-methyl-D-aspartate receptor (NMDAR) antibody was thought to be the cause of anti-NMDAR encephalitis, with manifestations similar to catatonia and schizophrenia. Anti-NMDAR antibody in neuropsychiatric patients who had catatonia before were investigated in a follow-up evaluation. The intensity of antibody immunofluorescence was quantified and compared with healthy controls. Nineteen patients (eight males and eleven females) agreed to be followed-up. Thirteen had the diagnosis of schizophrenia, two had the diagnosis of major depressive disorder, two had bipolar disorder, one had postpartum depression, and one had herpes simplex encephalitis. No patient had catatonia during the follow-up. Nineteen sex-matched healthy controls were recruited. Using Mann-Whitney U test, patients had greater intensity of anti-NMDAR antibody immunofluorescence than the healthy controls (121,979 ± 86,526 vs. 47,692 ± 26,102, p = 0.003). No correlation was found between immunofluorescence intensity and catatonia scales or symptom severity scores. Neuropsychiatric patients with past catatonia showed greater anti-NMDAR antibody response than the healthy controls. NMDAR dysfunction might play a role in the mechanism underlying catatonia. Further studies are needed to confirm this finding.

Self-assemblies of luminescent rare earth compounds in capsules and multilayers.

PubMed

Zhang, Renjie; Shang, Juanjuan; Xin, Jing; Xie, Beibei; Li, Ya; Möhwald, Helmuth

2014-05-01

This review addresses luminescent rare earth compounds assembled in microcapsules as well as in planar films fabricated by the layer-by-layer (LbL) technique, the Langmuir-Blodgett (LB) method and in self-assembled monolayers. Chemical precipitation, electrostatic, van der Waals interactions and covalent bonds are involved in the assembly of these compounds. Self-organized ring patterns of rare earth complexes in Langmuir monolayers and on planar surfaces with stripe patterns, as well as fluorescence enhancement due to donor-acceptor pairs, microcavities, enrichment of rare earth compounds, and shell protection against water are described. Recent information on the tuning of luminescence intensity and multicolors by the excitation wavelength and the ratio of rare earth ions, respectively, are also reviewed. Potential applications of luminescent rare earth complex assemblies serving as biological probes, temperature and gas sensors are pointed out. Copyright © 2014 Elsevier B.V. All rights reserved.

Time-activity relationships to VOC personal exposure factors

NASA Astrophysics Data System (ADS)

Edwards, Rufus D.; Schweizer, Christian; Llacqua, Vito; Lai, Hak Kan; Jantunen, Matti; Bayer-Oglesby, Lucy; Künzli, Nino

Social and demographic factors have been found to play a significant role in differences between time-activity patterns of population subgroups. Since time-activity patterns largely influence personal exposure to compounds as individuals move across microenvironments, exposure subgroups within the population may be defined by factors that influence daily activity patterns. Socio-demographic and environmental factors that define time-activity subgroups also define quantifiable differences in VOC personal exposures to different sources and individual compounds in the Expolis study. Significant differences in exposures to traffic-related compounds ethylbenzene, m- and p-xylene and o-xylene were observed in relation to gender, number of children and living alone. Categorization of exposures further indicated time exposed to traffic at work and time in a car as important determinants. Increased exposures to decane, nonane and undecane were observed for males, housewives and self-employed. Categorization of exposures indicated exposure subgroups related to workshop use and living downtown. Higher exposures to 3-carene and α-pinene commonly found in household cleaning products and fragrances were associated with more children, while exposures to traffic compounds ethylbenzene, m- and p-xylene and o-xylene were reduced with more children. Considerable unexplained variation remained in categorization of exposures associated with home product use and fragrances, due to individual behavior and product choice. More targeted data collection methods in VOC exposure studies for these sources should be used. Living alone was associated with decreased exposures to 2-methyl-1-propanol and 1-butanol, and traffic-related compounds. Identification of these subgroups may help to reduce the large amount of unexplained variation in VOC exposure studies. Further they may help in assessing impacts of urban planning that result in changes in behavior of individuals, resulting in shifts in the patterns of exposure experienced by the population.

Indirect micro-immunofluorescence test for detecting type-specific antibodies to herpes simplex virus.

PubMed

Forsey, T; Darougar, S

1980-02-01

A rapid indirect micro-immunofluorescence test capable of detecting and differentiating type-specific antibodies to herpes simplex virus is described. The test proved highly sensitive and, in 80 patients with active herpes ocular infection, antibody was detected in 94%. No anti-herpes antibody was detected in a control group of 20 patients with adenovirus infections. Testing of animal sera prepared against herpes simplex virus types 1 and 2 and of human sera from cases of ocular and genital herpes infections showed that the test can differentiate antibodies to the infecting serotypes. Specimens of whole blood, taken by fingerprick, and eye secretions, both collected on cellulose sponges, could be tested by indirect micro-immunofluorescence. Anti-herpes IgG, IgM, and IgA can also be detected.

Biochemical changes in desmosomes of bovine muzzle epidermis during differentiation.

PubMed

Konohana, A; Konohana, I; Roberts, G P; Marks, R

1987-10-01

Biochemical changes taking place in desmosomes during differentiation have been studied. Bovine muzzle epidermis was sliced horizontally into 6 layers, 0.2 mm thick, and desmosomes were isolated from each layer. These were then analyzed by polyacrylamide gel electrophoresis. The electrophoretic patterns of desmosomal proteins from the 6 layers were found to be qualitatively similar to each other, but there was an increase in the ratio of the amount of 150 kD glycoprotein (desmoglein I) relative to 240 and 210 kD proteins (desmoplakins) in the upper layers of the epidermis. This finding was supported by the similar increase observed in electrophoretic patterns of proteins extracted directly from each layer of the epidermis in electrophoretic sample buffer. In order to study the fate of desmosomal components in the stratum corneum, serial skin surface biopsies were stained with antisera against desmosomal components using indirect immunofluorescence techniques. This experiment showed that desmosomal proteins and glycoproteins persist in the stratum corneum but quantitatively decrease in the outer layers. This decrease may play a significant role in desquamation.

Distribution of calmodulin in pea seedlings: immunocytochemical localization in plumules and root apices

NASA Technical Reports Server (NTRS)

Dauwalder, M.; Roux, S. J.; Hardison, L.

1986-01-01

Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in young etiolated pea (Pisum sativum L.) seedlings. A fairly uniform staining was seen in the nucleoplasm and background cytoplasm of most cell types. Cell walls and nucleoli were not stained. In addition, patterned staining reactions were seen in many cells. In cells of the plumule, punctate staining of the cytoplasm was common, and in part this stain appeared to be associated with the plastids. A very distinctive staining of amyloplasts was seen in the columella of the root cap. Staining associated with cytoskeletal elements could be shown in division stages. By metaphase, staining of the spindle region was quite evident. In epidermal cells of the stem and along the underside of the leaf there was an intense staining of the vacuolar contents. Guard cells lacked this vacuolar stain. Vacuolar staining was sometimes seen in cells of the stele, but the most distinctive pattern in the stele was associated with young conducting cells of the xylem. These staining patterns are consistent with the idea that the interactions of plastids and the cytoskeletal may be one of the Ca(2+)-mediated steps in the response of plants to environmental stimuli. Nuclear functions may also be controlled, at least in part, by Ca2+.

Histopathological spectrum of childhood nephrotic syndrome in Pakistan.

PubMed

Mubarak, Muhammed; Lanewala, Ali; Kazi, Javed Iqbal; Akhter, Fazal; Sher, Atika; Fayyaz, Amir; Bhatti, Sajid

2009-12-01

There is no information in international literature on the pattern of glomerulopathies in children with idiopathic nephrotic syndrome (INS) in Pakistan. We undertook this study to determine the pattern of glomerulopathies based on renal biopsies studied by light (LM), immunofluorescence (IF), and electron microscopy (EM). The study was conducted at Sindh Institute of Urology and Transplantation (SIUT), Karachi over 12 years (1996-2008). All children (

Molecular Networking and Pattern-Based Genome Mining Improves Discovery of Biosynthetic Gene Clusters and their Products from Salinispora Species

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

Duncan, Katherine R.; Crüsemann, Max; Lechner, Anna

Genome sequencing has revealed that bacteria contain many more biosynthetic gene clusters than predicted based on the number of secondary metabolites discovered to date. While this biosynthetic reservoir has fostered interest in new tools for natural product discovery, there remains a gap between gene cluster detection and compound discovery. In this paper, we apply molecular networking and the new concept of pattern-based genome mining to 35 Salinispora strains, including 30 for which draft genome sequences were either available or obtained for this study. The results provide a method to simultaneously compare large numbers of complex microbial extracts, which facilitated themore » identification of media components, known compounds and their derivatives, and new compounds that could be prioritized for structure elucidation. Finally, these efforts revealed considerable metabolite diversity and led to several molecular family-gene cluster pairings, of which the quinomycin-type depsipeptide retimycin A was characterized and linked to gene cluster NRPS40 using pattern-based bioinformatic approaches.« less

Molecular Networking and Pattern-Based Genome Mining Improves Discovery of Biosynthetic Gene Clusters and their Products from Salinispora Species

DOE PAGES

Duncan, Katherine R.; Crüsemann, Max; Lechner, Anna; ...

2015-04-09

Genome sequencing has revealed that bacteria contain many more biosynthetic gene clusters than predicted based on the number of secondary metabolites discovered to date. While this biosynthetic reservoir has fostered interest in new tools for natural product discovery, there remains a gap between gene cluster detection and compound discovery. In this paper, we apply molecular networking and the new concept of pattern-based genome mining to 35 Salinispora strains, including 30 for which draft genome sequences were either available or obtained for this study. The results provide a method to simultaneously compare large numbers of complex microbial extracts, which facilitated themore » identification of media components, known compounds and their derivatives, and new compounds that could be prioritized for structure elucidation. Finally, these efforts revealed considerable metabolite diversity and led to several molecular family-gene cluster pairings, of which the quinomycin-type depsipeptide retimycin A was characterized and linked to gene cluster NRPS40 using pattern-based bioinformatic approaches.« less

Molecular Networking and Pattern-Based Genome Mining Improves discovery of biosynthetic gene clusters and their products from Salinispora species

PubMed Central

Duncan, Katherine R.; Crüsemann, Max; Lechner, Anna; Sarkar, Anindita; Li, Jie; Ziemert, Nadine; Wang, Mingxun; Bandeira, Nuno; Moore, Bradley S.; Dorrestein, Pieter C.; Jensen, Paul R.

2015-01-01

Summary Genome sequencing has revealed that bacteria contain many more biosynthetic gene clusters than predicted based on the number of secondary metabolites discovered to date. While this biosynthetic reservoir has fostered interest in new tools for natural product discovery, there remains a gap between gene cluster detection and compound discovery. Here we apply molecular networking and the new concept of pattern-based genome mining to 35 Salinispora strains including 30 for which draft genome sequences were either available or obtained for this study. The results provide a method to simultaneously compare large numbers of complex microbial extracts, which facilitated the identification of media components, known compounds and their derivatives, and new compounds that could be prioritized for structure elucidation. These efforts revealed considerable metabolite diversity and led to several molecular family-gene cluster pairings, of which the quinomycin-type depsipeptide retimycin A was characterized and linked to gene cluster NRPS40 using pattern-based bioinformatic approaches. PMID:25865308

Automated analysis and reannotation of subcellular locations in confocal images from the Human Protein Atlas.

PubMed

Li, Jieyue; Newberg, Justin Y; Uhlén, Mathias; Lundberg, Emma; Murphy, Robert F

2012-01-01

The Human Protein Atlas contains immunofluorescence images showing subcellular locations for thousands of proteins. These are currently annotated by visual inspection. In this paper, we describe automated approaches to analyze the images and their use to improve annotation. We began by training classifiers to recognize the annotated patterns. By ranking proteins according to the confidence of the classifier, we generated a list of proteins that were strong candidates for reexamination. In parallel, we applied hierarchical clustering to group proteins and identified proteins whose annotations were inconsistent with the remainder of the proteins in their cluster. These proteins were reexamined by the original annotators, and a significant fraction had their annotations changed. The results demonstrate that automated approaches can provide an important complement to visual annotation.

Impact of various factors on radioactivity distribution in different DBS papers.

PubMed

Ren, Xiao; Paehler, Tobias; Zimmer, Manfred; Guo, Zuyu; Zane, Patricia; Emmons, Gary T

2010-08-01

Dried blood spot (DBS) sampling could potentially become the preferred blood collection technique in toxicological and clinical studies. Autoradiography was performed to study compound distribution within a dbs under different conditions using five papers, 31ETF, Grade 226, 903(®), FTA(®) and FTA(®) Elute. The results showed an uneven distribution in all papers with common distribution patterns regardless of compounds: decreased concentrations along the edge, the volcano effect in the middle and the speckle pattern in the center. Treated papers were more readily influenced by environmental factors. Autoradiography enables visualization of a compound's distribution and can guide bioanalytical assay development by allowing convenient evaluation of factors, such as choice of paper, spotting volume, punch size, punch location, temperature and humidity.

Compounds of emerging concern in the San Antonio River Basin, Texas, 2011–12

USGS Publications Warehouse

Lambert, Rebecca B.; Opsahl, Stephen P.

2016-11-16

The City of San Antonio and the surrounding municipalities in Bexar County, Texas, are among the fastest growing cities in the Nation. Increases in residential and commercial development are changing runoff patterns and likely will increase chemical loads into streams. The U.S. Geological Survey, in cooperation with the San Antonio River Authority, evaluated the concentrations and distributional patterns of selected “compounds of emerging concern” (CECs) by collecting and analyzing water-quality samples from 20 sites in the San Antonio River Basin, Tex., during 2011–12. On the basis of their chemical composition or similar uses, the CECs discussed in this fact sheet are wastewater compounds, pharmaceutical compounds (hereinafter referred to as “pharmaceuticals”), and steroidal hormone and sterol compounds (hereinafter referred to as “steroidal hormones and sterols”). Three synoptic sampling events were completed during 2011–12 to analyze for CECs in the San Antonio River Basin. Samples were analyzed for 54 wastewater compounds, 13 pharmaceuticals, 17 steroidal hormones, and 4 sterols. Overall, the concentrations of all CECs analyzed for during this study were low, generally close to or less than the laboratory reporting level.

Alkylcyclohexanes in environmental geochemistry

USGS Publications Warehouse

Hostettler, F.D.; Kvenvolden, K.A.

2002-01-01

The n-alkylated cyclohexanes (CHs) are a homologous series of hydrocarbon compounds that are commonly present in crude oil and refinery products such as diesel fuel. These compounds exhibit specific distribution patterns for different fuel types, providing useful fingerprints for characterizing petroleum products, especially after degradation of n-alkanes has occurred. However, there are no published data to show how these compounds are altered in the environment after long-term spillage of petroleum products. This paper presents two case studies of oil spills that demonstrate the changing distribution patterns resulting from long-term anaerobic microbial degradation. These spills are the 1979 crude-oil spill in Bemidji, Minnesota, and a chronic diesel-fuel spillage from 1953-1991 at Mandan, North Dakota. The alkyl CHs in both spilled oil products are affected by similar biodegradative processes in which the compounds undergo a consistent pattern of loss from the high molecular weight end of the homolog distribution. Degradation results in a measurable increase in the concentrations of the homologs in the lower molecular weight range, a gradual lowering in carbon number of the homolog maximum, and a gradual decrease of the total homolog range from the high molecular weight end. This pattern is the opposite of low-end loss expected with weathering and aerobic biodegradation. The enhancement of the low molecular mass alkyl CH homologs, if not recognized as a degradative pathway of diesel fuel in an anaerobic environment, can potentially be misinterpreted in fuel-oil fingerprinting as deriving from lower distillation-range fuels or admixture of diesel with other fuels.

Root Exudation of Phytochemicals in Arabidopsis Follows Specific Patterns That Are Developmentally Programmed and Correlate with Soil Microbial Functions

PubMed Central

Sugiyama, Akifumi; Manter, Daniel K.; Vivanco, Jorge M.

2013-01-01

Plant roots constantly secrete compounds into the soil to interact with neighboring organisms presumably to gain certain functional advantages at different stages of development. Accordingly, it has been hypothesized that the phytochemical composition present in the root exudates changes over the course of the lifespan of a plant. Here, root exudates of in vitro grown Arabidopsis plants were collected at different developmental stages and analyzed using GC-MS. Principle component analysis revealed that the composition of root exudates varied at each developmental stage. Cumulative secretion levels of sugars and sugar alcohols were higher in early time points and decreased through development. In contrast, the cumulative secretion levels of amino acids and phenolics increased over time. The expression in roots of genes involved in biosynthesis and transportation of compounds represented in the root exudates were consistent with patterns of root exudation. Correlation analyses were performed of the in vitro root exudation patterns with the functional capacity of the rhizosphere microbiome to metabolize these compounds at different developmental stages of Arabidopsis grown in natural soils. Pyrosequencing of rhizosphere mRNA revealed strong correlations (p<0.05) between microbial functional genes involved in the metabolism of carbohydrates, amino acids and secondary metabolites with the corresponding compounds released by the roots at particular stages of plant development. In summary, our results suggest that the root exudation process of phytochemicals follows a developmental pattern that is genetically programmed. PMID:23383346

A study of the aetiology of gastritis following gastric surgery. I. Immunofluorescent studies of the gastric mucosa.

PubMed Central

Chapel, H M; Hoare, A M

1979-01-01

The gastritis which follows surgical trauma is probably not of autoimmune origin. Although identical with the gastritis of pernicious anaemia (PA) upon routine histological examination, immunofluorescent examination of post-operative gastritis differs in that IgA-containing plasma cells alone are found, in contrast to the predominantly IgG-containing plasma cells in PA. PMID:389496

Comparative study of indirect immunofluorescence, enzyme-linked immunosorbent assay, and the Tzanck smear test for the diagnosis of pemphigus.

PubMed

Zhou, Tingting; Fang, Siyue; Li, Chunlei; Hua, Hong

2016-11-01

Pemphigus is one of the potentially fatal autoimmune blistering diseases. An early and accurate diagnosis is important for prognosis and therapy. It may be difficult to diagnosis based on clinical grounds alone. Direct and indirect immunofluorescence, enzyme-linked immunosorbent assay, the Tzanck smear test, or histopathology are all available for the diagnosis of pemphigus. However, there are no generally accepted diagnostic criteria for the diagnosis of this condition at present. To evaluate the diagnostic value of indirect immunofluorescence, enzyme-linked immunosorbent assay, and the Tzanck smear test for the diagnosis of pemphigus in dental clinics. A single-center retrospective study was conducted, and the clinical data of 33 patients with pemphigus and 61 controls were collected and analyzed from the Department of Oral Medicine, Peking University School of Stomatology, during 2010-2014. The sensitivities and specificities of indirect immunofluorescence, enzyme-linked immunosorbent assay, and the Tzanck smear test were calculated and compared in two groups. Sensitivities for the Tzanck smear test, indirect immunofluorescence, and enzyme-linked immunosorbent assay were 96.7%, 84.8%, and 84.8%, respectively, whereas the specificities of these tests were 60%, 91.8%, and 96.7%, respectively. The serial tests for the Tzanck smear test and enzyme-linked immunosorbent assay showed 82% sensitivity and 98.7% specificity. The serial test for the Tzanck smear test and enzyme-linked immunosorbent assay may represent a simple, rapid, and reliable way to definitive diagnosis of pemphigus. It is recommended as a common test for the diagnosis of pemphigus in dental clinics. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Kinetic-dependent enzyme-linked immunosorbent assay for detection of antibodies to Legionella pneumophila.

PubMed Central

Sampson, J S; Wilkinson, H W; Tsang, V C; Brake, B J

1983-01-01

A semiautomated, kinetic-dependent, enzyme-linked immunosorbent assay (K-ELISA) was adapted to detect serum antibodies to Legionella pneumophila. In a comparative study, 158 human serum samples (79 pairs) were tested by K-ELISA and the standard indirect immunofluorescence assay for determination of antibody levels to L. pneumophila serogroup 1. K-ELISA determinations were made by using a serogroup-specific antigen or a preparation (unfractionated antigen) which contained both common antigen and serogroup-specific reactivity. There was good correlation between the immunofluorescence assay and the K-ELISA by using either antigen, although greater correlation was achieved with the unfractionated antigen (coefficients of correlation, 0.894 with unfractionated antigen and 0.841 with serogroup-specific antigen). These results indicate that the K-ELISA is a reliable alternative to the immunofluorescence assay for serologically diagnosing legionellosis. PMID:6361052

Kinetic-dependent enzyme-linked immunosorbent assay for detection of antibodies to Legionella pneumophila.

PubMed

Sampson, J S; Wilkinson, H W; Tsang, V C; Brake, B J

1983-12-01

A semiautomated, kinetic-dependent, enzyme-linked immunosorbent assay (K-ELISA) was adapted to detect serum antibodies to Legionella pneumophila. In a comparative study, 158 human serum samples (79 pairs) were tested by K-ELISA and the standard indirect immunofluorescence assay for determination of antibody levels to L. pneumophila serogroup 1. K-ELISA determinations were made by using a serogroup-specific antigen or a preparation (unfractionated antigen) which contained both common antigen and serogroup-specific reactivity. There was good correlation between the immunofluorescence assay and the K-ELISA by using either antigen, although greater correlation was achieved with the unfractionated antigen (coefficients of correlation, 0.894 with unfractionated antigen and 0.841 with serogroup-specific antigen). These results indicate that the K-ELISA is a reliable alternative to the immunofluorescence assay for serologically diagnosing legionellosis.

Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

PubMed

Schmitz, H

1981-01-01

An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

Analysis of the Cellular Stress Response During Ebola Virus Infection by Immunofluorescence.

PubMed

Nelson, Emily V; Schmidt, Kristina M

2017-01-01

In this chapter, the use of immunofluorescence analysis as a tool to examine stress granule (SG) formation in Ebola virus (EBOV)-infected cells is described. The following protocol focuses on the process of inducing and analyzing the cellular stress response, including treatment of cells with inducers and inhibitors of the SG formation, and also describes EBOV infection, DNA transfection, and the usage of different cell lines.

The Modulation of Fibrosis in Scleroderma by 3-Deoxyglucosone

DTIC Science & Technology

2010-06-01

stained and analyzed for expression of GADD153 in the nucleus by immunofluorescence analysis using Cy3- conjugated secondary antibody . Mean...with the anti-Nox4 polyclonal antibody and Cy2-conjugated secondary antibody . Images were taken at 40 X magnification on an epi-fluorescence...GADD153 in the nucleus by immunofluorescence using a Cy3-conjugated secondary antibody . Representative images were taken at 40 X magnification on an

Volatiles Emitted at Different Flowering Stages of Jasminum sambac and Expression of Genes Related to α-Farnesene Biosynthesis.

PubMed

Yu, Ying; Lyu, Shiheng; Chen, Dan; Lin, Yi; Chen, Jianjun; Chen, Guixin; Ye, Naixing

2017-03-29

Fresh jasmine flowers have been used to make jasmine teas in China, but there has been no complete information about volatile organic compound emissions in relation to flower developmental stages and no science-based knowledge about which floral stage should be used for the infusion. This study monitored volatile organic compounds emitted from living flowers of Jasminum sambac (L.) Ait. 'Bifoliatum' at five developmental stages and also from excised flowers. Among the compounds identified, α-farnesene, linalool, and benzyl acetate were most abundant. Since α-farnesene is synthesized through the Mevalonate pathway, four genes encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), farnesyl pyrophosphate synthase, and terpene synthase were isolated. Their expression patterns in living flowers at the five stages and in excised flowers coincided with the emission patterns of α-farnesene. Application of lovastatin, a HMGR inhibitor, significantly reduced the expression of the genes and greatly decreased the emission of α-farnesene. The sweet scent was diminished from lovastatin-treated flowers as well. These results indicate that α-farnesene is an important compound emitted from jasmine flowers, and its emission patterns suggest that flowers at the opening stage or flower buds 8 h after excision should be used for the infusion of tea leaves.

Influence of organophosphate poisoning on human dendritic cells.

PubMed

Schäfer, Marina; Koppe, Franziska; Stenger, Bernhard; Brochhausen, Christoph; Schmidt, Annette; Steinritz, Dirk; Thiermann, Horst; Kirkpatrick, Charles James; Pohl, Christine

2013-12-05

Organophosphourus compounds (OPC, including nerve agents and pesticides) exhibit acute toxicity by inhibition of acetylcholinesterase. Lung affections are frequent complications and a risk factor for death. In addition, epidemiological studies reported immunological alterations after OPC exposure. In our experiments we investigated the effects of organophosphourus pesticides dimethoate and chlorpyrifos on dendritic cells (DC) that are essential for the initial immune response, especially in the pulmonary system. DC, differentiated from the monocyte cell line THP-1 by using various cytokines (IL-4, GM-CSF, TNF-α, Ionomycin), were exposed to organophosphourus compounds at different concentrations for a 24h time period. DC were characterized by flow cytometry and immunofluorescence using typical dendritic cell markers (e.g., CD11c, CD209 and CD83). After OPC exposure we investigated cell death, the secretion profile of inflammatory mediators, changes of DC morphology, and the effect on protein kinase signalling pathways. Our results revealed a successful differentiation of THP-1 into DC. OPC exposure caused a significant concentration-dependent influence on DC: Dendrites of the DC were shortened and damaged, DC-specific cell surface markers (i.e., CD83and CD209) decreased dramatically after chlorpyrifos exposure. Interestingly, the effects caused by dimethoate were in general less pronounced. The organophosphourus compounds affected the release of inflammatory cytokines, such as IL-1ß and IL-8. The anti-inflammatory cytokine IL-10 was significantly down regulated. Protein kinases like the Akt family or ERK, which are essential for cell survival and proliferation, were inhibited by both OPC. These findings indicate that the tested organophosphourus compounds induced significant changes in cell morphology, inhibited anti-inflammatory cytokines and influenced important protein signalling pathways which are involved in regulation of apoptosis. Thus our results highlight novel aspects -apparently independent of AChE inhibition- of OPC poisoning with regard to lung toxicity. Our findings contribute to the basic understanding of pulmonary complications caused by OPC poisoning. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Trypanosoma cruzi in marsupial didelphids (Philander frenata and Didelhis marsupialis): differences in the humoral immune response in natural and experimental infections.

PubMed

Legey, Ana Paula; Pinho, Ana Paula; Xavier, Samanta C C; Marchevsky, Renato; Carreira, João Carlos; Leon, Leonor L; Jansen, Ana Maria

2003-01-01

Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence.

Novel Bis-(arylsulfonamide) hydroxamate-Based Selective MMP Inhibitors

PubMed Central

Subramaniam, Rajesh; Haldar, Manas K.; Tobwala, Shakila; Ganguly, Bratati; Srivastava, D. K.; Mallik, Sanku

2008-01-01

A series of bis-(arylsulfonamide) hydroxamate inhibitors were synthesized. These compounds exhibit good potency against MMP-7 and MMP-9 depending on the nature, steric bulk and substitution pattern of the substituents in the benzene ring. In general, the preliminary structure-activity relationships (SAR) suggest that among the DAPA hydroxamates (i) electron-rich benzene rings of the sulfonamides may produce better inhibitors than electron-poor analogs. However, potential H-bond acceptors can reverse the trend depending on the isozyme; (ii) isozyme-selectivity between MMP-7 and -9 can be conferred through steric bulk and substitution pattern of the substituents in the benzene ring and (iii) the MMP-10 inhibition pattern of the compounds paralleled that for MMP-9. PMID:18442906

Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents.

PubMed

Rotter, V; Boss, M A; Baltimore, D

1981-04-01

Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.

Unfolded protein response activation in cataracts.

PubMed

Torres-Bernal, Beatriz E; Torres-Bernal, Luis Fernando; Gutiérrez-Campos, Rafael R; Kershenobich Stalnikowitz, David D; Barba-Gallardo, Luis Fernando; Chayet, Arturo A; Ventura-Juárez, Javier

2014-10-01

To analyze the expression of 78 kDa glucose-regulated protein (GRP78) and activating transcription factor 6 (ATF6), 2 factors in the unfolded protein response (UPR), in age-related and diabetes-associated cataract. Universidad Autónoma de Aguascalientes, Aguascalientes, México. Experimental study. The qualitative and quantitative expression of GRP78 and ATF6 were measured in surgical samples from 11 senile cataracts, 9 diabetic-associated cataracts, and 3 normal lenses. Both proteins were detected by immunofluorescence and immunogold-conjugated antibodies. Quantitative morphometry was used to analyze the differences in GRP78 and ATF6 between samples. The Mann-Whitney test was used for statistical analysis. Scanning electron microscopy showed the characteristic organization of fibers in normal lenses with regular alignment and interdigitation between them. On the other hand, lenses from eyes with senile or diabetic cataract showed the same pattern of misalignment and disorganization of the fibers. Both proteins were detected through immunofluorescence in senile and diabetic cataracts, but not in normal lenses. Immunogold-conjugated antibodies and transmission electron microscopy showed that GRP78 and ATF6 grains were 30% higher and 35% higher, respectively, in diabetic cataracts than in senile cataracts (P<.05). These data show for the first time in humans that GRP78 and ATF6 are present in lens fibers of senile cataracts and diabetic cataracts, establishing that the UPR may be important in the process of cataractogenesis. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Cutaneous sensory and autonomic denervation in CADASIL.

PubMed

Nolano, Maria; Provitera, Vincenzo; Donadio, Vincenzo; Caporaso, Giuseppe; Stancanelli, Annamaria; Califano, Francesca; Pianese, Luigi; Liguori, Rocco; Santoro, Lucio; Ragno, Michele

2016-03-15

To assess the involvement of the peripheral nervous system in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) by means of immunofluorescence and confocal analysis of punch skin biopsies. We recruited 14 unrelated patients with CADASIL (M/F = 9/5; age 53.9 ± 10.5 years) and 52 healthy controls (M/F = 31/21; age 53.8 ± 9.8). Patients underwent clinical and neuroradiologic assessment. Three-millimeter punch skin biopsies were taken from the fingertip, the thigh, and the distal leg and processed using indirect immunofluorescence and a panel of primary antibodies to mark vessels and sensory and autonomic nerve fibers. Intraepidermal nerve fibers (IENF), Meissner corpuscles (MC), and sudomotor, vasomotor, and pilomotor nerves were assessed using confocal microscopy. In patients, compared to controls, we found a severe loss of IENF at the distal leg (p < 0.01), at the thigh (p < 0.01), and at the fingertip (p < 0.01) with a non-length-dependent pattern and a loss of MC (p < 0.01). A severe sudomotor, vasomotor, and pilomotor nerve fiber loss was found by semiquantitative evaluation. Along with nerve loss, a severe derangement of the vascular bed was observed. In our patient population, sensory and autonomic denervation did not correlate with age, sex, type of mutation, or MRI involvement. We found an involvement of the peripheral nervous system in patients with CADASIL through the assessment of cutaneous somatic and autonomic nerves. The neurovascular derangement observed in the skin may reflect, although to a lesser extent, what happens in the CNS. © 2016 American Academy of Neurology.

Pemphigus foliaceus in dogs: a review of 37 cases.

PubMed

Ihrke, P J; Stannard, A A; Ardans, A A; Griffin, C E

1985-01-01

Thirty-seven dogs with pemphigus foliaceus were seen over a span of 9 years in a veterinary medical teaching hospital. Four breeds of dogs (Bearded Collie, Akita, Newfoundland, Schipperke) were at significant elevated risk when compared with both the dermatology canine case population and the hospital canine population. The mean age of onset was 4.2 years. The dorsal part of the muzzle was the most common site of initial involvement in over 50% of the dogs, and lesions of the head were seen first in 81% of the dogs. Disease progression was gradual (greater than 3 months) in 73% of the dogs. Somewhat bilaterally symmetric scaling, crusting, and alopecia were seen in all of the dogs. Vesicles, pustules, and bullae were not seen commonly, but target lesions with peripheral collarettes were seen frequently. Most dogs had characteristic footpad lesions, with erythematous swelling at the pad margins, cracking, and villous hypertrophy. Generalized exfoliative dermatitis was seen in dogs with widespread disease. Pruritus was noted in less than one half of the dogs. Typical histopathologic findings included subcorneal and intragranular cell layer epidermal pustules, or intrafollicular pustules with prominent acantholysis. Direct immunofluorescence in an intercellular pattern was noted in 76% of the dogs tested and indirect immunofluorescence was noted in 75% of a much smaller sample. Thirty-nine percent of the dogs responded to corticosteroid therapy alone, and 50% and 55% responded, respectively, to prednisone and cytotoxic drugs, and to prednisone with aurothioglucose. Aurothioglucose was successful alone in 27% of the dogs. One-year survival was achieved in 53% of the dogs.

Age differences in visual search for compound patterns: long- versus short-range grouping.

PubMed

Burack, J A; Enns, J T; Iarocci, G; Randolph, B

2000-11-01

Visual search for compound patterns was examined in observers aged 6, 8, 10, and 22 years. The main question was whether age-related improvement in search rate (response time slope over number of items) was different for patterns defined by short- versus long-range spatial relations. Perceptual access to each type of relation was varied by using elements of same contrast (easy to access) or mixed contrast (hard to access). The results showed large improvements with age in search rate for long-range targets; search rate for short-range targets was fairly constant across age. This pattern held regardless of whether perceptual access to a target was easy or hard, supporting the hypothesis that different processes are involved in perceptual grouping at these two levels. The results also point to important links between ontogenic and microgenic change in perception (H. Werner, 1948, 1957).

The cranberry flavonoids PAC DP-9 and quercetin aglycone induce cytotoxicity and cell cycle arrest and increase cisplatin sensitivity in ovarian cancer cells

PubMed Central

WANG, YIFEI; HAN, ALEX; CHEN, EVA; SINGH, RAKESH K.; CHICHESTER, CLINTON O.; MOORE, RICHARD G.; SINGH, AJAY P.; VORSA, NICHOLI

2015-01-01

Cranberry flavonoids (flavonols and flavan-3-ols), in addition to their antioxidant properties, have been shown to possess potential in vitro activity against several cancers. However, the difficulty of isolating cranberry compounds has largely limited anticancer research to crude fractions without well-defined compound composition. In this study, individual cranberry flavonoids were isolated to the highest purity achieved so far using gravity and high performance column chromatography and LC-MS characterization. MTS assay indicated differential cell viability reduction of SKOV-3 and OVCAR-8 ovarian cancer cells treated with individual cranberry flavonoids. Treatment with quercetin aglycone and PAC DP-9, which exhibited the strongest activity, induced apoptosis, led to caspase-3 activation and PARP deactivation, and increased sensitivity to cisplatin. Furthermore, immunofluorescence microscopy and western blot study revealed reduced expression and activation of epidermal growth factor receptor (EGFR) in PAC DP-9 treated SKOV-3 cells. In addition, quercetin aglycone and PAC DP-9 deactivated MAPK-ERK pathway, induced downregulation of cyclin D1, DNA-PK, phosphohistone H3 and upregulation of p21, and arrested cell cycle progression. Overall, this study demonstrates promising in vitro cytotoxic and anti-proliferative properties of two newly characterized cranberry flavonoids, quercetin aglycone and PAC DP-9, against ovarian cancer cells. PMID:25776829

SAMPLE EXTRACTION AND GC-MS ANALYSIS FOR POLAR VOLATILE ORGANIC COMPOUNDS (PVOCS) IN LIQUID BIOLOGICAL MEDIA

EPA Science Inventory

Current approaches for assessing the cumulative exposures and effects from broad classes of environmental stressors incorporate the measurement of specific groups of endogenous compounds in human biological fluids. Recent focus has been on interpreting patterns of differentially...

GLP-1 secretion is stimulated by 1,10-phenanthroline via colocalized T2R5 signal transduction in human enteroendocrine L cell

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

Park, Jiyoung; Kim, Ki-Suk; Kim, Kang-Hoon

Glucagon-like peptide-1 (GLP-1) hormone is known to regulate blood glucose by an insulinotropic effect and increases proliferation as and also prevents apoptosis of pancreatic β cells. We know that GLP-1 is secreted by nutrients such as fatty acids and sweet compounds but also bitter compounds via stimulation of G-protein coupled receptors (GPCRs) in the gut. Among these, bitter compounds are multiply-contained in phytochemicals or artificial materials and perceived as ligands of various bitter taste receptors. We hypothesized that GLP-1 hormone is secreted through stimulation of a single bitter taste receptor by 1,10-phenanthroline which is known agonist of taste receptor typemore » 2 member 5 (T2R5). To prove this hypothesis, we used the representatively well-known 1,10-phenanthroline as ligand of single receptor and evaluated the existence of T2R5 by double-labeling immunofluorescence and then 1,10-phenanthroline is able to secrete GLP-1 hormone through stimulation of T2R5 in human enteroendocrine cells. Consequently, we verify that GLP-1 hormone is colocalized with T2R5 in the human duodenum and ileum tissue and is secreted by 1,10-phenanthroline via T2R5 signal transduction in differentiated human enteroendocrine L cells. - Highlights: • Taste receptor type 2 member 5 (T2R5) is colocalized with GLP-1 hormone in human enteroendocrine cells. • GLP-1 secretion is stimulated by 1,10-phenanthroline via stimulation of T2R5. • Inhibition of the bitter taste pathway reduce GLP-1 secretion.« less

Compound kinetochores of the Indian muntjac. Evolution by linear fusion of unit kinetochores.

PubMed

Brinkley, B R; Valdivia, M M; Tousson, A; Brenner, S L

1984-01-01

The chromosomes of the Indian muntjac (Muntiacus muntjak vaginalis) are unique among mammals due to their low diploid number (2N = 6 female, 7 male) and large size. It has been proposed that the karyotype of this small Asiatic deer evolved from a related deer the Chinese muntjac (Muntiacus reevesi) with a diploid chromosome number of 2n = 46 consisting of small telocentric chromosomes. In this study we utilized a kinetochore-specific antiserum derived from human patients with the autoimmune disease scleroderma CREST as an immunofluorescent probe to examine kinetochores of the two muntjac species. Since CREST antiserum binds to kinetochores of mitotic chromosomes as well as prekinetochores in interphase nuclei, it was possible to identify and compare kinetochore morphology throughout the cell cycle. Our observations indicated that the kinetochores of the Indian muntjac are composed of a linear beadlike array of smaller subunits that become revealed during interphase. The kinetochores of the Chinese muntjac consisted of minute fluorescent dots located at the tips of the 46 telocentric chromosomes. During interphase, however, the kinetochores of the Chinese muntjac clustered into small aggregates reminiscent of the beadlike arrays seen in the Indian muntjac. Morphometric measurements of fluorescence indicated an equivalent amount of stained material in the two species. Our observations indicate that the kinetochores of the Indian muntjac are compound structures composed of linear arrays of smaller units the size of the individual kinetochores seen on metaphase chromosomes of the Chinese muntjac. Our study supports the notion that the kinetochores of the Indian muntjac evolved by linear fusion of unit kinetochores of the Chinese muntjac. Moreover, it is concluded that the evolution of compound kinetochores may have been facilitated by the non-random aggregation of interphase kinetochores in the nuclei of the ancestral species.

Novel inhibitors of the cellular renin-angiotensin system components, poricoic acids, target Smad3 phosphorylation and Wnt/β-catenin pathway against renal fibrosis.

PubMed

Wang, Ming; Chen, Dan-Qian; Chen, Lin; Cao, Gang; Zhao, Hui; Liu, Dan; Vaziri, Nosratola D; Guo, Yan; Zhao, Ying-Yong

2018-07-01

Tubulo-interstitial fibrosis is the final pathway in the progression of chronic kidney disease (CKD) to kidney failure. The renin-angiotensin system (RAS) plays a major role in CKD progression. Hence, we determined the efficacy of novel RAS inhibitors isolated from Poria cocos against renal fibrosis. Effects of three novel tetracyclic triterpenoid compounds, poricoic acid ZC (PZC), poricoic acid ZD (PZD) and poricoic acid ZE (PZE), were investigated on TGFβ1- and angiotensin II (AngII)-treated HK-2 cells and unilateral ureteral obstruction (UUO) in mice. Immunofluorescence staining, quantitative real-time PCR, siRNA, co-immunoprecipitation and Western blot analyses were used to evaluate expression of key molecules in RAS, Wnt/β-catenin and TGFβ/Smad pathways. Addition of the above compounds to culture media and their administration to UUO mice: (i) significantly attenuated epithelial-to-mesenchymal transition and extracellular matrix production in TGFβ1- and AngII-treated HK-2 cells and UUO mice by inhibiting Wnt/β-catenin pathway activation and Smad3 phosphorylation; (ii) selectively inhibited Smad3 phosphorylation by blocking the interaction of TGFBR1 with Smad3; and (iii) specifically inhibited Smad3 activation. PZC and PZD showed a strong inhibitory effect on all RAS components, and PZE showed a strong inhibitory effect on renin. Furthermore, the secolanostane tetracyclic triterpenoids, PZC and PZD, showed a stronger inhibitory effect than the lanostane tetracyclic triterpenoid PZE. Therefore, compounds with secolanostance skeleton showed stronger bioactivity than those with lanostance skeleton. The secolanostane tetracyclic triterpenoids effectively blocked RAS by simultaneously targeting multiple RAS components and lanostane tetracyclic triterpenoids inhibited renin and protected against tubulo-interstitial fibrosis. © 2018 The British Pharmacological Society.

Reactive carbonyl compounds impair wound healing by vimentin collapse and loss of the primary cilium.

PubMed

Rodríguez-Ribera, Lara; Slattery, Craig; Mc Morrow, Tara; Marcos, Ricard; Pastor, Susana

2017-10-01

In renal pathologies tubulo-interstitial fibrosis results from an aberrant wound-healing ability where the normal epithelial tissue is substituted for scar tissue caused by accumulation of extracellular matrix proteins (ECM). During the wound-healing process, epithelial cells may undergo epithelial-mesenchymal transition (EMT) acquiring a mesenchymal-like phenotype that allows cells to migrate and re-epithelialize the wound site. It has been reported that chronic inflammation and uremic milieu are involved in wound-healing and enhanced kidney damage in chronic kidney disease (CKD) patients. In this study we evaluated reactive carbonyl compounds (RCC) effects on renal wound healing. The compounds resulting from carbonyl stress evaluated in this study were glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and 4-hydroxy-hexenal (HHE). Wound repair ability was evaluated by the wound healing assay using HK-2 cells. EMT was evaluated by morphological, protein and transcriptional changes using microscopy, western blot, zymography and RT-qPCR. Changes in the vimentin network and primary cilia were assessed by immunofluorescence. Our data demonstrated that MDA and GO delay wound closure mediated by vimentin disruption, which caused collagen I mRNA decrease, and deciliation. In contrast, HHE treatment (and MGO to a minor degree) induced morphological changes and increased mesenchymal marker expression and gelatinase activity in HK-2 cells. In this study, we have demonstrated for the first time that exposure to RCC differentially affects wound healing in proximal tubular epithelia. A better comprehension of effects of uremic toxins on wound healing and fibrosis and migration is necessary to seek mechanisms to slow down renal fibrosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immunomicrospheres - Reagents for cell labeling and separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Dreyer, W. J.

1980-01-01

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Cytotoxicities and genotoxicities of cements based on calcium silicate and of dental formocresol.

PubMed

Ko, Hyunjung; Jeong, Youngdan; Kim, Miri

2017-03-01

Increasing interest is being paid to the toxicities of dental materials. The purpose of this study was to determine the cytotoxicities and genotoxicities of endodontic compounds to Chinese hamster ovary (CHO-K1) reproductive cells. Cultured CHO-K1 cells were treated with dental formocresol, two types of calcium hydroxide paste, and two types of mineral trioxide aggregate cement for 24h. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed on each culture, and the micronucleus frequency was determined by performing a micronucleus assay. Alkaline comet assay and γ-H2AX immunofluorescence assay were used to detect DNA damage. Out of the five materials tested, only dental formocresol significantly increased DNA damage. The mineral trioxide aggregate cements based on calcium silicate were not found to be potentially genotoxic. The data suggest that dental formocresol should not be recommended for use in vital pulp therapy on young teeth. Copyright © 2017 Elsevier B.V. All rights reserved.

Poria cocos polysaccharide attenuates RANKL-induced osteoclastogenesis by suppressing NFATc1 activity and phosphorylation of ERK and STAT3.

PubMed

Song, Dezhi; Cao, Zhen; Tickner, Jennifer; Qiu, Heng; Wang, Chao; Chen, Kai; Wang, Ziyi; Guo, Chunyu; Dong, Shiwu; Xu, Jiake

2018-06-01

Pathological fractures caused by osteolytic lesions seriously threaten the health of patients. Osteoclasts play important roles in bone resorption whose hyperfunction are closely related to osteolytic lesions. Studies on osteoclast differentiation and function assist in the prevention of excessive bone loss associated diseases. We screened a variety of natural compounds with anti-inflammatory effect and found that poria cocos polysaccharide (PCP) inhibited RANKL-induced osteoclast formation and bone resorption via TRAcP staining, immunofluorescence, RT-PCR and western blot. PCP down-regulated phosphorylation of STAT3, P38, ERK and JNK, and thus repressed the expression of NFAcT1 and c-Fos during RANKL-induced osteoclastogenesis. Besides, the expression of bone resorption related genes such as TRAcP and CTSK was suppressed by PCP. The results suggest that PCP can be invoked as a candidate for the treatment of osteolytic diseases by inhibiting osteoclastogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Effects of grinding and humidification on the transformation of conglomerate to racemic compound in optically active drugs.

PubMed

Piyarom, S; Yonemochi, E; Oguchi, T; Yamamoto, K

1997-04-01

The effects of grinding and humidification on the transformation of conglomerate to racemic compound have been investigated by X-ray powder diffraction (XPD), differential scanning calorimetry (DSC) and infrared (IR) spectroscopy for leucine, norleucine, valine, serine, tartaric acid and malic acid. Racemic physical mixtures were prepared by physical mixing of equimolar quantities of D and I. crystals using a mortar and pestle. Ground mixtures were obtained by grinding the physical mixtures with a vibrational mill. Humidification was performed by storing the physical mixtures and the ground mixtures in a desiccator containing saturated aqueous salt solutions at 40 degrees C. When physical mixtures of malic acid, tartaric acid and serine were ground, the XPD peaks of the racemic compounds were observed. The XPD patterns of humidified physical mixtures of these compounds also showed the formation of the racemic compounds. This indicated that grinding or humidification of malic acid, tartaric acid and serine induced the transformation of conglomerate to racemic compound crystals. When, on the other hand, the physical mixtures of valine, leucine and norleucine were ground, peaks of racemic compounds were not detected in the XPD pattern. After humidification of the ground mixtures of valine, leucine and norleucine, however, the XPD peaks of racemic compounds were observed. DSC and IR studies revealed consistent results. We concluded that grinding or humidification of malic acid, tartaric acid and serine could induce the transformation of a conglomerate to racemic compound. In contrast, humidifying after grinding was needed to bring about the transformation in leucine, norleucine and valine.

When experience meets language statistics: Individual variability in processing English compound words

PubMed Central

Falkauskas, Kaitlin; Kuperman, Victor

2015-01-01

Statistical patterns of language use demonstrably affect language comprehension and language production. This study set out to determine whether the variable amount of exposure to such patterns leads to individual differences in reading behaviour as measured via eye-movements. Previous studies have demonstrated that more proficient readers are less influenced by distributional biases in language (e.g. frequency, predictability, transitional probability) than poor readers. We hypothesized that a probabilistic bias that is characteristic of written but not spoken language would preferentially affect readers with greater exposure to printed materials in general and to the specific pattern engendering the bias. Readers of varying reading experience were presented with sentences including English compound words that can occur in two spelling formats with differing probabilities: concatenated (windowsill, used 40% of the time) or spaced (window sill, 60%). Linear mixed effects multiple regression models fitted to the eye-movement measures showed that the probabilistic bias towards the presented spelling had a stronger facilitatory effect on compounds that occurred more frequently (in any spelling) or belonged to larger morphological families, and on readers with higher scores on a test of exposure-to-print. Thus, the amount of support towards the compound’s spelling is effectively exploited when reading, but only when the spelling patterns are entrenched in an individual’s mental lexicon via overall exposure to print and to compounds with alternating spelling. We argue that research on the interplay of language use and structure is incomplete without proper characterization of how particular individuals, with varying levels of experience and skill, learn these language structures. PMID:26076328

Rapid Identification of Dengue Virus Serotypes Using Monoclonal Antibodies in an Indirect Immunofluorescence Test.

DTIC Science & Technology

1982-06-18

areas !i). Presently, the only certain method of identification is through the use of rigidly standardized reference antiserum in a virus plaque...low passaged or unpassaged dengue virus from humans or insects using an indirect immunofluorescence 71 test. MATERIALS AND METHODS :, j Cell cultures...streptomycin. Maintanance medium for infected cell cultures consisted of the appropriate growth medium containing 0.4% bovine plasma albumin instead of FBS

A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

PubMed Central

Forment, Josep V.; Jackson, Stephen P.

2016-01-01

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy. PMID:26226461

Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples

NASA Astrophysics Data System (ADS)

Li, Longhui; Rao, Gong; Lv, Xiaohua; Chen, Ruixi; Cheng, Xiaofeng; Wang, Xiaojun; Zeng, Shaoqun; Liu, Xiuli

2018-02-01

Resin embedding is widely used and facilitates microscopic imaging of biological tissues. In contrast, quenching of fluorescence during embedding process hinders the application of resin embedding for imaging of fluorescence-labeled samples. For samples expressing fluorescent proteins, it has been demonstrated that the weakened fluorescence could be recovered by reactivating the fluorophore with alkaline buffer. We extended this idea to immunofluorescence-labeling technology. We showed that the fluorescence of pH-sensitive fluorescein isothiocyanate (FITC) was quenched after resin embedding but reactivated after treating by alkaline buffer. We observed 138.5% fluorescence preservation ratio of reactivated state, sixfold compared with the quenched state in embedding resin, which indicated its application for fluorescence imaging of high signal-to-background ratio. Furthermore, we analyzed the chemical reactivation mechanism of FITC fluorophore. This work would show a way for high-resolution imaging of immunofluorescence-labeled samples embedded in resin.

An erythrodermic variant of pemphigus foliaceus with puzzling histologic and immunopathologic features.

PubMed

Peterson, Jennifer D; Worobec, Sophie M; Chan, Lawrence S

2007-01-01

Pemphigus foliaceus is an autoimmune blistering disorder that affects the skin owing to autoantibodies against desmoglein 1. We employed clinical, histologic, immunopathologic, and serum laboratory studies to investigate a case of an erythrodermic variant of pemphigus foliaceus in an elderly man following treatment with bisoprolol-hydrochlorothiazide. Early histopathology revealed psoriasiform dermatitis, but later biopsies showed subcorneal and granular layer separation with neutrophilic infiltrate. Direct immunofluorescence showed intercellular deposits of immunoglobulin G throughout the epidermis, granular staining of C3 along the basement membrane zone, and fibrin and C3 deposition around the blood vessels. Indirect immunofluorescence on monkey esophagus showed a titer of greater than 1:1,280. Indirect immunofluorescence on rat bladder, antinuclear antibody, lupus panel, and kidney function panel were all negative. There are no reports in the literature of pemphigus foliaceus being induced by bisoprolol, but reports exist of propanolol resulting in drug-induced pemphigus foliaceus.

Seeking the Signs of Life: Assessing the Presence of Biosignatures in the Returned Sample Suite

NASA Astrophysics Data System (ADS)

iMOST Team; Des Marais, D. J.; Grady, M. M.; Shaheen, R.; Steele, A.; Westall, F.; Altieri, F.; Amelin, Y.; Ammannito, E.; Anand, M.; Beaty, D. W.; Benning, L. G.; Bishop, J. L.; Borg, L. E.; Boucher, D.; Brucato, J. R.; Busemann, H.; Campbell, K. A.; Carrier, B. L.; Czaja, A. D.; Debaille, V.; Dixon, M.; Ehlmann, B. L.; Farmer, J. D.; Fernandez-Remolar, D. C.; Fogarty, J.; Glavin, D. P.; Goreva, Y. S.; Hallis, L. J.; Harrington, A. D.; Hausrath, E. M.; Herd, C. D. K.; Horgan, B.; Humayun, M.; Kleine, T.; Kleinhenz, J.; Mangold, N.; Mackelprang, R.; Mayhew, L. E.; McCubbin, F. M.; McCoy, J. T.; McLennan, S. M.; McSween, H. Y.; Moser, D. E.; Moynier, F.; Mustard, J. F.; Niles, P. B.; Ori, G. G.; Raulin, F.; Rettberg, P.; Rucker, M. A.; Schmitz, N.; Sefton-Nash, E.; Sephton, M. A.; Shuster, D. L.; Siljestrom, S.; Smith, C. L.; Spry, J. A.; Swindle, T. D.; ten Kate, I. L.; Tosca, N. J.; Usui, T.; Van Kranendonk, M. J.; Wadhwa, M.; Weiss, B. P.; Werner, S. C.; Wheeler, R. M.; Zipfel, J.; Zorzano, M. P.

2018-04-01

Biosignatures are objects, substances, and/or patterns whose origins require life. They occur as organic compounds, stable isotope patterns, minerals, and morphologies. Each type requires particular modes of preservation and analytical measurements.

Molecular markers indicate different dynamics of leaves and roots during litter decomposition

NASA Astrophysics Data System (ADS)

Altmann, Jens; Jansen, Boris; Palviainen, Marjo; Kalbitz, Karsten

2010-05-01

Up to now there is only a poor understanding of the sources contributing to organic carbon in forest soils, especially the contribution of leaves and roots. Studies of the last 2 decades have shown that methods like pyrolysis and CuO oxidation are suitable tools to trace back the main contributors of organic matter in water, sediments and soils. Lignin derived monomers, extractable lipids, cutin and suberin derived compounds have been used frequently for identification of plant material. However, for the selection of suitable biomarker the decomposition patterns and stability of these compounds are of high importance but they are only poorly understood. In this study we focused on following questions: (I) Which compounds are characteristic to identify certain plant parts and plant species? (II) How stable are these compounds during the first 3 years of litter decomposition? We studied the chemical composition of samples from a 3-year litterbag decomposition experiment with roots and leaves of spruce, pine and birch which was done in Finland. Additionally to mass loss, carbon and nitrogen contents, free lipids were extracted; by alkaline hydrolysis non extractable lipids were gained. The extracts were analyzed afterwards by GC-MS, the insoluble residues were analyzed by curie-point Pyrolysis GC-MS. In addition to the identification and quantification of a variety of different compounds and compound ratios we used statistical classification methods to get deeper insights into the patterns of leaf and root-derived biomarkers during litter decomposition. The mass loss was largely different between the litter species and we always observed larger mass loss for leaf-derived litter in comparison to root derived litter. This trend was also observed by molecular analysis. The increase of the ratio of vanillic acid to vanillin was correlated to the mass loss of the samples over time. This shows that the degree of decomposition of plant material was linked with the degree of lignin degradation. Preliminary results show, that we were able to distinguish the different species and plant parts using various approaches, e.g., abundance and patterns of different substances and different ratios of compounds. The polyesters suberin and cutin were particularly useful to differentiate between roots and leaves. We conclude that knowledge of the decomposition patterns of molecular markers will largely improve the identification power of organic matter sources in soils.

RADIAL PATTERNS OF CARBON ISOTOPES IN THE XYLEM EXTRACTIVES AND CELLULOSE OF DOUGLAS-FIR

EPA Science Inventory

Heartwood extractives (nonstructural wood components) are believed to be formed from a combination of compounds present in the adjacent sapwood and materials imported from the phloem. The roles of local compounds and imported material in heartwood formation could have important i...

COMPARISON OF THE SINK CHARACTERISTICS OF THREE FULL-SCALE ENVIRONMENTAL CHAMBERS

EPA Science Inventory

The paper gives results of an investigation of the interaction of vapor-phase organic compounds with the interior surfaces of three large dynamic test chambers. A pattern of adsorption and reemission of the test compounds was observed in all three chambers. Quantitative compari...

Central nervous system vasculitis caused by propylthiouracil therapy: a case report and literature review.

PubMed

Vanek, C; Samuels, M H

2005-01-01

Antineutrophil cytoplasmic antibodies (ANCA) are associated with vasculitis, including vasculitis induced by drugs such as the thionamides. The affected organ systems in thionamide-induced vasculitis have been primarily renal, musculoskeletal, and dermatologic. We describe the first case of thionamide-induced central nervous system vasculitis presenting as confusion, with complete resolution after discontinuation of propylthiouracil. We review the literature and summarize 42 additional cases of thionamide-induced ANCA-positive vasculitis since 1992. Propylthiouracil was responsible in 93% of cases and the predominant ANCA pattern on immunofluorescent staining was perinuclear (p-ANCA). Clinical improvement occurred after drug discontinuation in 93%, steroid therapy was used in some cases. The mean duration of treatment with thionamides was 35 months prior to presentation. Long-term medical treatment with thionamides for hyperthyroidism may increase the risk of this severe side effect.

An experimental approach towards the development of an in vitro cortical-thalamic co-culture model.

PubMed

Kanagasabapathi, Thirukumaran T; Massobrio, Paolo; Tedesco, Mariateresa; Martinoia, Sergio; Wadman, Wytse J; Decré, Michel M J

2011-01-01

In this paper, we propose an experimental approach to develop an in vitro dissociated cortical-thalamic co-culture model using a dual compartment neurofluidic device. The device has two compartments separated by 10 μm wide and 3 μm high microchannels. The microchannels provide a physical isolation of neurons allowing only neurites to grow between the compartments. Long-term viable co-culture was maintained in the compartmented device, neurite growth through the microchannels was verified using immunofluorescence staining, and electrophysiological recordings from the co-culture system was investigated. Preliminary analysis of spontaneous activities from the co-culture shows a distinctively different firing pattern associated with cultures of individual cell types and further analysis is proposed for a deeper understanding of the dynamics involved in the network connectivity in such a co-culture system.

The clinical impact of Anti-DFS70 antibodies in undifferentiated connective tissue disease: case reports and a review of the literature.

PubMed

Infantino, M; Meacci, F; Grossi, V; Manfredi, M; Li Gobbi, F; Sarzi-Puttini, P; Atzeni, F; Benucci, M

2017-02-01

Anti-nuclear antibody (ANA) positivity suggests CTD but can also lead to a diagnosis of UCTD when a patient does not fulfill the CTD diagnostic criteria. An anti-dense fine speckled (DFS) immunofluorescence (IIF) pattern can be observed when using an ANA test on HEp-2 cells and is due to the presence of antibodies to the nuclear DFS70 antigen that has rarely found in CTD. Serological testing for anti-DFS70 antibodies could therefore play a very interesting negative predictive role in stratifying patients on the basis of the evolution of UCTD to CTD. We described two patients ANA and anti-DFS70 positive in which the use of new method allowing the immunoadsorption of anti-DFS70 antibodies has permitted to exclude the incorrect diagnosis of CTD.

Molecular basis of autosomal dominant neurohypophyseal diabetes insipidus. Cellular toxicity caused by the accumulation of mutant vasopressin precursors within the endoplasmic reticulum.

PubMed Central

Ito, M; Jameson, J L; Ito, M

1997-01-01

Mutations in the arginine vasopressin (AVP) gene cause autosomal dominant familial neurohypophyseal diabetes insipidus (FNDI). The dominant inheritance pattern has been postulated to reflect neuronal toxicity of the mutant proteins, but the mechanism for such cytotoxicity is unknown. In this study, wild-type or several different mutant AVP genes were stably expressed in neuro2A neuroblastoma cells. When cells were treated with valproic acid to induce neuronal differentiation, each of the mutants caused reduced viability. Metabolic labeling revealed diminished intracellular trafficking of mutant AVP precursors and confirmed inefficient secretion of immunoreactive AVP. Immunofluorescence studies demonstrated marked accumulation of mutant AVP precursors within the endoplasmic reticulum. These studies suggest that the cellular toxicity in FNDI may be caused by the intracellular accumulation of mutant precursor proteins. PMID:9109434

Initial Genomics of the Human Nucleolus

PubMed Central

Németh, Attila; Conesa, Ana; Santoyo-Lopez, Javier; Medina, Ignacio; Montaner, David; Péterfia, Bálint; Solovei, Irina; Cremer, Thomas; Dopazo, Joaquin; Längst, Gernot

2010-01-01

We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs) in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD–localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD–specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture. PMID:20361057

Human herpesvirus 8 seroprevalence among internationally adopted children coming from Eastern Europe.

PubMed

Viviano, Enza; Vitale, Francesco; Perna, Anna Maria; Cataldo, Francesco; Firenze, Alberto; Romano, Nino

2009-01-01

The seroprevalence of Human Herpes Virus 8 (HHV8) and its transmission pattern were assessed testing serum samples of 120 internationally adopted children (aged 1-15 years) coming from Eastern Europe. Determinations of IgG antibodies against both latent and lytic HHV-8 antigens were performed by indirect immunofluorescence assay. Antibodies were detected only for lytic antigen of the virus in 12.5% of children with a seroprevalence significatively higher (19.6%) in young children (age 1-6). No correlation was observed between HHV8 seropositivity and serological markers for hepatitis A, B and C viruses and Human Immunodeficiency virus. In conclusion, our findings suggest that HHV8 infection is widespread in some populations from the East Europe, and that person to person contacts among children could be considered the predominant mode of HHV8 transmission in younger age.

The comparative clinical study of efficacy of Gamisoyo-San (Jiaweixiaoyaosan) on generalized anxiety disorder according to differently manufactured preparations: multicenter, randomized, double blind, placebo controlled trial.

PubMed

Park, Dae-Myung; Kim, Seok-Hwan; Park, Yang-Chun; Kang, Wee-Chang; Lee, Sang-Ryong; Jung, In-Chul

2014-12-02

Gamisoyo-San (GSS) is a well-known Traditional Korean Medicine shown to be effective on mood disorders. The purpose of this research is to examine the effect of Gamisoyo-San on generalized anxiety disorder by its differently manufactured preparations. Multicenter, randomized, double-blinded, placebo-controlled study was set for 147 patients with generalized anxiety disorder recruited from November 1st 2009 to December 16th 2010. They were given Gamisoyo-San individual extract mixture (extraction done for each crude materia medica separately) or Gamisoyo-San multi-compound extract (extraction done for whole materia medica at once) or controlled medication. Hamilton Rating Scale for Anxiety (HAM-A), Korean State-Trait Anxiety Inventory (K-STAI), Penn State Worry Questionnaire (PSWQ), Korean Beck Depression Inventroy (K-BDI), Symptom Checklist-90-Revised (SCL-90-R), and Korean WHO Quality of Life Scale Abbreviated Version (WHOQOL-BREF) were evaluated. We also applied Pattern Identification tool for 'JingJi and ZhengChong (, Traditional Korean Medicine term which correlates with generalized anxiety disorder)' to patients to evaluate different responses among 9 patterns. HAM-A scores of Gamisoyo-San multi-compound extract group showed greater decrease compared to Gamisoyo-San individual extract mixture group and placebo group, but the difference was insignificant. WHOQOL-BREF scores of Gamisoyo-San multi-compound extract group showed significant increase compared to Gamisoyo-San individual extract mixture group and placebo group. In Heart blood deficiency pattern, the Gamisoyo-San multi-compound extract group showed significant decrease in K-BDI compared to the Gamisoyo-San individual extract mixture group. Gamisoyo-San did not improve anxiety level of GAD patients. However, it can be useful to improve quality of life, and reduce depressive, obsessive-compulsive, somatic symptoms of generalized anxiety disorder. Gamisoyo-San multi-compound seemed more effective than Gamisoyo-San individual extract mixture, especially in Heart blood deficiency pattern. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Diverse Neurotoxicants Target the Differentiation of Embryonic Neural Stem Cells into Neuronal and Glial Phenotypes

PubMed Central

Slotkin, Theodore A.; Skavicus, Samantha; Card, Jennifer; Levin, Edward D.; Seidler, Frederic J.

2016-01-01

The large number of compounds that need to be tested for developmental neurotoxicity drives the need to establish in vitro models to evaluate specific neurotoxic endpoints. We used neural stem cells derived from rat neuroepithelium on embryonic day 14 to evaluate the impact of diverse toxicants on their ability to differentiate into glia and neurons: a glucocorticoid (dexamethasone), organophosphate insecticides (chlorpyrifos, diazinon, parathion), insecticides targeting the GABAA receptor (dieldrin, fipronil), heavy metals (Ni2+, Ag+), nicotine and tobacco smoke extract. We found three broad groupings of effects. One diverse set of compounds, dexamethasone, the organophosphate pesticides, Ni2+ and nicotine, suppressed expression of the glial phenotype while having little or no effect on the neuronal phenotype. The second pattern was restricted to the pesticides acting on GABAA receptors. These compounds promoted the glial phenotype and suppressed the neuronal phenotype. Notably, the actions of compounds eliciting either of these differentiation patterns were clearly unrelated to deficits in cell numbers: dexamethasone, dieldrin and fipronil all reduced cell numbers, whereas organophosphates and Ni2+ had no effect. The third pattern, shared by Ag+ and tobacco smoke extract, clearly delineated cytotoxicity, characterized major cell loss with suppression of differentiation into both glial and neuronal phenotypes; but here again, there was some selectivity in that glia were suppressed more than neurons. Our results, from this survey with diverse compounds, point to convergence of neurotoxicant effects on a specific “decision node” that controls the emergence of neurons and glia from neural stem cells. PMID:27816694

High Carbon Use Efficiency is Not Explained by Production of Storage Compounds

NASA Astrophysics Data System (ADS)

Dijkstra, Paul; van Groenigen, Kees-Jan

2015-04-01

The efficiency with which microbes use substrate to make new microbial biomass (Carbon Use Efficiency or CUE; mol C / mol C) is an important variable in soil and ecosystem C cycling models. Estimates of CUE in soil microbial communities vary widely. It has been hypothesized that high values of CUE are associated with production of storage compounds following a sudden increases in substrate availability during CUE measurements. In that case, these high CUE values would not be representative for balanced microbial growth (i.e. the production of all compounds needed to make new microbial cells). To test this hypothesis, we added position-specific 13C-labeled glucose isotopomers in parallel incubations of a ponderosa pine and piñon-juniper soil. We compared the measured pattern of CO2 release for the six glucose C atoms with patterns of CO2 production expected for balanced growth with a low, medium, or high CUE, and with CO2 production patterns associated with production of storage compounds (glycogen, lipids, or polyhydroxybutyrate). The measured position-specific CO2 production did not match that for production of glycogen, lipids, or polyhydroxybutyrate, but agreed closely with that expected for balanced growth at high CUE and high pentose phosphate pathway activity. We conclude that soil microbial communities utilize glucose substrate for biomass growth with high CUE, and that addition of small amounts of 13C-labeled glucose tracers do not affect CUE or induce storage compounds production. We submit that the measurement of position-specific CO2 production offers a quick and easy way to test biochemically explicit hypotheses concerning microbial growth metabolism.

Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy.

PubMed

Sun, Yulong; Ip, Philbert; Chakrabartty, Avijit

2017-09-03

Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue sample. A great hindrance to the acquisition of high quality immunofluorescence images is endogenous autofluorescence of the tissue caused by aging pigments such as lipofuscin or by common sample preparation processes such as aldehyde fixation. This protocol describes how background fluorescence can be greatly reduced through photobleaching using white phosphor light emitting diode (LED) arrays prior to treatment with fluorescent probes. The broad-spectrum emission of white phosphor LEDs allow for bleaching of fluorophores across a range of emission peaks. The photobleaching apparatus can be constructed from off-the-shelf components at very low cost and offers an accessible alternative to commercially available chemical quenchers. A photobleaching pre-treatment of the tissue followed by conventional immunofluorescence staining generates images free of background autofluorescence. Compared to established chemical quenchers which reduced probe as well as background signals, photobleaching treatment had no effect on probe fluorescence intensity while it effectively reduced background and lipofuscin fluorescence. Although photobleaching requires more time for pre-treatment, higher intensity LED arrays may be used to reduce photobleaching time. This simple method can potentially be applied to a variety of tissues, particularly postmitotic tissues that accumulate lipofuscin such as the brain and cardiac or skeletal muscles.

Stress Placement on Phrases and Compounds in English

ERIC Educational Resources Information Center

Yurtbasi, Metin

2017-01-01

The three types of stresses namely "word stress," "compound stress" and "phrasal stress" are the key elements to determine the exact means of conveying a specific intent in an utterance. Therefore during perception and production of such meaning-carrying codes, being able to use the right stress pattern is vitally…

Synthesis Road Map Problems in Organic Chemistry

ERIC Educational Resources Information Center

Schaller, Chris P.; Graham, Kate J.; Jones, T. Nicholas

2014-01-01

Road map problems ask students to integrate their knowledge of organic reactions with pattern recognition skills to "fill in the blanks" in the synthesis of an organic compound. Students are asked to identify familiar organic reactions in unfamiliar contexts. A practical context, such as a medicinally useful target compound, helps…

Lexical Processes in the Recognition of Japanese Horizontal and Vertical Compounds

ERIC Educational Resources Information Center

Miwa, Koji; Dijkstra, Ton

2017-01-01

This lexical decision eye-tracking study investigated whether horizontal and vertical readings elicit comparable behavioral patterns and whether reading directions modulate lexical processes. Response times and eye movements were recorded during a lexical decision task with Japanese bimorphemic compound words presented vertically. The data were…

Emergence of Intraverbal Responding Following Tact Instruction with Compound Stimuli

ERIC Educational Resources Information Center

Devine, Bailey; Carp, Charlotte L.; Hiett, Kiley A.; Petursdottir, Anna Ingeborg

2016-01-01

Effective intraverbal responding often requires control by multiple elements of a verbal stimulus. The purpose of this study was to examine the emergence of such intraverbal relations following tact instruction with compound stimuli and to analyze any resulting error patterns. Participants were seven typically developing children between 3 and…

Normal and compound poisson approximations for pattern occurrences in NGS reads.

PubMed

Zhai, Zhiyuan; Reinert, Gesine; Song, Kai; Waterman, Michael S; Luan, Yihui; Sun, Fengzhu

2012-06-01

Next generation sequencing (NGS) technologies are now widely used in many biological studies. In NGS, sequence reads are randomly sampled from the genome sequence of interest. Most computational approaches for NGS data first map the reads to the genome and then analyze the data based on the mapped reads. Since many organisms have unknown genome sequences and many reads cannot be uniquely mapped to the genomes even if the genome sequences are known, alternative analytical methods are needed for the study of NGS data. Here we suggest using word patterns to analyze NGS data. Word pattern counting (the study of the probabilistic distribution of the number of occurrences of word patterns in one or multiple long sequences) has played an important role in molecular sequence analysis. However, no studies are available on the distribution of the number of occurrences of word patterns in NGS reads. In this article, we build probabilistic models for the background sequence and the sampling process of the sequence reads from the genome. Based on the models, we provide normal and compound Poisson approximations for the number of occurrences of word patterns from the sequence reads, with bounds on the approximation error. The main challenge is to consider the randomness in generating the long background sequence, as well as in the sampling of the reads using NGS. We show the accuracy of these approximations under a variety of conditions for different patterns with various characteristics. Under realistic assumptions, the compound Poisson approximation seems to outperform the normal approximation in most situations. These approximate distributions can be used to evaluate the statistical significance of the occurrence of patterns from NGS data. The theory and the computational algorithm for calculating the approximate distributions are then used to analyze ChIP-Seq data using transcription factor GABP. Software is available online (www-rcf.usc.edu/∼fsun/Programs/NGS_motif_power/NGS_motif_power.html). In addition, Supplementary Material can be found online (www.liebertonline.com/cmb).

Significance of microhabitat heterogeneity in the spatial pattern and size-class structure of Anastatica hierochuntica L.

NASA Astrophysics Data System (ADS)

Hegazy, Ahmad K.; Kabiel, Hanan F.

2007-05-01

Anastatica hierochuntica L. (Brassicaceae) is a desert monocarpic annual species characterized by a topochory/ombrohydrochory type of seed dispersal. The hygrochastic nature of the dry skeletons (dead individuals) permits controlling seed dispersal by rain events. The amount of dispersed seeds is proportional to the intensity of rainfall. When light showers occur, seeds are released and remain in the site. Seeds dispersed in the vicinity of the mother or source plant (primary type of seed dispersal) resulted in clumped pattern and complicated interrelationships among size-classes of the population. Following heavy rainfall, most seeds are released and transported into small patches and shallow depressions which collect runoff water. The dead A. hierochuntica skeletons demonstrate site-dependent size-class structure, spatial pattern and spatial interrelationships in different microhabitats. Four microhabitat types have been sampled: runnels, patches and simple and compound depressions in two sites (gravel and sand). Ripley's K-function was used to analyze the spatial pattern in populations of A. hierochuntica skeletons in the study microhabitats. Clumped patterns were observed in nearly all of the study microhabitats. Populations of A. hierochuntica in the sand site were more productive than in the gravel site and usually had more individuals in the larger size-classes. In the compound-depression microhabitat, the degree of clumping decreased from the core zone to the intermediate zone then shifted into overdispersed pattern in the outer zone. At the within size-class level, the clumped pattern dominated in small size classes but shifted into random and overdispersed patterns in the larger size classes. Aggregation between small and large size-classes was not well-defined but large individuals were found closer to the smaller individuals than to those of their own class. In relation to the phytomass and the size-class structure, the outer zone of the simple depression and the outer and intermediate zones of the compound depression microhabitats were the most productive sites.

Mayo Clinic/Renal Pathology Society Consensus Report on Pathologic Classification, Diagnosis, and Reporting of GN.

PubMed

Sethi, Sanjeev; Haas, Mark; Markowitz, Glen S; D'Agati, Vivette D; Rennke, Helmut G; Jennette, J Charles; Bajema, Ingeborg M; Alpers, Charles E; Chang, Anthony; Cornell, Lynn D; Cosio, Fernando G; Fogo, Agnes B; Glassock, Richard J; Hariharan, Sundaram; Kambham, Neeraja; Lager, Donna J; Leung, Nelson; Mengel, Michael; Nath, Karl A; Roberts, Ian S; Rovin, Brad H; Seshan, Surya V; Smith, Richard J H; Walker, Patrick D; Winearls, Christopher G; Appel, Gerald B; Alexander, Mariam P; Cattran, Daniel C; Casado, Carmen Avila; Cook, H Terence; De Vriese, An S; Radhakrishnan, Jai; Racusen, Lorraine C; Ronco, Pierre; Fervenza, Fernando C

2016-05-01

Renal pathologists and nephrologists met on February 20, 2015 to establish an etiology/pathogenesis-based system for classification and diagnosis of GN, with a major aim of standardizing the kidney biopsy report of GN. On the basis of etiology/pathogenesis, GN is classified into the following five pathogenic types, each with specific disease entities: immune-complex GN, pauci-immune GN, antiglomerular basement membrane GN, monoclonal Ig GN, and C3 glomerulopathy. The pathogenesis-based classification forms the basis of the kidney biopsy report. To standardize the report, the diagnosis consists of a primary diagnosis and a secondary diagnosis. The primary diagnosis should include the disease entity/pathogenic type (if disease entity is not known) followed in order by pattern of injury (mixed patterns may be present); score/grade/class for disease entities, such as IgA nephropathy, lupus nephritis, and ANCA GN; and additional features as detailed herein. A pattern diagnosis as the sole primary diagnosis is not recommended. Secondary diagnoses should be reported separately and include coexisting lesions that do not form the primary diagnosis. Guidelines for the report format, light microscopy, immunofluorescence microscopy, electron microscopy, and ancillary studies are also provided. In summary, this consensus report emphasizes a pathogenesis-based classification of GN and provides guidelines for the standardized reporting of GN. Copyright © 2016 by the American Society of Nephrology.

Changes in patterns of persistent halogenated compounds through a pelagic food web in the Baltic Sea.

PubMed

Stephansen, Diana A; Svendsen, Tore C; Vorkamp, Katrin; Frier, Jens-Ole

2012-02-01

The concentrations and patterns of persistent halogenated compounds (PHCs), including polychlorinated biphenyls (PCBs), DDT, hexachlorocyclohexanes (HCHs), hexachlorobenzene (HCB) and polybrominated diphenyl ethers (PBDEs) were examined in a pelagic food web from the southern Baltic Sea consisting of sediment, zooplankton, sprat, Atlantic salmon and anadromous brown trout. Lipid-normalized concentrations generally increased from low trophic levels to high trophic levels, with the exception of HCHs. Due to high concentrations of PBDEs in some zooplankton samples, biomagnification of BDE-47 was only observed for salmon/sprat and trout/sprat. Sprat collected individually and from salmon stomach had significantly different lipid-normalized concentrations and varied in their PHC pattern as well, possibly indicating a large natural variation within the Baltic Sea. The highest lipid-normalized concentrations were found in brown trout. Salmon and brown trout were similar in their PHC pattern suggesting similar food sources. Variation in PHC patterns among trophic levels was not smaller than that among geographically distinct locations, confirming the importance of comparable trophic levels for the assessment of PHC patterns, e.g. for tracing migratory fish. Copyright © 2011 Elsevier Ltd. All rights reserved.

Pure laparoscopic hepatectomy with augmented reality-assisted indocyanine green fluorescence versus open hepatectomy for hepatocellular carcinoma with liver cirrhosis: A propensity analysis at a single center.

PubMed

Cheung, Tan To; Ma, Ka Wing; She, Wong Hoi; Dai, Wing Chiu; Tsang, Simon Hing Yin; Chan, Albert Chi Yan; Chok, Kenneth Siu Ho; Lo, Chung Mau

2018-05-10

Laparoscopic hepatectomy is considered an acceptable treatment of choice in selected patients with primary hepatocellular carcinoma (HCC). Whether indocyanine green (ICG) immunofluorescence, a new technology, may improve surgery outcomes has yet to be tested. The aim of the present study was to investigate and compare the effect of ICG fluorescence imaging on the outcomes of pure laparoscopic hepatectomy and open hepatectomy for primary HCC with background cirrhosis. From January 2015 to June 2016, 20 patients with HCC and liver cirrhosis underwent laparoscopic hepatectomy with ICG immunofluorescence. The outcomes of pure laparoscopic hepatectomy with ICG immunofluorescence were compared with those of open hepatectomy. To avoid selection bias, patients were propensity score matched in a ratio of 1 : 6, with 20 patients in the laparoscopic group and 120 in the open group. The laparoscopic group had 20 patients, and the open group had 120 patients. The laparoscopic group had less blood loss (125 vs 450 mL, P < 0.001), a shorter operation time (200 vs 250 min, P = 0.003), and a shorter hospital stay (5 vs 6 days, P < 0.001). The complication rate was 0% in the laparoscopic group compared to 15.0% in the open group (P = 0.135). All patients in the laparoscopic group had negative margin involvement. Four patients (3.3%) in the open resection group had positive margin involvement. Two patients in the ICG immunofluorescence group had additional lesions identified and resected during operation. Pure laparoscopic hepatectomy with ICG immunofluorescence for primary HCC can be carried out safely with favorable short-term outcomes even in cirrhotic patients. Better identification of the bile duct structure and better assessment of the tumor resection margin and perfusion are advantages of this new technique. © 2018 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and John Wiley & Sons Australia, Ltd.

Fate and degradation kinetics of nonylphenol compounds in aerobic batch digesters.

PubMed

Ömeroğlu, Seçil; Sanin, F Dilek

2014-11-01

Nonylphenol (NP) compounds are toxic and persistent chemicals that are not fully degraded either in natural or engineered systems. Current knowledge indicates that these compounds concentrate in sewage sludge. Therefore, investigating the degradation patterns and types of metabolites formed during sludge treatment are important for land application of sewage sludge. Unfortunately, the information on the fate of nonylphenol compounds in sludge treatment is very limited. This study aims to investigate the biodegradation patterns of nonylphenol diethoxylate (NP2EO) in aerobic batch digesters. For this purpose, two NP2EO spiked and two control laboratory aerobic batch digesters were operated. The spiked digester contained 3 mg/L NP2EO in the whole reactor content. The compounds of interest (parent compound and expected metabolites) were extracted with sonication and analyzed by gas chromatography-mass spectrometry (GC-MS) as a function of time. Results showed that, following the day of spike, NP2EO degraded rapidly. The metabolites observed were nonylphenol monoethoxylate (NP1EO), NP and dominantly, nonylphenoxy acetic acid (NP1EC). The mass balance over the reactors indicated that the total mass spiked was highly accounted for by the products analyzed. The time dependent analysis indicated that the parent compound degradation and daughter product formation followed first order kinetics. The digester performance parameters analyzed (VS and COD reduction) indicated that the spike of NP2EO did not affect the digester performance. Published by Elsevier Ltd.

Immunocytochemistry of the amphibian embryo--from overview to ultrastructure.

PubMed

Kurth, Thomas

2003-06-01

Amphibian embryos are standard research objects to study pattern formation and morphogenesis. Due to their external development and robust nature, experimental manipulations such as microinjections or transplantations can be easily performed. However, most immunocytochemical approaches addressing the specific localization of proteins are hampered by the fragility of the large and yolky embryonic cells which render high resolution staining difficult. Immunocytochemical data are therefore often restricted to either overall patterns in whole embryo preparations or to immunofluorescent localization with limited resolution on sections. High resolution or ultrastructural protein localization data are rare and can be achieved only with time consuming procedures. Here, a comparative study of immunocytochemical methods suitable for light and electron microscopy using different kinds of plastic resins is presented. Three main approaches are described: preembedding staining of whole embryos, postembedding staining of ultrathin sections and preembedding staining of vibratome sections. All the procedures are designed to study protein expression in early amphibian embryos en gros as well as en detail and the described techniques are suitable to combine two or three levels of resolution on the very same biological specimen. Examples are presented and advantages and disadvantages of the different protocols are discussed.

Interpretation of ANA Indirect Immunofluorescence Test Outside the Darkroom Using NOVA View Compared to Manual Microscopy

PubMed Central

Copple, Susan S.; Jaskowski, Troy D.; Giles, Rashelle; Hill, Harry R.

2014-01-01

Objective. To evaluate NOVA View with focus on reading archived images versus microscope based manual interpretation of ANA HEp-2 slides by an experienced, certified medical technologist. Methods. 369 well defined sera from: 44 rheumatoid arthritis, 50 systemic lupus erythematosus, 35 scleroderma, 19 Sjögren's syndrome, and 10 polymyositis patients as well as 99 healthy controls were examined. In addition, 12 defined sera from the Centers for Disease Control and 100 random patient sera sent to ARUP Laboratories for ANA HEp-2 IIF testing were included. Samples were read using the archived images on NOVA View and compared to results obtained from manual reading. Results. At a 1 : 40/1 : 80 dilution the resulting comparison demonstrated 94.8%/92.9% positive, 97.4%/97.4% negative, and 96.5%/96.2% total agreements between manual IIF and NOVA View archived images. Agreement of identifiable patterns between methods was 97%, with PCNA and mixed patterns undetermined. Conclusion. Excellent agreements were obtained between reading archived images on NOVA View and manually on a fluorescent microscope. In addition, workflow benefits were observed which need to be analyzed in future studies. PMID:24741573

Cellular and Nuclear Alignment Analysis for Determining Epithelial Cell Chirality

PubMed Central

Raymond, Michael J.; Ray, Poulomi; Kaur, Gurleen; Singh, Ajay V.; Wan, Leo Q.

2015-01-01

Left-right (LR) asymmetry is a biologically conserved property in living organisms that can be observed in the asymmetrical arrangement of organs and tissues and in tissue morphogenesis, such as the directional looping of the gastrointestinal tract and heart. The expression of LR asymmetry in embryonic tissues can be appreciated in biased cell alignment. Previously an in vitro chirality assay was reported by patterning multiple cells on microscale defined geometries and quantified the cell phenotype–dependent LR asymmetry, or cell chirality. However, morphology and chirality of individual cells on micropatterned surfaces has not been well characterized. Here, a Python-based algorithm was developed to identify and quantify immunofluorescence stained individual epithelial cells on multicellular patterns. This approach not only produces results similar to the image intensity gradient-based method reported previously, but also can capture properties of single cells such as area and aspect ratio. We also found that cell nuclei exhibited biased alignment. Around 35% cells were misaligned and were typically smaller and less elongated. This new imaging analysis approach is an effective tool for measuring single cell chirality inside multicellular structures and can potentially help unveil biophysical mechanisms underlying cellular chiral bias both in vitro and in vivo. PMID:26294010

The detection of basal cell determinants in human basal cell carcinomas using two different monoclonal antibodies.

PubMed

Habets, J M; Tank, B; Vuzevski, V D; van Reede, E C; Stolz, E; van Joost, T

1987-01-01

This report deals with the reaction pattern(s) of two monoclonal antibodies (MoAbs) with normal skin and basal cell carcinomas (BCC). Using indirect immunoperoxidase (IIP) and indirect immunofluorescence (IIF) techniques, MoAb 12 G7 was observed to react with a determinant related to the cell membrane of the epidermal basal cells. In the IIP technique MoAb 12 G7 showed a positive reaction with 32 out of 34 BCC (94%), while in IIF all the 14 BCC that were studied were positive. In most cases only the cells at the periphery of the tumour nests were stained. MoAb 253 B7 reacted with cytoplasmic determinant(s) of the epidermal basal cells both in the IIF as well as in the IIP techniques. Using the IIP technique only 5 out of 34 BCC (15%) showed a positive reaction with this MoAb. Four of the 5 positively staining tumours showed aggressive histological features. Using IIF technique only 2 out of 14 BCC were positive. The results presented in this communication are discussed with regard to the possible expression of selective differentiation and tumor-associated determinant(s) in BCC.

SSCC TD: A Serial and Simultaneous Configural-Cue Compound Stimuli Representation for Temporal Difference Learning

PubMed Central

Mondragón, Esther; Gray, Jonathan; Alonso, Eduardo; Bonardi, Charlotte; Jennings, Dómhnall J.

2014-01-01

This paper presents a novel representational framework for the Temporal Difference (TD) model of learning, which allows the computation of configural stimuli – cumulative compounds of stimuli that generate perceptual emergents known as configural cues. This Simultaneous and Serial Configural-cue Compound Stimuli Temporal Difference model (SSCC TD) can model both simultaneous and serial stimulus compounds, as well as compounds including the experimental context. This modification significantly broadens the range of phenomena which the TD paradigm can explain, and allows it to predict phenomena which traditional TD solutions cannot, particularly effects that depend on compound stimuli functioning as a whole, such as pattern learning and serial structural discriminations, and context-related effects. PMID:25054799

Effects of UV-B radiation on phenolic composition and deposition patterns and leaf physiology in three Eastern tree species

NASA Astrophysics Data System (ADS)

Sullivan, Joseph H.; Gitz, Dennis C.; Peek, Michael S.; McElrone, Andrew J.

2002-01-01

Quantitative changes in foliar chemistry in response to UVB radiation are frequently reported but less is known about the qualitative changes in putative UV-screening compounds. It has also not been conclusively shown whether qualitative differences in screening compounds or differences in localization patterns influences the sensitivity of plants to damage from UVB radiation. In this study we evaluated the chemical composition and deposition patterns of UV-absorbing compounds in three tree species and assayed these species for possible effects on gas exchange and photosynthetic carbon assimilation. Branches of mature trees of sweetgum (Liquidambar styraciflua), tulip poplar (Liriodendron tulipifera) and red maple (Acer rubrum) were exposed to supplemental levels of UVB radiation over three growing seasons. Controls for UVA were also measured by exposing branches to supplemental UVA only, and additional branches not irradiated were also used for controls. These species demonstrated contrasting chemical composition and deposition patterns with poplar being the most responsive in terms of epidermal accumulation of phenolics including flavonols and chlorogenic acid and hydroxycinnamates. Sweetgum and red maple showed increases primarily in hydroxycinnamates, particularly in the mesophyll in red maple. Leaf area was marginally influenced by UV exposure level. Assimilation was generally not reduced by UVB radiation in these species and was enhanced in red maple by both UVB and UVA and by UVA in sweetgum. These finding are consistent with a hypothesis that epidermal attenuation of UVB would only be reduced in poplar, which accumulated the additional epidermal screening compounds. It is possible that photosynthetic efficiency was enhanced in red maple by the increased absorption of blue light within the mesophyll. Stomatal conductance was generally reduced, and this led to an increase in water use efficiency in red maple and poplar.

Mouse hepatitis virus immunofluorescence in formalin- or Bouin's-fixed tissues using trypsin digestion.

PubMed

Brownstein, D G; Barthold, S W

1982-02-01

Mouse hepatitis viral antigens were demonstrated by immunofluorescence in formalin- and Bouin's-fixed tissues processed routinely for histopathology followed by partial digestion with trypsin. Staining was superior in tissues fixed in formalin and was not diminished in tissue sections from paraffin blocks stored at room temperature as long as 2 years. The relative ease of this procedure and the commercial availability of reagents makes this a useful technique for the definitive diagnosis of mouse hepatitis virus infection.

Bacillus Anthracis Spores of the bclA Mutant Exhibit Increased Adherence to Epithelial Cells, Fibroblasts, and Endothelial Cells but not to Macrophages

DTIC Science & Technology

2007-09-01

immunofluorescence (IFM) and light microscopy. Samples were fixed in forma- lin, stained with immunofluorescent dyes (as described below) or spore stain ( malachite ...BEC. Adherence was assessed by microscopic observation of the infected cells stained with malachite green and counterstaining of the BEC. For enzymatic...this significant difference, BEC infected with spores were stained with malachite green and counter- stained with Wright-Giemsa (Fig. 1B and C). This

Efficient differentiation of mouse embryonic stem cells into motor neurons.

PubMed

Wu, Chia-Yen; Whye, Dosh; Mason, Robert W; Wang, Wenlan

2012-06-09

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

Dependence of Golgi apparatus integrity on nitric oxide in vascular cells: implications in pulmonary arterial hypertension

PubMed Central

Lee, Jason E.; Patel, Kirit; Almodóvar, Sharilyn; Tuder, Rubin M.; Flores, Sonia C.

2011-01-01

Although reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), its consequences on organellar structure and function within vascular cells is largely unexplored. We investigated the effect of reduced NO on the structure of the Golgi apparatus as assayed by giantin or GM130 immunofluorescence in human pulmonary arterial endothelial (HPAECs) and smooth muscle (HPASMCs) cells, bovine PAECs, and human EA.hy926 endothelial cells. Golgi structure was also investigated in cells in tissue sections of pulmonary vascular lesions in idiopathic PAH (IPAH) and in macaques infected with a chimeric simian immunodeficiency virus containing the human immunodeficiency virus (HIV)-nef gene (SHIV-nef) with subcellular three-dimensional (3D) immunoimaging. Compounds with NO scavenging activity including 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), methylene blue, N-acetylcysteine, and hemoglobin markedly fragmented the Golgi in all cell types evaluated as did monocrotaline pyrrole, while LY-83583, sildenafil, fasudil, Y-27632, Tiron, Tempol, or H2O2 did not. Golgi fragmentation by NO scavengers was inhibited by diethylamine NONOate, was evident in HPAECs after selective knockdown of endothelial nitric oxide synthase using small interfering RNA (siRNA), was independent of microtubule organization, required the GTPase dynamin 2, and was accompanied by depletion of α-soluble N-ethylmaleimide-sensitive factor (NSF) acceptor protein (α-SNAP) from Golgi membranes and codispersal of the SNAP receptor (SNARE) Vti1a with giantin. Golgi fragmentation was confirmed in endothelial and smooth muscle cells in pulmonary arterial lesions in IPAH and the SHIV-nef-infected macaque with subcellular 3D immunoimaging. In SHIV-nef-infected macaques Golgi fragmentation was observed in cells containing HIV-nef-bearing endosomes. The observed Golgi fragmentation suggests that NO plays a significant role in modulating global protein trafficking patterns that contribute to changes in the cell surface landscape and functional signaling in vascular cells. PMID:21217069

MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death.

PubMed

Blank, Norbert; Burger, Renate; Duerr, Birgit; Bakker, Frank; Wohlfarth, Anika; Dumitriu, Ingrid; Kalden, Joachim R; Herrmann, Martin

2002-08-01

Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126. Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining. U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses. These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered. Copyright 2002 Wiley-Liss, Inc.

A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors.

PubMed

Koh, James; Hogue, Joyce A; Sosa, Julie A

2016-01-01

The functional impact of intratumoral heterogeneity has been difficult to assess in the absence of a means to interrogate dynamic, live-cell biochemical events in the native tissue context of a human tumor. Conventional histological methods can reveal morphology and static biomarker expression patterns but do not provide a means to probe and evaluate tumor functional behavior and live-cell responsiveness to experimentally controlled stimuli. Here, we describe an approach that couples vibratome-mediated viable tissue sectioning with live-cell confocal microscopy imaging to visualize human parathyroid adenoma tumor cell responsiveness to extracellular calcium challenge. Tumor sections prepared as 300 micron-thick tissue slices retain viability throughout a >24 hour observation period and retain the native architecture of the parental tumor. Live-cell observation of biochemical signaling in response to extracellular calcium challenge in the intact tissue slices reveals discrete, heterogeneous kinetic waveform categories of calcium agonist reactivity within each tumor. Plotting the proportion of maximally responsive tumor cells as a function of calcium concentration yields a sigmoid dose-response curve with a calculated calcium EC50 value significantly elevated above published reference values for wild-type calcium-sensing receptor (CASR) sensitivity. Subsequent fixation and immunofluorescence analysis of the functionally evaluated tissue specimens allows alignment and mapping of the physical characteristics of individual cells within the tumor to specific calcium response behaviors. Evaluation of the relative abundance of intracellular PTH in tissue slices challenged with variable calcium concentrations demonstrates that production of the hormone can be dynamically manipulated ex vivo. The capability of visualizing live human tumor tissue behavior in response to experimentally controlled conditions opens a wide range of possibilities for personalized ex vivo therapeutic testing. This highly adaptable system provides a unique platform for live-cell ex vivo provocative testing of human tumor responsiveness to a range of physiological agonists or candidate therapeutic compounds.

Time-Dependent Migratory Behaviors in the Long-Term Studies of Fibroblast Durotaxis on a Hydrogel Substrate Fabricated with a Soft Band

PubMed Central

2015-01-01

Durotaxis, biased cell movement up a stiffness gradient on culture substrates, is one of the useful taxis behaviors for manipulating cell migration on engineered biomaterial surfaces. In this study, long-term durotaxis was investigated on gelatinous substrates containing a soft band of 20, 50, and 150 μm in width fabricated using photolithographic elasticity patterning; sharp elasticity boundaries with a gradient strength of 300 kPa/50 μm were achieved. Time-dependent migratory behaviors of 3T3 fibroblast cells were observed during a time period of 3 days. During the first day, most of the cells were strongly repelled by the soft band independent of bandwidth, exhibiting the typical durotaxis behavior. However, the repellency by the soft band diminished, and more cells crossed the soft band or exhibited other mixed migratory behaviors during the course of the observation. It was found that durotaxis strength is weakened on the substrate with the narrowest soft band and that adherent affinity-induced entrapment becomes apparent on the widest soft band with time. Factors, such as changes in surface topography, elasticity, and/or chemistry, likely contributing to the apparent diminishing durotaxis during the extended culture were examined. Immunofluorescence analysis indicated preferential collagen deposition onto the soft band, which is derived from secretion by fibroblast cells, resulting in the increasing contribution of haptotaxis toward the soft band over time. The deposited collagen did not affect surface topography or surface elasticity but did change surface chemistry, especially on the soft band. The observed time-dependent durotaxis behaviors are the result of the mixed mechanical and chemical cues. In the studies and applications of cell migratory behavior under a controlled stimulus, it is important to thoroughly examine other (hidden) compounding stimuli in order to be able to accurately interpret data and to design suitable biomaterials to manipulate cell migration. PMID:24851722

Gene expression pattern recognition algorithm inferences to classify samples exposed to chemical agents

NASA Astrophysics Data System (ADS)

Bushel, Pierre R.; Bennett, Lee; Hamadeh, Hisham; Green, James; Ableson, Alan; Misener, Steve; Paules, Richard; Afshari, Cynthia

2002-06-01

We present an analysis of pattern recognition procedures used to predict the classes of samples exposed to pharmacologic agents by comparing gene expression patterns from samples treated with two classes of compounds. Rat liver mRNA samples following exposure for 24 hours with phenobarbital or peroxisome proliferators were analyzed using a 1700 rat cDNA microarray platform. Sets of genes that were consistently differentially expressed in the rat liver samples following treatment were stored in the MicroArray Project System (MAPS) database. MAPS identified 238 genes in common that possessed a low probability (P < 0.01) of being randomly detected as differentially expressed at the 95% confidence level. Hierarchical cluster analysis on the 238 genes clustered specific gene expression profiles that separated samples based on exposure to a particular class of compound.

Centriole distribution during tripolar mitosis in Chinese hamster ovary cells

PubMed Central

1984-01-01

During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole. PMID:6373793

Long-term hydrocephalus alters the cytoarchitecture of the adult subventricular zone.

PubMed

Campos-Ordoñez, Tania; Herranz-Pérez, Vicente; Chaichana, Kaisorn L; Rincon-Torroella, Jordina; Rigamonti, Daniele; García-Verdugo, Jose M; Quiñones-Hinojosa, Alfredo; Gonzalez-Perez, Oscar

2014-11-01

Hydrocephalus can develop secondarily to a disturbance in production, flow and/or absorption of cerebrospinal fluid. Experimental models of hydrocephalus, especially subacute and chronic hydrocephalus, are few and limited, and the effects of hydrocephalus on the subventricular zone are unclear. The aim of this study was to analyze the effects of long-term obstructive hydrocephalus on the subventricular zone, which is the neurogenic niche lining the lateral ventricles. We developed a new method to induce hydrocephalus by obstructing the aqueduct of Sylvius in the mouse brain, thus simulating aqueductal stenosis in humans. In 120-day-old rodents (n=18 per group), the degree of ventricular dilatation and cellular composition of the subventricular zone were studied by immunofluorescence and transmission electron microscopy. In adult patients (age>18years), the sizes of the subventricular zone, corpus callosum, and internal capsule were analyzed by magnetic resonance images obtained from patients with and without aqueductal stenosis (n=25 per group). Mice with 60-day hydrocephalus had a reduced number of Ki67+ and doublecortin+cells on immunofluorescence, as well as decreased number of neural progenitors and neuroblasts in the subventricular zone on electron microscopy analysis as compared to non-hydrocephalic mice. Remarkably, a number of extracellular matrix structures (fractones) contacting the ventricular lumen and blood vessels were also observed around the subventricular zone in mice with hydrocephalus. In humans, the widths of the subventricular zone, corpus callosum, and internal capsule in patients with aqueductal stenosis were significantly smaller than age and gender-matched patients without aqueductal stenosis. In summary, supratentorial hydrocephalus reduces the proliferation rate of neural progenitors and modifies the cytoarchitecture and extracellular matrix compounds of the subventricular zone. In humans, this similar process reduces the subventricular niche as well as the width of corpus callosum and internal capsule. Copyright © 2014 Elsevier Inc. All rights reserved.

Distinct molecular structures and hydrogen bond patterns of α,α-diethyl-substituted cyclic imide, lactam, and acetamide derivatives in the crystalline phase

NASA Astrophysics Data System (ADS)

Krivoshein, Arcadius V.; Ordonez, Carlos; Khrustalev, Victor N.; Timofeeva, Tatiana V.

2016-10-01

α,α-Dialkyl- and α-alkyl-α-aryl-substituted cyclic imides, lactams, and acetamides show promising anticonvulsant, anxiolytic, and anesthetic activities. While a number of crystal structures of various α-substituted cyclic imides, lactams, and acetamides were reported, no in-depth comparison of crystal structures and solid-state properties of structurally matched compounds have been carried out so far. In this paper, we report molecular structure and intermolecular interactions of three α,α-diethyl-substituted compounds - 3,3-diethylpyrrolidine-2,5-dione, 3,3-diethylpyrrolidin-2-one, and 2,2-diethylacetamide - in the crystalline phase, as studied using single-crystal X-ray diffraction and IR spectroscopy. We found considerable differences in the patterns of H-bonding and packing of the molecules in crystals. These differences correlate with the compounds' melting points and are of significance to physical pharmacy and formulation development of neuroactive drugs.

Sensory characteristics and volatile profiles of parsley ( Petroselinum crispum [Mill.] Nym.) in correlation to resistance properties against Septoria Blight ( Septoria petroselini ).

PubMed

Ulrich, Detlef; Bruchmüller, Tobias; Krüger, Hans; Marthe, Frank

2011-10-12

Sixteen different genotypes of parsley, including two cultivars, six populations, and eight inbred lines, were investigated regarding their sensory characteristics in relation to the volatile patterns and resistance to Septoria petroselini . The sensory quality was determined by a combination of profile analysis and preference test, whereas the volatile patterns were analyzed by headspace-SPME-GC of leaf homogenates with subsequent nontargeted data processing to prevent a possible overlooking of volatile compounds. The more resistant genotypes are characterized by several negative sensory characteristics such as bitter, grassy, herbaceous, pungent, chemical, and harsh. In contrast, the contents of some volatile compounds correlate highly and significantly either with resistance (e.g., hexanal and α-copaene) or with susceptibility (e.g., p-menthenol). Some of these compounds with very strong correlation to resistance are still unidentified and are presumed to act as resistance markers.

LYRATE Is a Key Regulator of Leaflet Initiation and Lamina Outgrowth in Tomato[C][W][OA

PubMed Central

David-Schwartz, Rakefet; Koenig, Daniel; Sinha, Neelima R.

2009-01-01

Development of the flattened laminar structure in plant leaves requires highly regulated cell division and expansion patterns. Although tight regulation of these processes is essential during leaf development, leaf shape is highly diverse across the plant kingdom, implying that patterning of growth must be amenable to evolutionary change. Here, we describe the molecular identification of the classical tomato (Solanum lycopersicum) mutant lyrate, which is impaired in outgrowth of leaflet primodia and laminar tissues during compound leaf development. We found that the lyrate phenotype results from a loss-of-function mutation of the tomato JAGGED homolog, a well-described positive regulator of cell division in lateral organs. We demonstrate that LYRATE coordinates lateral outgrowth in the compound leaves of tomato by interacting with both the KNOX and auxin transcriptional networks and suggest that evolutionary changes in LYRATE expression may contribute to the fundamental difference between compound and simple leaves. PMID:19820188

Structural and magnetic behavior of (Ni, Cu) substituted Nd0.67Sr0.33MnO3 perovskite compounds

NASA Astrophysics Data System (ADS)

Arun, B.; Sudakshina, B.; Akshay, V. R.; Chandrasekhar, K. Devi; Yang, H. D.; Vasundhara, M.

2018-05-01

Structural and magnetic phase transition of Ni and Cu substituted Nd0.67Sr0.33MnO3 perovskite compounds have been investigated. The Rietveld refinement of X-ray powder diffraction patterns confirms that both compounds have crystallized into an orthorhombic structure with Pbnm space group same as that of Nd0.67Sr0.33MnO3 compound. X-ray absorption spectra studies completely ruled out the possibility of existence of any impurities. Both compounds do not obey the Curie-Weiss law indicates the presence of some ferromagnetic clusters within the paramagnetic matrix. Ni substituted compound shows a lower value of TC and Cu substituted compound shows a higher value of TC than that of the parent. Non-saturating tendency of magnetization is more prominently seen in the case of Cu substituted compound, indicating an increase in the AFM component.

Differential detection of nuclear envelope autoantibodies in primary biliary cirrhosis using routine and alternative methods

PubMed Central

2010-01-01

Background Detection of autoantibodies giving nuclear rim pattern by immunofluorescence (anti-nuclear envelope antibodies - ANEA) in sera from patients with primary biliary cirrhosis (PBC) is a useful tool for the diagnosis and prognosis of the disease. Differences in the prevalence of ANEA in PBC sera so far reported have been attributed to the methodology used for the detection as well as to ethnic/geographical variations. Therefore, we evaluated the prevalence of ANEA in sera of Greek patients with PBC by using methods widely used by clinical laboratories and a combination of techniques and materials. Methods We screened 103 sera by immunoblotting on nuclear envelopes and indirect immunofluorescence (IIF) using cells and purified nuclei. Reactivities against specific autoantigens were assessed using purified proteins, ELISA, immunoprecipitation and mass spectrometry. Results We found higher prevalence of ANEA when sera were assayed by IIF on purified nuclei or cultured cells (50%) compared to Hep2 commercially available slides (15%). Anti-gp210 antibodies were identified in 22.3% and 33% of sera using ELISA for the C-terminal of gp210 or both ELISA and immunoprecipitation, respectively. Immunoblotting on nuclear envelopes revealed that immunoreactivity for the 210 kDa zone is related to anti-gp210 antibodies (p < 0.0001). Moreover, we found that sera had antibodies for lamins A (6.8%), B (1%) and C (1%) and LBR (8.7%), whereas none at all had detectable anti-p62 antibodies. Conclusions The prevalence of ANEA or anti-gp210 antibodies is under-estimated in PBC sera which are analyzed by conventional commercially available IIF or ELISA, respectively. Therefore, new substrates for IIF and ELISA should be included by clinical laboratories in the analysis of ANEA in autoimmune sera. PMID:20205958

Characterization of pituitary cells targeted by antipituitary antibodies in patients with isolated autoimmune diseases without pituitary insufficiency may help to foresee the kind of future hypopituitarism.

PubMed

De Bellis, A; Dello Iacovo, A; Bellastella, G; Savoia, A; Cozzolino, D; Sinisi, A A; Bizzarro, A; Bellastella, A; Giugliano, D

2014-10-01

Detection of antipituitary antibodies (APA) at high levels and with a particular immunofluorescence pattern in patients with autoimmune polyendocrine syndromes may indicate a possible future autoimmune pituitary involvement. This longitudinal study was aimed at characterizing in patients with a single organ-specific autoimmune disease the pituitary cells targeted by APA at start, verifying whether this characterization allows to foresee the kind of possible subsequent hypopituitarism. Thirty-six APA positive and 40 APA negative patients with isolated autoimmune diseases participated in the study. None of them had pituitary dysfunction at entry. Characterization by four-layer immunofluorescence of pituitary cells targeted by APA in APA positive patients at entry and study of pituitary function in all patients were performed every 6 months during a 5 year follow-up. Antipituitary antibodies immunostained selectively one type of pituitary-secreting cells in 21 patients (58.3 %, group 1), and several types of pituitary cells in the remaining 15 (41.7 %, group 2). All patients in group 1 showed subsequently a pituitary insufficiency, corresponding to the type of cells targeted by APA in 18 of them (85.7 %). Only 8 out of 15 patients in group 2 (53.3 %) showed a hypopituitarism, isolated in 7 and combined in the other one. None of APA negative patients showed hypopituitarism. The characterization of pituitary cells targeted by APA in patients with isolated autoimmune diseases, when the pituitary function is still normal, may help to foresee the kind of subsequent hypopituitarism, especially when APA immunostained selectively only one type of pituitary cells. A careful follow-up of pituitary function in these patients is advisable to allow an early diagnosis of hypopituitarism, even in subclinical phase and a consequent timely replacement therapy.

Isolation of human monoclonal antibodies to the envelope e2 protein of hepatitis C virus and their characterization.

PubMed

Shimizu, Yohko K; Hijikata, Minako; Oshima, Masamichi; Shimizu, Kazufumi; Alter, Harvey J; Purcell, Robert H; Yoshikura, Hiroshi; Hotta, Hak

2013-01-01

We isolated and characterized two human monoclonal antibodies to the envelope E2 protein of hepatitis C virus (HCV). Lymphoblastoid cell lines stably producing antibodies were obtained by immortalizing peripheral blood mononuclear cells of a patient with chronic hepatitis C using Epstein-Barr virus. Screening for antibody-positive clones was carried out by immunofluorescence with Huh7 cells expressing the E2 protein of HCV strain H (genotype 1a) isolated from the same patient. Isotype of resulting antibodies, #37 and #55, was IgG1/kappa and IgG1/lambda, respectively. Epitope mapping revealed that #37 and #55 recognize conformational epitopes spanning amino acids 429 to 652 and 508 to 607, respectively. By immunofluorescence using virus-infected Huh7.5 cells as targets both antibodies were reactive with all of the nine different HCV genotypes/subtypes tested. The antibodies showed a different pattern of immuno-staining; while #37 gave granular reactions mostly located in the periphery of the nucleus, #55 gave diffuse staining throughout the cytoplasm. Both antibodies were shown by immuno-gold electron microscopy to bind to intact viral particles. In a neutralization assay (focus-forming unit reduction using chimeric infectious HCV containing structural proteins derived from genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a, and 7a), #55 inhibited the infection of all HCV genotypes tested but genotype 7a to a lesser extent. #37 did not neutralize any of these viruses. As a broadly cross-neutralizing human antibody, #55 may be useful for passive immunotherapy of HCV infection.

Immunofluorescent staining reveals hypermethylation of microchromosomes in the central bearded dragon, Pogona vitticeps.

PubMed

Domaschenz, Renae; Livernois, Alexandra M; Rao, Sudha; Ezaz, Tariq; Deakin, Janine E

2015-01-01

Studies of model organisms have demonstrated that DNA cytosine methylation and histone modifications are key regulators of gene expression in biological processes. Comparatively little is known about the presence and distribution of epigenetic marks in non-model amniotes such as non-avian reptiles whose genomes are typically packaged into chromosomes of distinct size classes. Studies of chicken karyotypes have associated the gene-richness and high GC content of microchromosomes with a distinct epigenetic landscape. To determine whether this is likely to be a common feature of amniote microchromosomes, we have analysed the distribution of epigenetic marks using immunofluorescence on metaphase chromosomes of the central bearded dragon (Pogona vitticeps). This study is the first to study the distribution of epigenetic marks on non-avian reptile chromosomes. We observed an enrichment of DNA cytosine methylation, active modifications H3K4me2 and H3K4me3, as well as the repressive mark H3K27me3 in telomeric regions on macro and microchromosomes. Microchromosomes were hypermethylated compared to macrochromosomes, as they are in chicken. However, differences between macro- and microchromosomes for histone modifications associated with actively transcribed or repressed DNA were either less distinct or not detectable. Hypermethylation of microchromosomes compared to macrochromosomes is a shared feature between P. vitticeps and avian species. The lack of the clear distinction between macro- and microchromosome staining patterns for active and repressive histone modifications makes it difficult to determine at this stage whether microchrosome hypermethylation is correlated with greater gene density as it is in aves, or associated with the greater GC content of P. vitticeps microchromosomes compared to macrochromosomes.

Monoclonal antibody against human ovarian tumor-associated antigens.

PubMed

Poels, L G; Peters, D; van Megen, Y; Vooijs, G P; Verheyen, R N; Willemen, A; van Niekerk, C C; Jap, P H; Mungyer, G; Kenemans, P

1986-05-01

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by 125I-labeled OV-TL 3 antibodies. Thus OV-TL 3 recognizes a common antigen for most ovarian carcinomas and may be a useful tool for rapid diagnosis of ovarian carcinomas.

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

Poels, L.G.; Peters, D.; van Megen, Y.

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derivedmore » from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by /sup 125/I-labeled OV-TL 3 antibodies.« less

Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

PubMed

Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

2016-01-01

Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

Childhood-onset bullous systemic lupus erythematosus.

PubMed

Lourenço, D M R; Gomes, R Cunha; Aikawa, N E; Campos, L M A; Romiti, R; Silva, C A

2014-11-01

Bullous systemic lupus erythematosus has rarely been described in pediatric lupus population and the real prevalence of childhood-onset bullous systemic lupus erythematosus has not been reported. From January 1983 to November 2013, 303 childhood-onset SLE (c-SLE) patients were followed at the Pediatric Rheumatology Unit of the Childreńs Institute of Hospital das Clínicas da Faculdade de Medicina Universidade da Universidade de São Paulo, three of them (1%) diagnosed as childhood-onset bullous systemic lupus erythematosus. All three cases presented tense vesiculobullous lesions unassociated with lupus erythematosus lesions, with the median duration of 60 days (30-60). All patients fulfilled bullous systemic lupus erythematosus criteria. Two had nephritis and serositis and presented specific autoantibodies. The histological pattern demonstrated subepidermal blisters with neutrophils-predominant infiltrates within the upper dermis. Direct immunofluorescence (DIF) showed deposits of IgG and complement along the epidermal basement membrane, in the presence or absence of IgA and/or IgM. A positive indirect immunofluorescence on salt-split skin demonstrating dermal binding was observed in two cases. All of them had moderate/severe disease activity at diagnosis with median Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) of 18 (14-24). Two patients received dapsone and one with severe nephritis received immunosuppressive drugs. In conclusion, in the last 30 years the prevalence of bullous lupus in childhood-onset lupus population was low (1%) in our tertiary University Hospital. A diagnosis of SLE should always be considered in children with recurrent tense vesiculobullous lesions with or without systemic manifestations. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

The small molecule CS1 inhibits mitosis and sister chromatid resolution in HeLa cells.

PubMed

Wu, Xingkang; Li, Zhenyu; Shen, Yuemao

2018-05-01

Mitosis, the most dramatic event in the cell cycle, involves the reorganization of virtually all cellular components. Antimitotic agents are useful for dissecting the mechanism of this reorganization. Previously, we found that the small molecule CS1 accumulates cells in G2/M phase [1], but the mechanism of its action remains unknown. Cell cycle analysis, live cell imaging and nuclear staining were used. Chromosomal morphology was detected by chromosome spreading. The effects of CS1 on microtubules were confirmed by tubulin polymerization, colchicine tubulin-binding, cellular tubulin polymerization and immunofluorescence assays and by analysis of microtubule dynamics and molecular modeling. Histone phosphoproteomics was performed using mass spectrometry. Cell signaling cascades were analyzed using immunofluorescence, immunoprecipitation, immunoblotting, siRNA knockdown and chemical inhibition of specific proteins. The small molecule CS1 was shown to be an antimitotic agent. CS1 potently inhibited microtubule polymerization via interaction with the colchicine-binding pocket of tubulin in vitro and inhibited the formation of the spindle apparatus by reducing the bulk of growing microtubules in HeLa cells, which led to activation of the spindle assembly checkpoint (SAC) and mitotic arrest of HeLa cells. Compared with colchicine, CS1 impaired the progression of sister chromatid resolution independent of cohesin dissociation, and this was reversed by the removal of CS1. Additionally, CS1 induced unique histone phosphorylation patterns distinct from those induced by colchicine. CS1 is a unique antimitotic small molecule and a powerful tool with unprecedented value over colchicine that makes it possible to specifically and conditionally perturb mitotic progression. Copyright © 2018 Elsevier B.V. All rights reserved.

A new mutation, gld, that produces lymphoproliferation and autoimmunity in C3H/HeJ mice.

PubMed

Roths, J B; Murphy, E D; Eicher, E M

1984-01-01

A newly discovered autosomal recessive mutation, generalized lymphoproliferative disease (gld), in the C3H/HeJ strain of mice, determines the development of early onset massive lymphoid hyperplasia with autoimmunity. Significant lymph node enlargement is apparent as early as 12 wk of age. By 20 wk, lymph nodes are 50-fold heavier than those of coisogenic C3H/HeJ-+/+ mice. There is a concomitant increase in the numbers of peripheral blood lymphocytes. Analysis of C3H-gld lymph node lymphocyte subsets by immunofluorescence indicates an increase in numbers of B cells, T cells, and null (Thy-1-, sIg-) lymphocytes by 6-, 15-, and 33-fold compared with congeneic control mice. Serologically, gld/gld mice develop antinuclear antibodies (including anti-dsDNA), thymocyte-binding autoantibody, and hypergammaglobulinemia with major increases in several immunoglobulin isotypes. Mutant gld mice live only one-half as long as normal controls (12 and 23 mo, respectively). Interstitial pneumonitis was found in virtually all C3H-gld mice autopsied when moribund. Although immune complexes were detected in the glomerulus by immunofluorescence techniques, only 14% of the autopsied mice had significant lupus-like nephritis. Vascular disease was not found. The pattern of early onset massive lymph node enlargement, hypergammaglobulinemia, and production of antinuclear autoantibodies resembles the basic abnormal phenotype induced by the lpr (lymphoproliferation) mutation. The mutations gld and lpr are not allelic. Linkage studies indicate that gld is located between Pep-3 and Lp on chromosome 1. This new mutation adds another genetically well-defined model to the list of murine lymphoproliferative/autoimmune disorders that may be exploited to gain a clearer understanding of immunoregulatory defects and for identifying common pathogenetic factors involved in systemic autoimmune diseases.

A new mutation, gld, that produces lymphoproliferation and autoimmunity in C3H/HeJ mice

PubMed Central

1984-01-01

A newly discovered autosomal recessive mutation, generalized lymphoproliferative disease (gld), in the C3H/HeJ strain of mice, determines the development of early onset massive lymphoid hyperplasia with autoimmunity. Significant lymph node enlargement is apparent as early as 12 wk of age. By 20 wk, lymph nodes are 50-fold heavier than those of coisogenic C3H/HeJ-+/+ mice. There is a concomitant increase in the numbers of peripheral blood lymphocytes. Analysis of C3H-gld lymph node lymphocyte subsets by immunofluorescence indicates an increase in numbers of B cells, T cells, and null (Thy-1-, sIg-) lymphocytes by 6-, 15-, and 33-fold compared with congeneic control mice. Serologically, gld/gld mice develop antinuclear antibodies (including anti-dsDNA), thymocyte-binding autoantibody, and hypergammaglobulinemia with major increases in several immunoglobulin isotypes. Mutant gld mice live only one-half as long as normal controls (12 and 23 mo, respectively). Interstitial pneumonitis was found in virtually all C3H-gld mice autopsied when moribund. Although immune complexes were detected in the glomerulus by immunofluorescence techniques, only 14% of the autopsied mice had significant lupus-like nephritis. Vascular disease was not found. The pattern of early onset massive lymph node enlargement, hypergammaglobulinemia, and production of antinuclear autoantibodies resembles the basic abnormal phenotype induced by the lpr (lymphoproliferation) mutation. The mutations gld and lpr are not allelic. Linkage studies indicate that gld is located between Pep-3 and Lp on chromosome 1. This new mutation adds another genetically well-defined model to the list of murine lymphoproliferative/autoimmune disorders that may be exploited to gain a clearer understanding of immunoregulatory defects and for identifying common pathogenetic factors involved in systemic autoimmune diseases. PMID:6693832

Establishment of a panel of in-house polyclonal antibodies for the diagnosis of enterovirus infections.

PubMed

Kotani, Osamu; Iwata-Yoshikawa, Naoko; Suzuki, Tadaki; Sato, Yuko; Nakajima, Noriko; Koike, Satoshi; Iwasaki, Takuya; Sata, Tetsutaro; Yamashita, Teruo; Minagawa, Hiroko; Taguchi, Fumihiro; Hasegawa, Hideki; Shimizu, Hiroyuki; Nagata, Noriyo

2015-04-01

The aim of this study was to establish a reliable method of virus detection for the diagnosis of critical enterovirus infections such as acute infective encephalitis, encephalomyelitis and myocarditis. Because histopathological and immunohistochemical analyses of paraffin-embedded tissues play an important role in recognizing infectious agents in tissue samples, six in-house polyclonal antibodies raised against three representative enteroviruses using an indirect immunofluorescence assay and immunohistochemistry were examined. This panel of polyclonal antibodies recognized three serotypes of enterovirus. Two of the polyclonal antibodies were raised against denatured virus particles from enterovirus A71, one was raised against the recombinant VP1 protein of coxsackievirus B3, and the other for poliovirus type 1 were raised against denatured virus particles, the recombinant VP1 protein and peptide 2C. Western blot analysis revealed that each of these antibodies recognized the corresponding viral antigen and none cross-reacted with non-enteroviruses within the family Picornaviridae. However, all cross-reacted to some extent with the antigens derived from other serotypes of enterovirus. Indirect immunofluorescence assay and immunohistochemistry revealed that the virus capsid and non-structural proteins were localized in the cytoplasm of affected culture cells, and skeletal muscles and neurons in neonatal mice experimentally-infected with human enterovirus. The antibodies also recognized antigens derived from recent clinical isolates of enterovirus A71, coxsackievirus B3 and poliovirus. In addition, immunohistochemistry revealed that representative antibodies tested showed the same recognition pattern according to each serotype. Thus, the panel of in-house anti-enterovirus polyclonal antibodies described herein will be an important tool for the screening and pathological diagnosis for enterovirus infections, and may be useful for the classification of different enterovirus serotypes, including coxsackieviruses A and B, echoviruses, enterovirus A71 and poliovirus. © 2014 Japanese Society of Neuropathology.

APR-246/PRIMA-1Met Inhibits and Reverses Squamous Metaplasia in Human Conjunctival Epithelium.

PubMed

Li, Jing; Li, Cheng; Wang, Guoliang; Liu, Zhen; Chen, Pei; Yang, Qichen; Dong, Nuo; Wu, Huping; Liu, Zuguo; Li, Wei

2016-02-01

Squamous metaplasia is a common pathologic condition in ocular surface diseases for which there is no therapeutic medication in clinic. In this study, we investigated the effect of a small molecule, APR-246/PRIMA-1(Met), on squamous metaplasia in human conjunctival epithelium. Human conjunctival explants were cultured for up to 12 days under airlifting conditions. Epithelial cell differentiation and proliferation were assessed by Cytokeratin 10 (K10), K14, K19, Pax6, MUC5AC, and p63 immunostaining patterns. β-catenin and TCF-4 immunofluorescent staining and real-time PCR characterized Wnt signaling pathway involvement. Pterygium clinical samples were cultured under airlifting conditions with or without APR-246 for 4 days. p63, K10, β-catenin, and TCF-4 expression in pterygial epithelium was determined by immunofluorescent staining and real-time PCR. Airlift conjunctival explants resulted in increased stratification and intrastromal epithelial invagination. Such pathology was accompanied by increases in K10, K14, and p63 expression, whereas K19 and Pax6 levels declined when compared to those in freshly isolated tissue. On the other hand, APR-246 reversed all of these declines in K10, K14, and p63 expression. Furthermore, K19 and Pax6 increased along with rises in goblet cell density. These effects of APR-246 were accompanied by near restoration of normal conjunctival epithelial histology. APR-246 also reversed squamous metaplasia in pterygial epithelium that had developed after 4 days in ex vivo culture. Reductions in squamous metaplasia induced by APR-246 suggest it may provide a novel therapeutic approach in different squamous metaplasia-associated ocular surface diseases.

Fatal infections with Balamuthia mandrillaris (a free-living amoeba) in gorillas and other Old World primates.

PubMed

Rideout, B A; Gardiner, C H; Stalis, I H; Zuba, J R; Hadfield, T; Visvesvara, G S

1997-01-01

Balamuthia mandrillaris is a newly described free-living amoeba capable of causing fatal meningoencephalitis in humans and animals. Because the number of human cases is rapidly increasing, this infection is now considered an important emerging disease by the medical community. A retrospective review of the pathology database for the Zoological Society of San Diego (the San Diego Zoo and San Diego Wild Animal Park) for the period July 1965 through December 1994 revealed five cases of amoebic meningoencephalitis, all in Old World primates. The infected animals were a 3-year, 10-month-old female mandrill (Papio sphinx), from which the original isolation of B. mandrillaris was made, a 5-year-old male white-cheeked gibbon (Hylobates concolor leucogenys), a 1-year-old female western lowland gorilla (Gorilla gorilla gorilla), a 13-year, 5-month-old male western lowland gorilla, and a 6-year-old female Kikuyu colobus monkey (Colobus guereza kikuyuensis). Two different disease patterns were identified: the gibbon, mandrill, and 1-year-old gorilla had an acute to subacute necrotizing amoebic meningoencephalitis with a short clinical course, and the adult gorilla and colobus monkey had a granulomatous amoebic meningoencephalitis with extraneural fibrogranulomatous inflammatory lesions and a long clinical course. Indirect immunofluorescent staining of amoebas in brain sections with a Balamuthia-specific polyclonal antibody was positive in all five animals. Indirect immunofluorescent staining for several species of Acanthamoeba, Naegleria fowleri, and Hartmanella vermiformis was negative. Direct examination of water and soil samples from the gorilla and former mandrill enclosures revealed unidentified amoebas in 11/27 samples, but intraperitoneal inoculations in mice failed to induce disease. Attempts to isolate amoebas from frozen tissues from the adult male gorilla were unsuccessful.

Pathology and diagnosis of renal non-AL amyloidosis.

PubMed

Sethi, Sanjeev; Theis, Jason D

2018-06-01

Renal amyloidosis is characterized by acellular Congo red positive deposits in the glomeruli, interstitium and/or arteries. Light chain restriction on immunofluorescence studies is present in AL-amyloidosis, the most common type of amyloidosis involving the kidney. The detection of Congo red positive deposits coupled with negative immunofluorescence studies is highly suggestive of non-AL amyloidosis. Some of the non-AL amyloidosis are common while others are relatively rare. The clinical features, laboratory and renal pathology findings are helpful in the diagnosis and typing of non-AL amyloidosis. Thus, ALECT2 amyloidosis is characterized by diffuse cortical interstitial amyloid deposits, AA amyloidosis shows vascular deposits in addition to the glomerular deposits, AFib amyloidosis is characterized by massive amyloid accumulation limited to the glomeruli resulting in the obliteration of glomerular architecture, AApoA1 and AApoAIV are characterized by large amyloid deposits restricted to the medulla, and AGel shows swirling patterns of amyloid fibrils on electron microscopy. While light microscopy is very helpful, accurate typing of non-AL amyloidosis then requires immunohistochemical or laser microdissection/mass spectrometry studies of the Congo red positive deposits. Immunohistochemical studies are available for some of the non-AL amyloidosis. On the other hand, mass spectrometry analysis is a one stop methodology for confirmation and typing of amyloidosis. The diagnosis and typing of amyloidosis by mass spectrometry is based on finding the signature amyloid peptides, apolipoprotein E and serum amyloid-P component, followed by detection of precursor amyloidogenic protein such as LECT2, fibrinogen-α, gelsolin, etc. To, summarize, non-AL amyloidosis is a group of amyloidosis with distinctive clinical, laboratory and renal pathology findings. Typing of the amyloidosis is best performed using mass spectrometry methodology. Accurate typing of non-AL amyloidosis is imperative for correct management, prognosis, and genetic counseling.

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies.

PubMed

Feng, Xuesong; Naz, Faiza; Juan, Aster H; Dell'Orso, Stefania; Sartorelli, Vittorio

2018-04-19

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

Pregnancy complicated with Alport syndrome: a good obstetric outcome and failure to diagnose an infant born to a mother with Alport syndrome by umbilical cord immunofluorescence staining.

PubMed

Matsubara, Shigeki; Ueda, Yoshihiko; Takahashi, Hisako; Nagai, Takashi; Kuwata, Tomoyuki; Muto, Shigeaki; Yamaguchi, Takehiko; Takizawa, Toshihiro; Suzuki, Mitsuaki

2009-12-01

Alport syndrome is a familial progressive nephritis. The most frequent type is X-linked Alport syndrome, caused by genetic abnormalities in the alpha 5 chain of type IV collagen. Skin biopsy is a useful tool for diagnosing this disease. It is not well known how this syndrome affects pregnancy and how it is affected by pregnancy, or whether the umbilical cord may provide material for detecting this collagen abnormality. We report a primigravida with Alport syndrome with mild proteinuria who gave birth abdominally to a term male infant without deteriorating renal function during pregnancy. The umbilical cord from not only this infant but also from an Alport (-) control infant showed negative immunofluorescence staining for the alpha 5 chain of type IV collagen. Women with Alport syndrome without renal dysfunction may follow an uneventful obstetrical course until term. The cord may not be suitable for diagnosing Alport syndrome with immunofluorescence staining.

Localization of connexin43 in rat kidney.

PubMed

Barajas, L; Liu, L; Tucker, M

1994-09-01

The localization of connexin43 (Cx 43) in rat kidney was investigated by the indirect immunofluorescence technique with polyclonal antisera raised against Cx 43. Cx 43 is a gap junction protein expressed in a variety of tissues. The typically punctuated gap junction immunofluorescence (GJI) was observed in the renal arterial and arteriolar system. In the renal artery the GJI was concentrated in the media. In the juxtamedullary nephrons, the GJI is particularly abundant in the vascular bundles. There is abundant GJI in the extraglomerular mesangium while in the afferent arteriole GJI appears decreased. Abundant GJI was observed in the inner medullary collecting ducts and pelvic epithelium. The localization of Cx 43 immunofluorescence observed in this study is only in partial agreement with the results of ultrastructural investigations on the distribution of gap junctions in the kidney. An extensive tight junctional system has been demonstrated in the collecting duct system. However, gap junctions have been reported to be absent. Further studies to resolve this discrepancy are required.

EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics.

PubMed

Krause, C; Ens, K; Fechner, K; Voigt, J; Fraune, J; Rohwäder, E; Hahn, M; Danckwardt, M; Feirer, C; Barth, E; Martinetz, T; Stöcker, W

2015-04-01

Antinuclear autoantibodies (ANA) are highly informative biomarkers in autoimmune diagnostics. The increasing demand for effective test systems, however, has led to the development of a confusingly large variety of different platforms. One of them, the indirect immunofluorescence (IIF), is regarded as the common gold standard for ANA screening, as described in a position statement by the American College of Rheumatology in 2009. Technological solutions have been developed aimed at standardization and automation of IIF to overcome methodological limitations and subjective bias in IIF interpretation. In this review, we present the EUROPattern Suite, a system for computer-aided immunofluorescence microscopy (CAIFM) including automated acquisition of digital images and evaluation of IIF results. The system was originally designed for ANA diagnostics on human epithelial cells, but its applications have been extended with the latest system update version 1.5 to the analysis of antineutrophil cytoplasmic antibodies (ANCA) and anti-dsDNA antibodies. © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Platform for Quantitative Evaluation of Spatial Intratumoral Heterogeneity in Multiplexed Fluorescence Images.

PubMed

Spagnolo, Daniel M; Al-Kofahi, Yousef; Zhu, Peihong; Lezon, Timothy R; Gough, Albert; Stern, Andrew M; Lee, Adrian V; Ginty, Fiona; Sarachan, Brion; Taylor, D Lansing; Chennubhotla, S Chakra

2017-11-01

We introduce THRIVE (Tumor Heterogeneity Research Interactive Visualization Environment), an open-source tool developed to assist cancer researchers in interactive hypothesis testing. The focus of this tool is to quantify spatial intratumoral heterogeneity (ITH), and the interactions between different cell phenotypes and noncellular constituents. Specifically, we foresee applications in phenotyping cells within tumor microenvironments, recognizing tumor boundaries, identifying degrees of immune infiltration and epithelial/stromal separation, and identification of heterotypic signaling networks underlying microdomains. The THRIVE platform provides an integrated workflow for analyzing whole-slide immunofluorescence images and tissue microarrays, including algorithms for segmentation, quantification, and heterogeneity analysis. THRIVE promotes flexible deployment, a maintainable code base using open-source libraries, and an extensible framework for customizing algorithms with ease. THRIVE was designed with highly multiplexed immunofluorescence images in mind, and, by providing a platform to efficiently analyze high-dimensional immunofluorescence signals, we hope to advance these data toward mainstream adoption in cancer research. Cancer Res; 77(21); e71-74. ©2017 AACR . ©2017 American Association for Cancer Research.

Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.

PubMed

Whittington, Niteace C; Wray, Susan

2017-10-23

Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Immunocytochemical methods to study the distribution of Orientia tsutsugamushi in Leptotrombidium (Acari: Trombiculidae) chiggers.

PubMed

Myint, K S; Linthicum, K J; Tanskul, P; Lerdthusnee, K; Vaughn, D W; Manomuth, C; Mongkolsirichaikul, D; Hansukjariya, P; Hastriter, M W

1998-07-01

Immunocytochemical methods were developed and tested for their ability to detect the distribution of Orientia tsutsugamushi in paraffin sections of adult chiggers (Leptotrombidium imphalum Vercammen-Grandjean & Langston). Rickettsial antigen was detected by application of a simple direct or amplified immunocytochemistry procedure and an indirect immunofluorescent procedure. In the direct procedure alkaline phosphatase conjugation to the mouse polyclonal antibody to the Karp strain was followed by the HistoMark Red test system to detect rickettsial antigen. The amplification procedure used a similar method but used an unlabeled primary antibody followed by secondary biotinylated antimouse IgG, streptavidin-alkaline phosphatase, and the HistoMark Red test system. The immunofluorescent procedure included a biotinylated secondary antibody followed by addition of a streptavidin-FITC conjugate. Specific tissue tropisms in infected chiggers were observed in the salivary glands, nervous tissue, and ovaries of adult female mites in all procedures; however, nonspecific fluorescence of the chigger limited definitive identification of tissue tropisms with the indirect immunofluorescent procedure.

PubMed Central

Lallier, R.; Olivier, G.; Chartier, P.; Turcotte, C.; Dionet, B.; Paulhus, P.

1981-01-01

Presence of the bacterial kidney disease in salmonid fish in Quebec During the summers of 1979 and 1980, wild and hatchery fish were analysed for the presence of the bacterial kidney disease (BKD) agent in salmonid fish in Quebec by the indirect immunofluorescence technique. The causative agent of BKD was detected in all hatcheries tested. Ten to 25% of the fish were positive. The presence of this agent was independent of age and species. We were unable to detect the BKD in fish from the rivers in the northern part of Quebec (over the 50th parallel). The first detection of BKD in Quebec was made in the spring of 1979 in a hatchery. A high mortality occurred in fish over a year old. The majority of the fish were positive by immunofluorescence and the bacteriun was isolated from some fish. Three weeks before the disease appeared, six ponds received fish from this hatchery. No mortality was observed in those ponds and in the middle of the summer the percentage of carrier fish by immunofluorescence was approximately 20%. PMID:7340922

Gene expression profiling in Ishikawa cells: A fingerprint for estrogen active compounds

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

Boehme, Kathleen; Simon, Stephanie; Mueller, Stefan O.

2009-04-01

Several anthropogenous and naturally occurring substances, referred to as estrogen active compounds (EACs), are able to interfere with hormone and in particular estrogen receptor signaling. EACs can either cause adverse health effects in humans and wildlife populations or have beneficial effects on estrogen-dependent diseases. The aim of this study was to examine global gene expression profiles in estrogen receptor (ER)-proficient Ishikawa plus and ER-deficient Ishikawa minus endometrial cancer cells treated with selected well-known EACs (Diethylstilbestrol, Genistein, Zearalenone, Resveratrol, Bisphenol A and o,p'-DDT). We also investigated the effect of the pure antiestrogen ICI 182,780 (ICI) on the expression patterns caused bymore » these compounds. Transcript levels were quantified 24 h after compound treatment using Illumina BeadChip Arrays. We identified 87 genes with similar expression changes in response to all EAC treatments in Ishikawa plus. ICI lowered the magnitude or reversed the expression of these genes, indicating ER dependent regulation. Apart from estrogenic gene regulation, Bisphenol A, o,p'-DDT, Zearalenone, Genistein and Resveratrol displayed similarities to ICI in their expression patterns, suggesting mixed estrogenic/antiestrogenic properties. In particular, the predominant antiestrogenic expression response of Resveratrol could be clearly distinguished from the other test compounds, indicating a distinct mechanism of action. Divergent gene expression patterns of the phytoestrogens, as well as weaker estrogenic gene expression regulation determined for the anthropogenous chemicals Bisphenol A and o,p'-DDT, warrants a careful assessment of potential detrimental and/or beneficial effects of EACs. The characteristic expression fingerprints and the identified subset of putative marker genes can be used for screening chemicals with an unknown mode of action and for predicting their potential to exert endocrine disrupting effects.« less

Differential Effects of Sex Pheromone Compounds on Adult Female Sea Lamprey (Petromyzon marinus) Locomotor Patterns.

PubMed

Walaszczyk, Erin J; Goheen, Benjamin B; Steibel, Juan Pedro; Li, Weiming

2016-06-01

Synchronization of male and female locomotor activity plays a critical role in ensuring reproductive success, especially in semelparous species. The goal of this study was to elucidate the effects of individual chemical signals, or pheromones, on the locomotor activity in the sea lamprey (Petromyzon marinus). In their native habitat, adult preovulated females (POF) and ovulated females (OF) are exposed to sex pheromone compounds that are released from spermiated males and attract females to nests during their migration and spawning periods. In this study, locomotor activity of individual POF and OF was measured hourly in controlled laboratory conditions using an automated video-tracking system. Differences in the activity between a baseline day (no treatment exposure) and a treatment day (sex pheromone compound or control exposure) were examined for daytime and nighttime periods. Results showed that different pheromone compound treatments affected both POF and OF sea lamprey (p < 0.05) but in different ways. Spermiated male washings (SMW) and one of its main components, 7α,12α,24-trihydroxy-5α-cholan-3-one 24 sulfate (3kPZS), decreased activity of POF during the nighttime. SMW also reduced activity in POF during the daytime. In contrast, SMW increased activity of OF during the daytime, and an additional compound found in SMW, petromyzonol sulfate (PZS), decreased the activity during the nighttime. In addition, we examined factors that allowed us to infer the overall locomotor patterns. SMW increased the maximum hourly activity during the daytime, decreased the maximum hourly activity during the nighttime, and reduced the percentage of nocturnal activity in OF. Our findings suggest that adult females have evolved to respond to different male compounds in regards to their locomotor activity before and after final maturation. This is a rare example of how species-wide chemosensory stimuli can affect not only the amounts of activity but also the overall locomotor pattern in a vertebrate species. © 2016 The Author(s).

In situ detection of the activation of Rac1 and RalA small GTPases in mouse adipocytes by immunofluorescent microscopy following in vivo and ex vivo insulin stimulation.

PubMed

Takenaka, Nobuyuki; Nihata, Yuma; Ueda, Sho; Satoh, Takaya

2017-11-01

Rac1 has been implicated in insulin-dependent glucose uptake by mechanisms involving plasma membrane translocation of the glucose transporter GLUT4 in skeletal muscle. Although the uptake of glucose is also stimulated by insulin in adipose tissue, the role for Rac1 in adipocyte insulin signaling remains controversial. As a step to reveal the role for Rac1 in adipocytes, we aimed to establish immunofluorescent microscopy to detect the intracellular distribution of activated Rac1. The epitope-tagged Rac1-binding domain of a Rac1-specific target was utilized as a probe that specifically recognizes the activated form of Rac1. Rac1 activation in response to ex vivo and in vivo insulin stimulations in primary adipocyte culture and mouse white adipose tissue, respectively, was successfully observed by immunofluorescent microscopy. These Rac1 activations were mediated by phosphoinositide 3-kinase. Another small GTPase RalA has also been implicated in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Similarly to Rac1, immunofluorescent microscopy using an activated RalA-specific polypeptide probe allowed us to detect intracellular distribution of insulin-activated RalA in adipocytes. These novel approaches to visualize the activation status of small GTPases in adipocytes will largely contribute to the understanding of signal transduction mechanisms particularly for insulin action. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel assay for detecting antibodies to cytochrome P4502D6, the molecular target of liver kidney microsomal antibody type 1.

PubMed

Kerkar, N; Ma, Y; Hussain, M; Muratori, L; Targett, C; Williams, R; Bianchi, F B; Mieli-Vergani, G; Vergani, D

1999-03-04

Liver Kidney Microsomal type 1 (LKM1) antibody, the diagnostic marker of autoimmune hepatitis type 2, is also found in a proportion of patients with hepatitis C virus infection (HCV). It is detected conventionally by the subjective immunofluorescence technique. Our aim was to establish a simple and objective enzyme-linked immunosorbent assay (ELISA) that measures antibodies to cytochrome P4502D6 (CYP2D6), the target of LKM1. An indirect ELISA using eukaryotically expressed CYP2D6 was designed. Absorbance values obtained against a reference microsomal preparation were subtracted from those obtained against a microsomal preparation over-expressing CYP2D6, thus removing the non-CYP2D6-specific reaction. Sera from 51 LKM1 positive patients (21 autoimmune hepatitis and 30 with HCV infection), 111 LKM1 negative patients with chronic liver disease (including 20 with HCV infection) and 43 healthy controls were tested. Of 51 patients positive by immunofluorescence, 48 were also positive by ELISA while all the 154 LKM1 negative subjects were also negative by ELISA. There was a high degree of association between IFL and ELISA as demonstrated by a kappa reliability value of 0.96. The absorbance values by ELISA correlated with immunofluorescence LKM1 titres both in autoimmune hepatitis (r = 0.74, p < 0.001) and HCV infection (r = 0.67, p < 0.001). The simple, objective ELISA described has the potential to replace the standard immunofluorescence technique.

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

PubMed Central

Yamada, Norishige; Ogawa, Akiyo; Ogawa, Yuya

2014-01-01

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation. PMID:25489864

Immunohistochemical Localization of AT1a, AT1b, and AT2 Angiotensin II Receptor Subtypes in the Rat Adrenal, Pituitary, and Brain with a Perspective Commentary

PubMed Central

Premer, Courtney; Lamondin, Courtney; Mitzey, Ann; Speth, Robert C.; Brownfield, Mark S.

2013-01-01

Angiotensin II increases blood pressure and stimulates thirst and sodium appetite in the brain. It also stimulates secretion of aldosterone from the adrenal zona glomerulosa and epinephrine from the adrenal medulla. The rat has 3 subtypes of angiotensin II receptors: AT1a, AT1b, and AT2. mRNAs for all three subtypes occur in the adrenal and brain. To immunohistochemically differentiate these receptor subtypes, rabbits were immunized with C-terminal fragments of these subtypes to generate receptor subtype-specific antibodies. Immunofluorescence revealed AT1a and AT2 receptors in adrenal zona glomerulosa and medulla. AT1b immunofluorescence was present in the zona glomerulosa, but not the medulla. Ultrastructural immunogold labeling for the AT1a receptor in glomerulosa and medullary cells localized it to plasma membrane, endocytic vesicles, multivesicular bodies, and the nucleus. AT1b and AT2, but not AT1a, immunofluorescence was observed in the anterior pituitary. Stellate cells were AT1b positive while ovoid cells were AT2 positive. In the brain, neurons were AT1a, AT1b, and AT2 positive, but glia was only AT1b positive. Highest levels of AT1a, AT1b, and AT2 receptor immunofluorescence were in the subfornical organ, median eminence, area postrema, paraventricular nucleus, and solitary tract nucleus. These studies complement those employing different techniques to characterize Ang II receptors. PMID:23573410

Profiles of gonadotropin-inhibitory hormone and melatonin during the sex change and maturation of cinnamon clownfish, Amphiprion melanopus.

PubMed

Choi, Young Jae; Habibi, Hamid R; Choi, Cheol Young

2016-06-24

The present study aimed to determine the relationship between melatonin and gonadotropin-inhibitory hormone (GnIH) and their effect on reproduction in cinnamon clownfish, Amphiprion melanopus. Accordingly, we investigated the expression pattern of GnIH, GnIH receptor (GnIH-R), and melatonin receptor (MT-R1) mRNA and protein, as well as the plasma levels of melatonin, during sex change in cinnamon clownfish. We found that GnIH and MT-R1 mRNA and melatonin activity were higher in fish with mature brain than in fish with developing gonads, and using double immunofluorescence staining, we found that both GnIH and MT-R1 proteins were co-expressed in the hypothalamus of cinnamon clownfish. These findings support the hypothesis that melatonin plays an important role in the negative regulation of maturation and GnIH regulation during reproduction. Copyright © 2016 Elsevier Inc. All rights reserved.

Repolarization of hepatocytes in culture.

PubMed

Talamini, M A; Kappus, B; Hubbard, A

1997-01-01

We have evaluated the biochemical, morphological, and functional redevelopment of polarity in freshly isolated hepatocytes cultured using a double layer collagen gel sandwich technique. Western blot analysis showed increased cellular levels of the cell adhesion protein uvomorulin as cultured hepatocytes repolarized. Immunofluorescence studies using antibodies against domain-specific membrane proteins showed polarity as early as 48 hours, although the pattern of the polymeric Immunoglobulin-A receptor (pIgA-R) differed from in vivo liver. Electron microscopy showed developing bile canaliculi at 1 day. However, the functional presence of tight junctions was absent at 1 day, but present at 5 days. We further showed functional polarity to be present at 4 days by documenting the ability of cultured hepatocytes to metabolize and excrete fluorescein diacetate into visible bile canaliculi. We conclude that hepatocytes cultured appropriately develop morphological and functional polarity. Hepatocyte culture is therefore a useful tool for the study of mechanisms responsible for the development of polarized function.

CHRONIC PERIPHERAL NERVE COMPRESSION DISRUPTS PARANODAL AXOGLIAL JUNCTIONS

PubMed Central

Otani, Yoshinori; Yermakov, Leonid M.; Dupree, Jeffrey L.; Susuki, Keiichiro

2016-01-01

Introduction Peripheral nerves are often exposed to mechanical stress leading to compression neuropathies. The pathophysiology underlying nerve dysfunction by chronic compression is largely unknown. Methods We analyzed molecular organization and fine structures at and near nodes of Ranvier in a compression neuropathy model in which a silastic tube was placed around the mouse sciatic nerve. Results Immunofluorescence study showed that clusters of cell adhesion complex forming paranodal axoglial junctions were dispersed with frequent overlap with juxtaparanodal components. These paranodal changes occurred without internodal myelin damage. The distribution and pattern of paranodal disruption suggests that these changes are the direct result of mechanical stress. Electron microscopy confirmed loss of paranodal axoglial junctions. Discussion Our data show that chronic nerve compression disrupts paranodal junctions and axonal domains required for proper peripheral nerve function. These results provide important clues toward better understanding of the pathophysiology underlying nerve dysfunction in compression neuropathies. PMID:27463510

SNOM and AFM microscopy techniques to study the effect of non-ionizing radiation on the morphological and biochemical properties of human keratinocytes cell line (HaCaT).

PubMed

Rieti, S; Manni, V; Lisi, A; Giuliani, L; Sacco, D; D'Emilia, E; Cricenti, A; Generosi, R; Luce, M; Grimaldi, S

2004-01-01

In this study we have employed atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM) techniques to study the effect of the interaction between human keratinocytes (HaCaT) and electromagnetic fields at low frequency. HaCaT cells were exposed to a sinusoidal magnetic field at a density of 50 Hz, 1 mT. AFM analysis revealed modification in shape and morphology in exposed cells with an increase in the areas of adhesion between cells. This latter finding was confirmed by SNOM indirect immunofluorescence analysis performed with a fluorescent antibody against the adhesion marker beta4 integrin, which revealed an increase of beta4 integrin segregation in the cell membrane of 50-Hz exposed cells, suggesting that a higher percentage of these cells shows a modified pattern of this adhesion marker.

Defining the limits of flowers: the challenge of distinguishing between the evolutionary products of simple versus compound strobili

PubMed Central

Rudall, Paula J.; Bateman, Richard M.

2010-01-01

Recent phylogenetic reconstructions suggest that axially condensed flower-like structures evolved iteratively in seed plants from either simple or compound strobili. The simple-strobilus model of flower evolution, widely applied to the angiosperm flower, interprets the inflorescence as a compound strobilus. The conifer cone and the gnetalean ‘flower’ are commonly interpreted as having evolved from a compound strobilus by extreme condensation and (at least in the case of male conifer cones) elimination of some structures present in the presumed ancestral compound strobilus. These two hypotheses have profoundly different implications for reconstructing the evolution of developmental genetic mechanisms in seed plants. If different flower-like structures evolved independently, there should intuitively be little commonality of patterning genes. However, reproductive units of some early-divergent angiosperms, including the extant genus Trithuria (Hydatellaceae) and the extinct genus Archaefructus (Archaefructaceae), apparently combine features considered typical of flowers and inflorescences. We re-evaluate several disparate strands of comparative data to explore whether flower-like structures could have arisen by co-option of flower-expressed patterning genes into independently evolved condensed inflorescences, or vice versa. We discuss the evolution of the inflorescence in both gymnosperms and angiosperms, emphasising the roles of heterotopy in dictating gender expression and heterochrony in permitting internodal compression. PMID:20047867

Defining the limits of flowers: the challenge of distinguishing between the evolutionary products of simple versus compound strobili.

PubMed

Rudall, Paula J; Bateman, Richard M

2010-02-12

Recent phylogenetic reconstructions suggest that axially condensed flower-like structures evolved iteratively in seed plants from either simple or compound strobili. The simple-strobilus model of flower evolution, widely applied to the angiosperm flower, interprets the inflorescence as a compound strobilus. The conifer cone and the gnetalean 'flower' are commonly interpreted as having evolved from a compound strobilus by extreme condensation and (at least in the case of male conifer cones) elimination of some structures present in the presumed ancestral compound strobilus. These two hypotheses have profoundly different implications for reconstructing the evolution of developmental genetic mechanisms in seed plants. If different flower-like structures evolved independently, there should intuitively be little commonality of patterning genes. However, reproductive units of some early-divergent angiosperms, including the extant genus Trithuria (Hydatellaceae) and the extinct genus Archaefructus (Archaefructaceae), apparently combine features considered typical of flowers and inflorescences. We re-evaluate several disparate strands of comparative data to explore whether flower-like structures could have arisen by co-option of flower-expressed patterning genes into independently evolved condensed inflorescences, or vice versa. We discuss the evolution of the inflorescence in both gymnosperms and angiosperms, emphasising the roles of heterotopy in dictating gender expression and heterochrony in permitting internodal compression.

A general analytical strategy for the characterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detection coupled to electrospray ionization and triple quadrupole mass spectrometry.

PubMed

Abad-García, Beatriz; Berrueta, Luis A; Garmón-Lobato, Sergio; Gallo, Blanca; Vicente, Francisca

2009-07-10

In the present study, a methodology based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer for the simultaneous identification of phenolic compounds in fruit juices has been developed. 72 available phenolic compound standards from diverse families present in fruits have been studied in order to analyze their fragmentation pattern. As a result, a general strategy for the characterization of unknown phenolic compounds in fruit juices was designed: (i) taking into account its UV-visible spectrum and elution order, assign the unknown polyphenol to a polyphenol class, (ii) identify the quasi-molecular ion using positive and negative MS spectra, being supported by adducts generated with solvent or sodium and molecular complexes, (iii) determinate the pattern of glycosylation in positive mode using ESI(+)-CID MS/MS product ion scan experiments, selecting the quasi-molecular ion as precursor ion, and finally, (iv) study the identity of the aglycone through ESI(+)-CID MS/MS product ion spectra from the protonated aglycone, [Y(0)](+). This strategy was successfully employed for the characterization of known and unknown phenolic compounds in juices from 17 different fruits.

Opsin evolution and expression in arthropod compound eyes and ocelli: insights from the cricket Gryllus bimaculatus.

PubMed

Henze, Miriam J; Dannenhauer, Kara; Kohler, Martin; Labhart, Thomas; Gesemann, Matthias

2012-08-30

Opsins are key proteins in animal photoreception. Together with a light-sensitive group, the chromophore, they form visual pigments which initiate the visual transduction cascade when photoactivated. The spectral absorption properties of visual pigments are mainly determined by their opsins, and thus opsins are crucial for understanding the adaptations of animal eyes. Studies on the phylogeny and expression pattern of opsins have received considerable attention, but our knowledge about insect visual opsins is still limited. Up to now, researchers have focused on holometabolous insects, while general conclusions require sampling from a broader range of taxa. We have therefore investigated visual opsins in the ocelli and compound eyes of the two-spotted cricket Gryllus bimaculatus, a hemimetabolous insect. Phylogenetic analyses place all identified cricket sequences within the three main visual opsin clades of insects. We assign three of these opsins to visual pigments found in the compound eyes with peak absorbances in the green (515 nm), blue (445 nm) and UV (332 nm) spectral range. Their expression pattern divides the retina into distinct regions: (1) the polarization-sensitive dorsal rim area with blue- and UV-opsin, (2) a newly-discovered ventral band of ommatidia with blue- and green-opsin and (3) the remainder of the compound eye with UV- and green-opsin. In addition, we provide evidence for two ocellar photopigments with peak absorbances in the green (511 nm) and UV (350 nm) spectral range, and with opsins that differ from those expressed in the compound eyes. Our data show that cricket eyes are spectrally more specialized than has previously been assumed, suggesting that similar adaptations in other insect species might have been overlooked. The arrangement of spectral receptor types within some ommatidia of the cricket compound eyes differs from the generally accepted pattern found in holometabolous insect taxa and awaits a functional explanation. From the opsin phylogeny, we conclude that gene duplications, which permitted differential opsin expression in insect ocelli and compound eyes, occurred independently in several insect lineages and are recent compared to the origin of the eyes themselves.

Opsin evolution and expression in Arthropod compound Eyes and Ocelli: Insights from the cricket Gryllus bimaculatus

PubMed Central

2012-01-01

Background Opsins are key proteins in animal photoreception. Together with a light-sensitive group, the chromophore, they form visual pigments which initiate the visual transduction cascade when photoactivated. The spectral absorption properties of visual pigments are mainly determined by their opsins, and thus opsins are crucial for understanding the adaptations of animal eyes. Studies on the phylogeny and expression pattern of opsins have received considerable attention, but our knowledge about insect visual opsins is still limited. Up to now, researchers have focused on holometabolous insects, while general conclusions require sampling from a broader range of taxa. We have therefore investigated visual opsins in the ocelli and compound eyes of the two-spotted cricket Gryllus bimaculatus, a hemimetabolous insect. Results Phylogenetic analyses place all identified cricket sequences within the three main visual opsin clades of insects. We assign three of these opsins to visual pigments found in the compound eyes with peak absorbances in the green (515 nm), blue (445 nm) and UV (332 nm) spectral range. Their expression pattern divides the retina into distinct regions: (1) the polarization-sensitive dorsal rim area with blue- and UV-opsin, (2) a newly-discovered ventral band of ommatidia with blue- and green-opsin and (3) the remainder of the compound eye with UV- and green-opsin. In addition, we provide evidence for two ocellar photopigments with peak absorbances in the green (511 nm) and UV (350 nm) spectral range, and with opsins that differ from those expressed in the compound eyes. Conclusions Our data show that cricket eyes are spectrally more specialized than has previously been assumed, suggesting that similar adaptations in other insect species might have been overlooked. The arrangement of spectral receptor types within some ommatidia of the cricket compound eyes differs from the generally accepted pattern found in holometabolous insect taxa and awaits a functional explanation. From the opsin phylogeny, we conclude that gene duplications, which permitted differential opsin expression in insect ocelli and compound eyes, occurred independently in several insect lineages and are recent compared to the origin of the eyes themselves. PMID:22935102

Spatial and temporal patterns of floral scent emission in Dianthus inoxianus and electroantennographic responses of its hawkmoth pollinator.

PubMed

Balao, Francisco; Herrera, Javier; Talavera, Salvador; Dötterl, Stefan

2011-05-01

Scent emission is important in nocturnal pollination systems, and plant species pollinated by nocturnal insects often present characteristic odor compositions and temporal patterns of emission. We investigated the temporal (day/night; flower lifetime) and spatial (different flower parts, nectar) pattern of flower scent emission in nocturnally pollinated Dianthusinoxianus, and determined which compounds elicit physiological responses on the antennae of the sphingid pollinator Hyles livornica. The scent of D.inoxianus comprises 68 volatile compounds, but is dominated by aliphatic 2-ketones and sesquiterpenoids, which altogether make up 82% of collected volatiles. Several major and minor compounds elicit electrophysiological responses in the antennae of H. livornica. Total odor emission does not vary along day and night hours, and neither does along the life of the flower. However, the proportion of compounds eliciting physiological responses varies between day and night. All flower parts as well as nectar release volatiles. The scent of isolated flower parts is dominated by fatty acid derivatives, whereas nectar is dominated by benzenoids. Dissection (= damage) of flowers induced a ca. 20-fold increase in the rate of emission of EAD-active volatiles, especially aliphatic 2-ketones. We suggest that aliphatic 2-ketones might contribute to pollinator attraction in D. inoxianus, even though they have been attributed an insect repellent function in other plant species. We also hypothesize that the benzenoids in nectar may act as an honest signal ('nectar guide') for pollinators. Copyright © 2011 Elsevier Ltd. All rights reserved.

Supervised de novo reconstruction of metabolic pathways from metabolome-scale compound sets

PubMed Central

Kotera, Masaaki; Tabei, Yasuo; Yamanishi, Yoshihiro; Tokimatsu, Toshiaki; Goto, Susumu

2013-01-01

Motivation: The metabolic pathway is an important biochemical reaction network involving enzymatic reactions among chemical compounds. However, it is assumed that a large number of metabolic pathways remain unknown, and many reactions are still missing even in known pathways. Therefore, the most important challenge in metabolomics is the automated de novo reconstruction of metabolic pathways, which includes the elucidation of previously unknown reactions to bridge the metabolic gaps. Results: In this article, we develop a novel method to reconstruct metabolic pathways from a large compound set in the reaction-filling framework. We define feature vectors representing the chemical transformation patterns of compound–compound pairs in enzymatic reactions using chemical fingerprints. We apply a sparsity-induced classifier to learn what we refer to as ‘enzymatic-reaction likeness’, i.e. whether compound pairs are possibly converted to each other by enzymatic reactions. The originality of our method lies in the search for potential reactions among many compounds at a time, in the extraction of reaction-related chemical transformation patterns and in the large-scale applicability owing to the computational efficiency. In the results, we demonstrate the usefulness of our proposed method on the de novo reconstruction of 134 metabolic pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG). Our comprehensively predicted reaction networks of 15 698 compounds enable us to suggest many potential pathways and to increase research productivity in metabolomics. Availability: Softwares are available on request. Supplementary material are available at http://web.kuicr.kyoto-u.ac.jp/supp/kot/ismb2013/. Contact: goto@kuicr.kyoto-u.ac.jp PMID:23812977

Extending breath analysis to the cellular level: current thoughts on the human microbiome and the expression of organic compounds in the human exposome

EPA Science Inventory

Human biomarkers are comprised of compounds from cellular metabolism, oxidative stress, and the microbiome of bacteria in the gut, genitourinary, and pulmonary tracts. When we examine patterns in human biomarkers to discern human health state or diagnose specific diseases, it is...

Modeling concentration patterns of agricultural and urban micropollutants in surface waters in catchment of mixed land use

NASA Astrophysics Data System (ADS)

Stamm, C.; Scheidegger, R.; Bader, H. P.

2012-04-01

Organic micropollutants detected in surface waters can originate from agricultural and urban sources. Depending on the use of the compounds, the temporal loss patterns vary substantially. Therefore models that simulate water quality in watersheds of mixed land use have to account for all relevant sources. We present here simulation results of a transport model that describes the dynamic of several biocidal compounds as well as the behaviour of human pharmaceuticals. The model consists of the sub-model Rexpo simulating the transfer of the compounds from the point of application to the stream in semi-lumped manner. The river sub-model, which is programmed in the Aquasim software, describes the fate of the compounds in the stream. Both sub-models are process-based. The Rexpo sub-model was calibrated at the scale of a small catchment of 25 km2, which is inhabited by about 12'000 people. Based on the resulting model parameters the loss dynamics of two herbicides (atrazine, isoproturon) and a compound of mixed urban and agricultural use (diuron) were predicted for two nested catchment of 212 and 1696 km2, respectively. The model output was compared to observed time-series of concentrations and loads obtained for the entire year 2009. Additionally, the fate of two pharmaceuticals with constant input (carbamazepine, diclofenac) was simulated for improving the understanding of possible degradation processes. The simulated loads and concentrations of the biocidal compounds differed by a factor of 2 to 3 from the observations. In general, the seasonal patterns were well captured by the model. However, a detailed analysis of the seasonality revealed substantial input uncertainty for the application of the compounds. The model results also demonstrated that for the dynamics of rain-driven losses of biocidal compounds the semi-lumped approach of the Rexpo sub-model was sufficient. Only for simulating the photolytic degradation of diclofenac in the stream the detailed representation of the routing in the stream was essential. Overall, the study demonstrated that the simulation of micropollutants at the watershed scale can be strongly hampered by input uncertainty regarding the use of the chemicals. Under such conditions the level of process-representation in the Rexpo sub-models is superfluous. For practical applications, one should address the question how to simply the approach while still maintaining the essential parts.

Low cost tooling material and process for graphite and Kevlar composites

NASA Technical Reports Server (NTRS)

Childs, William I.

1987-01-01

An Extruded Sheet Tooling Compound (ESTC) was developed for use in quickly building low cost molds for fabricating composites. The ESTC is a very highly mineral-filled resin system formed into a 6 mm thick sheet. The sheet is laid on the pattern, vacuum (bag) is applied to remove air from the pattern surface, and the assembly is heat cured. The formed ESTC is then backed and/or framed and ready for use. The cured ESTC exhibits low coefficient of thermal expansion and maintains strength at temperatures of 180 to 200 C. Tools were made and used successfully for: Compression molding of high strength epoxy sheet molding compound, stamping of aluminum, resin transfer molding of polyester, and liquid resin molding of polyester. Several variations of ESTC can be made for specific requirements. Higher thermal conductivity can be achieved by using an aluminum particle filler. Room temperature gel is possible to allow use of foam patterns.

Comparison of growth inhibition and immunofluorescence tests in serotyping clinical isolates of Ureaplasma urealyticum.

PubMed Central

Piot, P

1977-01-01

Typestrains of the eight serotypes of Ureaplasma urealyticum and 39 genital isolates were typed by the growth inhibition (GI) and indirect immunofluorescence (IF) tests. The GI test proved to be very specific and simple, demanding large volumes of serum. It was less sensitive than the IF test, which produced more cross-reactions, was economical in serum, and able to detect mixed infections. Of the 39 isolates, 27 were serotyped by GI and 34 by IF. Mixed cultures occurred in as many as 36% of the isolates. PMID:326348

Application of photostable quantum dots for indirect immunofluorescent detection of specific bacterial serotypes on small marine animals

NASA Astrophysics Data System (ADS)

Decho, Alan W.; Beckman, Erin M.; Chandler, G. Thomas; Kawaguchi, Tomohiro

2008-06-01

An indirect immunofluorescence approach was developed using semiconductor quantum dot nanocrystals to label and detect a specific bacterial serotype of the bacterial human pathogen Vibrio parahaemolyticus, attached to small marine animals (i.e. benthic harpacticoid copepods), which are suspected pathogen carriers. This photostable labeling method using nanotechnology will potentially allow specific serotypes of other bacterial pathogens to be detected with high sensitivity in a range of systems, and can be easily applied for sensitive detection to other Vibrio species such as Vibrio cholerae.

ISOLATION AND DETECTION OF GIARDIA CYSTS FROM WATER USING DIRECT IMMUNOFLUORESCENCE.

USGS Publications Warehouse

Sorenson, Stephen K.; Riggs, John L.; Dileanis, Peter D.; Suk, Thomas J.

1986-01-01

A water-sampling apparatus used for the isolation and detection of Giardia cysts in water has been designed and tested. The sampling apparatus uses one of a variety of pumps or waterline pressure to move water through a filter. Two of the optional pumps are lightweight enough to make the apparatus portable and thus suitable for sampling in remote areas. This technique of sample processing produces good cyst recovery in much less time than is required with previously established methods. Giardia cysts are identified using direct immunofluorescence.

Biofunctionalization of silicone rubber with microgroove-patterned surface and carbon-ion implantation to enhance biocompatibility and reduce capsule formation.

PubMed

Lei, Ze-Yuan; Liu, Ting; Li, Wei-Juan; Shi, Xiao-Hua; Fan, Dong-Li

Silicone rubber implants have been widely used to repair soft tissue defects and deformities. However, poor biocompatibility can elicit capsule formation, usually resulting in prosthesis contracture and displacement in long-term usage. To overcome this problem, this study investigated the properties of silicone rubber materials with or without a microgroove-patterned surface and with or without carbon (C)-ion implantation. Atomic force microscopy, X-ray photoelectron spectroscopy, and a water contact angle test were used to characterize surface morphology and physicochemical properties. Cytocompatibility was investigated by a cell adhesion experiment, immunofluorescence staining, a Cell Counting Kit-8 assay, and scanning electron microscopy in vitro. Histocompatibility was evaluated by studying the inflammatory response and fiber capsule formation that developed after subcutaneous implantation in rats for 7 days, 15 days, and 30 days in vivo. Parallel microgrooves were found on the surfaces of patterned silicone rubber (P-SR) and patterned C-ion-implanted silicone rubber (PC-SR). Irregular larger peaks and deeper valleys were present on the surface of silicone rubber implanted with C ions (C-SR). The silicone rubber surfaces with microgroove patterns had stable physical and chemical properties and exhibited moderate hydrophobicity. PC-SR exhibited moderately increased dermal fibroblast cell adhesion and growth, and its surface microstructure promoted orderly cell growth. Histocompatibility experiments on animals showed that both the anti-inflammatory and antifibrosis properties of PC-SR were slightly better than those of the other materials, and there was also a lower capsular contracture rate and less collagen deposition around implants made from PC-SR. Although the surface chemical properties, dermal fibroblast cell growth, and cell adhesion were not changed by microgroove pattern modification, a more orderly cell arrangement was obtained, leading to enhanced biocompatibility and reduced capsule formation. Thus, this approach to the modification of silicone rubber, in combination with C-ion implantation, should be considered for further investigation and application.

Long-term transport behavior of psychoactive compounds in sewage-affected groundwater

NASA Astrophysics Data System (ADS)

Nham, Hang Thuy Thi; Greskowiak, Janek; Hamann, Enrico; Meffe, Raffaella; Hass, Ulrike; Massmann, Gudrun

2016-11-01

The present study provides a model-based characterization of the long-term transport behavior of five psychoactive compounds (meprobamate, pyrithyldione, primidone, phenobarbital and phenylethylmalonamide) introduced into groundwater via sewage irrigation in Berlin, Germany. Compounds are still present in the groundwater despite the sewage farm closure in the year 1980. Due to the limited information on (i) compound concentrations in the source water and (ii) substance properties, a total of 180 cross-sectional model realizations for each compound were carried out, covering a large range of possible parameter combinations. Results were compared with the present-day contamination patterns in the aquifer and the most likely scenarios were identified based on a number of model performance criteria. The simulation results show that (i) compounds are highly persistent under the present field conditions, and (ii) sorption is insignificant. Thus, back-diffusion from low permeability zones appears as the main reason for the compound retardation.

Identification of volatile and semivolatile compounds in chemical ionization GC-MS using a mass-to-structure (MTS) Search Engine with integral isotope pattern ranking.

PubMed

Liao, Wenta; Draper, William M

2013-02-21

The mass-to-structure or MTS Search Engine is an Access 2010 database containing theoretical molecular mass information for 19,438 compounds assembled from common sources such as the Merck Index, pesticide and pharmaceutical compilations, and chemical catalogues. This database, which contains no experimental mass spectral data, was developed as an aid to identification of compounds in atmospheric pressure ionization (API)-LC-MS. This paper describes a powerful upgrade to this database, a fully integrated utility for filtering or ranking candidates based on isotope ratios and patterns. The new MTS Search Engine is applied here to the identification of volatile and semivolatile compounds including pesticides, nitrosoamines and other pollutants. Methane and isobutane chemical ionization (CI) GC-MS spectra were obtained from unit mass resolution mass spectrometers to determine MH(+) masses and isotope ratios. Isotopes were measured accurately with errors of <4% and <6%, respectively, for A + 1 and A + 2 peaks. Deconvolution of interfering isotope clusters (e.g., M(+) and [M - H](+)) was required for accurate determination of the A + 1 isotope in halogenated compounds. Integrating the isotope data greatly improved the speed and accuracy of the database identifications. The database accurately identified unknowns from isobutane CI spectra in 100% of cases where as many as 40 candidates satisfied the mass tolerance. The paper describes the development and basic operation of the new MTS Search Engine and details performance testing with over 50 model compounds.

3D multiplexed immunoplasmonics microscopy

NASA Astrophysics Data System (ADS)

Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

2016-07-01

Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence.Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence. Electronic supplementary information (ESI) available: Characterization of functionalized nanoparticles by UV-visible-NIR spectroscopy, standard dark field microscopy and reflected light microscopy. Immunofluorescence of cells. See DOI: 10.1039/c6nr01257d

Reduced-HMGB1 suppresses poly(I:C)-induced inflammation in keratinocytes.

PubMed

Mori, Hideki; Murakami, Masamoto; Tsuda, Teruko; Kameda, Kenji; Utsunomiya, Ryo; Masuda, Kana; Shiraishi, Ken; Dai, Xiuju; Tohyama, Mikiko; Nakaoka, Hiroki; Sayama, Koji

2018-05-01

High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes DNA and facilitates gene transcription. Additionally, cell stress or death induces the release of HMGB1 outside the cell membrane, where HMGB1 functions as an alarmin, causing an inflammatory response in combination with other cytokines, damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs). To evaluate the effect of reduced-HMGB1 (previously termed chemoattractive-HMGB1) on polyinosine-polycytidylic acid [poly(I:C)]-induced inflammation in normal human keratinocytes (NHKs). We focused on downstream components of the poly(I:C)-Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) pathways, including IκBα, nuclear factor (NF)-κB p65, mitogen-activated protein kinase (MAPK), and interferon regulatory factor 3 (IRF3), and assessed whether these pathways are involved in the suppression of poly(I:C)-induced inflammation in NHKs by HMGB1. An immunoprecipitation was performed to know whether HMGB1 could bind to poly(I:C), and immunofluorescence staining and flow cytometric analysis were performed to check whether reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis). Application of exogenous HMGB1 before, but not after, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs. In addition, reduced-HMGB1, but not disulfide-HMGB1, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs, suggesting the importance of the redox status of exogenous HMGB1. Pre-treatment with reduced-HMGB1 inhibited the phosphorylation of IκBα, NF-κB p65, and IRF3 induced by poly(I:C) stimulation in NHKs; however, phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was unaffected. Disulfide-HMGB1 formed a complex with poly(I:C), as did reduced- and oxidized-HMGB1, albeit to a lesser extent. Immunofluorescence staining and flow cytometric analysis indicated that reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis). These findings suggest that pre-treatment with reduced-HMGB1 ameliorates poly(I:C)-mediated inflammation in NHKs. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Gene expression changes in the retina following subretinal injection of human neural progenitor cells into a rodent model for retinal degeneration.

PubMed

Jones, Melissa K; Lu, Bin; Saghizadeh, Mehrnoosh; Wang, Shaomei

2016-01-01

Retinal degenerative diseases (RDDs) affect millions of people and are the leading cause of vision loss. Although treatment options for RDDs are limited, stem and progenitor cell-based therapies have great potential to halt or slow the progression of vision loss. Our previous studies have shown that a single subretinal injection of human forebrain derived neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) retinal degenerate rat offers long-term preservation of photoreceptors and visual function. Furthermore, neural progenitor cells are currently in clinical trials for treating age-related macular degeneration; however, the molecular mechanisms of stem cell-based therapies are largely unknown. This is the first study to analyze gene expression changes in the retina of RCS rats following subretinal injection of hNPCs using high-throughput sequencing. RNA-seq data of retinas from RCS rats injected with hNPCs (RCS(hNPCs)) were compared to sham surgery in RCS (RCS(sham)) and wild-type Long Evans (LE(sham)) rats. Differential gene expression patterns were determined with in silico analysis and confirmed with qRT-PCR. Function, biologic, cellular component, and pathway analyses were performed on differentially expressed genes and investigated with immunofluorescent staining experiments. Analysis of the gene expression data sets identified 1,215 genes that were differentially expressed between RCS(sham) and LE(sham) samples. Additionally, 283 genes were differentially expressed between the RCS(hNPCs) and RCS(sham) samples. Comparison of these two gene sets identified 68 genes with inverse expression (termed rescue genes), including Pdc, Rp1, and Cdc42ep5. Functional, biologic, and cellular component analyses indicate that the immune response is enhanced in RCS(sham). Pathway analysis of the differential expression gene sets identified three affected pathways in RCS(hNPCs), which all play roles in phagocytosis signaling. Immunofluorescent staining detected the increased presence of macrophages and microglia in RCS(sham) retinas, which decreased in RCS(hNPCs) retinas similar to the patterns detected in LE(sham). The results from this study provide evidence of the gene expression changes that occur following treatment with hNPCs in the degenerating retina. This information can be used in future studies to potentially enhance or predict responses to hNPC and other stem cell therapies for retinal degenerative diseases.

Simultaneous prediction of enzyme orthologs from chemical transformation patterns for de novo metabolic pathway reconstruction

PubMed Central

Tabei, Yasuo; Yamanishi, Yoshihiro; Kotera, Masaaki

2016-01-01

Motivation: Metabolic pathways are an important class of molecular networks consisting of compounds, enzymes and their interactions. The understanding of global metabolic pathways is extremely important for various applications in ecology and pharmacology. However, large parts of metabolic pathways remain unknown, and most organism-specific pathways contain many missing enzymes. Results: In this study we propose a novel method to predict the enzyme orthologs that catalyze the putative reactions to facilitate the de novo reconstruction of metabolic pathways from metabolome-scale compound sets. The algorithm detects the chemical transformation patterns of substrate–product pairs using chemical graph alignments, and constructs a set of enzyme-specific classifiers to simultaneously predict all the enzyme orthologs that could catalyze the putative reactions of the substrate–product pairs in the joint learning framework. The originality of the method lies in its ability to make predictions for thousands of enzyme orthologs simultaneously, as well as its extraction of enzyme-specific chemical transformation patterns of substrate–product pairs. We demonstrate the usefulness of the proposed method by applying it to some ten thousands of metabolic compounds, and analyze the extracted chemical transformation patterns that provide insights into the characteristics and specificities of enzymes. The proposed method will open the door to both primary (central) and secondary metabolism in genomics research, increasing research productivity to tackle a wide variety of environmental and public health matters. Availability and Implementation: Contact: maskot@bio.titech.ac.jp PMID:27307627

Cuttlefish Ink Melanin Encapsulated in Nanolipid Bubbles and Applied Through a Micro-Needling Procedure Easily Stains White Hair Facilitating Photoepilation.

PubMed

Trelles, Mario A; Almudever, Patricia; Alcolea, Justo M; Cortijo, Julio; Serrano, Gabriel; Expósito, Inmaculada; Royo, Josefina; Leclère, Franck Marie

2016-05-01

Photothermolysis of unwanted hair depends on the presence of melanin in the hair follicle as the chromophore, but is not effective in patients with non-pigmented, melanin-sparse hair shafts and follicles. This split-scalp, double-blind study was to monitor the efficacy of melanin bound in nanosomes to inject exogenous melanin into the hair follicles thus potentiating successful photothermolysis.
Twelve patients, phototypes II-III, with white or very fair hair, were treated with a compound containing melanin encapsulated in nanosomes (Melaser^®) together with a fluorescent marker. Two equal 6 cm² areas were marked on each side of the occiput of the subjects. The compound was applied to a randomly selected experimental side on each patient (area A), and a saline solution applied in the same manner to the contralateral control side (area B). Penetration of the melanin into the hair follicle was assessed using optical and fluorescence microscopy. Also, condition of hair structure was checked in vivo after standard laser settings used for epilation.
A slight transient erythema was observed in those areas where the compound was applied with some perifollicular edema. No such effects were noticed in those areas where saline solution was applied. No persistent complications such as scarring, hypo- or hyperpigmentation were observed in any of the experimental or control areas. Under fluorescence microscopy, the hair structures in the areas to which the compound had been applied showed a clear melanin deposit confirmed by the immunofluorescence intensity, which was highest at 2 hours after application. By optical microscopy, external melanin was deposited in hair follicles. Tests with standard settings for epilation were efficacious in damaging melanin-marked white hair.
This study strongly suggests the safety and efficacy of the application of nanosomes encapsulating melanin for the introduction of melanin into hair follicles. Changes noticed in the hair structure compromising its viability indicated potential application of this external melanin marker for white hair photoepilation.

J Drugs Dermatol. 2016;15(5):615-625.

[Behavioral response of Anopheles albimanus to volatile compounds collected inside houses from the south of Chiapas, Mexico].

PubMed

Ríos-Delgado, Silvany Mayoly; Rodríguez-Ramírez, Américo David; Cruz-López, Leopoldo; Escobar-Pérez, Luis Alonso; Aburto-Juárez, Ma de Lourdes; Torres-Estrada, José Luis

2008-01-01

To determine effects of volatile compounds in homes on the behavioral response of Anopheles albimanus. The study was conducted in January 2006, in the village of Nueva Independencia village, Suchiate, Chiapas. Volatile compounds were collected inside homes and the extracts were tested on unfed females in a Y-olfactometer. Extracts were analyzed in a gas chromatography-mass spectrometry system (GC-MS). Twenty eight extracts were obtained, twelve presented attraction and two repellency responses. GC-MS analyses of the extracts indicated variation in the volatile compound present in the extracts, but could not associated specific compounds with any particular effect. Within homes, volatiles presented attraction and repellency responses to An. albimanus. A definate pattern concerning the presence of a characteristic chemical compound and the observed response was not found.

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea.

PubMed

Huang, Lin-Chien; Thorne, Peter R; Housley, Gary D; Montgomery, Johanna M

2007-08-01

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.

Analysis of mammalian proteins involved in chromatin modification reveals new metaphase centromeric proteins and distinct chromosomal distribution patterns.

PubMed

Craig, Jeffrey M; Earle, Elizabeth; Canham, Paul; Wong, Lee H; Anderson, Melissa; Choo, K H Andy

2003-12-01

We have examined the metaphase chromosomal localization of 15 proteins that have previously been described as involved in mammalian chromatin modification and/or transcriptional modulation. Immunofluorescence data indicate that all the proteins localize to human and mouse centromeres, a neocentromere, and the active centromere of a dicentric chromosome, with six of these proteins (Sin3A, PCAF, MYST, MBD2, ORC2, P300/CBP) being demonstrated at mammalian centromeres for the first time. Most of these proteins fall into two distinct chromosomal distribution patterns: (a) kinetochore-associated proteins (Sin3A, PCAF, MYST and BAF180), which colocalize with metaphase kinetochores, but not any of the pericentric and other major heterochromatic regions; and (b) heterochromatin-associated proteins (MeCP2, MBD1, MBD2, ATRX, HP1alpha, HDAC1, HDAC2, DNMT1 and DNMT3b), which colocalize with centromeric/pericentric heterochromatin and all other major heterochromatic sites. A heterogeneous third group (c) consists of the origin recognition complex subunit ORC2 and the histone acetyltransferase P300/CBP, which associate generally with kinetochores in humans and centromeric/pericentric heterochromatin in mouse, with some minor differences in localization. These observations indicate an extensive sharing of protein components involved in chromatin modification at gene loci, centromeres and various chromosomal heterochromatic landmarks. The definition of distinct patterns of chromosomal distribution for these proteins provides a useful basis for the further investigation of the broad-ranging roles of these proteins.

Promyelocytic leukaemia zinc finger maintains self-renewal of male germline stem cells (mGSCs) and its expression pattern in dairy goat testis.

PubMed

Song, W; Zhu, H; Li, M; Li, N; Wu, J; Mu, H; Yao, X; Han, W; Liu, W; Hua, J

2013-08-01

Previous studies have shown that promyelocytic leukaemia zinc finger (PLZF) is a spermatogonia-specific transcription factor in the testis, required to regulate self-renewal and maintenance of the spermatogonia stem cell. Up to now, expression and function of PLZF in the goat testis has not been known. The objectives of this study were to investigate PLZF expression pattern in the dairy goat and its effect on male goat germline stem cell (mGSC) self-renewal and differentiation. Testis development and expression patterns of PLZF in the dairy goat were analysed by haematoxylin and eosin staining, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, effects of PLZF overexpression on mGSC self-renewal and differentiation were evaluated by quantitative RT-PCR (QRT-PCR), immunofluorescence and BrdU incorporation assay. Promyelocytic leukaemia zinc finger was essential for dairy goat testis development and expression of several proliferation and pluripotency-associated proteins including OCT4, C-MYC were upregulated by PLZF overexpression. The study demonstrated that PLZF played a key role in maintaining self-renewal of mGSCs and its overexpression enhanced expression of proliferation-associated genes. Promyelocytic leukaemia zinc finger could function in the dairy goat as well as in other species in maintaining self-renewal of germline stem cells and this study provides a model to study the mechanism on self-renewal and differentiation of mGSCs in livestock. © 2013 John Wiley & Sons Ltd.

Baicalin maintains late-stage functional cardiomyocytes in embryoid bodies derived from murine embryonic stem cells.

PubMed

Tang, Meilin; Yin, Mengmeng; Tang, Ming; Liang, Huamin; Yu, Chong; Hu, Xinwu; Luo, Hongyan; Baudis, Birte; Haustein, Moritz; Khalil, Markus; Sarić, Tomo; Hescheler, Jürgen; Xi, Jiaoya

2013-01-01

Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells. The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence. Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to β-adrenergic and muscarinic stimulations. Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells. Copyright © 2013 S. Karger AG, Basel.

An Immunological Strategy To Monitor In Situ the Phosphate Starvation State in Thiobacillus ferrooxidans

PubMed Central

Varela, Patricia; Levicán, Gloria; Rivera, Francisco; Jerez, Carlos A.

1998-01-01

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. During the process of ore bioleaching, the microorganisms are subjected to several stressing conditions, including the lack of some essential nutrients, which can affect the rates and yields of bioleaching. When T. ferrooxidans is starved for phosphate, the cells respond by inducing the synthesis of several proteins, some of which are outer membrane proteins of high molecular weight (70,000 to 80,000). These proteins were considered to be potential markers of the phosphate starvation state of these microorganisms. We developed a single-cell immunofluorescence assay that allowed monitoring of the phosphate starvation condition of this biomining microorganism by measuring the increased expression of the surface proteins. In the presence of low levels of arsenate (2 mM), the growth of phosphate-starved T. ferrooxidans cells was greatly inhibited compared to that of control nonstarved cells. Therefore, the determination of the phosphorus nutritional state is particularly relevant when arsenic compounds are solubilized during the bioleaching of different ores. PMID:9835593

Arsenite inhibits mitotic division and perturbs spindle dynamics in HeLa S3 cells.

PubMed

Huang, S C; Lee, T C

1998-05-01

Arsenical compounds, known to be human carcinogens, were shown to disturb cell cycle progression and induce cytogenetic alterations in a variety of cell systems. We report here that a 24 h treatment of arsenite induced mitotic accumulation in human cell lines. HeLa S3 and KB cells were most susceptible: 35% of the total cell population was arrested at the mitotic stage after treatment with 5 microM sodium arsenite in HeLa S3 cells and after 10 microM in KB cells. Under a microscope, we observed abnormal mitotic figures in arsenite-arrested mitotic cells, including deranged chromosome congression, elongated polar distance of mitotic spindle, and enhanced microtubule immunofluorescence. The spindle microtubules of arsenite-arrested mitotic cells were more resistant to nocodazole-induced dissolution than those of control mitotic cells. According to turbidity assay, arsenite at concentrations below 100 microM significantly enhanced polymerization of tubulins. Since spindle dynamics play a crucial role in mitotic progression, our results suggest that arsenite-induced mitotic arrest may be due to arsenite's effects on attenuation of spindle dynamics.

Asymmetric Distribution of GFAP in Glioma Multipotent Cells

PubMed Central

Guichet, Pierre-Olivier; Guelfi, Sophie; Ripoll, Chantal; Teigell, Marisa; Sabourin, Jean-Charles; Bauchet, Luc; Rigau, Valérie; Rothhut, Bernard; Hugnot, Jean-Philippe

2016-01-01

Asymmetric division (AD) is a fundamental mechanism whereby unequal inheritance of various cellular compounds during mitosis generates unequal fate in the two daughter cells. Unequal repartitions of transcription factors, receptors as well as mRNA have been abundantly described in AD. In contrast, the involvement of intermediate filaments in this process is still largely unknown. AD occurs in stem cells during development but was also recently observed in cancer stem cells. Here, we demonstrate the asymmetric distribution of the main astrocytic intermediate filament, namely the glial fibrillary acid protein (GFAP), in mitotic glioma multipotent cells isolated from glioblastoma (GBM), the most frequent type of brain tumor. Unequal mitotic repartition of GFAP was also observed in mice non-tumoral neural stem cells indicating that this process occurs across species and is not restricted to cancerous cells. Immunofluorescence and videomicroscopy were used to capture these rare and transient events. Considering the role of intermediate filaments in cytoplasm organization and cell signaling, we propose that asymmetric distribution of GFAP could possibly participate in the regulation of normal and cancerous neural stem cell fate. PMID:26953813

Micro Language Planning and Cultural Renaissance in Botswana

ERIC Educational Resources Information Center

Alimi, Modupe M.

2016-01-01

Many African countries exhibit complex patterns of language use because of linguistic pluralism. The situation is often compounded by the presence of at least one foreign language that is either the official or second language. The language situation in Botswana depicts this complex pattern. Out of the 26 languages spoken in the country, including…

Assimilate partitioning in avocado, Persea americana

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

Finazzo, S.; Davenport, T.L.

1986-04-01

Assimilate partitioning is being studied in avocado, Persea americana cv. Millborrow in relation to fruit set. Single leaves on girdled branches of 10 year old trees were radiolabeled for 1 hr with 13..mu..Ci of /sup 14/CO/sub 2/. The source leaves were sampled during the experiment to measure translocation rates. At harvest the sink tissues were dissected and the incorporated radioactivity was measured. The translocation of /sup 14/C-labelled compounds to other leaves was minimal. Incorporation of label into fruitlets varied with the tissue and the stage of development. Sink (fruitlets) nearest to the labelled leaf and sharing the same phyllotaxy incorporatedmore » the most /sup 14/C. Source leaves for single non-abscising fruitlets retained 3X more /sup 14/C-labelled compounds than did source leaves for 2 or more fruitlets at 31 hrs. post-labelling. Export of label decreased appreciably when fruitlets abscised. If fruitlets abscised within 4 days of labeling then the translocation pattern was similar to the pattern for single fruitlets. If the fruitlet abscised later, the translocation pattern was intermediate between the single and double fruitlet pattern.« less

Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks.

PubMed

Galay, Remil Linggatong; Matsuo, Tomohide; Hernandez, Emmanuel Pacia; Talactac, Melbourne Rio; Kusakisako, Kodai; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

2018-04-01

Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel histochemical method of simultaneous detection by a single- or double-immunofluorescence and Bielschowsky's silver staining in teased rat sciatic nerves.

PubMed

Segura-Anaya, Edith; Flores-Miranda, Rommel; Martínez-Gómez, Alejandro; Dent, Myrna A R

2018-07-01

The Golgi silver method has been widely used in neuroscience for the study of normal and pathological morphology of neurons. The method has been steadily improved and Bielschowsky's silver staining method (BSSM) is widely used in various pathological conditions, like Alzheimer's disease. In this work, teased sciatic nerves were silver impregnated using BSSM. We also developed simultaneous staining by silver impregnation and single- or double-immunofluorescence of the same section in teased nerve preparations. We immunostained against non-myelinating Schwann cells and different myelinating Schwann cell domains. BSSM teased nerves show a strong staining of axons (black) and a gold-brown staining of myelinating and non-myelinating Schwann cells. We were also able to stain by immunofluorescence these BSSM teased nerves with specific molecular markers against non-myelinating Schwann cells, also against non-compact myelin such as the Schmidt-Lanterman incisures or paranodal regions and compact myelin, but not axons. In peripheral nerves, several silver impregnation methods have been used to stain nerves in paraffin sections, but not in teased nerves to enable the assessment of isolated nerve fibers. In conclusion, BSSM gives accurate information of nerve morphology and combining the procedure with immunofluorescence it would be very useful to study the molecular nerve domain organization of the nerve fibers, and to study the molecular pathology of axon degeneration, or myelin disorders, or of any peripheral neuropathy, also to study demyelination diseases in the central nervous system. Copyright © 2018. Published by Elsevier B.V.

The value of tests of cure following cervical chlamydial infection.

PubMed

White, D J; Mann, C H; Matthews, R S; Leeming, J G; Clay, J C

1993-01-01

Test of cure (TOC) was performed 2, 4 and 6 weeks after treatment for cervical chlamydia infection with 10-14 days of Deteclo one tablet twice daily, erythromycin 500 mg twice daily or doxycycline 100 mg twice daily. Testing was by chlamydia culture and IDEIA (DAKO diagnostics Ltd). Discrepant results were subsequently checked by immunofluorescence (Syva MicroTrak) of both sets of left over transport media. Two hundred and three patients attended on at least one occasion; 189, 146 and 107 at 2, 4 and 6 weeks respectively. Of these 127, 70 and 34, respectively, denied sexual intercourse or had consistently used condoms. Fourteen were positive over the study period by either or both methods of detection. Of 8 culture positive results 3 were negative by IDEIA. Two of these had elementary bodies (EBs) on immunofluorescence of both sets of saved transport media. One had EBs on immunofluorescence of the saved culture transport medium only. None of the 6 IDEIA positive, culture negative patients had immunofluorescent EBs in the IDEIA transport media although one had EBs in the saved culture transport medium. One IDEIA suspicious, culture negative patient had EBs in both sets of saved transport media. There was no significant difference in the rate of chlamydia detection from patients admitting to or denying unprotected intercourse. TOC has a low yield in cases of cervical chlamydial infection when there has been careful contact tracing and treatment has been completed. If TOC is performed culture should be used if available and where antigen detection methods are used confirmation should be sought for any positive results.

In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization.

PubMed

Oglesbee, M; Jackwood, D; Perrine, K; Axthelm, M; Krakowka, S; Rice, J

1986-11-01

A cDNA library was prepared from canine distemper viral (CDV) messenger RNA (mRNA) derived from Vero cells lytically infected with the Onderstepoort strain (Ond) of CDV. A 300 base pair insert was identified which, by Northern blot analysis and Sanger sequence data, was shown to be specific to the nucleocapsid gene. The nucleocapsid (NC) clone was radiolabelled with 32P using nick translation and used to detect viral RNA in both dot-blot and in situ preparations of Vero cells lytically infected with Onderstepoort CDV (Ond-CDV) and immortalized mink lung cells persistently infected with racoon origin CDV (CCL64-RCDV). Dot-blot hybridization results paralleled immunofluorescent results in the lytically infected cells. In 18 persistently infected cell lines from the RCDV-CCL64 parental stock, 13 lines were positive and two were negative on both immunofluorescence and dot-blot hybridization analysis for CDV antigen and RNA, respectively. Viral nucleic acid was detected in these persistently infected cells, where as few as 1.9% of the members of a line were positive on immunofluorescence. A dot-blot autoradiographic signal was obtained in three lines which were negative for CDV antigen. CDV RNA was detected in both lytically and persistently infected cell lines by in situ hybridization, where decreasing probe length was important in increasing the sensitivity of this assay. Viral RNA was detected in over 90% of the lytically infected cells, where only 70% were positive for viral antigen by immunofluorescence.

Cell-based quantification of biomarkers from an ultra-fast microfluidic immunofluorescent staining: application to human breast cancer cell lines

NASA Astrophysics Data System (ADS)

Migliozzi, D.; Nguyen, H. T.; Gijs, M. A. M.

2018-02-01

Immunohistochemistry (IHC) is one of the main techniques currently used in the clinics for biomarker characterization. It consists in colorimetric labeling with specific antibodies followed by microscopy analysis. The results are then used for diagnosis and therapeutic targeting. Well-known drawbacks of such protocols are their limited accuracy and precision, which prevent the clinicians from having quantitative and robust IHC results. With our work, we combined rapid microfluidic immunofluorescent staining with efficient image-based cell segmentation and signal quantification to increase the robustness of both experimental and analytical protocols. The experimental protocol is very simple and based on fast-fluidic-exchange in a microfluidic chamber created on top of the formalin-fixed-paraffin-embedded (FFPE) slide by clamping it a silicon chip with a polydimethyl siloxane (PDMS) sealing ring. The image-processing protocol is based on enhancement and subsequent thresholding of the local contrast of the obtained fluorescence image. As a case study, given that the human epidermal growth factor receptor 2 (HER2) protein is often used as a biomarker for breast cancer, we applied our method to HER2+ and HER2- cell lines. We report very fast (5 minutes) immunofluorescence staining of both HER2 and cytokeratin (a marker used to define the tumor region) on FFPE slides. The image-processing program can segment cells correctly and give a cell-based quantitative immunofluorescent signal. With this method, we found a reproducible well-defined separation for the HER2-to-cytokeratin ratio for positive and negative control samples.

Coordinated induction of cell survival signaling in the inflamed microenvironment of the prostate.

PubMed

McIlwain, David W; Zoetemelk, Marloes; Myers, Jason D; Edwards, Marshé T; Snider, Brandy M; Jerde, Travis J

2016-06-01

Both prostate cancer and benign prostatic hyperplasia are associated with inflammatory microenvironments. Inflammation is damaging to tissues, but it is unclear how the inflammatory microenvironment protects specialized epithelial cells that function to proliferate and repair the tissue. The objective of this study is to characterize the cell death and cell survival response of the prostatic epithelium in response to inflammation. We assessed induction of cell death (TNF, TRAIL, TWEAK, FasL) and cell survival factors (IGFs, hedgehogs, IL-6, FGFs, and TGFs) in inflamed and control mouse prostates by ELISA. Cell death mechanisms were determined by immunoblotting and immunofluorescence for cleavage of caspases and TUNEL. Survival pathway activation was assessed by immunoblotting and immunofluorescence for Mcl-1, Bcl-2, Bcl-XL, and survivin. Autophagy was determined by immunoblotting and immunofluorescence for free and membrane associated light chain 3 (LC-3). Cleavage of all four caspases was significantly increased during the first 2 days of inflammation, and survival protein expression was substantially increased subsequently, maximizing at 3 days. By 5 days of inflammation, 50% of prostatic epithelial cells expressed survivin. Autophagy was also evident during the recovery phase (3 days). Finally, immunofluorescent staining of human specimens indicates strong activation of survival proteins juxtaposed to inflammation in inflamed prostate specimens. The prostate responds to deleterious inflammation with induction of cell survival mechanisms, most notably survivin and autophagy, demonstrating a coordinated induction of survival factors that protects and expands a specialized set of prostatic epithelial cells as part of the repair and recovery process during inflammation. © 2016 Wiley Periodicals, Inc.

The Acquisition of Compound vs. Phrasal Stress: The Role of Prosodic Constituents

ERIC Educational Resources Information Center

Vogel, Irene; Raimy, Eric

2002-01-01

This paper investigates the acquisition of compound vs. phrasal stress ("hot dog" vs. "hot dog") in English. This has previously been shown to be acquired quite late, in contrast to recent research showing that infants both perceive and prefer rhythmic patterns in their own language. Subjects (40 children in four groups the averages ages of which…

A strategy to improve the identification reliability of the chemical constituents by high-resolution mass spectrometry-based isomer structure prediction combined with a quantitative structure retention relationship analysis: Phthalide compounds in Chuanxiong as a test case.

PubMed

Zhang, Qingqing; Huo, Mengqi; Zhang, Yanling; Qiao, Yanjiang; Gao, Xiaoyan

2018-06-01

High-resolution mass spectrometry (HRMS) provides a powerful tool for the rapid analysis and identification of compounds in herbs. However, the diversity and large differences in the content of the chemical constituents in herbal medicines, especially isomerisms, are a great challenge for mass spectrometry-based structural identification. In the current study, a new strategy for the structural characterization of potential new phthalide compounds was proposed by isomer structure predictions combined with a quantitative structure-retention relationship (QSRR) analysis using phthalide compounds in Chuanxiong as an example. This strategy consists of three steps. First, the structures of phthalide compounds were reasonably predicted on the basis of the structure features and MS/MS fragmentation patterns: (1) the collected raw HRMS data were preliminarily screened by an in-house database; (2) the MS/MS fragmentation patterns of the analogous compounds were summarized; (3) the reported phthalide compounds were identified, and the structures of the isomers were reasonably predicted. Second, the QSRR model was established and verified using representative phthalide compound standards. Finally, the retention times of the predicted isomers were calculated by the QSRR model, and the structures of these peaks were rationally characterized by matching retention times of the detected chromatographic peaks and the predicted isomers. A multiple linear regression QSRR model in which 6 physicochemical variables were screened was built using 23 phthalide standards. The retention times of the phthalide isomers in Chuanxiong were well predicted by the QSRR model combined with reasonable structure predictions (R 2 =0.955). A total of 81 peaks were detected from Chuanxiong and assigned to reasonable structures, and 26 potential new phthalide compounds were structurally characterized. This strategy can improve the identification efficiency and reliability of homologues in complex materials. Copyright © 2018 Elsevier B.V. All rights reserved.

Factors controlling spatial and temporal patterns of multiple pesticide compounds in groundwater (Hesbaye chalk aquifer, Belgium).

PubMed

Hakoun, Vivien; Orban, Philippe; Dassargues, Alain; Brouyère, Serge

2017-04-01

Factors governing spatial and temporal patterns of pesticide compounds (pesticides and metabolites) concentrations in chalk aquifers remain unclear due to complex flow processes and multiple sources. To uncover which factors govern pesticide compound concentrations in a chalk aquifer, we develop a methodology based on time series analyses, uni- and multivariate statistics accounting for concentrations below detection limits. The methodology is applied to long records (1996-2013) of a restricted compound (bentazone), three banned compounds (atrazine, diuron and simazine) and two metabolites (deethylatrazine (DEA) and 2,6-dichlorobenzamide (BAM)) sampled in the Hesbaye chalk aquifer in Belgium. In the confined area, all compounds had non-detects fractions >80%. By contrast, maximum concentrations exceeded EU's drinking-water standard (100 ng L -1 ) in the unconfined area. This contrast confirms that recent recharge and polluted water did not reach the confined area, yet. Multivariate analyses based on variables representative of the hydrogeological setting revealed higher diuron and simazine concentrations in the southeast of the unconfined area, where urban activities dominate land use and where the aquifer lacks protection from a less permeable layer of hardened chalk. At individual sites, positive correlations (up to τ=0.48 for bentazone) between pesticide compound concentrations and multi-annual groundwater level fluctuations confirm occurrences of remobilization. A downward temporal trend of atrazine concentrations likely reflects decreasing use of this compound over the last 28 years. However, the lack of a break in concentrations time series and maximum concentrations of atrazine, simazine, DEA and BAM exceeding EU's standard post-ban years provide evidence of persistence. Contrasting upward trends in bentazone concentrations show that a time lag is required for restriction measures to be efficient. These results shed light on factors governing pesticide compound concentrations in chalk aquifers. The developed methodology is not restricted to chalk aquifers, it could be transposed to study other pollutants with concentrations below detection limits. Copyright © 2017 Elsevier Ltd. All rights reserved.

In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor ▿

PubMed Central

Wamsley, Heather L.; Barbet, Anthony F.

2008-01-01

Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potentially cryptic sites of Anaplasma sp. infection in mammalian tissues, a sensitive and specific isothermal in situ technique to detect localized Anaplasma gene sequences by using rolling-circle amplification of circularizable, linear, oligonucleotide probes (padlock probes) was developed. Cytospin preparations of uninfected or Anaplasma-infected cell cultures were examined using this technique. Via fluorescence microscopy, the technique described here, and a combination of differential interference contrast microscopy and von Willebrand factor immunofluorescence, Anaplasma phagocytophilum and Anaplasma marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for the detection of a microorganism and forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected mammalian and arthropod hosts and in cell cultures. PMID:18495855

Double-label immunofluorescence method for simultaneous detection of adenovirus and herpes simplex virus from the eye.

PubMed

Walpita, P; Darougar, S

1989-07-01

The development and application of a double-label immunofluorescence method which has the potential to screen for single or dual infections from any site, in single shell vial cultures, is described. In this study, a total of 1,141 ocular specimens were inoculated in shell vials, centrifuged at 15,000 X g for 1 h, incubated at 37 degrees C for 48 h, and fixed in methanol at room temperature for 15 min. The virus inclusions were detected by staining with a double-label indirect immunofluorescence procedure using mixtures of appropriate first antibodies, followed by fluorescein- and rhodamine-conjugated second antibodies. Each specimen was also inoculated in parallel by the conventional virus isolation method. The sensitivity and specificity of the double-label shell vial procedure were comparable to those with the conventional method, and the former test took only 48 h to complete. The test offers a rapid and simple single-vial procedure which allows for individual or simultaneous detection of multiple pathogens. It results in savings in time and cost over the conventional virus isolation method and other shell vial procedures.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

PubMed Central

Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

2016-01-01

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

The use of immunofluorescence techniques for the laboratory diagnosis of transmissible gastroenteritis of swine.

PubMed Central

Solorzano, R F; Morin, M; Morehouse, L G

1978-01-01

Over a four year period, 74 of 250 field outbreaks of enteric disease (30%) and 110 of 440 swine (25%) were positive for transmissible gastroenteritis by immunofluorescence procedures. Of 141 swine from herds positive for transmissible gastroenteritis 110 (78%) were positive by fluorescent antibody techniques. The fastest, easiest to perform and most effective procedure was the examination of frozen sections of the jejunum from acutely ill animals by the fluorescent antibody tissue section technique. Only two herds were found to be positive by the fluorescent antibody tissue culture technique which were negative by fluorescent antibody tissue section technique. A considerable number of outbreaks, 21 of 74 (28%), of transmissible gastroenteritis were detected by immunofluorescence in swine over two weeks of age. The majority of outbreaks of transmissible gastroenteritis, 50 of 74 (68%), occurred in Missouri during the months of January through April and 63 of 74 (85%) during the months of December through May. The recurrence of the disease in a number of counties over a four-year period suggest the possibility of endemic foci. PMID:217505

[Extramembranous glomerulonephritis of acquired syphilis in a patient recently infected by the hepatitis B virus. Demonstration of the treponemal antigen in the kidney by indirect immunofluorescence].

PubMed

Schillinger, F; Montagnac, R; Goclowski, C; Dine, G; Alessandri, E; Hopfner, C; Birembaut, P

1983-01-22

A 38 years old male homosexual with active secondary syphilis presented with pure nephrotic syndrome while HBs and HBe tests were positive without clinical hepatitis. He had circulating immune complexes, IgG--IgM cryoglobulinemia and high IgA, IgM and IgE levels; the C3 and C4 complement constituents were normal. Examination of renal biopsy sections under light, fluorescent and electronic microscopy showed stage I membranous glomerulonephritis the syphilitic origin of which was confirmed by indirect immunofluorescence and by rapid cure under penicillin treatment. This case calls for the following comments: (1) glomerular deposits are extramembranous rather than subendothelial in syphilitic nephrosis, a disease now classified among circulating immune complexes diseases; (2) in the kidney, the treponema antigen can be demonstrated by indirect immunofluorescence and the anti-treponema antibody, by elution; (3) the outcome of the nephrotic syndrome is always favourable, either spontaneously or after penicillin treatment; (4) syphilis and HBs antigens are frequently associated, particularly in homosexual patients; one should be looked for when the other is discovered.

The reactivity of phenolic and non-phenolic residual kraft lignin model compounds with Mn(II)-peroxidase from Lentinula edodes.

PubMed

Crestini, C; D'Annibale, A; Sermanni, G G; Saladino, R

2000-02-01

Three phenolic model compounds representing bonding patterns of residual kraft lignin were incubated with manganese peroxidase from Lentinula edodes. Extensive degradation of all the phenolic models, mainly occurring via side-chain benzylic oxidation, was observed. Among the tested model compounds the diphenylmethane alpha-5 phenolic model was found to be the most reactive, yielding several products showing oxidation and fragmentation at the bridging position. The non-phenolic 5-5' biphenyl and 5-5' diphenylmethane models were found unreactive.

TreeNetViz: revealing patterns of networks over tree structures.

PubMed

Gou, Liang; Zhang, Xiaolong Luke

2011-12-01

Network data often contain important attributes from various dimensions such as social affiliations and areas of expertise in a social network. If such attributes exhibit a tree structure, visualizing a compound graph consisting of tree and network structures becomes complicated. How to visually reveal patterns of a network over a tree has not been fully studied. In this paper, we propose a compound graph model, TreeNet, to support visualization and analysis of a network at multiple levels of aggregation over a tree. We also present a visualization design, TreeNetViz, to offer the multiscale and cross-scale exploration and interaction of a TreeNet graph. TreeNetViz uses a Radial, Space-Filling (RSF) visualization to represent the tree structure, a circle layout with novel optimization to show aggregated networks derived from TreeNet, and an edge bundling technique to reduce visual complexity. Our circular layout algorithm reduces both total edge-crossings and edge length and also considers hierarchical structure constraints and edge weight in a TreeNet graph. These experiments illustrate that the algorithm can reduce visual cluttering in TreeNet graphs. Our case study also shows that TreeNetViz has the potential to support the analysis of a compound graph by revealing multiscale and cross-scale network patterns. © 2011 IEEE

Numerical solutions for patterns statistics on Markov chains.

PubMed

Nuel, Gregory

2006-01-01

We propose here a review of the methods available to compute pattern statistics on text generated by a Markov source. Theoretical, but also numerical aspects are detailed for a wide range of techniques (exact, Gaussian, large deviations, binomial and compound Poisson). The SPatt package (Statistics for Pattern, free software available at http://stat.genopole.cnrs.fr/spatt) implementing all these methods is then used to compare all these approaches in terms of computational time and reliability in the most complete pattern statistics benchmark available at the present time.

Effects of nitrogen nutrition on the synthesis and deposition of the ω-gliadins of wheat.

PubMed

Wan, Yongfang; Gritsch, Cristina Sanchis; Hawkesford, Malcolm J; Shewry, Peter R

2014-03-01

The ω-gliadin storage proteins of wheat are of interest in relation to their impact on grain processing properties and their role in food allergy, particularly the ω-5 sub-group and wheat-dependent exercise-induced anaphylaxis. The ω-gliadins are also known to be responsive to nitrogen application. This study therefore compares the effects of cultivar and nitrogen availability on the synthesis and deposition of ω-gliadins in wheat grown under field conditions in the UK, including temporal and spatial analyses at the protein and transcript levels. SDS-PAGE, western blotting and N-terminal amino acid sequencing were used to compare the patterns of ω-gliadin components in mature grain of six British wheat (Triticum aestivum) cultivars and their accumulation during the development of grain grown in field plots with varying nitrogen supply. Changes in gene expression during development were determined using real-time reverse transcription-PCR (RT-PCR). Spatial patterns of gene expression and protein accumulation were determined by in situ hybridization and immunofluorescence microscopy, respectively. Two patterns of ω-gliadins were identified in the six cultivars, including both monomeric 'gliadin' proteins and subunits present in polymeric 'glutenin' fractions. Increasing the level of nitrogen fertilizer in field plots resulted in increased expression of ω-gliadin transcripts and increased proportions of ω-5 gliadins. Nitrogen supply also affected the spatial patterns of ω-gliadin synthesis and deposition, which were differentially increased in the outer layers of the starchy endosperm with high levels of nitrogen. Wheat ω-gliadins vary in amount and composition between cultivars, and in their response to nitrogen supply. Their spatial distribution is also affected by nitrogen supply, being most highly concentrated in the sub-aleurone cells of the starchy endosperm under higher nitrogen availability.

Analysis of a Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of mesodermal patterning

PubMed Central

Harfe, Brian D.; Gomes, Ana Vaz; Kenyon, Cynthia; Liu, Jun; Krause, Michael; Fire, Andrew

1998-01-01

Mesodermal development is a multistep process in which cells become increasingly specialized to form specific tissue types. In Drosophila and mammals, proper segregation and patterning of the mesoderm involves the bHLH factor Twist. We investigated the activity of a Twist-related factor, CeTwist, during Caenorhabditis elegans mesoderm development. Embryonic mesoderm in C. elegans derives from a number of distinct founder cells that are specified during the early lineages; in contrast, a single blast cell (M) is responsible for all nongonadal mesoderm formation during postembryonic development. Using immunofluorescence and reporter fusions, we determined the activity pattern of the gene encoding CeTwist. No activity was observed during specification of mesodermal lineages in the early embryo; instead, the gene was active within the M lineage and in a number of mesodermal cells with nonstriated muscle fates. A role for CeTwist in postembryonic mesodermal cell fate specification was indicated by ectopic expression and genetic interference assays. These experiments showed that CeTwist was responsible for activating two target genes normally expressed in specific subsets of nonstriated muscles derived from the M lineage. In vitro and in vivo assays suggested that CeTwist cooperates with the C. elegans E/Daughterless homolog in directly activating these targets. The two target genes that we have studied, ceh-24 and egl-15, encode an NK-2 class homeodomain and an FGF receptor (FGFR) homolog, respectively. Twist activates FGFR and NK-homeodomain target genes during mesodermal patterning of Drosophila and similar target interactions have been proposed to modulate mesenchymal growth during closure of the vertebrate skull. These results suggest the possibility that a conserved pathway may be used for diverse functions in mesodermal specification. PMID:9716413

Cocaine- and amphetamine-regulated transcript and calcium binding proteins immunoreactivity in the subicular complex of the guinea pig.

PubMed

Wasilewska, Barbara; Najdzion, Janusz; Równiak, Maciej; Bogus-Nowakowska, Krystyna; Hermanowicz, Beata; Kolenkiewicz, Małgorzata; Żakowski, Witold; Robak, Anna

2016-03-01

In this study we present the distribution and colocalization pattern of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins: calbindin (CB), calretinin (CR) and parvalbumin (PV) in the subicular complex (SC) of the guinea pig. The subiculum (S) and presubiculum (PrS) showed higher CART-immunoreactivity (-IR) than the parasubiculum (PaS) as far as the perikarya and neuropil were concerned. CART- IR cells were mainly observed in the pyramidal layer and occasionally in the molecular layer of the S. In the PrS and PaS, single CART-IR perikarya were dispersed, however with a tendency to be found only in superficial layers. CART-IR fibers were observed throughout the entire guinea pig subicular neuropil. Double-labeling immunofluorescence showed that CART-IR perikarya, as well as fibers, did not stain positively for any of the three CaBPs. CART-IR fibers were only located near the CB-, CR-, PV-IR perikarya, whereas CART-IR fibers occasionally intersected fibers containing one of the three CaBPs. The distribution pattern of CART was more similar to that of CB and CR than to that of PV. In the PrS, the CART, CB and CR immunoreactivity showed a laminar distribution pattern. In the case of the PV, this distribution pattern in the PrS was much less prominent than that of CART, CB and CR. We conclude that a heterogeneous distribution of the CART and CaBPs in the guinea pig SC is in keeping with findings from other mammals, however species specific differences have been observed. Copyright © 2015 Elsevier GmbH. All rights reserved.

Determination of size and sign of hetero-nuclear coupling constants from 2D 19F-13C correlation spectra.

PubMed

Ampt, Kirsten A M; Aspers, Ruud L E G; Dvortsak, Peter; van der Werf, Ramon M; Wijmenga, Sybren S; Jaeger, Martin

2012-02-01

Fluorinated organic compounds have become increasingly important within the polymer and the pharmaceutical industry as well as for clinical applications. For the structural elucidation of such compounds, NMR experiments with fluorine detection are of great value due to the favorable NMR properties of the fluorine nucleus. For the investigation of three fluorinated compounds, triple resonance 2D HSQC and HMBC experiments were adopted to fluorine detection with carbon and/or proton decoupling to yield F-C, F-C{H}, F-C{C(acq)} and F-C{H,C(acq)} variants. Analysis of E.COSY type cross-peak patterns in the F-C correlation spectra led, apart from the chemical shift assignments, to determination of size and signs of the J(CH), J(CF), and J(HF) coupling constants. In addition, the fully coupled F-C HMQC spectrum of steroid 1 was interpreted in terms of E.COSY type patterns. This example shows how coupling constants due to different nuclei can be determined together with their relative signs from a single spectrum. The analysis of cross-peak patterns, as presented here, not only provides relatively straightforward routes to the determination of size and sign of hetero-nuclear J-couplings in fluorinated compounds, it also provides new and easy ways for the determination of residual dipolar couplings and thus for structure elucidation. The examples and results presented in this study may contribute to a better interpretation and understanding of various F-C correlation experiments and thereby stimulate their utilization. Copyright © 2011 Elsevier Inc. All rights reserved.

Spatio-temporal patterns of mass fluxes of micropollutants in Swiss rivers of catchments with different land use

NASA Astrophysics Data System (ADS)

Stamm, Christian; van der Voet, Jürgen; Singer, Heinz

2010-05-01

It is known from many studies that a large number of micropollutants like pesticides, household products or pharmaceuticals can be found in water bodies. However, there is a general lack of systematic monitoring data that allow for distinguishing between possible sources, detecting temporal trends, or evaluating effects of possible mitigation measures. Including micropollutants in existing monitoring programs is not a trivial task for several reasons (e.g., sorption to sampling equipment, hydrolysis, detection limits etc.). Here, we present systematic concentration and load data for 12 substances (7 pesticides and/or biocides, 3 pharmaceuticals, and 2 anti-corrosives) obtained from a one-year sampling campaign within the "National Long-term Surveillance of Swiss Rivers" (NADUF) programme. Six (partially) nested sampling stations were selected to monitor these compounds in weekly or bi-weekly, flow-proportional samples over one year. Due to the high sensitivity of the LC-MSMS method all compounds could be quantified in almost all samples. Only at the reference site without any effluent from waste water treatment plants and hardly any arable farming, the concentrations were always below the limits of detection of a few ng/L. At all other sites, concentrations generally ranged between 10 and 200 ng/L. Only, the anticorrosive agent benzotriazole often exceeded 1000 ng/L. According to the use of the compounds, different temporal load patterns can be expected. In general, the data confirmed these patterns with almost constant loads of pharmaceuticals at most sites, increased herbicides loads during the periods of agricultural use and positive correlations with discharge year round for biocides used in material protection. However, at some sites the expectations were not met for all compounds. The pain-killer diclofenac for example showed strongly declining loads during the summer months at sites influenced by lake water. This compound is not stable in the epilimnion of lakes, where it has a residence time of several weeks, while it flows through the river system within a few days. This example illustrates how compound properties, season and spatial location may interact and control the occurrence of micropollutants in a stream. The spatial nesting of study catchments made it possible to check the data for plausibility and consistency: we present data on cumulative mass balances downstream and test whether the load development along the river network corresponds to the spatial distribution of possible compound sources (e.g., acreage of arable fields, number of inhabitants etc.). Overall, the data show that monitoring of micropollutants may be achieved even without changing an existing monitoring programme. However, given the generally low concentrations in the composite samples of the NADUF programme compounds with lower use and/or lower stability may fall below the limit of reliable quantification or even detection. A proper interpretation of the data relies on additional (spatio-temporal) information like land use data or precipitation patterns.

Secondary ion mass spectrometry imaging and multivariate data analysis reveal co-aggregation patterns of Populus trichocarpa leaf surface compounds on a micrometer scale.

PubMed

Kulkarni, Purva; Dost, Mina; Bulut, Özgül Demir; Welle, Alexander; Böcker, Sebastian; Boland, Wilhelm; Svatoš, Aleš

2018-01-01

Spatially resolved analysis of a multitude of compound classes has become feasible with the rapid advancement in mass spectrometry imaging strategies. In this study, we present a protocol that combines high lateral resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging with a multivariate data analysis (MVA) approach to probe the complex leaf surface chemistry of Populus trichocarpa. Here, epicuticular waxes (EWs) found on the adaxial leaf surface of P. trichocarpa were blotted on silicon wafers and imaged using TOF-SIMS at 10 μm and 1 μm lateral resolution. Intense M +● and M -● molecular ions were clearly visible, which made it possible to resolve the individual compound classes present in EWs. Series of long-chain aliphatic saturated alcohols (C 21 -C 30 ), hydrocarbons (C 25 -C 33 ) and wax esters (WEs; C 44 -C 48 ) were clearly observed. These data correlated with the 7 Li-chelation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, which yielded mostly molecular adduct ions of the analyzed compounds. Subsequently, MVA was used to interrogate the TOF-SIMS dataset for identifying hidden patterns on the leaf's surface based on its chemical profile. After the application of principal component analysis (PCA), a small number of principal components (PCs) were found to be sufficient to explain maximum variance in the data. To further confirm the contributions from pure components, a five-factor multivariate curve resolution (MCR) model was applied. Two distinct patterns of small islets, here termed 'crystals', were apparent from the resulting score plots. Based on PCA and MCR results, the crystals were found to be formed by C 23 or C 29 alcohols. Other less obvious patterns observed in the PCs revealed that the adaxial leaf surface is coated with a relatively homogenous layer of alcohols, hydrocarbons and WEs. The ultra-high-resolution TOF-SIMS imaging combined with the MVA approach helped to highlight the diverse patterns underlying the leaf's surface. Currently, the methods available to analyze the surface chemistry of waxes in conjunction with the spatial information related to the distribution of compounds are limited. This study uses tools that may provide important biological insights into the composition of the wax layer, how this layer is repaired after mechanical damage or insect feeding, and which transport mechanisms are involved in deploying wax constituents to specific regions on the leaf surface. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Filamentous actin organization in the unfertilized sea urchin egg cortex.

PubMed

Henson, J H; Begg, D A

1988-06-01

We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2-3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.

Differential distribution of blood and lymphatic vessels in the murine cornea.

PubMed

Ecoiffier, Tatiana; Yuen, Don; Chen, Lu

2010-05-01

Because of its unique characteristics, the cornea has been widely used for blood and lymphatic vessel research. However, whether limbal or corneal vessels are evenly distributed under normal or inflamed conditions has never been studied. The purpose of this study was to investigate this question and to examine whether and how the distribution patterns change during corneal inflammatory lymphangiogenesis (LG) and hemangiogenesis (HG). Corneal inflammatory LG and HG were induced in two most commonly used mouse strains, BALB/c and C57BL/6 (6-8 weeks of age), by a standardized two-suture placement model. Oriented flat-mount corneas together with the limbal tissues were used for immunofluorescence microscope studies. Blood and lymphatic vessels under normal and inflamed conditions were analyzed and quantified to compare their distributions. The data demonstrate, for the first time, greater distribution of both blood and lymphatic vessels in the nasal side in normal murine limbal areas. This nasal-dominant pattern was maintained during corneal inflammatory LG, whereas it was lost for HG. Blood and lymphatic vessels are not evenly distributed in normal limbal areas. Furthermore, corneal LG and HG respond differently to inflammatory stimuli. These new findings will shed some light on corneal physiology and pathogenesis and on the development of experimental models and therapeutic strategies for corneal diseases.

The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm.

PubMed

Henson, J H; Cole, D G; Roesener, C D; Capuano, S; Mendola, R J; Scholey, J M

1997-01-01

We have utilized immunoblotting and light microscopic immunofluorescent staining methods to examine the expression and localization of sea urchin kinesin-II, a heterotrimeric plus end-directed microtubule motor protein (previously referred to as KRP(85/95)), in sea urchin and sand dollar sperm. We demonstrate the presence of the 85 K and 115 K subunits of kinesin-II in sperm and localize these proteins to the sperm flagella and midpiece. The kinesin-II localization pattern is punctate and discontinuous, and in the flagella it is quite distinct from the continuous labeling present in sperm labeled with anti-flagellar dynein. The kinesin-II staining is largely insensitive to prefixation detergent extraction, suggesting that it is not associated with membranous elements in the sperm. In the midpiece the kinesin-II staining is similar to the pattern present in sperm labeled with an anti-centrosomal antibody. To our knowledge, this is the first localization of kinesin-like proteins in mature sperm and corroborates the recent identification and localization of kinesin-like proteins in the flagella and basal body of the unicellular green alga Chlamydomonas. We hypothesize that kinesin-II in the sperm may play functional roles in intraflagellar transport and/or the formation of flagella during spermatogenesis.

Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.

PubMed

Doublier, S; Ruotsalainen, V; Salvidio, G; Lupia, E; Biancone, L; Conaldi, P G; Reponen, P; Tryggvason, K; Camussi, G

2001-05-01

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

Nephrin Redistribution on Podocytes Is a Potential Mechanism for Proteinuria in Patients with Primary Acquired Nephrotic Syndrome

PubMed Central

Doublier, Sophie; Ruotsalainen, Vesa; Salvidio, Gennaro; Lupia, Enrico; Biancone, Luigi; Conaldi, Pier Giulio; Reponen, Paula; Tryggvason, Karl; Camussi, Giovanni

2001-01-01

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG4, plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-α, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site. PMID:11337370

Changing pattern of the subcellular distribution of erythroblast macrophage protein (Emp) during macrophage differentiation.

PubMed

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp) mediates the attachment of erythroid cells to macrophages and is required for normal differentiation of both cell lineages. In erythroid cells, Emp is believed to be involved in nuclear extrusion, however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data show that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture shows that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data show that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation and suggest that Emp may be involved in multiple cellular functions.

*CHANGING PATTERN OF THE SUBCELLULAR DISTRIBUTION OF ERYTHROBLAST MACROPHAGE PROTEIN (EMP) DURING MACROPHAGE DIFFERENTIATION

PubMed Central

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp), mediates the attachment of erythroid cells to macrophages, and is required for normal differentiation of both cell lineages. In erythroid cells Emp is believed to be involved in nuclear extrusion however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data shows that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture show that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data shows that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation, and suggests that Emp may be involved in multiple cellular functions. PMID:17071116

[Fractal research of neurite growth in immunofluorescent images].

PubMed

Tang, Min; Wang, Huinan

2008-12-01

Fractal dimension has been widely used in medical images processing and analysis. The neurite growth of cultured dorsal root ganglion (DRG) was detected by fluorescent immunocytochemistry treated with nerve regeneration factor (0.1, 0.5, 2.0 mg/L). A novel method based on triangular prism surface area (TPSA) was introduced and adopted to calculate the fractal dimension of the two-dimensional immunofluorescent images. Experimental results demonstrate that this method is easy to understand and convenient to operate, and the quantititve results are concordant with the observational findings under microscope. This method can be guidelines for analyzing and deciding experimental results.

Advanced Diagnostic Techniques in Autoimmune Bullous Diseases

PubMed Central

Jindal, Anuradha; Rao, Raghavendra; Bhogal, Balbir S

2017-01-01

Autoimmune blistering diseases are diverse group of conditions characterized by blisters in the skin with or without mucosal lesions. There may be great degree of clinical and histopathological overlap; hence, advanced immunological tests may be necessary for more precise diagnosis of these conditions. Direct immunofluorescence microscopy is the gold standard tests to demonstrate the tissue-bound antibodies and should be done in all cases. Magnitude of antibody level in patient’ serum can be assessed by indirect immunofluorescence and enzyme linked immunosorbent assay. In this article we have reviewed the various techniques that are available in the diagnosis of autoimmune blistering diseases. PMID:28584369

Patterns of poverty exposure and children's trajectories of externalizing and internalizing behaviors.

PubMed

Comeau, Jinette; Boyle, Michael H

2018-04-01

Using data from the Child Supplement of the National Longitudinal Survey of Youth, we compare trajectories of externalizing and internalizing behaviors among children exposed to five patterns of poverty from birth to age 14: always or never poor - stable patterns; a single transition into or out of poverty, or repeated fluctuations in and out of poverty - changing patterns. We also examine how low maternal education and single parenthood interact with these poverty exposures to compound their adverse effects. Finally, we compare the magnitude of effects associated with the patterns of poverty exposure, as well as their interactions with low maternal education and single parenthood, on trajectories of externalizing and internalizing behaviors to determine if they are significantly different. Results reveal that initial levels and rates of change in children's trajectories of externalizing and internalizing behaviors are similar across the three changing patterns of poverty exposure, leading us to combine them into a single group representing intermittent poverty. Initial disparities between children who are never poor and their counterparts who are always or intermittently poor are constant over time for internalizing behaviors and grow in magnitude for externalizing behaviors. The cumulative negative effect of poverty exposure over time is stronger for externalizing vs. internalizing behaviors. Low maternal education compounds the adverse effects of persistent poverty, an effect that is similar for externalizing and internalizing behaviors.

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

PubMed

Zareba, Ilona; Surazynski, Arkadiusz; Chrusciel, Marcin; Miltyk, Wojciech; Doroszko, Milena; Rahman, Nafis; Palka, Jerzy

2017-01-01

The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

Novel compounds TAD-1822-7-F2 and F5 inhibited HeLa cells growth through the JAK/Stat signaling pathway.

PubMed

Yang, Tianfeng; Shi, Xianpeng; Kang, Yuan; Zhu, Man; Fan, Mengying; Zhang, Dongdong; Zhang, Yanmin

2018-07-01

Cervical carcinoma remains the second most common malignancy with a high mortality rate among women worldwide. TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) are novel compounds synthesized on the chemical structure of taspine derivatives, and show an effective suppression for HeLa cells. Our study aims to confirm the potential targets of F2 and F5, and investigate the underlying mechanism of the inhibitory effect on HeLa cells. In this study, Real Time Cell Analysis and crystal violet staining assay were conducted to investigate the effect of F2 and F5 on HeLa cells proliferation. And the analytical methods of surface plasmon resonance and quartz crystal microbalance were established and employed to study the interaction between F2 and F5 and potential target protein JAK2, suggesting that both compounds have strong interaction with the JAK2 protein. Western blot analysis, immunofluorescence staining study and PCR was conducted to investigate the molecules of JAK/Stat signaling pathway. Interestingly, F2 and F5 showed diverse regulation for signaling molecules because of their different chemical structure. F2 increased the expression of JAK2 and downregulated the level of P-JAK1 and P-JAK2, and decreased P-Stat3 (Ser727). While F5 could increase the expression of JAK2 and naturally decrease the phosphorylation of JAK1 and Tyk2, and decreased the expression of P-Stat6. Moreover, F2 and F5 showed the same downregulation on the P-Stat3 (Tyr705). Therefore, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, which suggested that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Diversity patterns, ecology and biological activities of fungal communities associated with the endemic macroalgae across the Antarctic peninsula

Treesearch

Laura E. Furbino; Valaria M. Godinho; Iara F. Santiago; Franciane M. Pellizari; Tania M.A. Alves; Carlos L. Zani; Policarpo A.S. Junior; Alvaro J. Romanha; Amanda G.O. Carvalho; Laura H.V.G. Gil; Carlos A. Rosa; Andrew M. Minnis; Luiz H. Rosa

2014-01-01

We surveyed diversity patterns and engaged in bioprospecting for bioactive compounds of fungi associated with the endemic macroalgae, Monostroma hariotii and Pyropia endiviifolia, in Antarctica. A total of 239 fungal isolates were obtained, which were identified to represent 48 taxa and 18 genera using molecular methods. The fungal...

Extracellular compounds produced by fungi associated with Botryosphaeria dieback induce differential defence gene expression patterns and necrosis in Vitis vinifera cv. Chardonnay cells.

PubMed

Ramírez-Suero, M; Bénard-Gellon, M; Chong, J; Laloue, H; Stempien, E; Abou-Mansour, E; Fontaine, F; Larignon, P; Mazet-Kieffer, F; Farine, S; Bertsch, C

2014-11-01

Three major grapevine trunk diseases, esca, botryosphaeria dieback and eutypa dieback, pose important economic problems for vineyards worldwide, and currently, no efficient treatment is available to control these diseases. The different fungi associated with grapevine trunk diseases can be isolated in the necrotic wood, but not in the symptomatic leaves. Other factors seem to be responsible for the foliar symptoms and may represent the link between wood and foliar symptoms. One hypothesis is that the extracellular compounds produced by the fungi associated with grapevine trunk diseases are responsible for pathogenicity.In the present work, we used Vitis vinifera cv. Chardonnay cells to test the aggressiveness of total extracellular compounds produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with botryosphaeria dieback. Additionally, the toxicity of purified mellein, a characteristic toxin present in the extracellular compounds of Botryosphaeriaceae, was assessed.Our results show that the total extracellular compounds produced by N. parvum induce more necrosis on Chardonnay calli and induce a different defence gene expression pattern than those of D. seriata. Mellein was produced by both fungi in amounts proportional to its aggressiveness. However, when purified mellein was added to the culture medium of calli, only a delayed necrosis and a lower-level expression of defence genes were observed. Extracellular compounds seem to be involved in the pathogenicity of the fungi associated with botryosphaeria dieback. However, the doses of mellein used in this study are 100 times higher than those found in the liquid fungal cultures: therefore, the possible function of this toxin is discussed.

A small molecule screen identifies a novel compound that induces a homeotic transformation in Hydra

PubMed Central

Glauber, Kristine M.; Dana, Catherine E.; Park, Steve S.; Colby, David A.; Noro, Yukihiko; Fujisawa, Toshitaka; Chamberlin, A. Richard; Steele, Robert E.

2013-01-01

Developmental processes such as morphogenesis, patterning and differentiation are continuously active in the adult Hydra polyp. We carried out a small molecule screen to identify compounds that affect patterning in Hydra. We identified a novel molecule, DAC-2-25, that causes a homeotic transformation of body column into tentacle zone. This transformation occurs in a progressive and polar fashion, beginning at the oral end of the animal. We have identified several strains that respond to DAC-2-25 and one that does not, and we used chimeras from these strains to identify the ectoderm as the target tissue for DAC-2-25. Using transgenic Hydra that express green fluorescent protein under the control of relevant promoters, we examined how DAC-2-25 affects tentacle patterning. Genes whose expression is associated with the tentacle zone are ectopically expressed upon exposure to DAC-2-25, whereas those associated with body column tissue are turned off as the tentacle zone expands. The expression patterns of the organizer-associated gene HyWnt3 and the hypostome-specific gene HyBra2 are unchanged. Structure-activity relationship studies have identified features of DAC-2-25 that are required for activity and potency. This study shows that small molecule screens in Hydra can be used to dissect patterning processes. PMID:24255098

A small molecule screen identifies a novel compound that induces a homeotic transformation in Hydra.

PubMed

Glauber, Kristine M; Dana, Catherine E; Park, Steve S; Colby, David A; Noro, Yukihiko; Fujisawa, Toshitaka; Chamberlin, A Richard; Steele, Robert E

2013-12-01

Developmental processes such as morphogenesis, patterning and differentiation are continuously active in the adult Hydra polyp. We carried out a small molecule screen to identify compounds that affect patterning in Hydra. We identified a novel molecule, DAC-2-25, that causes a homeotic transformation of body column into tentacle zone. This transformation occurs in a progressive and polar fashion, beginning at the oral end of the animal. We have identified several strains that respond to DAC-2-25 and one that does not, and we used chimeras from these strains to identify the ectoderm as the target tissue for DAC-2-25. Using transgenic Hydra that express green fluorescent protein under the control of relevant promoters, we examined how DAC-2-25 affects tentacle patterning. Genes whose expression is associated with the tentacle zone are ectopically expressed upon exposure to DAC-2-25, whereas those associated with body column tissue are turned off as the tentacle zone expands. The expression patterns of the organizer-associated gene HyWnt3 and the hypostome-specific gene HyBra2 are unchanged. Structure-activity relationship studies have identified features of DAC-2-25 that are required for activity and potency. This study shows that small molecule screens in Hydra can be used to dissect patterning processes.

Consumer palatability scores and volatile beef flavor compounds of five USDA quality grades and four muscles.

PubMed

Legako, J F; Brooks, J C; O'Quinn, T G; Hagan, T D J; Polkinghorne, R; Farmer, L J; Miller, M F

2015-02-01

Proximate data, consumer palatability scores and volatile compounds were investigated for four beef muscles (Longissimus lumborum, Psoas major, Semimembranosus and Gluteus medius) and five USDA quality grades(Prime, Upper 2/3 Choice, Low Choice, Select, and Standard). Quality grade did not directly affect consumer scores or volatiles but interactions (P < 0.05) between muscle and grade were determined. Consumer scores and volatiles differed (P < 0.05) between muscles. Consumers scored Psoas major highest for tenderness, juiciness, flavor liking and overall liking, followed by Longissimus lumborum, Gluteus medius, and Semimembranosus (P < 0.05). Principal component analysis revealed clustering of compound classes, formed by related mechanisms. Volatile n-aldehydes were inversely related to percent fat. Increases in lipid oxidation compounds were associated with Gluteus medius and Semimembranosus, while greater quantities of sulfur-containing compounds were associated with Psoas major. Relationships between palatability scores and volatile compound classes suggest that differences in the pattern of volatile compounds may play a valuable role in explaining consumer liking.

Clinical and immunological studies of 49 cases of various types of intercellular IgA dermatosis and 13 cases of classical subcorneal pustular dermatosis examined at Kurume University.

PubMed

Hashimoto, T; Teye, K; Ishii, N

2017-01-01

Intercellular IgA dermatosis (IAD) is a subset of autoimmune bullous disease exclusively with IgA antikeratinocyte cell-surface antibodies. The classification and pathogenesis of this condition are still obscure. To classify IAD and study its pathogenesis. From our cohort of 5402 cases of autoimmune bullous disease, we selected 49 cases of various types of intercellular IgA dermatosis (IAD) and 13 cases of classical subcorneal pustular dermatosis (SPD), for which sera and information were available. We studied these cases clinically and immunologically. There were 17 SPD-type IAD, 12 intraepidermal neutrophilic IgA dermatosis (IEN)-type IAD, two IgA-pemphigus vegetans, four IgA-pemphigus foliaceus, six IgA-pemphigus vulgaris and eight unclassified IAD cases. There was no sex predominance, and the average age at disease onset was 45·9 years. Clinically, bullous and pustular skin lesions developed on various sites, particularly intertriginous areas. Histopathology showed intraepidermal blisters or pustules at the upper epidermis in the SPD-type and at the midepidermis in the IEN-type. Immunological studies revealed that direct immunofluorescence, indirect immunofluorescence of normal human skin and enzyme-linked immunosorbent assays (ELISAs) of recombinant proteins of desmogleins and desmocollins frequently showed positive results, although no antigens were detected in many cases. All cases of classical SPD, which showed no positive immunological results, were indistinguishable clinically and histopathologically from SPD-type IAD. The present study of the largest cohort of cases of IAD showed that the major subtypes are SPD and IEN, and that the combination of indirect immunofluorescence and ELISAs of desmogleins and desmocollins, in addition to direct immunofluorescence, was useful for the diagnosis of IAD and its subtypes. © 2016 British Association of Dermatologists.

Hepatitis B Core Antigen in Hepatocytes of Chronic Hepatitis B: Comparison between Indirect Immunofluorescence and Immunoperoxidase Method

PubMed Central

Tabassum, Shahina; Al-Mahtab, Mamun; Nessa, Afzalun; Jahan, Munira; Shamim Kabir, Chowdhury Mohammad; Kamal, Mohammad; Cesar Aguilar, Julio

2015-01-01

Background Hepatitis B virus (HBV) infection has many faces. Precore and core promoter mutants resemble inactive carrier status. The identification of hepatitis B core antigen (HBcAg) in hepatocytes may have variable clinical significance. The present study was undertaken to detect HBcAg in chronic hepatitis B (CHB) patients and to assess the efficacy of detection system by indirect immunofluorescence (IIF) and indirect immunoperoxidase (IIP). Materials and methods The study was done in 70 chronic HBV-infected patients. Out of 70 patients, eight (11.4%) were hepatitis B e antigen (HBeAg) positive and 62 (88.57%) were HBeAg negative. Hepatitis B core antigen was detected by indirect immunofluorescence (IIF) and indirect immunoperoxidase (IIP) methods in liver tissue. Results All HBeAg positive patients expressed HBcAg by both IIF and IIP methods. Out of 62 patients with HBeAg-negative CHB, HBcAg was detected by IIF in 55 (88.7%) patients and by IIP in 51 (82.26%) patients. A positive relation among viral load and HBcAg detection was also found. This was more evident in the case of HBeAg negative patients and showed a positive relation with HBV DNA levels. Conclusion Hepatitis B core antigen can be detected using the IIF from formalin fixed paraffin block preparation and also by IIP method. This seems to reflect the magnitudes of HBV replication in CHB. How to cite this article Raihan R, Tabassum S, Al-Mahtab M, Nessa A, Jahan M, Kabir CMS, Kamal M, Aguilar JC. Hepatitis B Core Antigen in Hepatocytes of Chronic Hepatitis B: Comparison between Indirect Immunofluorescence and Immunoperoxidase Method. Euroasian J Hepato-Gastroenterol 2015;5(1):7-10. PMID:29201677

Discovery and Optimization of Novel 5-Indolyl-7-arylimidazo[1,2-a]pyridine-8-carbonitrile Derivatives as Potent Antitubulin Agents Targeting Colchicine-binding Site

NASA Astrophysics Data System (ADS)

Zhai, Xin; Wang, Xiaoqiang; Wang, Jiao; Liu, Jin; Zuo, Daiying; Jiang, Nan; Zeng, Tianfang; Yang, Xiuxiu; Jing, Tongfei; Gong, Ping

2017-02-01

Aiming at development of potent antitubulin agents targeting colchicine-binding site, a series of novel 5-indolyl-7-arylimidazo[1,2-a]pyridine-8-carbonitrilederivatives (5a-5v and 7a-7h) were designed based on bioisosterism and hybridization strategies. All these compounds were concisely synthesized via a three-step process and examined against five human cancer cell lines (HT-29, A549, MKN-45, MDA-MB-231 and SMMC-7721) along with a normal human cell (L02) in vitro. A structure-activity relationships (SARs) study was carried out and optimization towards this series of compounds in cellular assay resulted in the discovery of 5k, which displayed similar or better antitumor potency against the tested cancer cells with IC50 value ranging from 0.02 to 1.22 μM superior to CA-4 and Crolibulin. Significantly, a cell cycle study disclosed the ability of 5k to arrest cell cycle at the G2/M phase, and immunofluorescence assay as well as a colchicine competition assay revealed that tubulin polymerization was disturbed by 5k by binding to the colchicine site. Moreover, the molecular modeling mode showed the posture of 5k and Crolibulin was similar in the colchcine-binding pocket of tubulin as identified with the SARs and pharmacological results. Together, all these results rationalized 5k might serve as a promising lead for a novel class of antitubulin agents for cancer treatments.

Pachymic acid promotes induction of autophagy related to IGF-1 signaling pathway in WI-38 cells.

PubMed

Lee, Su-Gyeong; Kim, Moon-Moo

2017-12-01

The insulin-like growth factor 1 (IGF-1) signaling pathway has spotlighted as a mechanism to elucidate aging associated with autophagy in recent years. Therefore, we have tried to screen an effective compound capable of inducing autophagy to delay aging process. The aim of this study is to investigate whether pachymic acid, a main compound in Poria cocos, induces autophagy in the aged cells. The aging of young cells was induced by treatment with IGF-1 at 50 ng/ml three times every two days. The effect of pachymic acid on cell viability was evaluated in human lung fibroblasts, WI-38 cells, using MTT assay. The induction of autophagy was detected using autophagy detection kit. The expression of proteins related to autophagy and IGF-1 signaling pathway was examined by western blot analysis and immunofluorescence assay. In this study, pachymic acid showed cytotoxic effect in a dose dependent manner and remarkably induced autophagy at the same time. Moreover, pachymic acid increased the expression of proteins related to autophagy such as LC3-II and Beclin1 and decreased the levels of mTor phosphorylation and p70S6K in the aged cells. In particular, pachymic acid increased the expression of p-PI3K, p-FoxO and Catalase. In addition, pachymic acid remarkably increased the expression of IGFBP-3. Above results suggest that pachymic acid could induce autophagy related to IGF-1 signaling pathway in the aged cells. Copyright © 2017 Elsevier GmbH. All rights reserved.

The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines

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

Flaskos, J.; Harris, W.; Sachana, M.

2007-03-15

Diazinon and cypermethrin are pesticides extensively used in sheep dipping. Diazinon is a known anti-cholinesterase, but there is limited information regarding its molecular mechanism of action. This paper describes the effects of diazinon and cypermethrin at a morphological and molecular level on differentiating mouse N2a neuroblastoma and rat C6 glioma cell lines. Concentrations up to 10 {mu}M of both compounds and their mixture had no effect on the viability of either cell line, as determined by methyl blue tetrazolium reduction and total protein assays. Microscopic analysis revealed that 1 {mu}M and 10 {mu}M diazinon but not cypermethrin inhibited the outgrowthmore » of axon-like processes in N2a cells after a 24-h exposure but neither compound affected process outgrowth by differentiating C6 cells at these concentrations. Under these conditions, 10 {mu}M diazinon inhibited AChE slightly compared to the control after a 4-h exposure but not after 24 h. Western blotting analysis showed that morphological changes were associated with reduced cross-reactivity with antibodies that recognize the neurofilament heavy chain (NFH), microtubule associated protein MAP 1B and HSP-70 compared to control cell extracts, whereas reactivity with anti-{alpha}-tubulin antibodies was unchanged. Aggregation of NFH was observed in cell bodies of diazinon-treated N2a cells, as determined by indirect immunofluorescence staining. These data demonstrate that diazinon specifically targets neurite outgrowth in neuronal cells and that this effect is associated with disruption of axonal cytoskeleton proteins, whereas cypermethrin has no effect on the same parameters.« less

Repurposed drug screen identifies cardiac glycosides as inhibitors of TGF-β-induced cancer-associated fibroblast differentiation.

PubMed

Coleman, David T; Gray, Alana L; Stephens, Charles A; Scott, Matthew L; Cardelli, James A

2016-05-31

The tumor microenvironment, primarily composed of myofibroblasts, directly influences the progression of solid tumors. Through secretion of growth factors, extracellular matrix deposition, and contractile mechanotransduction, myofibroblasts, or cancer-associated fibroblasts (CAFs), support angiogenesis and cancer cell invasion and metastasis. The differentiation of fibroblasts to CAFs is primarily induced by TGF-β from cancer cells. To discover agents capable of blocking CAF differentiation, we developed a high content immunofluorescence-based assay to screen repurposed chemical libraries utilizing fibronectin expression as an initial CAF marker. Screening of the Prestwick chemical library and NIH Clinical Collection repurposed drug library, totaling over 1700 compounds, identified cardiac glycosides as particularly potent CAF blocking agents. Cardiac glycosides are traditionally used to regulate intracellular calcium by inhibiting the Na+/K+ ATPase to control cardiac contractility. Herein, we report that multiple cardiac glycoside compounds, including digoxin, are able to inhibit TGF-β-induced fibronectin expression at low nanomolar concentrations without undesirable cell toxicity. We found this inhibition to hold true for multiple fibroblast cell lines. Using real-time qPCR, we determined that digoxin prevented induction of multiple CAF markers. Furthermore, we report that digoxin is able to prevent TGF-β-induced fibroblast contraction of extracellular matrix, a major phenotypic consequence of CAF differentiation. Assessing the mechanism of inhibition, we found digoxin reduced SMAD promoter activity downstream of TGF-β, and we provide data that the effect is through inhibition of its known target, the Na+/K+ ATPase. These findings support a critical role for calcium signaling during CAF differentiation and highlight a novel, repurposable modality for cancer therapy.

Fate and transport of cyanobacteria and associated toxins and taste-and-odor compounds from upstream reservoir releases in the Kansas River, Kansas, September and October 2011

USGS Publications Warehouse

Graham, Jennifer L.; Ziegler, Andrew C.; Loving, Brian L.; Loftin, Keith A.

2012-01-01

Cyanobacteria cause a multitude of water-quality concerns, including the potential to produce toxins and taste-and-odor compounds. Toxins and taste-and-odor compounds may cause substantial economic and public health concerns and are of particular interest in lakes, reservoirs, and rivers that are used for drinking-water supply, recreation, or aquaculture. The Kansas River is a primary source of drinking water for about 800,000 people in northeastern Kansas. Water released from Milford Lake to the Kansas River during a toxic cyanobacterial bloom in late August 2011 prompted concerns about cyanobacteria and associated toxins and taste-and-odor compounds in downstream drinking-water supplies. During September and October 2011 water-quality samples were collected to characterize the transport of cyanobacteria and associated compounds from upstream reservoirs to the Kansas River. This study is one of the first to quantitatively document the transport of cyanobacteria and associated compounds during reservoir releases and improves understanding of the fate and transport of cyanotoxins and taste-and-odor compounds downstream from reservoirs. Milford Lake was the only reservoir in the study area with an ongoing cyanobacterial bloom during reservoir releases. Concentrations of cyanobacteria and associated toxins and taste-and-odor compounds in Milford Lake (upstream from the dam) were not necessarily indicative of outflow conditions (below the dam). Total microcystin concentrations, one of the most commonly occurring cyanobacterial toxins, in Milford Lake were 650 to 7,500 times higher than the Kansas Department of Health and Environment guidance level for a public health warning (20 micrograms per liter) for most of September 2011. By comparison, total microcystin concentrations in the Milford Lake outflow generally were less than 10 percent of the concentrations in surface accumulations, and never exceeded 20 micrograms per liter. The Republican River, downstream from Milford Lake, was the only Kansas River tributary with detectable microcystin concentrations throughout the study period, and concentrations exceeded 1 microgram per liter for most of September 2011. Microcystin was detected periodically in other tributaries, but concentrations were low (less than 0.3 micrograms per liter). In contrast, the taste-and-odor compounds geosmin and 2-methylisoborneol (MIB) were detected in all tributaries located immediately downstream from reservoirs and total concentrations generally exceeded the human detection threshold (5 to 10 nanograms per liter) from September through mid-October. Microcystin, geosmin, and MIB were not detected in the Smoky Hill River upstream from the confluence with the Republican River that forms the Kansas River. Within a week after initial reservoir releases, microcystin, geosmin, and MIB were detected throughout a 173-mile reach of the Kansas River; these compounds remained detectable throughout the reach until mid-October. Losses to groundwater when streamflows in the Kansas River were increasing indicate the potential for reservoir releases to affect groundwater quality as well as surface-water quality. Total microcystin concentrations in the Kansas River generally were highest within about 24 miles of the confluence of the Smoky Hill and Republican Rivers, and decreased downstream; concentrations exceeded 1 microgram per liter in the Kansas River upstream from Topeka during the first 2 weeks of September. Patterns in microcystin occurrence and concentration at Kansas River tributary and main-stem sites indicate that Milford Lake was the source of microcystin in the Kansas River; however, the source of taste-and-odor compounds was not as evident, possibly because multiple tributaries contributed taste-and-odor compounds to the Kansas River. Microcystin and taste-and-odor compounds co-occurred in 56 percent of samples collected, indicating co-occurrence was common. Despite frequent co-occurrence, the spatial and temporal patterns in microcystin, geosmin, and MIB were unique and did not necessarily match patterns in cyanobacterial abundance. Use of a single compound or cyanobacterial abundance alone cannot necessarily be used as an indicator of the presence or concentration of these compounds. Measured concentrations of cyanobacteria and associated compounds were substantially higher than expected concentrations based on simple dilution models at some sites and substantially lower at others, though spatial and temporal patterns were unique for individual compounds. Data were not collected in such a way to determine whether differences between measured and expected concentrations were statistically significant. Results, however, indicate that simple dilution models were not sufficient to describe the downstream transport of cyanobacteria and associated compounds in the Kansas River.

The effects of using stimuli from three different dimensions on autoshaping with a complex negative patterning discrimination.

PubMed

Pearce, John M; George, David N

2002-10-01

In two experiments pigeons received a complex negative patterning discrimination, using autoshaping, in which food was made available after three stimuli if they were presented alone (A, B, C), or in pairs (AB, AC, BC), but not when they were all presented together (ABC). Subjects also received a positive patterning discrimination in which three additional stimuli were not followed by food when presented alone (D, E, F), or in pairs (DE, DF, EF), but they were followed by food when presented together (DEF). Stimuli A and D belonged to one dimension, B and E to a second dimension, and D and F to a third dimension. For both problems, the discrimination between the individual stimuli and the triple-element compounds developed more readily than that between the pairs of stimuli and the triple-element compound. The results are consistent with predictions that can be derived from a configural theory of conditioning.

Interleukin (IL)-8 immunoreactivity of injured axons and surrounding oligodendrocytes in traumatic head injury.

PubMed

Hayashi, Takahito; Ago, Kazutoshi; Nakamae, Takuma; Higo, Eri; Ogata, Mamoru

2016-06-01

Interleukin (IL)-8 has been suggested to be a positive regulator of myelination in the central nervous system, in addition to its principal role as a chemokine for neutrophils. Immunostaining for beta-amyloid precursor protein (AβPP) is an effective tool for detecting traumatic axonal injury, although AβPP immunoreactivity can also indicate axonal injury due to hypoxic causes. In this study, we examined IL-8 and AβPP immunoreactivity in sections of corpus callosum obtained from deceased patients with blunt head injury and from equivalent control tissue. AβPP immunoreactivity was detected in injured axons, such as axonal bulbs and varicose axons, in 24 of 44 head injury cases. These AβPP immunoreactive cases had survived for more than 3h. The AβPP immunostaining pattern can be classified into two types: traumatic (Pattern 1) and non-traumatic (Pattern 2) axonal injuries, which we described previously [Hayashi et al. Int. J. Legal Med. 129 (2015) 1085-1090]. Three of 44 control cases also showed AβPP immunoreactive injured axons as Pattern 2. In contrast, IL-8 immunoreactivity was detected in 7 AβPP immunoreactive and in 2 non-AβPP immunoreactive head injury cases, but was not detected in any of the 44 control cases, including the 3 AβPP immunoreactive control cases. The IL-8 immunoreactive cases had survived from 3 to 24 days, whereas those cases who survived less than 3 days (n=29) and who survived 90 days (n=1) were not IL-8 immunoreactive. Moreover, IL-8 was detected as Pattern 1 axons only. In addition, double immunofluorescence analysis showed that IL-8 is expressed by oligodendrocytes surrounding injured axons. In conclusion, our results suggest that immunohistochemical detection of IL-8 may be useful as a complementary diagnostic marker of traumatic axonal injury. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Protein subcellular location pattern classification in cellular images using latent discriminative models.

PubMed

Li, Jieyue; Xiong, Liang; Schneider, Jeff; Murphy, Robert F

2012-06-15

Knowledge of the subcellular location of a protein is crucial for understanding its functions. The subcellular pattern of a protein is typically represented as the set of cellular components in which it is located, and an important task is to determine this set from microscope images. In this article, we address this classification problem using confocal immunofluorescence images from the Human Protein Atlas (HPA) project. The HPA contains images of cells stained for many proteins; each is also stained for three reference components, but there are many other components that are invisible. Given one such cell, the task is to classify the pattern type of the stained protein. We first randomly select local image regions within the cells, and then extract various carefully designed features from these regions. This region-based approach enables us to explicitly study the relationship between proteins and different cell components, as well as the interactions between these components. To achieve these two goals, we propose two discriminative models that extend logistic regression with structured latent variables. The first model allows the same protein pattern class to be expressed differently according to the underlying components in different regions. The second model further captures the spatial dependencies between the components within the same cell so that we can better infer these components. To learn these models, we propose a fast approximate algorithm for inference, and then use gradient-based methods to maximize the data likelihood. In the experiments, we show that the proposed models help improve the classification accuracies on synthetic data and real cellular images. The best overall accuracy we report in this article for classifying 942 proteins into 13 classes of patterns is about 84.6%, which to our knowledge is the best so far. In addition, the dependencies learned are consistent with prior knowledge of cell organization. http://murphylab.web.cmu.edu/software/.

Zebrafish: an exciting model for investigating the spatio-temporal pattern of enteric nervous system development.

PubMed

Doodnath, Reshma; Dervan, Adrian; Wride, Michael A; Puri, Prem

2010-12-01

Recently, the zebrafish (Danio rerio) has been shown to be an excellent model for human paediatric research. Advantages over other models include its small size, externally visually accessible development and ease of experimental manipulation. The enteric nervous system (ENS) consists of neurons and enteric glia. Glial cells permit cell bodies and processes of neurons to be arranged and maintained in a proper spatial arrangement, and are essential in the maintenance of basic physiological functions of neurons. Glial fibrillary acidic protein (GFAP) is expressed in astrocytes, but also expressed outside of the central nervous system. The aim of this study was to investigate the spatio-temporal pattern of GFAP expression in developing zebrafish ENS from 24 h post-fertilization (hpf), using transgenic fish that express green fluorescent protein (GFP). Zebrafish embryos were collected from transgenic GFP Tg(GFAP:GFP)(mi2001) adult zebrafish from 24 to 120 hpf, fixed and processed for whole mount immunohistochemistry. Antibodies to Phox2b were used to identify enteric neurons. Specimens were mounted on slides and imaging was performed using a fluorescent laser confocal microscope. GFAP:GFP labelling outside the spinal cord was identified in embryos from 48 hpf. The patterning was intracellular and consisted of elongated profiles that appeared to migrate away from the spinal cord into the periphery. At 72 and 96 hpf, GFAP:GFP was expressed dorsally and ventrally to the intestinal tract. At 120 hpf, GFAP:GFP was expressed throughout the intestinal wall, and clusters of enteric neurons were identified using Phox2b immunofluorescence along the pathway of GFAP:GFP positive processes, indicative of a migratory pathway of ENS precursors from the spinal cord into the intestine. The pattern of migration of GFAP:GFP expressing cells outside the spinal cord suggests an organized, early developing migratory pathway to the ENS. This shows for the first time that Tg(GFAP:GFP)(mi2001) zebrafish model is an ideal one to study spatio-temporal patterning of early ENS development.

Biologically inspired artificial compound eyes.

PubMed

Jeong, Ki-Hun; Kim, Jaeyoun; Lee, Luke P

2006-04-28

This work presents the fabrication of biologically inspired artificial compound eyes. The artificial ommatidium, like that of an insect's compound eyes, consists of a refractive polymer microlens, a light-guiding polymer cone, and a self-aligned waveguide to collect light with a small angular acceptance. The ommatidia are omnidirectionally arranged along a hemispherical polymer dome such that they provide a wide field of view similar to that of a natural compound eye. The spherical configuration of the microlenses is accomplished by reconfigurable microtemplating, that is, polymer replication using the deformed elastomer membrane with microlens patterns. The formation of polymer waveguides self-aligned with microlenses is also realized by a self-writing process in a photosensitive polymer resin. The angular acceptance is directly measured by three-dimensional optical sectioning with a confocal microscope, and the detailed optical characteristics are studied in comparison with a natural compound eye.

Dimeric Matrine-Type Alkaloids from the Roots of Sophora flavescens and Their Anti-Hepatitis B Virus Activities.

PubMed

Zhang, Yu-Bo; Zhan, Li-Qin; Li, Guo-Qiang; Wang, Feng; Wang, Ying; Li, Yao-Lan; Ye, Wen-Cai; Wang, Guo-Cai

2016-08-05

Six unusual matrine-type alkaloid dimers, flavesines A-F (1-6, respectively), together with three proposed biosynthetic intermediates (7-9) were isolated from the roots of Sophora flavescens. Compounds 1-5 were the first natural matrine-type alkaloid dimers, and compound 6 represented an unprecedented dimerization pattern constructed by matrine and (-)-cytisine. Their structures were elucidated by NMR, MS, single-crystal X-ray diffraction, and a chemical method. The hypothetical biogenetic pathways of 1-6 were also proposed. Compounds 1-9 exhibited inhibitory activities against hepatitis B virus.

Reading compounds in neglect dyslexia: the headedness effect.

PubMed

Semenza, Carlo; Arcara, Giorgio; Facchini, Silvia; Meneghello, Francesca; Ferraro, Marco; Passarini, Laura; Pilosio, Cristina; Vigato, Giovanna; Mondini, Sara

2011-09-01

Reading compound words was studied in neglect dyslexia in order to assess the influence of 'headedness'. The 'head' of a compound is the component that determines the grammatical category, the syntactic (e.g., the gender) and the semantic properties of the compound as a whole. For example, in the word 'blackberry'berry is the compound's head. The question was addressed of whether or not the privileged status of the head constituent influences processing and determines behavioural patterns in the breakdown of spatial attention in neglect. Italian right-headed (e.g. capobanda, band leader) and left-headed compounds (e.g. astronave, spaceship) were administered to 18 participants affected by neglect dyslexia. Left-headed compounds were read better than right-headed compounds. This result was not due to factors such as frequency, familiarity, age of acquisition or imageability, since these effects were controlled. It is suggested that attention is captured by the head component after implicit reading of the whole word. The head would require a relatively lighter processing load than the modifier and benefit from top-down facilitation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Apparatus for detecting and recognizing analytes based on their crystallization patterns

DOEpatents

Morozov, Victor; Bailey, Charles L.; Vsevolodov, Nikolai N.; Elliott, Adam

2010-12-14

The invention contemplates apparatuses for recognition of proteins and other biological molecules by imaging morphology, size and distribution of crystalline and amorphous dry residues in droplets (further referred to as "crystallization patterns") containing predetermined amount of certain crystal-forming organic compounds (reporters) to which protein to be analyzed is added. Changes in the crystallization patterns of a number of amino-acids can be used as a "signature" of a protein added. Also, changes in the crystallization patterns, as well as the character of such changes, can be used as recognition elements in analysis of protein molecules.

Volatiles from the xylarialean fungus Hypoxylon invadens.

PubMed

Dickschat, Jeroen S; Wang, Tao; Stadler, Marc

2018-01-01

The volatiles emitted by agar plate cultures of the xylarialean fungus Hypoxylon invadens were investigated by use of a closed loop stripping apparatus in combination with GC-MS. Several aromatic compounds were found that could only be identified by comparison to all possible constitutional isomers with different ring substitution patterns. For the set of identified compounds a plausible biosynthetic scheme was suggested that gives further support for the assigned structures.

Penicillipyrones A and B, meroterpenoids from a marine-derived Penicillium sp. fungus.

PubMed

Liao, Lijuan; Lee, Jung-Ho; You, Minjung; Choi, Tae Joon; Park, Wanki; Lee, Sang Kook; Oh, Dong-Chan; Oh, Ki-Bong; Shin, Jongheon

2014-02-28

Penicillipyrones A (1) and B (2), two novel meroterpenoids, were isolated from the marine-derived fungus Penicillium sp. On the basis of the results of combined spectroscopic analyses, these compounds were structurally elucidated to be sesquiterpene γ-pyrones from a new skeletal class derived from a unique linkage pattern between the drimane sesquiterpene and pyrone moieties. Compound 2 elicited significant induction of quinone reductase.

Synthesis and Mass Spectra of Butenyltin Compounds

DTIC Science & Technology

1989-03-01

activities or operations. One instance of this concern has focused on the Navy’s use of tributyltin compounds as antifoulants on ships’ hulls. Such...butene, and 1-chloro- 2-butene were obtained from Pfaltz & Bauer (Waterbury, CT). Resublimed magne- sium chips, tetrabutyltin and tributyltin bromide...preferential elimination of R minus R to form RSn ’from R3Sn + The fragmentation patterns of tributyltin bromide and tributenyltin bromides are dominated

Outdoor, Indoor, and Personal Exposure to VOCs in Children

PubMed Central

Adgate, John L.; Church, Timothy R.; Ryan, Andrew D.; Ramachandran, Gurumurthy; Fredrickson, Ann L.; Stock, Thomas H.; Morandi, Maria T.; Sexton, Ken

2004-01-01

We measured volatile organic compound (VOC) exposures in multiple locations for a diverse population of children who attended two inner-city schools in Minneapolis, Minnesota. Fifteen common VOCs were measured at four locations: outdoors (O), indoors at school (S), indoors at home (H), and in personal samples (P). Concentrations of most VOCs followed the general pattern O ≈ S < P ≤ H across the measured microenvironments. The S and O environments had the smallest and H the largest influence on personal exposure to most compounds. A time-weighted model of P exposure using all measured microenvironments and time–activity data provided little additional explanatory power beyond that provided by using the H measurement alone. Although H and P concentrations of most VOCs measured in this study were similar to or lower than levels measured in recent personal monitoring studies of adults and children in the United States, p-dichlorobenzene was the notable exception to this pattern, with upper-bound exposures more than 100 times greater than those found in other studies of children. Median and upper-bound H and P exposures were well above health benchmarks for several compounds, so outdoor measurements likely underestimate long-term health risks from children’s exposure to these compounds. PMID:15471730

Characterization of organic aerosol in fine particles in a mega-city of South China: Molecular composition, seasonal variation, and size distribution

NASA Astrophysics Data System (ADS)

Huang, Xiao-Feng; Chen, Dong-Lei; Lan, Zi-Juan; Feng, Ning; He, Ling-Yan; Yu, Guang-He; Luan, Sheng-Ji

2012-10-01

A one-year-long observation on major organic compounds in PM2.5 was performed in a coastal mega-city in South China, Shenzhen, in order to gain information of their ambient concentration levels and the implications for sources. The compounds identified included alkanes, PAHs, hopanes, fatty acids and dicarboxylic acids, whose annual average concentrations during the year were 56.0, 14.8, 2.51, 253, and 25.2 ng m- 3, respectively. The seasonal molecular distributions of these organic compounds were discussed to explore their contributing sources in Shenzhen. Conclusively, alkanes and PAHs had the dominant source of fossil fuel combustion, although alkanes also had significant contribution from plant wax (~ 16%). The hopane series distributions further indicated that vehicle emissions were the dominant fossil fuel combustion source for PM2.5 in Shenzhen. Cooking emissions were inferred to be the most possible main source for fatty acids, while both primary and secondary origins were implied for azelaic acid, the dominant one in the dicarboxylic acids identified. Most of the organic compounds analyzed showed a size distribution pattern peaking at 0.32-0.56 or 0.56-1 μm in the accumulation mode, except that the cooking-related organic acids showed implication of a coarse mode-dominated pattern.

Cultivation of Cimicifuga racemosa (L.) nuttal and quality of CR extract BNO 1055.

PubMed

Popp, Michael; Schenk, Regina; Abel, Gudrun

2003-03-14

For Cimicifuga racemosa, well-founded investigations concerning multiplication, germination of seeds and field cultivation have not yet been published. Defined origins or varieties with certain agronomic properties and a specific pattern of active compounds are not commercially available. Special challenges are found with regard to growing of young plantlets from seeds. Comprehensive investigations have been started to find optimal conditions for all steps of the whole process to establish cultivation for Cimicifuga. Aim is to get defined varieties or sources with desirable agronomic characteristics and specific reproducible compound patterns in order to reach homogeneous plant raw material. For analytical tests, validated HPLC and TLC methods were used. Results from germination experiments with different temperature regimens show that the time for germination can be shortened from about 20 months to about 6 months. Gibberellic acid had positive influence on the development of the embryo. Content of triterpenglycosides and phenolic compounds was highest in May and June and decreased then from July until September. The quality of the ethanolic extract BNO 1055 (contained in Klimadynon(R) and Menofem(R)) differs from that of an isopropanolic extract. Comparison was carried out by means of TLC pattern of triterpenglycosides and phenolic compounds. Extensive systematic research on cultivation parameters with regard to all stages from the seeds to the herbal drug enables commercial field cultivation of Cimicifuga. Controlled cultivation (according to good agricultural practice or GAP) ensures the availability of homogenous standardized raw material. For pharmacological and clinical studies, standardized extracts and finished herbal medicinal products are required. Results of these studies are never transferable to other products and therefore valid only for the tested extracts/products.

Natural and Human Influences on Water Quality in a Shallow Regional Unconsolidated Aquifer, Northern Atlantic Coastal Plain

USGS Publications Warehouse

Ator, Scott W.

2008-01-01

Data collected from more than 400 wells in the surficial unconfined aquifer in the Northern Atlantic Coastal Plain (New York through North Carolina) were compiled and analyzed to improve understanding of multiple natural and human influences on water quality in such shallow regional aquifers. Geochemical patterns were identified and described through principal components analysis on major ions, and correlation and logistic regression were used to relate observed concentrations of nitrate and selected pesticide compounds (atrazine, metolachlor, simazine, and deethylatrazine, an atrazine degradate) and volatile organic compounds (chloroform, 1,1,1-trichloroethane, tetrachlorethene, and methyl tert-butyl ether) to likely influences, such as observed geochemical patterns, land use, hydrogeology, and soils. Variability in major-ion concentrations is primarily related to ionic strength and redox condition. Concentrations of nitrate, pesticides, and volatile organic compounds are related to natural conditions, as well as the distribution of likely sources reflected in land use. Nitrate is most common in aerobic ground water and in relatively well-drained areas, for example; concentrations greater than 0.4 milligrams per liter may result from a variety of human activities, although concentrations greater than 3 milligrams per liter are more likely in agricultural areas. Atrazine, deethylatrazine, and metolachlor also are related to geochemical patterns, likely because ground-water geochemistry reflects hydrogeologic and soil conditions affecting pesticide transport to the water table. Results demonstrate the value of geochemical information along with the distribution of sources and other influences to understanding the regional occurrence of selected compounds in ground water. Such influences are not unique to the Northern Atlantic Coastal Plain, and thus observations and interpretations are relevant to broader areas.

Evolution of a strategy for preparing bioactive small molecules by sequential multicomponent assembly processes, cyclizations, and diversification.

PubMed

Sahn, James J; Granger, Brett A; Martin, Stephen F

2014-10-21

A strategy for generating diverse collections of small molecules has been developed that features a multicomponent assembly process (MCAP) to efficiently construct a variety of intermediates possessing an aryl aminomethyl subunit. These key compounds are then transformed via selective ring-forming reactions into heterocyclic scaffolds, each of which possesses suitable functional handles for further derivatizations and palladium-catalyzed cross coupling reactions. The modular nature of this approach enables the facile construction of libraries of polycyclic compounds bearing a broad range of substituents and substitution patterns for biological evaluation. Screening of several compound libraries thus produced has revealed a large subset of compounds that exhibit a broad spectrum of medicinally-relevant activities.

Chemical Compounds Toxic to Invertebrates Isolated from Marine Cyanobacteria of Potential Relevance to the Agricultural Industry

PubMed Central

Essack, Magbubah; Alzubaidy, Hanin S.; Bajic, Vladimir B.; Archer, John A. C.

2014-01-01

In spite of advances in invertebrate pest management, the agricultural industry is suffering from impeded pest control exacerbated by global climate changes that have altered rain patterns to favour opportunistic breeding. Thus, novel naturally derived chemical compounds toxic to both terrestrial and aquatic invertebrates are of interest, as potential pesticides. In this regard, marine cyanobacterium-derived metabolites that are toxic to both terrestrial and aquatic invertebrates continue to be a promising, but neglected, source of potential pesticides. A PubMed query combined with hand-curation of the information from retrieved articles allowed for the identification of 36 cyanobacteria-derived chemical compounds experimentally confirmed as being toxic to invertebrates. These compounds are discussed in this review. PMID:25356733

Chemical compounds toxic to invertebrates isolated from marine cyanobacteria of potential relevance to the agricultural industry.

PubMed

Essack, Magbubah; Alzubaidy, Hanin S; Bajic, Vladimir B; Archer, John A C

2014-10-29

In spite of advances in invertebrate pest management, the agricultural industry is suffering from impeded pest control exacerbated by global climate changes that have altered rain patterns to favour opportunistic breeding. Thus, novel naturally derived chemical compounds toxic to both terrestrial and aquatic invertebrates are of interest, as potential pesticides. In this regard, marine cyanobacterium-derived metabolites that are toxic to both terrestrial and aquatic invertebrates continue to be a promising, but neglected, source of potential pesticides. A PubMed query combined with hand-curation of the information from retrieved articles allowed for the identification of 36 cyanobacteria-derived chemical compounds experimentally confirmed as being toxic to invertebrates. These compounds are discussed in this review.

LC/QTOF-MS fragmentation of N-nitrosodimethylamine precursors in drinking water supplies is predictable and aids their identification.

PubMed

Hanigan, David; Ferrer, Imma; Thurman, E Michael; Herckes, Pierre; Westerhoff, Paul

2017-02-05

N-Nitrosodimethylamine (NDMA) is carcinogenic in rodents and occurs in chloraminated drinking water and wastewater effluents. NDMA forms via reactions between chloramines and mostly unidentified, N-containing organic matter. We developed a mass spectrometry technique to identify NDMA precursors by analyzing 25 model compounds with LC/QTOF-MS. We searched isolates of 11 drinking water sources and 1 wastewater using a custom MATLAB ® program and extracted ion chromatograms for two fragmentation patterns that were specific to the model compounds. Once a diagnostic fragment was discovered, we conducted MS/MS during a subsequent injection to confirm the precursor ion. Using non-target searches and two diagnostic fragmentation patterns, we discovered 158 potential NDMA precursors. Of these, 16 were identified using accurate mass combined with fragment and retention time matches of analytical standards when available. Five of these sixteen NDMA precursors were previously unidentified in the literature, three of which were metabolites of pharmaceuticals. Except methadone, the newly identified precursors all had NDMA molar yields of less than 5%, indicating that NDMA formation could be additive from multiple compounds, each with low yield. We demonstrate that the method is applicable to other disinfection by-product precursors by predicting and verifying the fragmentation patterns for one nitrosodiethylamine precursor. Copyright © 2016. Published by Elsevier B.V.

Kidney lesions in Rocky Mountain spotted fever: a light-, immunofluorescence-, and electron-microscopic study.

PubMed Central

Bradford, W. D.; Croker, B. P.; Tisher, C. C.

1979-01-01

The essential pathologic lesion in Rocky Mountain spotted fever (RMSF) is a vasculitis that may involve the kidneys as well as the heart, brain, skin, and subcutaneous tissues. Histopathologic information concerning the response of the kidneys in RMSF is rather limited, however. In this study renal tissue from 17 children who died of RMSF was examined by light, electron, and immunofluorescence microscopy. A lymphocytic or mixed inflammation, or both, involving vessels and interstitium of the kidney was found in all patients. In addition, 10 patients had histologic evidence of acute tubular necrosis, and another 3 had glomerular lesions consisting of focal segmental tuft necrosis or increased cellularity secondary to neutophilic infiltration, or both. Immunofluorescence- and electron-microscopic studies failed to demonstrate immune-complex deposition within glomeruli, a finding that suggests that immunoglobulin and classic immune complexes were not involved in the pathogenesis of the renal lesions at the time of death. These findings suggest the possibility that the pathogenesis of the renal lesion in RMSF may be due to a direct action of the organism (Rickettsia rickettsii) on the vessel wall. Images Figure 2 Figure 1 PMID:525676

Proof of the quantitative potential of immunofluorescence by mass spectrometry.

PubMed

Toki, Maria I; Cecchi, Fabiola; Hembrough, Todd; Syrigos, Konstantinos N; Rimm, David L

2017-03-01

Protein expression in formalin-fixed, paraffin-embedded patient tissue is routinely measured by Immunohistochemistry (IHC). However, IHC has been shown to be subject to variability in sensitivity, specificity and reproducibility, and is generally, at best, considered semi-quantitative. Mass spectrometry (MS) is considered by many to be the criterion standard for protein measurement, offering high sensitivity, specificity, and objective molecular quantification. Here, we seek to show that quantitative immunofluorescence (QIF) with standardization can achieve quantitative results comparable to MS. Epidermal growth factor receptor (EGFR) was measured by quantitative immunofluorescence in 15 cell lines with a wide range of EGFR expression, using different primary antibody concentrations, including the optimal signal-to-noise concentration after quantitative titration. QIF target measurement was then compared to the absolute EGFR concentration measured by Liquid Tissue-selected reaction monitoring mass spectrometry. The best agreement between the two assays was found when the EGFR primary antibody was used at the optimal signal-to-noise concentration, revealing a strong linear regression (R 2 =0.88). This demonstrates that quantitative optimization of titration by calculation of signal-to-noise ratio allows QIF to be standardized to MS and can therefore be used to assess absolute protein concentration in a linear and reproducible manner.

A direct view by immunofluorescent comet assay (IFCA) of DNA damage induced by nicking and cutting enzymes, ionizing (137)Cs radiation, UV-A laser microbeam irradiation and the radiomimetic drug bleomycin.

PubMed

Grigaravicius, Paulius; Rapp, Alexander; Greulich, Karl Otto

2009-03-01

In DNA repair research, DNA damage is induced by different agents, depending on the technical facilities of the investigating researchers. A quantitative comparison of different investigations is therefore often difficult. By using a modified variant of the neutral comet assay, where the histone H1 is detected by immunofluorescence [immunofluorescent comet assay (IFCA)], we achieve previously unprecedented resolution in the detection of fragmented chromatin and show that trillions of ultraviolet A photons (of a few eV), billions of bleomycin (BLM) molecules and thousands of gamma quanta (of 662 keV) generate, in first order, similar damage in the chromatin of HeLa cells. A somewhat more detailed inspection shows that the damage caused by 20 Gy ionizing radiation and by a single laser pulse of 10 microJ are comparable, while the damage caused by 12 microg/ml BLM depends highly on the individual cell. Taken together, this work provides a detailed view of DNA fragmentation induced by different treatments and allows comparing them to some extent, especially with respect to the neutral comet assay.

Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

PubMed

Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

2016-02-01

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies. © 2016 The Histochemical Society.

Experimental rabies in skunks: effects of immunosuppression induced by cyclophosphamide.

PubMed Central

Charlton, K M; Casey, G A; Campbell, J B

1984-01-01

Striped skunks (Mephitis mephitis) were inoculated with street rabies virus and immunosuppressed with several doses of cyclophosphamide. Control skunks were inoculated with street virus only. The skunks were killed in terminal stages of the disease and several tissues were collected for examination by immunofluorescence, light microscopy and viral titration. Sera collected at euthanasia from most of the principals did not contain detectable rabies neutralizing antibodies, whereas high titers occurred terminally in controls. Immunofluorescence was much more entensive in submandibular salivary glands of cyclophosphamide-treated than control skunks. Similarly, virus was isolated from this tissue more consistently and at higher titer from principals than from controls. Immunofluorescence was extensive in brains of all skunks (both groups), but virus was isolated consistently only from brains of cyclophosphamide-treated skunks. Most of the cyclophosphamide-treated skunks had very few inflammatory cells in brain and cerebrospinal ganglia. Neuronal degeneration occurred in dorsal root ganglia of both principals and controls. The results suggest that the immune response has no effect on the development of rabies-induced aggressive behavior, that the immune response may inhibit salivary gland infection and that it is not essential for the development of neuronal degeneration in dorsal root ganglia. PMID:6370390

[Laboratory diagnosis of genital herpes--direct immunofluorescence method].

PubMed

Majewska, Anna; Romejko-Wolniewicz, Ewa; Zareba-Szczudlik, Julia; Kilijańczyk, Marek; Gajewska, Małgorzata; Młynarczyk, Grazyna

2013-07-01

Aim of the study was to determine clinical usefulness of direct immunofluorescence method in the laboratory diagnosis of genital herpes in women. Overall 187 anogenital swabs were collected from 120 women. Using a dacron-tipped applicator 83 swabs were collected from women suspected of genital herpes and 104 from patients with no signs of genital infection. All samples were tested using cell culture (Vero cell line) and then direct immunofluorescence method (DIF) for the identification of antigens of herpes simplex viruses: HSV-1 and HSV-2. Characteristic cytopathic effect (CPE), indicative of alphaherpesvirus infection, was observed in 43.4% of cultures with clinical specimens collected from women with suspected genital herpes and in 29.8% of cultures of clinical specimens taken from patients with no clinical symptoms of genital herpes. Herpes simplex viruses were determined in 73 samples by direct immunofluorescence method after amplification of the virus in cell culture. The DIF test confirmed the diagnosis based on the microscopic CPE observation in 85%. In 15% of samples (taken from pregnant women without clinical signs of infection) we reported positive immunofluorescence in the absence of CPE. The frequency of antigen detection was statistically significantly higher in samples that were positive by culture study (chi-square test with Yates's correction, p < 0.01). This method proved to be highly sensitive (97%) in women with clinically suspected infection. High negative predictive value (99%) proves the clinical utility of the DIF in these group of patients. In asymptomatic infections, viral antigens were detected most frequently in the swabs from the cervical canal, and in cases of suspected genital herpes in swabs taken from the vestibule of the vagina and the vulva. However, there was no statistically significant difference in the frequency of detection of Herpes Simplex Virus antigens in specimens from different parts of the genital tract in both groups of women (chi-square test, p > 0.05). In our study HHV-1 was the main causative agent of genital herpes. The growing worldwide prevalence of genital herpes, challenges with the clinical diagnosis, and availability of effective antiviral therapy are the main reasons for a growing interest in rapid, proper laboratory diagnosis of infected patients. Optimal testing diagnostic algorithm depends on patient population, clinical circumstances and availability. Our results indicated that combination of laboratory tests may help to establish the diagnosis if genital herpes is suspected but there are no typical signs.

Method of patterning an aerogel

DOEpatents

Reed, Scott T [Edgewood, NM

2012-07-24

A method for producing a pattern in an aerogel disposed as a coating on a substrate comprises exposing the aerogel coating to the vapors of a hydrophobic silane compound, masking the aerogel coating with a shadow photomask and irradiating the aerogel coating with ultraviolet (UV) irradiation. The exposure to UV through the shadow mask creates a pattern of hydrophobic and hydrophilic regions in the aerogel coating. Etching away the hydrophilic regions of the aerogel coating, preferably with a 1 molar solution of sodium hydroxide, leaves the unwetted and unetched hydrophobic regions of the aerogel layer on the substrate, replicating the pattern of the photomask. The hydrophobic aerogel pattern can be further exposed to UV irradiation if desired, to create a hydrophilic aerogel pattern.

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine.

PubMed

Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

2016-02-15

To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

HPASubC: A suite of tools for user subclassification of human protein atlas tissue images.

PubMed

Cornish, Toby C; Chakravarti, Aravinda; Kapoor, Ashish; Halushka, Marc K

2015-01-01

The human protein atlas (HPA) is a powerful proteomic tool for visualizing the distribution of protein expression across most human tissues and many common malignancies. The HPA includes immunohistochemically-stained images from tissue microarrays (TMAs) that cover 48 tissue types and 20 common malignancies. The TMA data are used to provide expression information at the tissue, cellular, and occasionally, subcellular level. The HPA also provides subcellular data from confocal immunofluorescence data on three cell lines. Despite the availability of localization data, many unique patterns of cellular and subcellular expression are not documented. To get at this more granular data, we have developed a suite of Python scripts, HPASubC, to aid in subcellular, and cell-type specific classification of HPA images. This method allows the user to download and optimize specific HPA TMA images for review. Then, using a playstation-style video game controller, a trained observer can rapidly step through 10's of 1000's of images to identify patterns of interest. We have successfully used this method to identify 703 endothelial cell (EC) and/or smooth muscle cell (SMCs) specific proteins discovered within 49,200 heart TMA images. This list will assist us in subdividing cardiac gene or protein array data into expression by one of the predominant cell types of the myocardium: Myocytes, SMCs or ECs. The opportunity to further characterize unique staining patterns across a range of human tissues and malignancies will accelerate our understanding of disease processes and point to novel markers for tissue evaluation in surgical pathology.

Analysis of biennial outbreak pattern of respiratory syncytial virus according to subtype (A and B) in the Zagreb region.

PubMed

Mlinaric-Galinovic, Gordana; Tabain, Irena; Kukovec, Tamara; Vojnovic, Gordana; Bozikov, Jadranka; Bogovic-Cepin, Jasna; Ivkovic-Jurekovic, Irena; Knezovic, Ivica; Tesovic, Goran; Welliver, Robert C

2012-06-01

The epidemic pattern of respiratory syncytial virus (RSV) in Croatia is biennial. In order to determine if the circulation of different RSV subtypes affects the outbreak cycle, the aim of the present study was to analyze the epidemic pattern of RSV in children in Croatia (Zagreb region) over a period of 3 consecutive years. The study group consisted of 696 inpatients, aged 0-5 years, who were hospitalized with acute respiratory tract infections caused by RSV, in Zagreb, in the period 1 January 2006-31 December 2008. The virus was identified in nasopharyngeal secretions using direct immunofluorescence. The virus subtype was determined on real-time polymerase chain reaction. Of 696 RSV infections identified in children, subtype A virus caused 374 infections, and subtype B, 318. Four patients had a dual RSV infection (subtypes A and B). The period of study was characterized by four epidemic waves of RSV infections: the first, smaller, in the spring of 2006; the second, larger, in December 2006/January 2007; the third in spring 2008, followed by a fourth outbreak beginning in November of 2008. The biennial virus cycles were persistent although the predominant RSV subtype in the first two epidemic waves was subtype B, and in the second two it was subtype A. Over a 3 year period of observation, the biennial RSV cycle in Croatia cannot be explained by a difference in the predominant circulating subtype of RSV. Other unknown factors account for the biennial cycle of RSV epidemics in Croatia. © 2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society.

HPASubC: A suite of tools for user subclassification of human protein atlas tissue images

PubMed Central

Cornish, Toby C.; Chakravarti, Aravinda; Kapoor, Ashish; Halushka, Marc K.

2015-01-01

Background: The human protein atlas (HPA) is a powerful proteomic tool for visualizing the distribution of protein expression across most human tissues and many common malignancies. The HPA includes immunohistochemically-stained images from tissue microarrays (TMAs) that cover 48 tissue types and 20 common malignancies. The TMA data are used to provide expression information at the tissue, cellular, and occasionally, subcellular level. The HPA also provides subcellular data from confocal immunofluorescence data on three cell lines. Despite the availability of localization data, many unique patterns of cellular and subcellular expression are not documented. Materials and Methods: To get at this more granular data, we have developed a suite of Python scripts, HPASubC, to aid in subcellular, and cell-type specific classification of HPA images. This method allows the user to download and optimize specific HPA TMA images for review. Then, using a playstation-style video game controller, a trained observer can rapidly step through 10's of 1000's of images to identify patterns of interest. Results: We have successfully used this method to identify 703 endothelial cell (EC) and/or smooth muscle cell (SMCs) specific proteins discovered within 49,200 heart TMA images. This list will assist us in subdividing cardiac gene or protein array data into expression by one of the predominant cell types of the myocardium: Myocytes, SMCs or ECs. Conclusions: The opportunity to further characterize unique staining patterns across a range of human tissues and malignancies will accelerate our understanding of disease processes and point to novel markers for tissue evaluation in surgical pathology. PMID:26167380

Clinical features and histological patterns of lupus nephritis in a single center of South India.

PubMed

Devadass, Clement Wilfred; Mysorekar, Vijaya Viswanath; Eshwarappa, Mahesh; Mekala, Lakshminarayanan; Siddaiah, M Gireesh; Channabasappa, K Gurudev

2016-01-01

Renal involvement occurs in up to 60% of patients with systemic lupus erythematosus (SLE) and signifies a poor prognosis. The class of lupus nephritis (LN), determined on renal biopsy evaluation, guides the therapeutic management and has prognostic connotations. Our aim is to determine the clinicolaboratory features and histopathological patterns of LN at presentation in our local (South Indian) population. The study was conducted in a tertiary care hospital in South India between 2009 and 2014 on SLE patients with clinical evidence of LN. The renal biopsies were examined by light and immunofluorescence microscopy and were classified according to the International Society of Nephrology/Renal Pathology Society Classification of LN. A total of 46 patients were included, with age range of 12-60 years and a female to male ratio of 8.2:1. Arthritis, dermatological manifestations, and fever occurred, respectively, in 43.5%, 39.1%, and 30.4% of the cases. Class IV LN was present in 17 (37.1%), Class III LN in ten (21.7%), Class II LN in nine (19.5%), Class V LN in eight (17.4%), Class I LN in one (2.2%), and Class VI LN in one (2.2%) patients. Antinuclear antibody (ANA) and dsDNA positivity were present, respectively, in 82.6% and 65.2% of the patients. The most common pattern of LN was Class IV LN followed by Class III LN. Relatively higher proportions of ANA and anti-dsDNA positivity were present in proliferative LN, and there was a high frequency of arthritis at presentation in our LN patients.

Seminal fluid protein depletion and replenishment in the fruit fly, Drosophila melanogaster: an ELISA-based method for tracking individual ejaculates

PubMed Central

Sirot, Laura K.; Wolfner, Mariana F.

2014-01-01

In many species, seminal fluid proteins (SFPs) affect female post-mating behavioral patterns, including sperm storage, egg laying, feeding, and remating. Yet, few studies have investigated the patterns of allocation, depletion, and replenishment of SFPs in male animals, despite the importance of these proteins to male and female reproductive success. To investigate such SFP dynamics, it is necessary to have a sensitive method for quantifying SFP levels in males and mated females. We developed such a method by adapting the enzyme-linked immunosorbent assay (ELISA) using anti-SFP antibodies. Here, we first use two Drosophila melanogaster SFPs (ovulin and sex peptide) to demonstrate that ELISAs provide accurate measures of SFP levels. We find that, consistent with previous data from Western blotting or immunofluorescence studies, levels of both ovulin and sex peptide decline in the mated female with time since mating, but they do so at different rates. We then use ELISAs to show that males become depleted of SFPs with repeated matings, but that previously mated males are able to transfer “virgin” levels of SFPs after 3 days of sexual inactivity. Finally, we demonstrate that ELISAs can detect SFPs from wild-caught D. melanogaster males and, thus, potentially can be used to track mating patterns in the wild. This method of measuring SFP dynamics can be used in a wide range of species to address questions related to male reproductive investment, female mating history, and variation in female post-mating behavioral changes. PMID:24733957

Roles of microtubules and cellulose microfibril assembly in the localization of secondary-cell-wall deposition in developing tracheary elements.

PubMed

Roberts, A W; Frost, A O; Roberts, E M; Haigler, C H

2004-12-01

The roles of cellulose microfibrils and cortical microtubules in establishing and maintaining the pattern of secondary-cell-wall deposition in tracheary elements were investigated with direct dyes to inhibit cellulose microfibril assembly and amiprophosmethyl to inhibit microtubule polymerization. When direct dyes were added to xylogenic cultures of Zinnia elegans L. mesophyll cells just before the onset of differentiation, the secondary cell wall was initially secreted as bands composed of discrete masses of stained material, consistent with immobilized sites of cellulose synthesis. The masses coalesced, forming truncated, sinuous or smeared thickenings, as secondary cell wall deposition continued. The absence of ordered cellulose microfibrils was confirmed by polarization microscopy and a lack of fluorescence dichroism as determined by laser scanning microscopy. Indirect immunofluorescence showed that cortical microtubules initially subtended the masses of dye-altered secondary cell wall material but soon became disorganized and disappeared. Although most of the secondary cell wall was deposited in the absence of subtending cortical microtubules in dye-treated cells, secretion remained confined to discrete regions of the plasma membrane. Examination of non-dye-treated cultures following application of microtubule inhibitors during various stages of secondary-cell-wall deposition revealed that the pattern became fixed at an early stage such that deposition remained localized in the absence of cortical microtubules. These observations indicate that cortical microtubules are required to establish, but not to maintain, patterned secondary-cell-wall deposition. Furthermore, cellulose microfibrils play a role in maintaining microtubule arrays and the integrity of the secondary-cell-wall bands during deposition.

Gold(III) bis(thiosemicarbazonate) compounds in breast cancer cells: Cytotoxicity and thioredoxin reductase targeting.

PubMed

Rodríguez-Fanjul, Vanessa; López-Torres, Elena; Mendiola, M Antonia; Pizarro, Ana María

2018-03-25

Gold(III) compounds have received increasing attention in cancer research. Three gold complexes of general formula [Au III L]Cl, where L is benzil bis(thiosemicarbazonate), compound 1, benzil bis(4-methyl-3-thiosemicarbazonate), compound 2, or benzil bis(4-cyclohexyl-3-thiosemicarbazonate), compound 3, have been synthesized and fully characterized, including the X-ray crystal structure of compound 3, confirming square-planar geometry around the gold(III) centre. Compound 1 showed moderate cytotoxicity and accumulation in MCF7 breast cancer cells but did not inhibit thioredoxin reductase (TrxR) activity and did not induce reactive oxygen species (ROS) production. Compound 2, the least cytotoxic, was found to be capable of modestly inhibiting TrxR activity and produced low levels of ROS in the MCF7 cell line. The most cytotoxic compound, 3, had the highest cellular accumulation and its distribution pattern showed a clear preference for the cytosol and mitochondria of MCF7 cells. It readily hampered intracellular TrxR activity leading to a dramatic alteration of the cellular redox state and to the induction of cell death. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Substrate-induced Nuclear Export and Peripheral Compartmentalization of Hepatic Glucokinase Correlates with Glycogen Deposition

PubMed Central

Shiota, Masa; Knobel, Susan M.; Piston, David W.; Cherrington, Alan D.; Magnuson, Mark A.

2001-01-01

Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopyand quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface. PMID:12369705

Subcellular distribution and mitogenic effect of basic fibroblast growth factor in mesenchymal uncommitted stem cells.

PubMed

Benavente, Claudia A; Sierralta, Walter D; Conget, Paulette A; Minguell, José J

2003-06-01

Uncommitted mesenchymal stem cells (MSC), upon commitment and differentiation give rise to several mature mesenchymal lineages. Although the involvement of specific growth factors, including FGF2, in the development of committed MSC is known, the effect of FGF2 on uncommitted progenitors remains unclear. We have analyzed on a comparative basis, the subcellular distribution and mitogenic effect of FGF2 in committed and uncommitted MSC prepared from human bone marrow. Indirect immunofluorescence studies showed strong nuclear FGF2 staining in both progenitors; however, cytoplasmic staining was only detected in committed cells. Western blot analysis revealed the presence of 22.5 and 21-22 kDa forms of FGF2 in the nucleus of both progenitors; however, their relative content was higher in uncommitted than in committed cells. Exogenous FGF2 stimulated proliferation and sustained quiescence in committed and uncommitted cells, respectively. These results show that both type of progenitors, apart from morphological and proliferative differences, display specific patterns of response to FGF2.

Chromosomal abnormalities, meiotic behavior and fertility in domestic animals.

PubMed

Villagómez, D A F; Pinton, A

2008-01-01

Since the advent of the surface microspreading technique for synaptonemal complex analysis, increasing interest in describing the synapsis patterns of chromosome abnormalities associated with fertility of domestic animals has been noticed during the past three decades. In spite of the number of scientific reports describing the occurrence of structural chromosome abnormalities, their meiotic behavior and gametic products, little is known in domestic animal species about the functional effects of such chromosome aberrations in the germ cell line of carriers. However, some interesting facts gained from recent and previous studies on the meiotic behavior of chromosome abnormalities of domestic animals permit us to discuss, in the frame of recent knowledge emerging from mouse and human investigations, the possible mechanism implicated in the well known association between meiotic disruption and chromosome pairing failure. New cytogenetic techniques, based on molecular and immunofluorescent analyses, are allowing a better description of meiotic processes, including gamete production. The present communication reviews the knowledge of the meiotic consequences of chromosome abnormalities in domestic animals. Copyright 2008 S. Karger AG, Basel.

Ring-like distribution of constitutive heterochromatin in bovine senescent cells.

PubMed

Pichugin, Andrey; Beaujean, Nathalie; Vignon, Xavier; Vassetzky, Yegor

2011-01-01

Cells that reach "Hayflick limit" of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining. We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH). Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli. Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.

Apo-14 is required for digestive system organogenesis during fish embryogenesis and larval development.

PubMed

Xia, Jian-Hong; Liu, Jing-Xia; Zhou, Li; Li, Zhi; Gui, Jian-Fang

2008-01-01

Apo-14 is a fish-specific apolipoprotein and its biological function remains unknown. In this study, CagApo-14 was cloned from gibel carp (Carassius auratus gibelio) and its expression pattern was investigated during embryogenesis and early larval development. The CagApo-14 transcript and its protein product were firstly localized in the yolk syncytial layer at a high level during embryogenesis, and then found to be restricted to the digestive system including liver and intestine in later embryos and early larvae. Immunofluorescence staining in larvae and adults indicated that Cag Apo-14 protein was predominantly synthesized in and excreted from sinusoidal endothelial cells of liver tissue. Morpholino knockdown of Cag Apo-14 resulted in severe disruption of digestive organs including liver, intestine, pancreas and swim bladder. Moreover, yolk lipid transportation and utilization were severely affected in the Cag Apo-14 morphants. Overall, this data indicates that Cag Apo-14 is required for digestive system organogenesis during fish embryogenesis and larval development.

Subcellular Localization of Patched and Smoothened, the Receptors for Sonic Hedgehog Signaling, in the Hippocampal Neuron

PubMed Central

Petralia, Ronald S.; Schwartz, Catherine M.; Wang, Ya-Xian; Mattson, Mark P.; Yao, Pamela J.

2011-01-01

Cumulative evidence suggests that, aside from patterning the embryonic neural tube, Sonic hedgehog (Shh) signaling plays important roles in the mature nervous system. In this study, we investigate the expression and localization of the Shh signaling receptors, Patched (Ptch) and Smoothened (Smo), in the hippocampal neurons of young and mature rats. Reverse transcriptase-polymerase chain reaction and immunoblotting analyses show that the expression of Ptch and Smo remains at a moderate level in young postnatal and adult brains. By using immunofluorescence light microscopy and immunoelectron microscopy, we examine the spatial distribution of Ptch and Smo within the hippocampal neurons. In young developing neurons, Ptch and Smo are present in the processes and are clustered at their growth cones. In mature neurons, Ptch and Smo are concentrated in dendrites, spines, and postsynaptic sites. Synaptic Ptch and Smo often co-exist with unusual structures—synaptic spinules and autophagosomes. Our results reveal the anatomical organization of the Shh receptors within both the young and the mature hippocampal neurons. PMID:21618238

Dynamic monitoring of membrane nanotubes formation induced by vaccinia virus on a high throughput microfluidic chip

NASA Astrophysics Data System (ADS)

Xiao, Min; Xu, Na; Wang, Cheng; Pang, Dai-Wen; Zhang, Zhi-Ling

2017-03-01

Membrane nanotubes (MNTs) are physical connections for intercellular communication and induced by various viruses. However, the formation of vaccinia virus (VACV)-induced MNTs has never been studied. In this report, VACV-induced MNTs formation process was monitored on a microfluidic chip equipped with a series of side chambers, which protected MNTs from fluidic shear stress. MNTs were formed between susceptible cells and be facilitated by VACV infection through three patterns. The formed MNTs varied with cell migration and virus concentration. The length of MNTs was positively correlated with the distance of cell migration. With increasing virus titer, the peak value of the ratio of MNT-carried cell appeared earlier. The immunofluorescence assay indicated that the rearrangement of actin fibers induced by VACV infection may lead to the formation of MNTs. This study presents evidence for the formation of MNTs induced by virus and helps us to understand the relationship between pathogens and MNTs.

Systemic lupus erythematosus and renal tubular acidosis associated with hyperthyroidism. Case Report.

PubMed

Deng, Datong; Sun, Li; Xia, Tongjia; Xu, Min; Wang, Youmin; Zhang, Qiu

2016-07-01

A case of a 42-year-old female with hyperthyroidism was subsequently diagnosed to have systemic lupus erythematosus with distal RTA. The clinical examination on admission showed swelling of the knee joints and the urinalysis showed pH 6.5, pro 3+. Her blood routine results were as follows: white blood cells 1.85×109/L, platelets 100×109/L, erythrocyte 3.06×1012/L. The serum potassium was 3.11 mmol/L, 24 hour urinary electrolyte: K 68.87 mmol/24 H, antinuclear antibodies (ANA) 1:1 000, speckled pattern. The anti-double stranded DNA antibody (anti-dsDNA), anti SS-A(52) antibody and anti SS-A(60) antibody were positive. The light microscopy and immunofluorescence showed diffuse proliferative lupus nephritis. These data were compatible with the diagnosis of systemic lupus erythematosus. The diagnosis of hyperthyroidism and distal RTA is clear. This report showed that other autoimmune disease in the diagnosis of hyperthyroidism should not be ignored.

Losartan Affects Glomerular AKT and mTOR Phosphorylation in an Experimental Model of Type 1 Diabetic Nephropathy

PubMed Central

Petrakis, Ioannis; Stylianou, Kostas; Katsarou, Theodora; Giannakakis, Konstantinos; Perakis, Kostas; Vardaki, Eleftheria; Stratigis, Spyridon; Ganotakis, Emmanuel; Papavasiliou, Stathis; Daphnis, Eugenios

2013-01-01

The AKT-mTOR pathway is activated in diabetic nephropathy. Renin-angiotensin system modulators exert beneficial effects on the diabetic kidney. We explored the action of losartan on AKT-mTOR phosphorylation in glomeruli and podocytes. Diabetes mellitus was induced to Sprague-Dawley rats by streptozotocin. Five months later, the rats were commenced on losartan and euthanized 2 months later. Kidneys were processed for immunofluorescence studies. Glomeruli were isolated for Western blot analysis. Diabetes increased activated forms of AKT and mTOR both in glomeruli and podocytes. In diabetic rats, losartan decreased phosphorylated/activated forms of AKT (Thr308) and mTOR (Ser2448) in glomeruli but decreased only activated mTOR in podocytes. However, in both glomeruli and podocytes of healthy animals, an inverse pattern was evident. In conclusion, a new body of evidence indicates the differential activation of AKT-mTOR in glomeruli and podocytes of healthy and diabetic animals in response to losartan. PMID:23456824

Tissue distribution of mesenchymal stem cell marker Stro-1.

PubMed

Lin, Guiting; Liu, Gang; Banie, Lia; Wang, Guifang; Ning, Hongxiu; Lue, Tom F; Lin, Ching-Shwun

2011-10-01

Stro-1 is the best-known mesenchymal stem cell marker. However, despite its bone marrow origin, its localization in bone marrow has never been demonstrated. By immunofluorescence staining, we show here that ∼ 0.74% of nucleated bone marrow cells expressed Stro-1. We also found that ∼ 8.7% of CD34-expressing cells expressed Stro-1, and more than 20% of Stro-1-expressing cells did not express CD34. In adipose tissue Stro-1 expression was identified in the endothelium of arterioles and capillaries. Stro-1 was also localized in the endothelium of some but not all adipose tissue veins. Endothelial expression of Stro-1 was also identified in blood vessels in penis and in leg muscles, but not in other tested tissues. In these other tissues, Stro-1 was scantly expressed near but not in blood vessels. These variable and endothelial expression patterns of Stro-1 point to a need to re-examine published data that relied on Stro-1 as a mesenchymal stem cell marker.

A map of protein dynamics during cell-cycle progression and cell-cycle exit

PubMed Central

Gookin, Sara; Min, Mingwei; Phadke, Harsha; Chung, Mingyu; Moser, Justin; Miller, Iain; Carter, Dylan

2017-01-01

The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence. PMID:28892491

Novel leukocyte protein, Trojan, differentially expressed during thymocyte development.

PubMed

Petrov, Petar; Motobu, Maki; Salmi, Jussi; Uchida, Tatsuya; Vainio, Olli

2010-04-01

"Trojan" is a novel cell surface protein, discovered from chicken embryonic thymocytes on the purpose to identify molecules involved in T cell differentiation. The molecule is predicted as a type I transmembrane protein having a Sushi and two fibronectin type III domains and a pair of intracellular phosphorylation sites. Its transcript expression is specific for lymphoid tissues and the presence of the protein on the surface of recirculating lymphocytes and macrophages was confirmed by immunofluorescence analysis. In thymus, about half of the double negative (CD4(-) CD8(-)) and CD8 single positive and the majority of CD4 single positive cells express Trojan with a relatively high intensity. However, only a minority of the double positive (CD4(+) CD8(+)) cells are positive for Trojan. This expression pattern, similar to that of some proteins with anti-apoptotic and function, like IL-7Ralpha, makes Trojan an attractive candidate of having an anti-apoptotic role. Copyright 2010 Elsevier Ltd. All rights reserved.

Seasonal Variations of Quantified Organic Compounds in PM10 over Seoul

NASA Astrophysics Data System (ADS)

Choi, N.; Lee, J.; Kim, Y. P.

2014-12-01

The concentrations of 87 individual organic matters in the PM10 samples, systematically collected on the roof of the School of Public Health building at Seoul National University (mixed commercial and residential area), Seoul, South Korea on a daily basis from April 2010 to April 2011, were quantified by mean of Gas Chromatography/Mass Spectrometry (GC/MS). The daily average concentrations of five organic groups, alkanes, PAHs, fatty acid, DCAs, and sugars were ranged from 498.40 ng m3 to 10.20 μg m3. The seasonal concentrations of the total quantified organic species were 1.73 μg m3 (Spring), 2.04 μg m3 (Summer), 3.11 μg m3 (Fall), and 3.60 μg m3 (Winter), respectively. All the organic groups showed higher average concentration in winter than in summer. However, some organic compounds among fatty acids, DCAs, and sugars showed reverse pattern. The seasonal concentration patterns and episode variation of individual organic compounds were studied to clarify the emission characteristics of organic matters in PM10.

Concentration patterns of agricultural pesticides and urban biocides in surface waters of a catchment of mixed land use

NASA Astrophysics Data System (ADS)

Stamm, C.; Wittmer, I.; Bader, H.-P.; Scheidegger, R.; Alder, A.; Lück, A.; Hanke, I.; Singer, H.

2009-04-01

Organic pesticides and biocides that are found in surface waters, can originate from agricultural and urban sources. For a long time, agricultural pesticides have received substantially more attention than biocidal compounds from urban use like material protection or in-can preservatives (cosmetics etc.). Recent studies however revealed that the amounts of urban biocides used may exceed those of agricultural pesticides. This study aims at comparing the input of several important pesticides and biocides into a small Swiss stream with a special focus on loss events triggered by rainfall. A set of 16 substances was selected to represent urban and agricultural sources. The selected substances are either only used as biocides (irgarol, isothiazolinones, IPBC), as pesticides (atrazine, sulcotrione, dichlofluanid, tolylfluanid) or have a mixed use (isoproturon, terbutryn, terbutylazine, mecoprop, diazinon, carbendazim) The study catchment has an area of 25 km2 and is inhabited by about 12'000 people. Four sampling sites were selected in the river system in order to reflect different urban and agricultural sources. Additionally, we sampled a combined sewer overflow, a rain sewer and the outflow of a wastewater treatment plant. At each site discharge was measured continuously from March to November 2007. During 16 rain events samples were taken by automatic devices at a high temporal resolution. The results, based on more than 500 analyzed samples, revealed distinct concentration patterns for different compounds and sources. Agricultural pesticides exhibited a strong seasonality as expected based on the application periods. During the first one or two rain events after application the concentrations reached up to several thousand ng/l during peak flow (atrazine, isoproturon). The temporal patterns of urban biocides were more diverse. Some compounds obviously stem from permanent sources independent of rainfall because they were found mostly in the outlet of the wastewater treatment plant throughout the year. The insecticide diazinon for example showed a background concentration in treated waste water of approximately 50 ng/l. Substances like mecoprop, which are used in urban areas (roof protection, private gardens) and agriculture showed a mixed pattern. At the time scale of single events two concentration peaks have been observed. One of them was due to the fast reaction of sewer overflows or rain sewers carrying urban storm water. The delayed peak was caused by fast flow from agricultural soils. Overall, the study revealed complex concentration patterns for the different compounds. Source identification was only possible by means of a comprehensive approach including different nested measuring sites, a broad range of different compounds that were complemented by tracer substances like caffeine or drugs and their metabolites (sulfamethoxazole, N4-acetylsulfamethoxazole, diclofenac) that can be non-ambiguously attributed to sources like treated or untreated wastewater.

Diterpenoid compounds and other lipids in deep-sea sediments and their geochemical significance

NASA Technical Reports Server (NTRS)

Simoneit, B. R. T.

1977-01-01

Cyclic diterpenoid compounds have been found by various investigators in the geosphere (e.g., fossil resins, coals, soil, shale, and deep-sea sediments). These compounds occur in significant amounts only in higher plants and are therefore potential markers of terrigenous plant lipids. Diterpenoids with the abietane skeleton (mainly dehydroabietic acid) have been identified in the lipids of sediment samples from the northeast Pacific Ocean, Black Sea, and North Atlantic Ocean. The presence of these resin-derived compounds was correlated with the terrigenous clay components and with the presence of pollen. The presence of polycyclic diterpenoids was also correlated with the distribution patterns and inferred sources of other sediment lipid constituents. Potamic transport, followed by turbidite redistribution, is the probable input mechanism of these resin-derived compounds to the deep-sea sediments. These diterpenoids appear to be excellent biological markers of resinous higher plants.

Environmental bacteria produce abundant and diverse antibiofilm compounds.

PubMed

Farmer, J T; Shimkevitch, A V; Reilly, P S; Mlynek, K D; Jensen, K S; Callahan, M T; Bushaw-Newton, K L; Kaplan, J B

2014-12-01

The aim of this study was to isolate novel antibiofilm compounds produced by environmental bacteria. Cell-free extracts were prepared from lawns of bacteria cultured on agar. A total of 126 bacteria isolated from soil, cave and river habitats were employed. Extracts were tested for their ability to inhibit Staphylococcus aureus biofilm in a 96-well microtitre plate assay. A total of 55/126 extracts (44%) significantly inhibited Staph. aureus biofilm. Seven extracts were selected for further analysis. The antibiofilm activities in all seven extracts exhibited unique patterns of molecular mass, chemical polarity, heat stability and spectrum of activity against Staph. aureus, Staphylococcus epidermidis and Pseudomonas fluorescens, suggesting that these seven antibiofilm activities were mediated by unique chemical compounds with different mechanisms of action. Environmental bacteria produce abundant and diverse antibiofilm compounds. Screening cell-free extracts is a useful method for identifying secreted compounds that regulate biofilm formation. Such compounds may represent a novel source of antibiofilm agents for technological development. © 2014 The Society for Applied Microbiology.

Subcellular localization patterns and their relationship to photodynamic activity of pyropheophorbide-a derivatives.

PubMed

MacDonald, I J; Morgan, J; Bellnier, D A; Paszkiewicz, G M; Whitaker, J E; Litchfield, D J; Dougherty, T J

1999-11-01

To determine if subcellular localization is important to photodynamic therapy (PDT) efficacy, an in vitro fluorescence microscopy study was conducted with a congeneric series of pyropheophorbide-a derivatives in human pharyngeal squamous cell carcinoma (FaDu) cells and murine radiation-induced fibrosarcoma (RIF) mutant cells. In the FaDu cells the octyl, decyl and dodecyl ether derivatives localized to the lysosomes at extracellular concentrations less than needed to produce a 50% cell kill (LD50). At extracellular concentrations equal or greater than the LD50 the compounds localized mainly to mitochondria. The propyl, pentyl, hexyl and heptyl ether derivatives localized mainly to the mitochondria at all concentrations studied. This suggested that mitochondria are a sensitive PDT target for these derivatives. Similar experiments were performed with two Photofrin-PDT resistant RIF cell lines, one of which was found to be resistant to hexyl ether derivative (C6) mediated-PDT and the other sensitive to C6-PDT relative to the parent line. At extracellular concentrations of C6 below the LD50 of each cell line, the mutants exhibited lysosomal localization. At concentrations above these values the patterns shifted to a mainly mitochondrial pattern. In these cell lines mitochondrial localization also correlated with PDT sensitivity. Localization to mitochondria or lysosomes appeared to be affected by the aggregation state of the congeners, all of which are highly aggregated in aqueous medium. Monomers apparently were the active fraction of these compounds because equalizing the extracellular monomer concentrations produced equivalent intracellular concentrations, photoxicity and localization patterns. Compounds that were mainly aggregates localized to the lysosomes where they were rendered less active. Mitochondria appear to be a sensitive target for pyropheophorbide-a-mediated photodamage, and the degree of aggregation seems to be a determinant of the localization site.

Distinct brain metabolic patterns separately associated with cognition, motor function, and aging in Parkinson's disease dementia.

PubMed

Ko, Ji Hyun; Katako, Audrey; Aljuaid, Maram; Goertzen, Andrew L; Borys, Andrew; Hobson, Douglas E; Kim, Seok Min; Lee, Chong Sik

2017-12-01

We explored whether patients with Parkinson's disease dementia (PDD) show a distinct spatial metabolic pattern that characterizes cognitive deficits in addition to motor dysfunction. Eighteen patients with PDD underwent 3 separate positron emission tomography sessions with [ 18 F]fluorodeoxyglucose (for glucose metabolism), fluorinated N-3-fluoropropyl-2-beta-carboxymethoxy-3-beta-(4-iodophenyl) nortropane (for dopamine transporter density) and Pittsburgh compound-B (for beta-amyloid load). We confirmed in PDD versus normal controls, overall hypometabolism in the posterior and prefrontal brain regions accompanied with hypermetabolism in subcortical structures and the cerebellar vermis. A multivariate network analysis then revealed 3 metabolic patterns that are separately associated with cognitive performance (p = 0.042), age (p = 0.042), and motor symptom severity (p = 0.039). The age-related pattern's association with aging was replicated in healthy controls (p = 0.047) and patients with Alzheimer's disease (p = 0.002). The cognition-related pattern's association with cognitive performance was observed, with a trend-level of correlation, in patients with dementia with Lewy bodies (p = 0.084) but not in patients with Alzheimer's disease (p = 0.974). We found no association with fluorinated N-3-fluoropropyl-2-beta-carboxymethoxy-3-beta-(4-iodophenyl) nortropane and Pittsburgh compound-B positron emission tomography with patients' cognitive performance. Copyright © 2017 Elsevier Inc. All rights reserved.

Patterned substrates and methods for nerve regeneration

DOEpatents

Mallapragada, Surya K.; Heath, Carole; Shanks, Howard; Miller, Cheryl A.; Jeftinija, Srdija

2004-01-13

Micropatterned substrates and methods for fabrication of artificial nerve regeneration conduits and methods for regenerating nerves are provided. Guidance compounds or cells are seeded in grooves formed on the patterned substrate. The substrates may also be provided with electrodes to provide electrical guidance cues to the regenerating nerve. The micropatterned substrates give physical, chemical, cellular and/or electrical guidance cues to promote nerve regeneration at the cellular level.

Surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications.

PubMed

Gupta, Ashim; Sharif, Kevin; Walters, Megan; Woods, Mia D; Potty, Anish; Main, Benjamin J; El-Amin, Saadiq F

2014-04-30

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities. The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 ºC or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

PubMed Central

Gupta, Ashim; Sharif, Kevin; Walters, Megan; Woods, Mia D.; Potty, Anish; Main, Benjamin J.; El-Amin, Saadiq F.

2014-01-01

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities. The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 ºC or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction. PMID:24836540

Expression of interleukin-33 and its receptor ST2 in periapical granulomas and radicular cysts.

PubMed

Velickovic, Milena; Pejnovic, Nada; Petrovic, Renata; Mitrovic, Slobodanka; Jeftic, Ilija; Kanjevac, Tatjana; Lukic, Aleksandra

2016-01-01

Interleukin-33 (IL-33) is a recently identified cytokine belonging to the IL-1 family and ligand for the IL-1 receptor-related protein ST2. IL-33/ST2 signaling plays a critical role in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. We aimed to investigate the expression patterns of IL-33 and ST2 in human periapical lesions. Periapical lesions (n = 36) and healthy periapical tissues (n = 10) were evaluated by immunohistochemistry using antibodies specific for human IL-33 and ST2. Lesion samples were further analyzed by double immunofluorescence to assess IL-33/ST2 co-expression. The numbers of IL-33- and ST2-positive fibroblasts were significantly higher in periapical lesions compared to healthy periapical tissues (both P < 0.05), while the numbers of IL-33- and ST2-positive endothelial cells were similar (both P > 0.05). There were no significant differences in the numbers of IL-33- and ST2-positive fibroblasts and endothelial cells between periapical granulomas and radicular cysts (all P > 0.05). Similarly, numbers of ST2-positive mononuclear cells did not differ between periapical granulomas and radicular cysts (P > 0.05). The majority of epithelial cells in radicular cysts were IL-33 positive, while the small proportion of epithelial cells was ST2 positive. Double immunofluorescence analysis revealed IL-33/ST2 co-expression in fibroblasts and endothelial cells. IL-33 and ST2 are expressed in periapical granulomas and radicular cysts. Increased numbers of IL-33- and ST2-positive fibroblasts in periapical lesions when compared to healthy periapical tissues suggest that IL-33/ST2 signaling may be involved in periapical inflammation and tissue fibrosis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Neurotrophic and neuroprotective potential of human limbus-derived mesenchymal stromal cells.

PubMed

Liang, Chang-Min; Weng, Shao-Ju; Tsai, Tung-Han; Li, I-Hsun; Lu, Pin-Hui; Ma, Kuo-Hsing; Tai, Ming-Cheng; Chen, Jiann-Torng; Cheng, Cheng-Yi; Huang, Yuahn-Sieh

2014-10-01

The purpose of this study was to examine neurotrophic and neuroprotective effects of limbus stroma-derived mesenchymal stromal cells (L-MSCs) on cortical neurons in vitro and in vivo. Cultured L-MSCs were characterized by flow cytometry and immunofluorescence through the use of specific MSC marker antibodies. Conditioned media were collected from normoxia- and hypoxia-treated L-MSCs to assess neurotrophic effects. Neuroprotective potentials were evaluated through the use of in vitro hypoxic cortical neuron culture and in vivo rat focal cerebral ischemia models. Neuronal morphology was confirmed by immunofluorescence with the use of anti-MAP2 antibody. Post-ischemic infarct volume and motor behavior were assayed by means of triphenyltetrazolium chloride staining and open-field testing, respectively. Human growth antibody arrays and enzyme-linked immunoassays were used to analyze trophic/growth factors contained in conditioned media. Isolated human L-MSCs highly expressed CD29, CD90 and CD105 but not CD34 and CD45. Mesenchymal lineage cell surface expression pattern and differentiation capacity were identical to MSCs derived form human bone marrow and adipose tissue. The L-MSC normoxic and hypoxic conditioned media both promoted neurite outgrowth in cultured cortical neurons. Hypoxic conditioned medium showed superior neurotrophic function and neuroprotective potential with reduced ischemic brain injury and improved functional recovery in rat focal cerebral ischemia models. Human growth factor arrays and enzyme-linked immunoassays measurements showed neuroprotective and growth-associated cytokines (vascular endothelial growth factor [VEGF], VEGFR3, brain-derived neurotrophic factor, insulin-like growth factor -2 and hepatocyte growth factor) contained in conditioned media. Hypoxic exposure caused VEGF and brain-derived neurotrophic factor upregulation, possibly contributing to neurotrophic and neuroprotective effects. L-MSCs can secrete various neurotrophic factors stimulating neurite outgrowth and protecting neurons against brain ischemic injury through paracrine mechanism. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Association between antinuclear antibody titers and connective tissue diseases in a Rheumatology Department.

PubMed

Menor Almagro, Raúl; Rodríguez Gutiérrez, Juan Francisco; Martín-Martínez, María Auxiliadora; Rodríguez Valls, María José; Aranda Valera, Concepción; de la Iglesia Salgado, José Luís

To determine the dilution titles at antinuclear antibodies (ANA) by indirect immunofluorescence observed in cell substrate HEp-2 and its association with the diagnosis of systemic connective tissue disease in ANA test requested by a Rheumatology Unit. Samples of patients attended for the first time in the rheumatology unit, without prior ANA test, between January 2010 and December 2012 were selected. The dilution titers, immunofluorescence patterns and antigen specificity were recorded. In January 2015 the diagnosis of the patients were evaluated and classified in systemic disease connective tissue (systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, undifferentiated connective, antiphospholipid syndrome, mixed connective tissue and inflammatory myophaty) or not systemic disease connective tissue. A total of 1282 ANA tests requested by the Rheumatology Unit in subjects without previous study, 293 were positive, predominance of women (81.9%). Patients with systemic connective tissue disease were recorded 105, and 188 without systemic connective tissue disease. For 1/640 dilutions the positive predictive value in the connective was 73.3% compared to 26.6% of non-connective, and for values ≥1/1,280 85% versus 15% respectively. When performing the multivariate analysis we observed a positive association between 1/320 dilution OR 3.069 (95% CI: 1.237-7.614; P=.016), 1/640 OR 12.570 (95% CI: 3.659-43.187; P=.000) and ≥1/1,280 OR 42.136 (95% CI: 8.604-206.345; P=.000). These results show association titles dilution ≥1/320 in ANA's first test requested by a Rheumatology Unit with patients with systemic connective tissue disease. The VPP in these patients was higher than previous studies requested by other medical specialties. This may indicate the importance of application of the test in a targeted way. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

Organic compounds and trace elements in the Pocomoke River and its tributaries

USGS Publications Warehouse

Miller, Cherie V.; Foster, Gregory D.; Huff, Thomas B.; Garbarino, John R.

1999-01-01

In response to concern about recent blooms of the dinoflagellate, Pfiesteria piscicida, samples of sediment and water were collected from the lower Pocomoke River Basin and were screened for trace elements, pesticides, and other organic compounds. A large group of steroid and fatty acid methyl-ester compounds was detected in streamwater using gas chromatography/mass spectroscopy in scan mode. Some of these steroid compounds have been identified and further quantified in bed-sediment extracts. Spatial patterns of the concentrations of cholesterol suggest that these compounds are linked to the runoff of animal wastes into the river. Many of the organic compounds found in the Pocomoke River sediments have not yet been identified, but at least several are in the class of hormone compounds related to estradiols and have the potential to promote endocrine-disrupting effects in aquatic life. Particulate forms of arsenic and zinc are slightly elevated above normal levels for streams, but the sources for these elements are still undetermined. Several pesticides were found in low, parts-per-trillion concentrations, but were within the ranges commonly found in streams of this region.

Water- and air-quality and surficial bed-sediment monitoring of the Sweetwater Reservoir watershed, San Diego County, California, 2003-09

USGS Publications Warehouse

Mendez, Gregory O.; Majewski, Michael S.; Foreman, William T.; Morita, Andrew Y.

2015-01-01

Sampling results show concentrations of the gasoline oxygenate methyl tert-butyl ether in water and air samples declined after it was phased out by the State of California in January 2004. The largest concentrations of gasoline hydrocarbons benzene and toluene in water were detected at or near the surface of the SWR. Isophorone and phenol were the two most frequently detected BNA compounds in water. Diuron, prometon, and simazine were the most frequently detected pesticide compounds in water. Concentrations of benzene and toluene in air samples were highest during the cooler months and had a consistent seasonal pattern over time. Ten PAH compounds were detected frequently in air samples. Twelve pesticide compounds were also detected in air samples. Surficial bed-sediment samples were analyzed for 53 PAHs; 22 of the compounds had one or more detections. Surficial bed-sediment samples were analyzed for 22 organic compounds; only 6 compounds had one or more detections. Surficial bed-sediment samples were analyzed for 37 metals.

Unique and Highly Selective Anticytomegalovirus Activities of Artemisinin-Derived Dimer Diphenyl Phosphate Stem from Combination of Dimer Unit and a Diphenyl Phosphate Moiety

PubMed Central

He, Ran; Forman, Michael; Mott, Bryan T.; Venkatadri, Rajkumar; Posner, Gary H.

2013-01-01

We report that the artemisinin-derived dimer diphenyl phosphate (DPP; dimer 838) is the most selective inhibitor of human cytomegalovirus (CMV) replication among a series of artemisinin-derived monomers and dimers. Dimer 838 was also unique in being an irreversible CMV inhibitor. The peroxide unit within artemisinins' chemical structures is critical to their activities, and its absence results in loss of anti-CMV activities. Surprisingly, the deoxy dimer of 838 retained modest anti-CMV activity, suggesting that the DPP moiety of dimer 838 contributes to its anti-CMV activities. DPP alone did not inhibit CMV replication, but triphenyl phosphate (TPP) had modest CMV inhibition, although its selectivity index was low. Artemisinin DPP derivatives dimer 838 and monomer diphenyl phosphate (compound 558) showed stronger CMV inhibition and a higher selectivity index than their analogs lacking the DPP unit. An add-on and removal assay revealed that removing DPP derivatives (compounds 558 and 838) but not the non-DPP backbones (artesunate and compound 606) at 24 h postinfection (hpi) already resulted in dominant CMV inhibition. CMV inhibition was fully irreversible with 838 and partially irreversible with 558, while non-DPP artemisinins were reversible inhibitors. While all artemisinin derivatives and TPP reduced the expression of the CMV immediate early 2 (IE2), UL44, and pp65 proteins at or after 48 hpi, only TPP inhibited the expression of both IE1 and IE2. Combination of a non-DPP dimer (compound 606) with TPP was synergistic in CMV inhibition, while ganciclovir and TPP were additive. Although TPP shared structural similarity with monomer DPP (compound 558) and dimer DPP (compound 838), its pattern of CMV inhibition was significantly different from the patterns of the artemisinins. These findings demonstrate that the DPP group contributes to the unique activities of compound 838. PMID:23774439

Patterns of presentation and clinical features of toxicity after reported use of ([2-aminopropyl]-2,3-dihydrobenzofurans), the 'benzofuran' compounds. A report from the United Kingdom National Poisons Information Service.

PubMed

Kamour, Ashraf; James, David; Lupton, David J; Cooper, Gillian; Eddleston, Micheal; Vale, Allister; Thompson, John P; Thanacoody, Ruben; Hill, Simon L; Thomas, Simon H L

2014-12-01

To characterise the patterns of presentation and clinical features of toxicity following reported recreational use of benzofuran compounds ((2-aminopropyl)-2,3-dihydrobenzofurans) in the UK, as reported to the National Poisons Information Service (NPIS), and to compare clinical features of toxicity with those after reported mephedrone use. NPIS patient-specific telephone enquiries and user sessions for TOXBASE(®), the NPIS online information database, related to (2-aminopropyl)-2,3-dihydrobenzofurans and associated synonyms were reviewed from March 2009 to August 2013. These data were compared with those of mephedrone, the recreational substance most frequently reported to NPIS, collected over the same period. There were 63 telephone enquiries concerning 66 patients and 806 TOXBASE(®) user sessions regarding benzofuran compounds during the period of study. The first telephone enquiry was made in July 2010 and the highest numbers of enquiries were received in August 2010 (33 calls, 112 TOXBASE(®) sessions). Patients were predominantly male (82%) with a median age of 29 years; 9 reported co-ingestion of other substances. Comparing the 57 patients who reported ingesting benzofuran compounds alone with 315 patients ingesting mephedrone alone, benzofurans were more often associated with stimulant features, including tachycardia, hypertension, mydriasis, palpitation, fever, increased sweating, and tremor, (72% vs. 38%, odds ratio [OR] 4.2, 95% confidence interval [CI] 2.27-7.85, P < 0.0001) and mental health disturbances (58% vs. 38%, OR 2.3, 95% CI 1.29-4.07, P = 0.006). Other features reported after benzofuran compound ingestion included gastrointestinal symptoms (16%), reduced level of consciousness (9%), chest pain (7%), and creatinine kinase elevation (5%). Reported ingestion of benzofuran compounds is associated with similar toxic effects to those of amphetamines and cathinones. Mental health disturbances and stimulant features were reported more frequently following reported ingestion of benzofuran compounds than after ingestion of mephedrone.