Culture of hESC-derived pancreatic progenitors in alginate-based scaffolds.

PubMed

Formo, Kjetil; Cho, Candy H-H; Vallier, Ludovic; Strand, Berit L

2015-12-01

The effect of alginate-based scaffolds with added basement membrane proteins on the in vitro development of hESC-derived pancreatic progenitors was investigated. Cell clusters were encapsulated in scaffolds containing the basement membrane proteins collagen IV, laminin, fibronectin, or extracellular matrix-derived peptides, and maintained in culture for up to 46 days. The cells remained viable throughout the experiment with no signs of central necrosis. Whereas nonencapsulated cells aggregated into larger clusters, some of which showed signs of morphological changes and tissue organization, the alginate matrix stabilized the cluster size and displayed more homogeneous cell morphologies, allowing culture for long periods of time. For all conditions tested, a stable or declining expression of insulin and PDX1 and an increase in glucagon and somatostatin over time indicated a progressive reduction in beta cell-related gene expression. Alginate scaffolds can provide a chemically defined, xeno-free and easily scalable alternative for culture of pancreatic progenitors. Although no increase in insulin and PDX1 gene expression after alginate-immobilized cell culture was seen in this study, further optimization of the matrix physicochemical and biological properties and of the medium composition may still be a relevant strategy to promote the stabilization or maturation of stem cell-derived beta cells. © 2015 Wiley Periodicals, Inc.

Oxygen transport and stem cell aggregation in stirred-suspension bioreactor cultures.

PubMed

Wu, Jincheng; Rostami, Mahboubeh Rahmati; Cadavid Olaya, Diana P; Tzanakakis, Emmanuel S

2014-01-01

Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation, viability and differentiation potential. Here, a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation, mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover, the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics.

Brain Aggregates: An Effective In Vitro Cell Culture System Modeling Neurodegenerative Diseases.

PubMed

Ahn, Misol; Kalume, Franck; Pitstick, Rose; Oehler, Abby; Carlson, George; DeArmond, Stephen J

2016-03-01

Drug discovery for neurodegenerative diseases is particularly challenging because of the discrepancies in drug effects between in vitro and in vivo studies. These discrepancies occur in part because current cell culture systems used for drug screening have many limitations. First, few cell culture systems accurately model human aging or neurodegenerative diseases. Second, drug efficacy may differ between dividing and stationary cells, the latter resembling nondividing neurons in the CNS. Brain aggregates (BrnAggs) derived from embryonic day 15 gestation mouse embryos may represent neuropathogenic processes in prion disease and reflect in vivo drug efficacy. Here, we report a new method for the production of BrnAggs suitable for drug screening and suggest that BrnAggs can model additional neurological diseases such as tauopathies. We also report a functional assay with BrnAggs by measuring electrophysiological activities. Our data suggest that BrnAggs could serve as an effective in vitro cell culture system for drug discovery for neurodegenerative diseases. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Novel Benzothiazole Derivatives as Fluorescent Probes for Detection of β-Amyloid and α-Synuclein Aggregates.

PubMed

Watanabe, Hiroyuki; Ono, Masahiro; Ariyoshi, Taisuke; Katayanagi, Rikako; Saji, Hideo

2017-08-16

Deposits of β-amyloid (Aβ) and α-synuclein (α-syn) are the hallmark of Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. The detection of these protein aggregates with fluorescent probes is particularly of interest for preclinical studies using fluorescence microscopy on human brain tissue. In this study, we newly designed and synthesized three push-pull benzothiazole (PP-BTA) derivatives as fluorescent probes for detection of Aβ and α-syn aggregates. Fluorescence intensity of all PP-BTA derivatives significantly increased upon binding to Aβ(1-42) and α-syn aggregates in solution. In in vitro saturation binding assays, PP-BTA derivatives demonstrated affinity for both Aβ(1-42) (K d = 40-148 nM) and α-syn (K d = 48-353 nM) aggregates. In particular, PP-BTA-4 clearly stained senile plaques composed of Aβ aggregates in the AD brain section. Moreover, it also labeled Lewy bodies composed of α-syn aggregates in the PD brain section. These results suggest that PP-BTA-4 may serve as a promising fluorescent probe for the detection of Aβ and α-syn aggregates.

Rational Topological Design for Fluorescence Enhancement upon Aggregation of Distyrylfuran Derivatives.

PubMed

Mallet, Charlotte; Moussallem, Chady; Faurie, Alexandre; Allain, Magali; Gohier, Frédéric; Skene, William G; Frère, Pierre

2015-05-18

A series of 2,5-distyrylfuran derivatives bearing pentafluorophenyl- and cyanovinyl units have been synthesized for aggregation-induced emission (AIE). The effect of the type and extent of the supramolecular connections on the AIE of the furan derivatives were examined and correlated with their X-ray crystal structures. It was found that the simultaneous presence of cyano and perfluorophenyl units strongly enhances the fluorescence upon aggregation. Single-crystal X-ray diffraction analysis confirmed that CH⋅⋅⋅F, F⋅⋅⋅F, CH⋅⋅⋅nitrile, Ar⋅⋅⋅ArF (Ar=aryl, ArF =fluoroaryl), and nitrile⋅⋅⋅ArF intra- and intermolecular interactions drive the topology of the molecule and that solid-state supramolecular contacts favor AIE of the furan derivatives. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

PubMed

Radtke, Andrea L; Herbst-Kralovetz, Melissa M

2012-04-03

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties

Increased formation of autophagosomes in ectromelia virus-infected primary culture of murine bone marrow-derived macrophages.

PubMed

Martyniszyn, L; Szulc-Dąbrowska, L; Boratyńska-Jasińska, A; Niemiałtowski, M

2013-01-01

Induction of autophagy by ectromelia virus (ECTV) in primary cultures of bone marrow-derived macrophages (BMDMs) was investigated. The results showed that ECTV infection of BMDMs resulted in increased formation of autophagosomes, increased level of LC3-II protein present in aggregates and extensive cytoplasmic vacuolization. These data indicate an increased autophagic activity in BMDMs during ECTV infection.

Cryopreservation of embryonic stem cell-derived multicellular neural aggregates labeled with micron-sized particles of iron oxide for magnetic resonance imaging.

PubMed

Yan, Yuanwei; Sart, Sébastien; Calixto Bejarano, Fabian; Muroski, Megan E; Strouse, Geoffrey F; Grant, Samuel C; Li, Yan

2015-01-01

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre-labeled neural cells, especially in three-dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC-derived multicellular NPC aggregates labeled with micron-sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70-80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post-cryopreservation. MRI analysis showed comparable detectability for the MPIO-labeled cells before and after cryopreservation indicated by T2 and T2* relaxation rates. Cryopreserving MPIO-labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation. © 2015 American Institute of Chemical Engineers.

Method for the production of cementitious compositions and aggregate derivatives from said compositions

DOEpatents

Minnick, L. John

1981-01-01

Method for the production of cementitious compositions and aggregate derivatives of said compositions, and cementitious compositions and aggregates produced by said method, wherein fluidized bed combustion residue and pozzolanic material, such as pulverized coal combustion system fly ash, are incorporated in a cementitious mix. The mix is cast into desired shape and cured. If desired, the shape may then be crushed so as to result in a fluidized bed combustion residue-fly ash aggregate material or the shape may be used by itself.

Long-Term Results of Cartilage Repair after Allogeneic Transplantation of Cartilaginous Aggregates Formed from Bone Marrow-Derived Cells for Large Osteochondral Defects in Rabbit Knees.

PubMed

Yoshioka, Tomokazu; Mishima, Hajime; Sakai, Shinsuke; Uemura, Toshimasa

2013-10-01

The purpose of this study was to evaluate the long-term results of cartilage repair after allogeneic transplantation of cartilaginous aggregates formed from bone marrow-derived cells. Bone marrow cells were harvested from 12-day-old rabbits. The cells were subjected to a monolayer culture, and the spindle-shaped cells attached to the flask surface were defined as bone marrow-derived mesenchymal cells. After the monolayer culture, a 3-dimensional cartilaginous aggregate was formed using a bioreactor with chondrogenesis. We created osteochondral defects, measuring 5 mm in diameter and 4 mm in depth, at the femoral trochlea of 10-week-old rabbits. Two groups were established, the transplanted group in which the cartilaginous aggregate was transplanted into the defect, and the control group in which the defect was left untreated. Twenty-six and 52 weeks after surgery, the rabbits were sacrificed and their tissue repair status was evaluated macroscopically (International Cartilage Repair Society [ICRS] score) and histologically (O'Driscoll score). The ICRS scores were as follows: at week 26, 7.2 ± 0.5 and 7.6 ± 0.8; at week 52, 7.6 ± 1.1 and 9.7 ± 0.7, for the transplanted and control groups, respectively. O'Driscoll scores were as follows: at week 26, 12.6 ± 1.9 and 10.1 ± 1.9; at week 52, 9.6 ± 3.0 and 14.0 ± 1.4, each for transplanted and control groups, respectively. No significant differences were observed between the groups. This study demonstrates that allogeneic transplantation of cartilaginous aggregates formed from bone marrow-derived cells produces comparable long-term results based on macroscopic and histological outcome measures when compared with osteochondral defects that are left untreated.

Long-Term Results of Cartilage Repair after Allogeneic Transplantation of Cartilaginous Aggregates Formed from Bone Marrow–Derived Cells for Large Osteochondral Defects in Rabbit Knees

PubMed Central

Mishima, Hajime; Sakai, Shinsuke; Uemura, Toshimasa

2013-01-01

Objective: The purpose of this study was to evaluate the long-term results of cartilage repair after allogeneic transplantation of cartilaginous aggregates formed from bone marrow–derived cells. Methods: Bone marrow cells were harvested from 12-day-old rabbits. The cells were subjected to a monolayer culture, and the spindle-shaped cells attached to the flask surface were defined as bone marrow–derived mesenchymal cells. After the monolayer culture, a 3-dimensional cartilaginous aggregate was formed using a bioreactor with chondrogenesis. We created osteochondral defects, measuring 5 mm in diameter and 4 mm in depth, at the femoral trochlea of 10-week-old rabbits. Two groups were established, the transplanted group in which the cartilaginous aggregate was transplanted into the defect, and the control group in which the defect was left untreated. Twenty-six and 52 weeks after surgery, the rabbits were sacrificed and their tissue repair status was evaluated macroscopically (International Cartilage Repair Society [ICRS] score) and histologically (O’Driscoll score). Results: The ICRS scores were as follows: at week 26, 7.2 ± 0.5 and 7.6 ± 0.8; at week 52, 7.6 ± 1.1 and 9.7 ± 0.7, for the transplanted and control groups, respectively. O’Driscoll scores were as follows: at week 26, 12.6 ± 1.9 and 10.1 ± 1.9; at week 52, 9.6 ± 3.0 and 14.0 ± 1.4, each for transplanted and control groups, respectively. No significant differences were observed between the groups. Conclusions: This study demonstrates that allogeneic transplantation of cartilaginous aggregates formed from bone marrow–derived cells produces comparable long-term results based on macroscopic and histological outcome measures when compared with osteochondral defects that are left untreated. PMID:26069678

Equine cloning: in vitro and in vivo development of aggregated embryos.

PubMed

Gambini, Andrés; Jarazo, Javier; Olivera, Ramiro; Salamone, Daniel F

2012-07-01

The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.

Red blood cell generation by three-dimensional aggregate cultivation of late erythroblasts.

PubMed

Lee, EunMi; Han, So Yeon; Choi, Hye Sook; Chun, Bokhwan; Hwang, Byunghee; Baek, Eun Jung

2015-02-01

Stem cell-derived erythroid cells hold great potential for the treatment of blood-loss anemia and for erythropoiesis research; however, cultures using conventional flat plates or bioreactors have failed to show promising results. By mimicking the in vivo bone marrow (BM) environment in which most erythroid cells are physically aggregated, we show that a three-dimensional (3D) aggregate culture system facilitates erythroid cell maturation and red blood cell (RBC) production more effectively than two-dimensional high-density cell cultivation. Late erythroblasts (polychromatic or orthochromatic erythroblasts) were differentiated from cord blood CD34(+) cells over 15 days and then allowed to form tight aggregates at a minimum density of 1×10(7) cells/mL for 2-3 days. To scale up the cell culture and to make the media supply efficient throughout the cell aggregates, several macroporous microcarriers and porous scaffolds were applied to the 3D culture system. In comparison to control culture conditions, erythroid cells in 3D aggregates were significantly more differentiated toward RBCs with significantly reduced nuclear dysplasia. When 3D culture was performed inside macroporous microcarriers, the cell culture scale was increased and cells exhibited enhanced differentiation and enucleation. Microcarriers with a pore diameter of approximately 400 μm produced more mature cells than those with a smaller pore diameter. In addition, this aggregate culture method minimized the culture space and media volume required. In conclusion, a 3D aggregate culture system can be used to generate transfusable human erythrocytes at the terminal maturation stage, mimicking the in vivo BM microenvironment. Porous structures can efficiently maximize the culture scale, enabling large-scale production of RBCs. These results enhance our understanding of the importance of physical contact among late erythroblasts for their final maturation into RBCs.

[Effect of dilution on aggregation of nanoparticles of polycarboxylic derivative of fullerene C60].

PubMed

Bobylev, A G; Pen'kov, N V; Troshin, P A; Gudkov, S V

2015-01-01

In this work, we investigated the effect of dilution on aggregation of nanoparticles of the polycarboxylic derivative of fullerene C60. It is shown that the diminution of the concentration of PCDF-1 in aqueous medium leads to a decreased amount of aggregates of fullerene and an increased amount of single molecules. This can potentially interfere with the biological activity of a compound on one molecule basis. Addition of organic and inorganic salts to the aqueous medium with fullerene derivative leads to intense disaggregation of PCDF-1. The data obtained suggest an explanation of non-stoichiometric nature of neutralization of reactive oxygen species by derivatives of fullerenes, as well as provide new insight into the physical meaning of the work on the impact of nanoparticles at ultra-low concentrations on biological objects.

Oil-derived marine aggregates - hot spots of polysaccharide degradation by specialized bacterial communities

NASA Astrophysics Data System (ADS)

Arnosti, Carol; Ziervogel, Kai; Yang, Tingting; Teske, Andreas

2016-07-01

Aggregates generated in the laboratory from incubations of seawater and surface-water oil collected in the initial phase of the Deepwater Horizon oil spill resemble the oil-aggregates observed in situ. Here, we investigated the enzyme activities and microbial community composition of laboratory generated oil-aggregates, focusing on the abilities of these communities to degrade polysaccharides, which are major components of marine organic matter and are abundant constituents of exopolymeric substances (EPS) generated by oil-associated bacteria in response to the presence of oil. The patterns of polysaccharide-hydrolyzing enzyme activities in oil aggregates were very different from those in the water surrounding the aggregates after formation, and in the surface water that did not contain the oil. Specific oil aggregate-associated hydrolysis rates were also considerably higher than in the water surrounding the aggregates. The differences in initial hydrolysis profiles, and in evolution of these profiles with time, points to specialized metabolic abilities among the oil-aggregate communities compared to oil-water and ambient water communities. The composition of the oil-aggregate community indicates a multifunctional microbial assemblage containing primary oil-degrading and exopolysaccharide-producing members of the Gammaproteobacteria, and diverse members of the Alphaproteobacteria, Bacteroidetes and Planktomycetales that most likely participate in the breakdown of oil-derived bacterial biopolymers. Formation and aging of oil-aggregates encourages the growth and transformation of microbial communities that are specialized in degradation of petroleum, as well as their secondary degradation products.

Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after space-flight

NASA Technical Reports Server (NTRS)

Gruener, R.; Roberts, R.; Reitstetter, R.

1994-01-01

We carried out parallel experiments first on the slow clinostat and then in space-flight to examine the effects of altered gravity on the aggregation of the nicotinic acetylcholine receptors and the structure of the cytoskeleton in cultured Xenopus embryonic muscle cells. By examining the concordance between results from space flight and the clinostat, we tested whether the slow clinostat is a relevant simulation paradigm. Space-flown cells showed marked changes in the distribution and organization of actin filaments and had a reduced incidence of acetylcholine receptor aggregates at the site of contact with polystyrene beads. Similar effects were found after clinostat rotation. The sensitivity of synaptic receptor aggregation and cytoskeletal morphology suggests that in the microgravity of space cell behavior may be importantly altered.

Hyaluronan preserves the proliferation and differentiation potentials of long-term cultured murine adipose-derived stromal cells

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

Chen, P.-Y.; Huang, Lynn L.H.; Hsieh, H.-J.

2007-08-17

For long-term culture, murine adipose-derived stromal cells (mADSCs) at latter passages demonstrated a marked decline in proliferative activity, exhibited senescent morphology and reduced differentiation potentials, particularly osteogenesis. To extend the lifespan of mADSCs, two culture conditions containing hyaluronan (HA) was compared in our study, one as a culture medium supplement (SHA), and the other where HA was pre-coated on culture surface (CHA). mADSCs cultivated with SHA exhibited a prolonged lifespan, reduced cellular senescence, and enhanced osteogenic potential compared to regular culture condition (control). Upon CHA treatment, mADSCs tended to form cell aggregates with gradual growth profiles, while their differentiation activitiesmore » remained similar to SHA groups. After transferring mADSCs from CHA to control surface, they were shown to have an extended lifespan and an increase of osteogenic potential. Our results suggested that HA can be useful for preserving the proliferation and differentiation potentials of long-term cultured mADSCs.« less

Cultural Consensus Theory: Aggregating Continuous Responses in a Finite Interval

NASA Astrophysics Data System (ADS)

Batchelder, William H.; Strashny, Alex; Romney, A. Kimball

Cultural consensus theory (CCT) consists of cognitive models for aggregating responses of "informants" to test items about some domain of their shared cultural knowledge. This paper develops a CCT model for items requiring bounded numerical responses, e.g. probability estimates, confidence judgments, or similarity judgments. The model assumes that each item generates a latent random representation in each informant, with mean equal to the consensus answer and variance depending jointly on the informant and the location of the consensus answer. The manifest responses may reflect biases of the informants. Markov Chain Monte Carlo (MCMC) methods were used to estimate the model, and simulation studies validated the approach. The model was applied to an existing cross-cultural dataset involving native Japanese and English speakers judging the similarity of emotion terms. The results sharpened earlier studies that showed that both cultures appear to have very similar cognitive representations of emotion terms.

Aggregation of Culture Expanded Human Mesenchymal Stem Cells in Microcarrier-based Bioreactor.

PubMed

Yuan, Xuegang; Tsai, Ang-Chen; Farrance, Iain; Rowley, Jon; Ma, Teng

2018-03-15

Three-dimensional aggregation of human mesenchymal stem cells (hMSCs) has been used to enhance their therapeutic properties but current fabrication protocols depend on laboratory methods and are not scalable. In this study, we developed thermal responsive poly(N-isopropylacrylamide) grafted microcarriers (PNIPAM-MCs), which supported expansion and thermal detachment of hMSCs at reduced temperature (23.0 °C). hMSCs were cultured on the PNIPAM-MCs in both spinner flask (SF) and PBS Vertical-Wheel (PBS-VW) bioreactors for expansion. At room temperature, hMSCs were detached as small cell sheets, which subsequently self-assembled into 3D hMSC aggregates in PBS-VW bioreactor and remain as single cells in SF bioreactor owing to different hydrodynamic conditions. hMSC aggregates generated from the bioreactor maintained comparable immunomodulation and cytokine secretion properties compared to the ones made from the AggreWell ® . The results of the current study demonstrate the feasibility of scale-up production of hMSC aggregates in the suspension bioreactor using thermal responsive microcarriers for integrated cell expansion and 3D aggregation in a close bioreactor system and highlight the critical role of hydrodynamics in self-assembly of detached hMSC in suspension.

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration

PubMed Central

2012-01-01

Introduction Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration. Methods Human and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined. Results Aggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs. Conclusions Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment. PMID:22676383

Evolution and role of corded cell aggregation in Mycobacterium tuberculosis cultures.

PubMed

Caceres, Neus; Vilaplana, Cristina; Prats, Clara; Marzo, Elena; Llopis, Isaac; Valls, Joaquim; Lopez, Daniel; Cardona, Pere-Joan

2013-11-01

The aim of this study was to evaluate the evolution and role of corded cell aggregation in Mycobacterium tuberculosis cultures according to growth time and conditions. Thus, in standard culture using aerated 7H9 Middlebrook broth supplemented with 0.05% Tween 80, a dramatic CFU decrease was observed at the end of the exponential phase. This phase was followed by a stable stationary phase that led to dissociation between the optical density (O.D.) and CFU values, together with the formation of opaque colonies in solid culture. Further analysis revealed that this was due to cording. Scanning electron microscopy showed that cording led to the formation of very stable coiled structures and corded cell aggregations which proved impossible to disrupt by any of the physical means tested. Modulation of cording with a high but non-toxic concentration of Tween 80 led to a slower growth rate, avoidance of a sudden drop-off to the stationary phase, the formation of weaker cording structures and the absence of opaque colonies, together with a lower survival at later time-points. An innovative automated image analysis technique has been devised to characterize the cording process. This analysis has led to important practical consequences for the elaboration of M. tuberculosis inocula and suggests the importance of biofilm formation in survival of the bacilli in the extracellular milieu. Copyright © 2013. Published by Elsevier Ltd.

Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions

PubMed Central

Gardner, Jameson K.; Herbst-Kralovetz, Melissa M.

2016-01-01

The key to better understanding complex virus-host interactions is the utilization of robust three-dimensional (3D) human cell cultures that effectively recapitulate native tissue architecture and model the microenvironment. A lack of physiologically-relevant animal models for many viruses has limited the elucidation of factors that influence viral pathogenesis and of complex host immune mechanisms. Conventional monolayer cell cultures may support viral infection, but are unable to form the tissue structures and complex microenvironments that mimic host physiology and, therefore, limiting their translational utility. The rotating wall vessel (RWV) bioreactor was designed by the National Aeronautics and Space Administration (NASA) to model microgravity and was later found to more accurately reproduce features of human tissue in vivo. Cells grown in RWV bioreactors develop in a low fluid-shear environment, which enables cells to form complex 3D tissue-like aggregates. A wide variety of human tissues (from neuronal to vaginal tissue) have been grown in RWV bioreactors and have been shown to support productive viral infection and physiological meaningful host responses. The in vivo-like characteristics and cellular features of the human 3D RWV-derived aggregates make them ideal model systems to effectively recapitulate pathophysiology and host responses necessary to conduct rigorous basic science, preclinical and translational studies. PMID:27834891

Cluster-cluster aggregation with particle replication and chemotaxy: a simple model for the growth of animal cells in culture

NASA Astrophysics Data System (ADS)

Alves, S. G.; Martins, M. L.

2010-09-01

Aggregation of animal cells in culture comprises a series of motility, collision and adhesion processes of basic relevance for tissue engineering, bioseparations, oncology research and in vitro drug testing. In the present paper, a cluster-cluster aggregation model with stochastic particle replication and chemotactically driven motility is investigated as a model for the growth of animal cells in culture. The focus is on the scaling laws governing the aggregation kinetics. Our simulations reveal that in the absence of chemotaxy the mean cluster size and the total number of clusters scale in time as stretched exponentials dependent on the particle replication rate. Also, the dynamical cluster size distribution functions are represented by a scaling relation in which the scaling function involves a stretched exponential of the time. The introduction of chemoattraction among the particles leads to distribution functions decaying as power laws with exponents that decrease in time. The fractal dimensions and size distributions of the simulated clusters are qualitatively discussed in terms of those determined experimentally for several normal and tumoral cell lines growing in culture. It is shown that particle replication and chemotaxy account for the simplest cluster size distributions of cellular aggregates observed in culture.

Design, synthesis of novel tryptophan derivatives for antiplatelet aggregation activity based on tripeptide pENW (pGlu-Asn-Trp).

PubMed

Xie, Zhouling; Feng, Sen; Wang, Ying; Cao, Chen; Huang, Jing; Chen, Yahui; Kong, Yi; Li, Zhiyu

2015-09-18

pENW, a three mer peptide derived from Agkistrodon acutus Guenther venom, has been found to be an antagonist of the GPIIb/IIIa receptor and shows antiplatelet aggregation activity. Based on pENW and a GPIIb/IIIa inhibitor Tirofiban, a series of tryptophan derivatives were designed, synthesized and evaluated for their antiplatelet aggregation activity induced by ADP. The most potent compound 87 was also tested for the bleeding time and antithrombotic activity in vivo in comparison with Tirofiban. The results indicated that 87 shows similar antiplatelet aggregation activity as Tirofiban to the aggregation of platelet induced by all of the four agonists, but has lower bleeding risk than Tirofiban, representing a promising lead compound for further study. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Plasticity and Aggregation of Juvenile Porcine Islets in Modified Culture: Preliminary Observations.

PubMed

Weegman, Bradley P; Taylor, Michael J; Baicu, Simona C; Mueller, Kate; O'brien, Timothy D; Wilson, John; Papas, Klearchos K

2016-10-01

Diabetes is a major health problem worldwide, and there is substantial interest in developing xenogeneic islet transplantation as a potential treatment. The potential to relieve the demand on an inadequate supply of human pancreata is dependent upon the efficiency of techniques for isolating and culturing islets from the source pancreata. Porcine islets are favored for xenotransplantation, but mature pigs (>2 years) present logistic and economic challenges, and young pigs (3-6 months) have not yet proven to be an adequate source. In this study, islets were isolated from 20 juvenile porcine pancreata (~3 months; 25 kg Yorkshire pigs) immediately following procurement or after 24 h of hypothermic machine perfusion (HMP) preservation. The resulting islet preparations were characterized using a battery of tests during culture in silicone rubber membrane flasks. Islet biology assessment included oxygen consumption, insulin secretion, histopathology, and in vivo function. Islet yields were highest from HMP-preserved pancreata (2,242 ± 449 IEQ/g). All preparations comprised a high proportion (>90%) of small islets (<100 μm), and purity was on average 63 ± 6%. Morphologically, islets appeared as clusters on day 0, loosely disaggregated structures at day 1, and transitioned to aggregated structures comprising both exocrine and endocrine cells by day 6. Histopathology confirmed both insulin and glucagon staining in cultures and grafts excised after transplantation in mice. Nuclear staining (Ki-67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates = 175 ± 16 nmol/min/mg DNA, and physiological function was intact by glucose stimulation after 6-8 days in culture. In vivo function was confirmed with blood glucose control achieved in nearly 50% (8/17) of transplants. Preparation and culture of juvenile porcine islets as a source for islet transplantation require specialized

Properties of concrete with tire derived aggregate and crumb rubber as a lighthweight substitute for mineral aggregates in the concrete mix

NASA Astrophysics Data System (ADS)

Siringi, Gideon Momanyi

Scrap tires continue to be a nuisance to the environment and this research proposes one way of recycling them as a lightweight aggregate which can substitute for mineral aggregates in concrete. Aggregates derived from scrap tires are often referred to as Tire Derived Aggregate (TDA). First, the focus is how much mineral aggregate can be replaced by these waste tires and how the properties of concrete are affected with the introduction of rubber. This is being mindful of the fact that for a new material to be acceptable as an engineering material, its properties and behavior has to be well understood, the materials must perform properly and be acceptable to the regulating agencies. The role played by the quantity of TDA and Crumb Rubber replacing coarse aggregate and fine aggregate respectively as well as different treatment and additives in concrete on its properties are examined. Conventional concrete (without TDA) and concrete containing TDA are compared by examining their compressive strength based on ASTM C39, workability based on ASTM C143, Splitting Tensile Strength based on ASTM C496, Modulus of Rupture (flexural strength) based on ASTM C78 and Bond strength of concrete developed with reinforcing steel based on ASTM C234.Through stress-strain plots, the rubberized concrete is compared in terms of change in ductility, toughness and Elastic Modulus. Results indicate that while replacement of mineral aggregates with TDA results in reduction in compressive strength, this may be mitigated by addition of silica fume or using a smaller size of TDA to obtain the desired strength. The greatest benefit of using TDA is in the development of a higher ductile product with lower density while utilizing recycled TDA. From the results, it is observed that 7-10% of weight of mineral aggregates can be replaced by an equal volume of TDA to produce concrete with compressive strength of up to 4000 psi (27.5 MPa). Rubberized concrete would have higher ductility and toughness with

Syntheses and characterization of novel oxoisoaporphine derivatives as dual inhibitors for cholinesterases and amyloid beta aggregation.

PubMed

Li, Yan-Ping; Ning, Fang-Xian; Yang, Meng-Bi; Li, Yong-Cheng; Nie, Min-Hua; Ou, Tian-Miao; Tan, Jia-Heng; Huang, Shi-Liang; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu

2011-05-01

A series of 3-substituted (5c-5f, 6c-6f) and 4-substituted (10a-10g) oxoisoaporphine derivatives were synthesized. It was found that all these synthetic compounds had IC50 values at micro or nano molar range for cholinesterase inhibition, and most of them could inhibit amyloid β (Aβ) self-induced aggregation with inhibition ratio from 31.8% to 57.6%. The structure-activity relationship studies revealed that the derivatives with higher selectivity on AChE also showed better inhibition on Aβ self-induced aggregation. The results from cell toxicity study indicated that most quaternary methiodide salts had higher IC50 values than the corresponding non-quaternary compounds. This study provided potentially important information for further development of oxoisoaporphine derivatives as lead compounds for the treatment of Alzheimer's disease. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Not just fractal surfaces, but surface fractal aggregates: Derivation of the expression for the structure factor and its applications

NASA Astrophysics Data System (ADS)

Besselink, R.; Stawski, T. M.; Van Driessche, A. E. S.; Benning, L. G.

2016-12-01

Densely packed surface fractal aggregates form in systems with high local volume fractions of particles with very short diffusion lengths, which effectively means that particles have little space to move. However, there are no prior mathematical models, which would describe scattering from such surface fractal aggregates and which would allow the subdivision between inter- and intraparticle interferences of such aggregates. Here, we show that by including a form factor function of the primary particles building the aggregate, a finite size of the surface fractal interfacial sub-surfaces can be derived from a structure factor term. This formalism allows us to define both a finite specific surface area for fractal aggregates and the fraction of particle interfacial sub-surfaces at the perimeter of an aggregate. The derived surface fractal model is validated by comparing it with an ab initio approach that involves the generation of a "brick-in-a-wall" von Koch type contour fractals. Moreover, we show that this approach explains observed scattering intensities from in situ experiments that followed gypsum (CaSO4 ṡ 2H2O) precipitation from highly supersaturated solutions. Our model of densely packed "brick-in-a-wall" surface fractal aggregates may well be the key precursor step in the formation of several types of mosaic- and meso-crystals.

Clinical outcomes after IVF or ICSI using human blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum.

PubMed

Itoi, Fumiaki; Asano, Yukiko; Shimizu, Masashi; Nagai, Rika; Saitou, Kanako; Honnma, Hiroyuki; Murata, Yasutaka

2017-04-01

In this study the clinical and neo-natal outcomes after transfer of blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum (SER) were compared between IVF and intracytoplasmic sperm injection (ICSI) cycles. Clinical and neo-natal outcomes of blastocysts in cycles with at least one SER metaphase II oocyte (SER + MII; SER + cycles) did not significantly differ between the two insemination methods. When SER + MII were cultured to day 5/6, fertilization, embryo cleavage and blastocyst rates were not significantly different between IVF and ICSI cycles. In vitrified-warmed blastocyst transfer cycles, the clinical pregnancy rates from SER + MII in IVF and ICSI did not significantly differ. In this study, 52 blastocysts (27 IVF and 25 ICSI) derived from SER + MII were transferred, yielding 15 newborns (5 IVF and 10 ICSI) and no malformations. Moreover, 300 blastocysts (175 IVF and 125 ICSI) derived from SER-MII were transferred, yielding 55 newborns (24 IVF and 31 ICSI cycles). Thus, blastocysts derived from SER + cycles exhibited an acceptable ongoing pregnancy rate after IVF (n = 125) or ICSI (n = 117) cycles. In conclusion, blastocysts from SER + MII in both IVF and ICSI cycles yield adequate ongoing pregnancy rates with neo-natal outcomes that do not differ from SER-MII. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Stem Cells in Aggregate Form to Enhance Chondrogenesis in Hydrogels

PubMed Central

Sridharan, BanuPriya; Lin, Staphany M.; Hwu, Alexander T.; Laflin, Amy D.; Detamore, Michael S.

2015-01-01

There are a variety of exciting hydrogel technologies being explored for cartilage regenerative medicine. Our overall goal is to explore whether using stem cells in an aggregate form may be advantageous in these applications. 3D stem cell aggregates hold great promise as they may recapitulate the in vivo skeletal tissue condensation, a property that is not typically observed in 2D culture. We considered two different stem cell sources, human umbilical cord Wharton’s jelly cells (hWJCs, currently being used in clinical trials) and rat bone marrow-derived mesenchymal stem cells (rBMSCs). The objective of the current study was to compare the influence of cell phenotype, aggregate size, and aggregate number on chondrogenic differentiation in a generic hydrogel (agarose) platform. Despite being differing cell sources, both rBMSC and hWJC aggregates were consistent in outperforming cell suspension control groups in biosynthesis and chondrogenesis. Higher cell density impacted biosynthesis favorably, and the number of aggregates positively influenced chondrogenesis. Therefore, we recommend that investigators employing hydrogels consider using cells in an aggregate form for enhanced chondrogenic performance. PMID:26719986

Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells

PubMed Central

1984-01-01

The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the

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

PubMed

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

1990-01-01

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

Circulating platelet aggregates damage endothelial cells in culture.

PubMed

Aluganti Narasimhulu, Chandrakala; Nandave, Mukesh; Bonilla, Diana; Singaravelu, Janani; Sai-Sudhakar, Chittoor B; Parthasarathy, Sampath

2017-06-01

Presence of circulating endothelial cells (CECs) in systemic circulation may be an indicator of endothelial damage and/or denudation, and the body's response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt/denude the endothelium and contribute to the presence of CEC and EC-derived particles (ECDP). Endothelial cells were grown in glass tubes and tagged with/without 0.5 μm fluorescent beads. These glass tubes were connected to a mini-pump variable-flow system to study the effect of circulating AgPlts on the endothelium. ECs in glass tube were exposed to medium alone, nonaggregated platelets (NAgPlts), AgPlts, and 90 micron polystyrene beads at a flow rate of 20 mL/min for various intervals. Collected effluents were cultured for 72 h to analyze the growth potential of dislodged but intact ECs. Endothelial damage was assessed by real time polymerase chain reaction (RT-PCR) for inflammatory genes and Western blot analysis for von Willebrand factor. No ECs and ECDP were observed in effluents collected after injecting medium alone and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs, releasing ECs and ECDP in effluents as the time increased. Effluents collected when endothelial cell damage was seen showed increased presence of von Willebrand factor as compared to control effluents. Furthermore, we analyzed the presence of ECs and ECDPs in heart failure subjects, as well as animal plasma samples. Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelium damage. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantification of the aggregation of magnetic nanoparticles with different polymeric coatings in cell culture medium

NASA Astrophysics Data System (ADS)

Eberbeck, D.; Kettering, M.; Bergemann, C.; Zirpel, P.; Hilger, I.; Trahms, L.

2010-10-01

The knowledge of the physico-chemical characteristics of magnetic nanoparticles (MNPs) is essential to enhance the efficacy of MNP-based therapeutic treatments (e.g. magnetic heating, magnetic drug targeting). According to the literature, the MNP uptake by cells may depend on the coating of MNPs, the surrounding medium as well as on the aggregation behaviour of the MNPs. Therefore, in this study, the aggregation behaviour of MNPs in various media was investigated. MNPs with different coatings were suspended in cell culture medium (CCM) containing fetal calf serum (FCS) and the distribution of the hydrodynamic sizes was measured by magnetorelaxometry (MRX). FCS as well as bovine serum albumin (BSA) buffer (phosphate buffered saline with 0.1% bovine serum albumin) may induce MNP aggregation. Its strength depends crucially on the type of coating. The degree of aggregation in CCM depends on its FCS content showing a clear, local maximum at FCS concentrations, where the IgG concentration (part of FCS) is of the order of the MNP number concentration. Thus, we attribute the observed aggregation behaviour to the mechanism of agglutination of MNPs by serum compartments as for example IgG. No aggregation was induced for MNPs coated with dextran, polyarabic acid or sodium phosphate, respectively, which were colloidally stable in CCM.

Azaphilones inhibit tau aggregation and dissolve tau aggregates in vitro.

PubMed

Paranjape, Smita R; Riley, Andrew P; Somoza, Amber D; Oakley, C Elizabeth; Wang, Clay C C; Prisinzano, Thomas E; Oakley, Berl R; Gamblin, T Chris

2015-05-20

The aggregation of the microtubule-associated protein tau is a seminal event in many neurodegenerative diseases, including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activities. We have previously screened Aspergillus nidulans secondary metabolites for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol. One aggregation inhibitor identified was asperbenzaldehyde, an intermediate in azaphilone biosynthesis. We therefore tested 11 azaphilone derivatives to determine their tau assembly inhibition properties in vitro. All compounds tested inhibited tau filament assembly to some extent, and four of the 11 compounds had the advantageous property of disassembling preformed tau aggregates in a dose-dependent fashion. The addition of these compounds to the tau aggregates reduced both the total length and number of tau polymers. The most potent compounds were tested in in vitro reactions to determine whether they interfere with tau's normal function of stabilizing microtubules (MTs). We found that they did not completely inhibit MT assembly in the presence of tau. These derivatives are very promising lead compounds for tau aggregation inhibitors and, more excitingly, for compounds that can disassemble pre-existing tau filaments. They also represent a new class of anti-tau aggregation compounds with a novel structural scaffold.

Marine Synechococcus Aggregation

NASA Astrophysics Data System (ADS)

Neuer, S.; Deng, W.; Cruz, B. N.; Monks, L.

2016-02-01

Cyanobacteria are considered to play an important role in the oceanic biological carbon pump, especially in oligotrophic regions. But as single cells are too small to sink, their carbon export has to be mediated by aggregate formation and possible consumption by zooplankton producing sinking fecal pellets. Here we report results on the aggregation of the ubiquitous marine pico-cyanobacterium Synechococcus as a model organism. We first investigated the mechanism behind such aggregation by studying the potential role of transparent exopolymeric particles (TEP) and the effects of nutrient (nitrogen or phosphorus) limitation on the TEP production and aggregate formation of these pico-cyanobacteria. We further studied the aggregation and subsequent settling in roller tanks and investigated the effects of the clays kaolinite and bentonite in a series of concentrations. Our results show that despite of the lowered growth rates, Synechococcus in nutrient limited cultures had larger cell-normalized TEP production, formed a greater volume of aggregates, and resulted in higher settling velocities compared to results from replete cultures. In addition, we found that despite their small size and lack of natural ballasting minerals, Synechococcus cells could still form aggregates and sink at measureable velocities in seawater. Clay minerals increased the number and reduced the size of aggregates, and their ballasting effects increased the sinking velocity and carbon export potential of aggregates. In comparison with the Synechococcus, we will also present results of the aggregation of the pico-cyanobacterium Prochlorococcus in roller tanks. These results contribute to our understanding in the physiology of marine Synechococcus as well as their role in the ecology and biogeochemistry in oligotrophic oceans.

A model for the kinetics of homotypic cellular aggregation under static conditions

NASA Technical Reports Server (NTRS)

Neelamegham, S.; Munn, L. L.; Zygourakis, K.; McIntire, L. V. (Principal Investigator)

1997-01-01

We present the formulation and testing of a mathematical model for the kinetics of homotypic cellular aggregation. The model considers cellular aggregation under no-flow conditions as a two-step process. Individual cells and cell aggregates 1) move on the tissue culture surface and 2) collide with other cells (or aggregates). These collisions lead to the formation of intercellular bonds. The aggregation kinetics are described by a system of coupled, nonlinear ordinary differential equations, and the collision frequency kernel is derived by extending Smoluchowski's colloidal flocculation theory to cell migration and aggregation on a two-dimensional surface. Our results indicate that aggregation rates strongly depend upon the motility of cells and cell aggregates, the frequency of cell-cell collisions, and the strength of intercellular bonds. Model predictions agree well with data from homotypic lymphocyte aggregation experiments using Jurkat cells activated by 33B6, an antibody to the beta 1 integrin. Since cell migration speeds and all the other model parameters can be independently measured, the aggregation model provides a quantitative methodology by which we can accurately evaluate the adhesivity and aggregation behavior of cells.

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

PubMed Central

Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

2016-01-01

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

Aggregation-Induced Emission of Multiphenyl-Substituted 1,3-Butadiene Derivatives: Synthesis, Property and Application.

PubMed

Zhang, Yahui; Mao, Huiling; Xu, Weiquan; Shi, Jianbing; Cai, Zhengxu; Tong, Bin; Dong, Yuping

2018-05-29

Organic functional materials, including conjugated molecules and fluorescent dyes, have been rapidly developed in recent years because they can be applied in many fields, such as solar cells, biosensing and bioimaging, and medical adjuvant therapy. Organic functional materials with aggregation-induced emission or aggregation-enhanced emission (AIE/AEE) characteristics have increasingly attracted attention due to their high quantum efficiency in the aggregated or solid state. A large variety of AIE/AEE materials have been designed and applied during the exponential growth of research interest in the abovementioned fields. Multiphenyl-substituted 1,3-butadiene (MPB), as a core structure that includes tetraphenyl-1,3-butadiene, hexaphenyl-1,3-butadiene and their derivatives, show a typical AIE/AEE feature and can be potentially used in all the abovementioned fields. This review summarizes the design principles, the corresponding syntheses, and the structure-property relationships of MPBs, as well as their excellent innovative functionalities and applications. This review will be very useful for scientists conducting chemistry, materials, and biomedical research in AIE/AEE-related fields. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells.

PubMed

Piotrowski-Daspit, Alexandra S; Simi, Allison K; Pang, Mei-Fong; Tien, Joe; Nelson, Celeste M

2017-01-01

Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

Method for Producing Non-Neoplastic, Three Dimensional, Mammalian Tissue and Cell Aggregates Under Microgravity Culture Conditions and the Products Produced Therefrom

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tracey L. (Inventor)

1996-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural, and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Resource recycling through artificial lightweight aggregates from sewage sludge and derived ash using boric acid flux to lower co-melting temperature.

PubMed

Hu, Shao-Hua; Hu, Shen-Chih; Fu, Yen-Pei

2012-02-01

This study focuses on artificial lightweight aggregates (ALWAs) formed from sewage sludge and ash at lowered co-melting temperatures using boric acid as the fluxing agent. The weight percentages of boric acid in the conditioned mixtures of sludge and ash were 13% and 22%, respectively. The ALWA derived from sewage sludge was synthesized under the following conditions: preheating at 400 degrees C 0.5 hr and a sintering temperature of 850 degrees C 1 hr. The analytical results of water adsorption, bulk density, apparent porosity, and compressive strength were 3.88%, 1.05 g/cm3, 3.93%, and 29.7 MPa, respectively. Scanning electron microscope (SEM) images of the ALWA show that the trends in water adsorption and apparent porosity were opposite to those of bulk density. This was due to the inner pores being sealed off by lower-melting-point material at the aggregates'surface. In the case of ash-derived aggregates, water adsorption, bulk density, apparent porosity, and compressive strength were 0.82%, 0.91 g/cm3, 0.82%, and 28.0 MPa, respectively. Both the sludge- and ash-derived aggregates meet the legal standards for ignition loss and soundness in Taiwan for construction or heat insulation materials.

The Cleavage Effect of Mesenchymal Stem Cell and Its Derived Matrix Metalloproteinase‐2 on Extracellular α‐Synuclein Aggregates in Parkinsonian Models

PubMed Central

Oh, Se Hee; Kim, Ha Na; Park, Hyun Jung; Shin, Jin Young; Kim, Dong Yeol

2016-01-01

Abstract Ample evidence has suggested that extracellular α‐synuclein aggregates would play key roles in the pathogenesis and progression of Parkinsonian disorders (PDs). In the present study, we investigated whether mesenchymal stem cells (MSCs) and their derived soluble factors could exert neuroprotective effects via proteolysis of extracellular α‐synuclein. When preformed α‐synuclein aggregates were incubated with MSC‐conditioned medium, α‐synuclein aggregates were disassembled, and insoluble and oligomeric forms of α‐synuclein were markedly decreased, thus leading to a significant increase in neuronal viability. In an animal study, MSC or MSC‐conditioned medium treatment decreased the expression of α‐synuclein oligomers and the induction of pathogenic α‐synuclein with an attenuation of apoptotic cell death signaling. Furthermore, we identified that matrix metalloproteinase‐2 (MMP‐2), a soluble factor derived from MSCs, played an important role in the degradation of extracellular α‐synuclein. Our data demonstrated that MSCs and their derived MMP‐2 exert neuroprotective properties through proteolysis of aggregated α‐synuclein in PD‐related microenvironments. Stem Cells Translational Medicine 2017;6:949–961 PMID:28297586

Engineered cartilage using primary chondrocytes cultured in a porous cartilage-derived matrix

PubMed Central

Cheng, Nai-Chen; Estes, Bradley T; Young, Tai-Horng; Guilak, Farshid

2011-01-01

Aim To investigate the cell growth, matrix accumulation and mechanical properties of neocartilage formed by human or porcine articular chondrocytes on a porous, porcine cartilage-derived matrix (CDM) for use in cartilage tissue engineering. Materials & methods We examined the physical properties, cell infiltration and matrix accumulation in different formulations of CDM and selected a CDM made of homogenized cartilage slurry as an appropriate scaffold for long-term culture of human and porcine articular chondrocytes. Results The CDM scaffold supported growth and proliferation of both human and porcine chondrocytes. Histology and immunohistochemistry showed abundant cartilage-specific macromolecule deposition at day 28. Human chondrocytes migrated throughout the CDM, showing a relatively homogeneous distribution of new tissue accumulation, whereas porcine chondrocytes tended to form a proteoglycan-rich layer primarily on the surfaces of the scaffold. Human chondrocyte-seeded scaffolds had a significantly lower aggregate modulus and hydraulic permeability at day 28. Conclusions These data show that a scaffold derived from native porcine articular cartilage can support neocartilage formation in the absence of exogenous growth factors. The overall characteristics and properties of the constructs depend on factors such as the concentration of CDM used, the porosity of the scaffold, and the species of chondrocytes. PMID:21175289

J-aggregation of a water-soluble tetracationic porphyrin in mixed LB films with a calix[8]arene carboxylic acid derivative.

PubMed

Miguel, Gustavo de; Pérez-Morales, Marta; Martín-Romero, María T; Muñoz, Eulogia; Richardson, Tim H; Camacho, Luis

2007-03-27

The molecular organization of a mixed film, containing a water-soluble tetracationic porphyrin (TMPyP) and a p-tert-butyl calix[8]arene octacarboxylic acid derivative (C8A), at the air-water interface and on a solid support (LB film), has been investigated. Although the TMPyP aggregation was not detected at the air-water interface, TMPyP J-aggregates have been found in the LB films (Y-type). Unlike tetraanionic porphyrins, for example TSPP, the TMPyP J-aggregates are not induced by a zwitterion formation. The TMPyP J-aggregation is a result of a "double comb" configuration, where porphyrins from opposite layers are interwoven in a linear infinite J-aggregate. Our results confirm that TMPyP molecules tend to self-aggregate strongly, provided the electrostatic repulsions of their peripheral groups are cancelled by the anionic groups of the C8A matrix.

Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas

USGS Publications Warehouse

Lu, Y.; Aguirre, A.A.; Work, Thierry M.; Balazs, G.H.; Nerurkar, V.R.; Yanagihara, R.

2000-01-01

Serial cultivation of cell lines derived from lung, testis, periorbital and tumor tissues of a green turtle (Chelonia mydas) with fibropapillomas resulted in the in vitro formation of tumor-like cell aggregates, ranging in size from 0.5 to 2.0 mm in diameter. Successful induction of tumor-like aggregates was achieved in a cell line derived from lung tissue of healthy green turtles, following inoculation with cell-free media from these tumor-bearing cell lines, suggesting the presence of a transmissible agent. Thin-section electron microscopy of the cell aggregates revealed massive collagen deposits and intranuclear naked viral particles, measuring 5095 nm in diameter. These findings, together with the morphological similarity between these tumor-like cell aggregates and the naturally occurring tumor, suggest a possible association between this novel virus and the disease. Further characterization of this small naked virus will clarify its role in etiology of green turtle fibropapilloma, a life-threatening disease of this endangered marine species.

Microencapsulation of dopamine neurons derived from human induced pluripotent stem cells.

PubMed

Konagaya, Shuhei; Iwata, Hiroo

2015-01-01

Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress. hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons. Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved. hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads. Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons. Copyright © 2014 Elsevier B.V. All rights reserved.

Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation

PubMed Central

Song, Wei; Lu, Yen-Chun; Frankel, Angela S.; An, Duo; Schwartz, Robert E.; Ma, Minglin

2015-01-01

Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies. PMID:26592180

Engaging Youth through African-Derived Dance and Culture

ERIC Educational Resources Information Center

Franklin, Kikora

2013-01-01

This article provides a brief history of African and African-derived dance and culture and highlights the physical health, dance education, historical, and cultural benefits of a school-based program that incorporates African dance as its core component. The article also includes the phases of the programming and brings attention to potential…

The cell aggregating propensity of probiotic actinobacterial isolates: isolation and characterization of the aggregation inducing peptide pheromone.

PubMed

Muthu Selvam, Ramu; Vinothini, Gopal; Palliyarai Thaiyammal, Sethuramalingam; Latha, Selvanathan; Chinnathambi, Arunachalam; Dhanasekaran, Dharumadurai; Padmanabhan, Parasuraman; Ali Alharbi, Sulaiman; Archunan, Govindaraju

2016-01-01

The auto-aggregating ability of a probiotic is a prerequisite for colonization and protection of the gastrointestinal tract, whereas co-aggregation provides a close interaction with pathogenic bacteria. Peptide pheromone mediated signaling has been studied in several systems. However, it has not yet been explored in prokaryotes, especially actinobacteria. Hence, in the present study, the diffusible aggregation promoting factor was purified from the culture supernatant of a potent actinobacterial probiont and characterized using 20 different actinobacterial cultures isolated from the gut region of chicken and goat. The results showed that the pheromone-like compound induces the aggregation propensity of treated isolates. The factor was found to be a heat stable, acidic pH resistant, low molecular weight peptide which enhances the biofilm forming ability of other actinobacterial isolates. The aggregation promoting factor represents a bacterial sex factor (pheromone) and its characterization confirms its usage in the probiotic formulation.

Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation.

PubMed

Shen, Francis H; Werner, Brian C; Liang, Haixiang; Shang, Hulan; Yang, Ning; Li, Xudong; Shimer, Adam L; Balian, Gary; Katz, Adam J

2013-01-01

Healthy mammalian cells in normal tissues are organized in complex three-dimensional (3D) networks that display nutrient and signaling gradients. Conventional techniques that grow cells in a 2D monolayer fail to reproduce the environment that is observed in vivo. In recent years, 3D culture systems have been used to mimic tumor microenvironments in cancer research and to emulate embryogenesis in stem cell cultures. However, there have been no studies exploring the ability for adipose-derived stromal (ADS) cells in a 3D culture system to undergo osteogenic differentiation. To characterize and investigate the in vitro and in vivo potential for human ADS cells in a novel 3D culture system to undergo osteogenic differentiation. Basic science and laboratory study. Human ADS cells were isolated and prepared as either a 2D monolayer or 3D multicellular aggregates (MAs). Multicellular aggregates were formed using the hanging droplet technique. Cells were treated in osteogenic medium in vitro, and cellular differentiation was investigated using gene expression, histology, and microCT at 1-, 2-, and 4-week time points. In vivo investigation involved creating a muscle pouch by developing the avascular muscular interval in the vastus lateralis of male athymic rats. Specimens were then pretreated with osteogenic medium and surgically implanted as (1) carrier (Matrigel) alone (control), (2) carrier with human ADS cells in monolayer, or (3) human ADS cells as MAs. In vivo evidence of osteogenic differentiation was evaluated with micro computed tomography and histologic sectioning at a 2-week time point. Human ADS cells cultured by the hanging droplet technique successfully formed MAs at the air-fluid interface. Adipose-derived stromal cells cultured in monolayer or as 3D MAs retain their ability to self-replicate and undergo multilineage differentiation as confirmed by increased runx2/Cbfa2, ALP, and OCN and increased matrix mineralization on histologic sectioning

Engineering a fibrocartilage spectrum through modulation of aggregate redifferentiation.

PubMed

Murphy, Meghan K; Masters, Taylor E; Hu, Jerry C; Athanasiou, Kyriacos A

2015-01-01

Expanded costochondral cells provide a clinically relevant cell source for engineering both fibrous and hyaline articular cartilage. Expanding chondrocytes in a monolayer results in a shift toward a proliferative, fibroblastic phenotype. Three-dimensional aggregate culture may, however, be used to recover chondrogenic matrix production. This study sought to engineer a spectrum of fibrous to hyaline neocartilage from a single cell source by varying the duration of three-dimensional culture following expansion. In third passage porcine costochondral cells, the effects of aggregate culture duration were assessed after 0, 8, 11, 14, and 21 days of aggregate culture and after 4 subsequent weeks of neocartilage formation. Varying the duration of aggregate redifferentiation generated a spectrum of fibrous to hyaline neocartilage. Within 8 days of aggregation, proliferation ceased, and collagen and glycosaminoglycan production increased, compared with monolayer cells. In self-assembled neocartilage, type II-to-I collagen ratio increased with increasing aggregate duration, yet glycosaminoglycan content varied minimally. Notably, 14 days of aggregate redifferentiation increased collagen content by 25%, tensile modulus by over 110%, and compressive moduli by over 50%, compared with tissue formed in the absence of redifferentiation. A spectrum of fibrous to hyaline cartilage was generated using a single, clinically relevant cell source, improving the translational potential of engineered cartilage.

Engineering a Fibrocartilage Spectrum Through Modulation of Aggregate Redifferentiation

PubMed Central

Murphy, Meghan K.; Masters, Taylor E.; Hu, Jerry C.; Athanasiou, Kyriacos A.

2015-01-01

Expanded costochondral cells provide a clinically relevant cell source for engineering both fibrous and hyaline articular cartilage. Expanding chondrocytes in monolayer results in a shift toward a proliferative, fibroblastic phenotype. Three-dimensional aggregate culture may, however, be used to recover chondrogenic matrix production. This study sought to engineer a spectrum of fibrous to hyaline neocartilage from a single cell source by varying the duration of three-dimensional culture following expansion. In third passage porcine costochondral cells, the effects of aggregate culture duration were assessed after 0, 8, 11, 14, and 21 days of aggregate culture and after 4 subsequent weeks of neocartilage formation. Varying the duration of aggregate redifferentiation generated a spectrum of fibrous to hyaline neocartilage. Within 8 days of aggregation, proliferation ceased, and collagen and glycosaminoglycan production increased, compared with monolayer cells. In self-assembled neocartilage, type II to I collagen ratio increased with increasing aggregate duration, yet glycosaminoglycan content varied minimally. Notably, 14 days of aggregate redifferentiation increased collagen content by 25%, tensile modulus by over 110%, and compressive moduli by over 50%, compared with tissue formed in the absence of redifferentiation. A spectrum of fibrous to hyaline cartilage was generated using a single, clinically relevant cell source, improving the translational potential of engineered cartilage. PMID:24380383

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

PubMed

Bai, Guang-Yu; Song, Si-Hang; Wang, Zhen-Dong; Shan, Zhi-Yan; Sun, Rui-Zhen; Liu, Chun-Jia; Wu, Yan-Shuang; Li, Tong; Lei, Lei

2016-04-01

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study. © 2016 Japanese Society of Developmental Biologists.

A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.

PubMed

Carterson, A J; Höner zu Bentrup, K; Ott, C M; Clarke, M S; Pierson, D L; Vanderburg, C R; Buchanan, K L; Nickerson, C A; Schurr, M J

2005-02-01

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.

Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes.

PubMed

Ishii, Misawa Niki; Yamamoto, Koji; Shoji, Masanobu; Asami, Asano; Kawamata, Yuji

2017-08-15

Accurate risk assessment for drug-induced seizure is expected to be performed before entering clinical studies because of its severity and fatal damage to drug development. Induced pluripotent stem cell (iPSC) technology has allowed the use of human neurons and glial cells in toxicology studies. Recently, several studies showed the advantage of co-culture system of human iPSC (hiPSC)-derived neurons with rodent/human primary astrocytes regarding neuronal functions. However, the application of hiPSC-derived neurons for seizure risk assessment has not yet been fully addressed, and not at all when co-cultured with hiPSC-derived astrocytes. Here, we characterized hiPSC-derived neurons co-cultured with hiPSC-derived astrocytes to discuss how hiPSC-derived neurons are useful to assess seizure risk of drugs. First, we detected the frequency of spikes and synchronized bursts hiPSC-derived neurons when co-cultured with hiPSC-derived astrocytes for 8 weeks. This synchronized burst was suppressed by the treatment with 6-cyano-7-nitroquinoxaline-2,3-dione, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, and D-(-)-2-amino-5-phosphonopentanoic acid, an N-Methyl-d-aspartate (NMDA) receptor antagonist. These data suggested that co-cultured hiPSC-derived neurons formed synaptic connections mediated by AMPA and NMDA receptors. We also demonstrated that co-cultured hiPSC-derived neurons showed epileptiform activity upon treatment with gabazine or kaliotoxin. Finally, we performed single-cell transcriptome analysis in hiPSC-derived neurons and found that hiPSC-derived astrocytes activated the pathways involved in the activities of AMPA and NMDA receptor functions, neuronal polarity, and axon guidance in hiPSC-derived neurons. These data suggested that hiPSC-derived astrocytes promoted the development of action potential, synaptic functions, and neuronal networks in hiPSC-derived neurons, and then these functional alterations result in the epileptiform

In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

PubMed

Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

2012-05-01

In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

PubMed

Choi, Young-Jun; Kim, Hyemin; Kim, Ji-Woo; Yoon, Seokjoo; Park, Han-Jin

2018-05-01

The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p < 0.001). Moreover, hepatocyte-specific functions, including albumin secretion and bile canaliculi formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p < 0.001). The enhanced activities of these hepatic esterases were confirmed by the cholinesterase activity assay and the increased susceptibility of HLCs to oseltamivir, which is metabolized by CES1. 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

Development of markers for cholinergic neurones in re-aggregate cultures of foetal rat whole brain in serum-containing and serum-free media: effects of triiodothyronine (T3).

PubMed Central

Atterwill, C. K.; Kingsbury, A.; Nicholls, J.; Prince, A.

1984-01-01

Development has been studied in re-aggregate cultures derived from the 16 day foetal rat brain and the effects of triiodothyronine (T3) investigated. Cultures were maintained in either a medium containing 10% serum (S+), or in serum-free culture medium (S-) or in serum-free medium containing 30nM T3. The muscarinic cholinoceptor, measured by specific binding of [3H]-quinuclidinyl benzitate ([3H]-QNB) at 9 and 14 days in vitro, was at a lower level in the serum-free cultured cells compared with those in serum-containing culture medium (S+). In cultures in the latter medium, receptor concentration at day 14 was of a similar magnitude to that in rat brain at an equivalent postnatal age. Binding increased with development from 9 to 14 days in vitro in the S+ medium but not in the S- medium. T3 treatment caused an 85% increase in [3H]-QNB binding compared with the cultures in S- medium at day 14 to a level equivalent to that found in the cells grown in S+ medium. This increase was reflected in the Bmax but not in the KD (approx. 0.1nM). Choline acetyltransferase (ChAT) activity developed more slowly in the S- medium than in the S+ medium where the specific activity approximated values obtained in vivo. T3 treatment of cultures grow in S- medium significantly enhanced the developmental rate of increase of ChAT activity. The characteristics of [3H]-choline uptake and metabolism in the cultures was examined. Uptake was strictly Na+-independent but was energy-dependent, and inhibited by 2, 4'-dinitrophenol (2, 4'-DNP) and cooling (0-4 degrees C). Neither iodoacetate nor ouabain had any effect on the amount of uptake. Hemicholinium (HC3) was a potent inhibitor of uptake (70% inhibition at 10 microM HC3). Metabolism studies showed virtually no conversion to [3H]-acetylcholine ([3H]-ACH) in reaggregates grown in either the S+, S- or T3 containing media. However, a small amount of [3H]-choline was incorporated into phosphorylcholine. T3 treatment had no effect on this metabolic

Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging

PubMed Central

Ishii, Tomohiro; Kawakami, Emiko; Endo, Kentaro; Misawa, Hidemi; Watabe, Kazuhiko

2017-01-01

TAR DNA-binding protein 43 (TDP-43) is a main constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We have previously demonstrated that adenovirus-transduced artificial TDP-43 cytoplasmic aggregates formation is enhanced by proteasome inhibition in vitro and in vivo. However, the relationship between cytoplasmic aggregate formation and cell death remains unclear. In the present study, rat neural stem cell lines stably transfected with EGFP- or Sirius-expression vectors under the control of tubulin beta III, glial fibrillary acidic protein, or 2′,3′-cyclic nucleotide 3′-phosphodiesterase promoter were differentiated into neurons, astrocytes, and oligodendrocytes, respectively, in the presence of retinoic acid. The differentiated cells were then transduced with adenoviruses expressing DsRed-tagged human wild type and C-terminal fragment TDP-43 under the condition of proteasome inhibition. Time-lapse imaging analyses revealed growing cytoplasmic aggregates in the transduced neuronal and glial cells, followed by collapse of the cell. The aggregates remained insoluble in culture media, consisted of sarkosyl-insoluble granular materials, and contained phosphorylated TDP-43. Moreover, the released aggregates were incorporated into neighboring neuronal cells, suggesting cell-to-cell spreading. The present study provides a novel tool for analyzing the detailed molecular mechanisms of TDP-43 proteinopathy in vitro. PMID:28599005

Curcumin Pyrazole and its derivative (N-(3-Nitrophenylpyrazole) Curcumin inhibit aggregation, disrupt fibrils and modulate toxicity of Wild type and Mutant α-Synuclein

PubMed Central

Ahsan, Nuzhat; Mishra, Satyendra; Jain, Manish Kumar; Surolia, Avadhesha; Gupta, Sarika

2015-01-01

Accumulating evidence suggests that deposition of neurotoxic α-synuclein aggregates in the brain during the development of neurodegenerative diseases like Parkinson’s disease can be curbed by anti-aggregation strategies that either disrupt or eliminate toxic aggregates. Curcumin, a dietary polyphenol exhibits anti-amyloid activity but the use of this polyphenol is limited owing to its instability. As chemical modifications in curcumin confiscate this limitation, such efforts are intensively performed to discover molecules with similar but enhanced stability and superior properties. This study focuses on the inhibitory effect of two stable analogs of curcumin viz. curcumin pyrazole and curcumin isoxazole and their derivatives against α-synuclein aggregation, fibrillization and toxicity. Employing biochemical, biophysical and cell based assays we discovered that curcumin pyrazole (3) and its derivative N-(3-Nitrophenylpyrazole) curcumin (15) exhibit remarkable potency in not only arresting fibrillization and disrupting preformed fibrils but also preventing formation of A11 conformation in the protein that imparts toxic effects. Compounds 3 and 15 also decreased neurotoxicity associated with fast aggregating A53T mutant form of α-synuclein. These two analogues of curcumin described here may therefore be useful therapeutic inhibitors for the treatment of α-synuclein amyloidosis and toxicity in Parkinson’s disease and other synucleinopathies. PMID:25985292

RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures.

PubMed

Morabito, Caterina; Steimberg, Nathalie; Mazzoleni, Giovanna; Guarnieri, Simone; Fanò-Illic, Giorgio; Mariggiò, Maria A

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

RCCS Bioreactor-Based Modelled Microgravity Induces Significant Changes on In Vitro 3D Neuroglial Cell Cultures

PubMed Central

Mazzoleni, Giovanna; Fanò-Illic, Giorgio; Mariggiò, Maria A.

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions. PMID:25654124

Delta-9-tetrahydrocannabinol accumulation, metabolism and cell-type-specific adverse effects in aggregating brain cell cultures

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

Monnet-Tschudi, Florianne; Hazekamp, Arno; Perret, Nicolas

Despite the widespread use of Cannabis as recreational drug or as medicine, little is known about its toxicity. The accumulation, metabolism and toxicity of THC were analyzed 10 days after a single treatment, and after repeated exposures during 10 days. Mixed-cell aggregate cultures of fetal rat telencephalon were used as in vitro model, as well as aggregates enriched either in neurons or in glial cells. It was found that THC accumulated preferentially in neurons, and that glia-neuron interactions decreased THC accumulation. The quantification of 11-OH-THC and of THC-COOH showed that brain aggregates were capable of THC metabolism. No cell-type differencemore » was found for the metabolite 11-OH-THC, whereas the THC-COOH content was higher in mixed-cell cultures. No cell death was found at THC concentrations of 2 {mu}M in single treatment and of 1 {mu}M and 2 {mu}M in repeated treatments. Neurons, and particularly GABAergic neurons, were most sensitive to THC. Only the GABAergic marker was affected after the single treatment, whereas the GABAergic, cholinergic and astrocytic markers were decreased after the repeated treatments. JWH 015, a CB2 receptor agonist, showed effects similar to THC, whereas ACEA, a CB1 receptor agonist, had no effect. The expression of the cytokine IL-6 was upregulated 48 h after the single treatment with 5 {mu}M of THC or JWH 015, whereas the expression of TNF-{alpha} remained unchanged. These results suggest that the adverse effects of THC were related either to THC accumulation or to cannabinoid receptor activation and associated with IL-6 upregulation.« less

Engineering cell aggregates through incorporated polymeric microparticles.

PubMed

Ahrens, Caroline C; Dong, Ziye; Li, Wei

2017-10-15

Ex vivo cell aggregates must overcome significant limitations in the transport of nutrients, drugs, and signaling proteins compared to vascularized native tissue. Further, engineered extracellular environments often fail to sufficiently replicate tethered signaling cues and the complex architecture of native tissue. Co-cultures of cells with microparticles (MPs) is a growing field directed towards overcoming many of these challenges by providing local and controlled presentation of both soluble and tethered proteins and small molecules. Further, co-cultured MPs offer a mechanism to better control aggregate architecture and even to report key characteristics of the local microenvironment such as pH or oxygen levels. Herein, we provide a brief introduction to established and developing strategies for MP production including the choice of MP materials, fabrication techniques, and techniques for incorporating additional functionality. In all cases, we emphasize the specific utility of each approach to form MPs useful for applications in cell aggregate co-culture. We review established techniques to integrate cells and MPs. We highlight those strategies that promote targeted heterogeneity or homogeneity, and we describe approaches to engineer cell-particle and particle-particle interactions that enhance aggregate stability and biological response. Finally, we review advances in key application areas of MP aggregates and future areas of development. Cell-scaled polymer microparticles (MPs) integrated into cellular aggregates have been shown to be a powerful tool to direct cell response. MPs have supported the development of healthy cartilage, islets, nerves, and vasculature by the maintenance of soluble gradients as well as by the local presentation of tethered cues and diffusing proteins and small molecules. MPs integrated with pluripotent stem cells have directed in vivo expansion and differentiation. Looking forward, MPs are expected to support both the

A zeta potential value determines the aggregate's size of penta-substituted [60]fullerene derivatives in aqueous suspension whereas positive charge is required for toxicity against bacterial cells.

PubMed

Deryabin, Dmitry G; Efremova, Ludmila V; Vasilchenko, Alexey S; Saidakova, Evgeniya V; Sizova, Elena A; Troshin, Pavel A; Zhilenkov, Alexander V; Khakina, Ekaterina A; Khakina, Ekaterina E

2015-08-08

The cause-effect relationships between physicochemical properties of amphiphilic [60]fullerene derivatives and their toxicity against bacterial cells have not yet been clarified. In this study, we report how the differences in the chemical structure of organic addends in 10 originally synthesized penta-substituted [60]fullerene derivatives modulate their zeta potential and aggregate's size in salt-free and salt-added aqueous suspensions as well as how these physicochemical characteristics affect the bioenergetics of freshwater Escherichia coli and marine Photobacterium phosphoreum bacteria. Dynamic light scattering, laser Doppler micro-electrophoresis, agarose gel electrophoresis, atomic force microscopy, and bioluminescence inhibition assay were used to characterize the fullerene aggregation behavior in aqueous solution and their interaction with the bacterial cell surface, following zeta potential changes and toxic effects. Dynamic light scattering results indicated the formation of self-assembled [60]fullerene aggregates in aqueous suspensions. The measurement of the zeta potential of the particles revealed that they have different surface charges. The relationship between these physicochemical characteristics was presented as an exponential regression that correctly described the dependence of the aggregate's size of penta-substituted [60]fullerene derivatives in salt-free aqueous suspension from zeta potential value. The prevalence of DLVO-related effects was shown in salt-added aqueous suspension that decreased zeta potential values and affected the aggregation of [60]fullerene derivatives expressed differently for individual compounds. A bioluminescence inhibition assay demonstrated that the toxic effect of [60]fullerene derivatives against E. coli cells was strictly determined by their positive zeta potential charge value being weakened against P. phosphoreum cells in an aquatic system of high salinity. Atomic force microscopy data suggested that the

Sequestration of synaptic proteins by alpha-synuclein aggregates leading to neurotoxicity is inhibited by small peptide

PubMed Central

Choi, Mal-Gi; Kim, Mi Jin; Kim, Do-Geun; Yu, Ri; Jang, You-Na

2018-01-01

α-Synuclein (α-syn) is a major component of Lewy bodies found in synucleinopathies including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). Under the pathological conditions, α-syn tends to generate a diverse form of aggregates showing toxicity to neuronal cells and able to transmit across cells. However, mechanisms by which α-syn aggregates affect cytotoxicity in neurons have not been fully elucidated. Here we report that α-syn aggregates preferentially sequester specific synaptic proteins such as vesicle-associated membrane protein 2 (VAMP2) and synaptosomal-associated protein 25 (SNAP25) through direct binding which is resistant to SDS. The sequestration effect of α-syn aggregates was shown in a cell-free system, cultured primary neurons, and PD mouse model. Furthermore, we identified a specific blocking peptide derived from VAMP2 which partially inhibited the sequestration by α-syn aggregates and contributed to reduced neurotoxicity. These results provide a mechanism of neurotoxicity mediated by α-syn aggregates and suggest that the blocking peptide interfering with the pathological role of α-syn aggregates could be useful for designing a potential therapeutic drug for the treatment of PD. PMID:29608598

Macroeconomic susceptibility, inflation, and aggregate supply

NASA Astrophysics Data System (ADS)

Hawkins, Raymond J.

2017-03-01

We unify aggregate-supply dynamics as a time-dependent susceptibility-mediated relationship between inflation and aggregate economic output. In addition to representing well various observations of inflation-output dynamics this parsimonious formalism provides a straightforward derivation of popular representations of aggregate-supply dynamics and a natural basis for economic-agent expectations as an element of inflation formation. Our formalism also illuminates questions of causality and time-correlation that challenge central banks for whom aggregate-supply dynamics is a key constraint in their goal of achieving macroeconomic stability.

New pyridine derivatives as inhibitors of acetylcholinesterase and amyloid aggregation.

PubMed

Pandolfi, Fabiana; De Vita, Daniela; Bortolami, Martina; Coluccia, Antonio; Di Santo, Roberto; Costi, Roberta; Andrisano, Vincenza; Alabiso, Francesco; Bergamini, Christian; Fato, Romana; Bartolini, Manuela; Scipione, Luigi

2017-12-01

A new series of pyridine derivatives with carbamic or amidic function has been designed and synthesized to act as cholinesterase inhibitors. The synthesized compounds were tested toward EeAChE and hAChE and toward eqBChE and hBChE. The carbamate 8 was the most potent hAChE inhibitor (IC 50  = 0.153 ± 0.016 μM) while the carbamate 11 was the most potent inhibitor of hBChE (IC 50  = 0.828 ± 0.067 μM). A molecular docking study indicated that the carbamate 8 was able to bind AChE by interacting with both CAS and PAS, in agreement with the mixed inhibition mechanism. Furthermore, the carbamates 8, 9 and 11 were able to inhibit Aβ 42 self-aggregation and possessed quite low toxicity against human astrocytoma T67 and HeLa cell lines, being the carbamate 8 the less toxic compound on both cell lines. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Growth hormone aggregates in the rat adenohypophysis

NASA Technical Reports Server (NTRS)

Farrington, M.; Hymer, W. C.

1990-01-01

Although it has been known for some time that GH aggregates are contained within the rat anterior pituitary gland, the role that they might play in pituitary function is unknown. The present study examines this issue using the technique of Western blotting, which permitted visualization of 11 GH variants with apparent mol wt ranging from 14-88K. Electroelution of the higher mol wt variants from gels followed by their chemical reduction with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. With the blot procedure we found 1) that GH aggregates greater than 44K were associated with a 40,000 x g sedimentable fraction; 2) that GH aggregates were not present in glands from thyroidectomized rats, but were in glands from the thyroidectomized rats injected with T4; 3) that GH aggregates were uniquely associated with a heavily granulated somatotroph subpopulation isolated by density gradient centrifugation; and 4) that high mol wt GH forms were released from the dense somatotrophs in culture, since treatment of the culture medium with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. Taken together, the results show that high mol wt GH aggregates are contained in secretory granules of certain somatotrophs and are also released in aggregate form from these cells in vitro.

Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification.

PubMed

Dang, Phuong N; Dwivedi, Neha; Phillips, Lauren M; Yu, Xiaohua; Herberg, Samuel; Bowerman, Caitlin; Solorio, Loran D; Murphy, William L; Alsberg, Eben

2016-02-01

Bone tissue engineering via endochondral ossification has been explored by chondrogenically priming cells using soluble mediators for at least 3 weeks to produce a hypertrophic cartilage template. Although recapitulation of endochondral ossification has been achieved, long-term in vitro culture is required for priming cells through repeated supplementation of inductive factors in the media. To address this challenge, a microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Sequential exogenous presentation of soluble transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) at various defined time courses resulted in varying degrees of chondrogenesis and osteogenesis as demonstrated by glycosaminoglycan and calcium content. The time course that best induced endochondral ossification was used to guide the development of the microparticle-based controlled delivery system for TGF-β1 and BMP-2. Gelatin microparticles capable of relatively rapid release of TGF-β1 and mineral-coated hydroxyapatite microparticles permitting more sustained release of BMP-2 were then incorporated within hMSC aggregates and cultured for 5 weeks following the predetermined time course for sequential presentation of bioactive signals. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated TGF-β1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity at week 2 and a greater degree of mineralization by week 5. Staining for types I and II collagen, osteopontin, and osteocalcin revealed the presence of cartilage and bone. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming. Significance: This study demonstrates the regulation of chondrogenesis

Intrinsic Lens Forming Potential of Mouse Lens Epithelial versus Newt Iris Pigment Epithelial Cells in Three-Dimensional Culture

PubMed Central

Nakamura, Kenta; Tsonis, Panagiotis A.

2014-01-01

Adult newts (Notophthalmus viridescens) are capable of complete lens regeneration that is mediated through dorsal iris pigment epithelial (IPE) cells transdifferentiation. In contrast, higher vertebrates such as mice demonstrate only limited lens regeneration in the presence of an intact lens capsule with remaining lens epithelial cells. To compare the intrinsic lens regeneration potential of newt IPE versus mouse lens epithelial cells (MLE), we have established a novel culture method that uses cell aggregation before culture in growth factor-reduced Matrigel™. Dorsal newt IPE aggregates demonstrated complete lens formation within 1 to 2 weeks of Matrigel culture without basic fibroblast growth factor (bFGF) supplementation, including the establishment of a peripheral cuboidal epithelial cell layer, and the appearance of central lens fibers that were positive for αA-crystallin. In contrast, the lens-forming potential of MLE cell aggregates cultured in Matrigel was incomplete and resulted in the formation of defined-size lentoids with partial optical transparency. While the peripheral cell layers of MLE aggregates were nucleated, cells in the center of aggregates demonstrated a nonapoptotic nuclear loss over a time period of 3 weeks that was representative of lens fiber formation. Matrigel culture supplementation with bFGF resulted in higher transparent bigger-size MLE aggregates that demonstrated increased appearance of βB1-crystallin expression. Our study demonstrates that bFGF is not required for induction of newt IPE aggregate-dependent lens formation in Matrigel, while the addition of bFGF seems to be beneficial for the formation of MLE aggregate-derived lens-like structures. In conclusion, the three-dimensional aggregate culture of IPE and MLE in Matrigel allows to a higher extent than older models the indepth study of the intrinsic lens-forming potential and the corresponding identification of lentogenic factors. PMID:23672748

[Design, synthesis and evaluation of bis-nicotine derivatives as inhibitors of cholinesterases and beta-amyloid aggregation].

PubMed

Luo, Wen; Zhao, Yong-mei; Tian, Run-guo; Su, Ya-bin; Hong, Chen

2013-11-01

A novel series of bis-nicotine derivatives (3a-3i) were designed, synthesized and evaluated as bivalent anti-Alzheimer's disease agents. The pharmacological results indicated that compounds 3e-3i inhibited both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the micromolar range (IC50, 2.28-117.86 micromol x L(-1) for AChE and 1.67-125 micromol x L(-1) for BChE), which was at the same potency as rivastigmine. A Lineweaver-Burk plot and molecular modeling study showed that these derivatives targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, these compounds could significantly inhibit the self-induced Abeta aggregation with inhibition activity (11.85%-62.14%) at the concentration of 20 micromol x L(-1).

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

PubMed Central

Gärdes, Astrid; Iversen, Morten H; Grossart, Hans-Peter; Passow, Uta; Ullrich, Matthias S

2011-01-01

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump. PMID:20827289

Multitarget Therapeutic Leads for Alzheimer's Disease: Quinolizidinyl Derivatives of Bi- and Tricyclic Systems as Dual Inhibitors of Cholinesterases and β-Amyloid (Aβ) Aggregation.

PubMed

Tonelli, Michele; Catto, Marco; Tasso, Bruno; Novelli, Federica; Canu, Caterina; Iusco, Giovanna; Pisani, Leonardo; Stradis, Angelo De; Denora, Nunzio; Sparatore, Anna; Boido, Vito; Carotti, Angelo; Sparatore, Fabio

2015-06-01

Multitarget therapeutic leads for Alzheimer's disease were designed on the models of compounds capable of maintaining or restoring cell protein homeostasis and of inhibiting β-amyloid (Aβ) oligomerization. Thirty-seven thioxanthen-9-one, xanthen-9-one, naphto- and anthraquinone derivatives were tested for the direct inhibition of Aβ(1-40) aggregation and for the inhibition of electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBChE). These compounds are characterized by basic side chains, mainly quinolizidinylalkyl moieties, linked to various bi- and tri-cyclic (hetero)aromatic systems. With very few exceptions, these compounds displayed inhibitory activity on both AChE and BChE and on the spontaneous aggregation of β-amyloid. In most cases, IC50 values were in the low micromolar and sub-micromolar range, but some compounds even reached nanomolar potency. The time course of amyloid aggregation in the presence of the most active derivative (IC50 =0.84 μM) revealed that these compounds might act as destabilizers of mature fibrils rather than mere inhibitors of fibrillization. Many compounds inhibited one or both cholinesterases and Aβ aggregation with similar potency, a fundamental requisite for the possible development of therapeutics exhibiting a multitarget mechanism of action. The described compounds thus represent interesting leads for the development of multitarget AD therapeutics. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.

PubMed

Han, Yi; Liu, Xing-Mao; Liu, Hong; Li, Shi-Chong; Wu, Ben-Chuan; Ye, Ling-Ling; Wang, Qu-Wei; Chen, Zhao-Lie

2006-11-01

Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 mum after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (mu). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.

Placental-derived stem cells: Culture, differentiation and challenges

PubMed Central

Oliveira, Maira S; Barreto-Filho, João B

2015-01-01

Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice. PMID:26029347

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages.

PubMed

Jin, Xueting; Kruth, Howard S

2016-06-30

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

PubMed

Eiraku, Mototsugu; Sasai, Yoshiki

2011-12-15

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

Temporally coordinated spiking activity of human induced pluripotent stem cell-derived neurons co-cultured with astrocytes.

PubMed

Kayama, Tasuku; Suzuki, Ikuro; Odawara, Aoi; Sasaki, Takuya; Ikegaya, Yuji

2018-01-01

In culture conditions, human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks, which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons, the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study, we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

Receptors for aggregated IgG on mouse lymphocytes: their presence on thymocytes, thymus-derived, and bone marrow-derived lymphocytes.

PubMed

Anderson, C L; Grey, H M

1974-05-01

An autoradiographic binding assay employing (125)I-labeled heat-aggregated mouse IgG2b myeloma protein (MOPC 141) was used to demonstrate receptors for IgG on 20-45% of Balb/c thymocytes and on 70-80% of splenocytes. Binding could also be shown with heat or BDB aggregates of another IgG2b (MOPC 195), with IgG1 and with human gamma-globulin, but not with aggregated chicken gamma-globulin, IgA, BSA, nor with aggregated Fab fragments of IgG2b. Optimum binding was obtained at 37 degrees C. Detection of binding was dependent upon aggregate size with complexes of more than 100 IgG molecules being optimal, aggregates of 6-25 detecting splenocytes but not thymocytes, and aggregates of less than 6 binding to a negligible extent. Comparison of grain counts on various cell types showed mastocytoma cells (P815) and macrophages averaging 40-50 grains/cell/day, allogeneically activated thymocytes 20-30, splenocytes 2-3, L5178 lymphoma cells 1, and positive thymocytes 0.6 grains/cell/day. Double labeling experiments for surface Ig, theta-antigen, and agg IgG receptor on mouse spleen cells indicated that a relatively high density of receptor was present on about 80% of B cells, 30% of T cells, and 60% of SIg(-), theta(-), null cells.

Dissolution and aggregation of Cu nanoparticles in culture media: effects of incubation temperature and particles size

NASA Astrophysics Data System (ADS)

Li, Lingxiangyu; Fernández-Cruz, María Luisa; Connolly, Mona; Schuster, Michael; Navas, José María

2015-01-01

Here, the effects of incubation temperature and particle size on the dissolution and aggregation behavior of copper nanoparticles (CuNPs) in culture media were investigated over 96 h, equivalent to the time period for acute cell toxicity tests. Three CuNPs with the nominal sizes of 25, 50, and 100 nm and one type of micro-sized particles (MPs, 500 nm) were examined in culture media used for human and fish hepatoma cell lines acute tests. A large decrease in sizes of CuNPs in the culture media was observed in the first 24 h incubation, and subsequently the sizes of CuNPs changed slightly over the following 72 h. Moreover, the decreasing rate in size was significantly dependent on the incubation temperature; the higher the incubation temperature, the larger the decreasing rate in size. In addition to that, we also found that the release of copper ions depended on the incubation temperature. Moreover, the dissolution rate of Cu particles increased very fast in the first 24 h, with a slight increase over the following 72 h.

Three-dimensional spheroid culture of human gingiva-derived mesenchymal stem cells enhances mitigation of chemotherapy-induced oral mucositis.

PubMed

Zhang, Qunzhou; Nguyen, Andrew L; Shi, Shihong; Hill, Colin; Wilder-Smith, Petra; Krasieva, Tatiana B; Le, Anh D

2012-04-10

Mesenchymal stem cells (MSCs) are capable of regenerative and immunomodulatory functions in cell-based therapies in a variety of human diseases and injuries; however, their therapeutic efficacy and potential side effects remain major obstacles in clinical applications. We report here a 3D spheroid culture approach to optimize stem cell properties and therapeutic effects of human gingiva-derived mesenchymal stem cells (GMSCs) in mitigation of experimental oral mucositis. Under growth condition of ultra-low attachment, GMSCs spontaneously aggregated into 3D spheroids and exhibited distinct early stem cell phenotype characterized by elevated expression Stro-1 and CXC chemokine receptor 4 (CXCR-4) as well as OCT-4 and Nanog, 2 important transcriptional factors relevant to stem cell properties, and decreased expression of MSC-associated markers, including CD29, CD90, and CD105. Functionally, spheroid GMSCs are capable of enhanced multipotency and augmented secretion of several chemokines and cytokines relevant to cell migration, survival, and angiogenesis. More importantly, spheroid GMSCs expressed increased levels of reactive oxygen species, hypoxia-inducible factor (HIF)-1 and -2α, and manganese superoxide dismutase, which correlated with improved resistance to oxidative stress-induced apoptosis. Using an in vivo murine model of chemotherapy-induced oral mucositis, we demonstrated that spheroid-derived GMSCs possessed better therapeutic efficacy than their adherent cells in reversing body weight loss and promoting the regeneration of disrupted epithelial lining of the mucositic tongues. These findings suggest that 3D spheroid culture allows early stemness preservation and potentially precondition GMSCs for enhanced mitigation of oral mucositis. © Mary Ann Liebert, Inc.

Trivalent MDCK cell culture-derived influenza vaccine Optaflu (Novartis Vaccines).

PubMed

Doroshenko, Alexander; Halperin, Scott A

2009-06-01

Annual influenza epidemics continue to have a considerable impact in both developed and developing countries. Vaccination remains the principal measure to prevent seasonal influenza and reduce associated morbidity and mortality. The WHO recommends using established mammalian cell culture lines as an alternative to egg-based substrates in the manufacture of influenza vaccine. In June 2007, the EMEA approved Optaflu, a Madin Darby canine kidney cell culture-derived influenza vaccine manufactured by Novartis Vaccines. This review examines the advantages and disadvantages of cell culture-based technology for influenza vaccine production, compares immunogenicity and safety data for Optaflu with that of currently marketed conventional egg-based influenza vaccines, and considers the prospects for wider use of cell culture-based influenza vaccines.

Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells.

PubMed

Ikeda, Kazuhiro; Nagata, Shogo; Okitsu, Teru; Takeuchi, Shoji

2017-06-06

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

Coupling of aggregation and immunogenicity in biotherapeutics: T- and B-cell immune epitopes may contain aggregation-prone regions.

PubMed

Kumar, Sandeep; Singh, Satish K; Wang, Xiaoling; Rup, Bonita; Gill, Davinder

2011-05-01

Biotherapeutics, including recombinant or plasma-derived human proteins and antibody-based molecules, have emerged as an important class of pharmaceuticals. Aggregation and immunogenicity are among the major bottlenecks during discovery and development of biotherapeutics. Computational tools that can predict aggregation prone regions as well as T- and B-cell immune epitopes from protein sequence and structure have become available recently. Here, we describe a potential coupling between aggregation and immunogenicity: T-cell and B-cell immune epitopes in therapeutic proteins may contain aggregation-prone regions. The details of biological mechanisms behind this observation remain to be understood. However, our observation opens up an exciting potential for rational design of de-immunized novel, as well as follow on biotherapeutics with reduced aggregation propensity.

Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures.

PubMed

Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2017-09-01

Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

[Regulation of age-dependent phenomena. Influence of C6-substituted purines on cell aggregation and cell migration in primary cultures of lense epithelial cells].

PubMed

Glässer, D; Iwig, M; Weber, E

1975-01-01

The existence of an age dependent latent period of cell emigration has been proved in the primary culture of epithelial cells of bovine lenses. The previously described aggregation phenomenon as well as the latent period of the cell emigration increase with the age of the sponsor animals. Extracellular adenine and other C6-substituted purines, isolated from the cells themselves and added to the medium, act the same way on the lens cells in the primary culture as the increasing age of the sponsor animals. Adenine stimulates cell aggregation and inhibits the adhesion of the cells to the substratum, the cell flattening and the cell migration. The adenine action has been proved down to a concentration of 3 X 10(-6) M. During the primary culture, the lens cells gradually los the adenine sensitivity. The adenine action also occurs on single cells, isolated by trypsination, it differs from the reaction of ouabain and can be removed at low concentration by washing procedures. The results favour the suggestion C6-substituted purines to be involved in cell ageing.

Understanding curcumin-induced modulation of protein aggregation.

PubMed

Ahmad, Basir; Borana, Mohanish S; Chaudhary, Ankur P

2017-07-01

Curcumin, a diarylheptanoid compound, found in spice turmeric is known to alter the aggregation of proteins and reduce the toxicity of the aggregates. This review looks at the molecular basis of modulating protein aggregation and toxicity of the aggregates. Foremost, we identify the interaction of curcumin and its derivatives with proteins/peptides and the effect of their interaction on the conformational stability and unfolding/folding pathway(s). The unfolding/folding processes generate partially folded/unfolded intermediate, which serve as aggregation precursor state. Secondly, we discuss the effect of curcumin binding on the kinetics parameters of the aggregation process, which give information about the mechanism of the aggregation inhibition. We describe, in addition, that curcumin can accelerate/promote fibril formation by binding to oligomeric intermediate(s) accumulated in the aggregation pathway. Finally, we discuss the correlation of curcumin-induced monomeric and/or oligomeric precursor states with aggregate structure and toxicity. On the basis of these discussions, we propose a model describing curcumin-induced inhibition/promotion of formation of amyloid-like fibrils. Copyright © 2016 Elsevier B.V. All rights reserved.

Biogrid--a microfluidic device for large-scale enzyme-free dissociation of stem cell aggregates.

PubMed

Wallman, Lars; Åkesson, Elisabet; Ceric, Dario; Andersson, Per Henrik; Day, Kelly; Hovatta, Outi; Falci, Scott; Laurell, Thomas; Sundström, Erik

2011-10-07

Culturing stem cells as free-floating aggregates in suspension facilitates large-scale production of cells in closed systems, for clinical use. To comply with GMP standards, the use of substances such as proteolytic enzymes should be avoided. Instead of enzymatic dissociation, the growing cell aggregates may be mechanically cut at passage, but available methods are not compatible with large-scale cell production and hence translation into the clinic becomes a severe bottle-neck. We have developed the Biogrid device, which consists of an array of micrometerscale knife edges, micro-fabricated in silicon, and a manifold in which the microgrid is placed across the central fluid channel. By connecting one side of the Biogrid to a syringe or a pump and the other side to the cell culture, the culture medium with suspended cell aggregates can be aspirated, forcing the aggregates through the microgrid, and ejected back to the cell culture container. Large aggregates are thereby dissociated into smaller fragments while small aggregates pass through the microgrid unaffected. As proof-of-concept, we demonstrate that the Biogrid device can be successfully used for repeated passage of human neural stem/progenitor cells cultured as so-called neurospheres, as well as for passage of suspension cultures of human embryonic stem cells. We also show that human neural stem/progenitor cells tolerate transient pressure changes far exceeding those that will occur in a fluidic system incorporating the Biogrid microgrids. Thus, by using the Biogrid device it is possible to mechanically passage large quantities of cells in suspension cultures in closed fluidic systems, without the use of proteolytic enzymes.

Roles of CD34+ cells and ALK5 signaling in the reconstruction of seminiferous tubule-like structures in 3-D re-aggregate culture of dissociated cells from neonatal mouse testes.

PubMed

Abe, Shin-Ichi; Abe, Kazuko; Zhang, Jidong; Harada, Tomoaki; Mizumoto, Go; Oshikawa, Hiroki; Akiyama, Haruhiko; Shimamura, Kenji

2017-01-01

Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFβ signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results

Biotransformation of tryptamine derivatives in mycelial cultures of Psilocybe.

PubMed

Gartz, J

1989-01-01

Mycelial cultures of Psilocybe cubensis capable of forming psilocybin and psilocin de novo display a high capacity for hydroxylation of tryptamine derivatives at the 4-position. A specific biotransformation of added synthetic N,N-diethyl-tryptamine was found. Thus high amounts of 4-hydroxy-N,N-diethyltryptamine (up to 3.3%) and a minor quantity of 4-phosphoryloxy-N,N-diethyltryptamine (0.01-0.8%) were isolated from fruiting bodies of Psilocybe cubensis in corresponding experiments. This is the first example of a directed biosynthesis of tryptamine substances by fungi. An effective biotransformation of N-methyltryptamine was also demonstrated with surface cultures of Psilocybe semilanceata. Baeocystin, a possible natural precursor of psilocybin, was detected and quantified in the biomasses. No alkaloids could be found in the culture medium.

Dynamic self-organization of microwell-aggregated cellular mixtures.

PubMed

Song, Wei; Tung, Chih-Kuan; Lu, Yen-Chun; Pardo, Yehudah; Wu, Mingming; Das, Moumita; Kao, Der-I; Chen, Shuibing; Ma, Minglin

2016-06-29

Cells with different cohesive properties self-assemble in a spatiotemporal and context-dependent manner. Previous studies on cell self-organization mainly focused on the spontaneous structural development within a short period of time during which the cell numbers remained constant. However the effect of cell proliferation over time on the self-organization of cells is largely unexplored. Here, we studied the spatiotemporal dynamics of self-organization of a co-culture of MDA-MB-231 and MCF10A cells seeded in a well defined space (i.e. non-adherent microfabricated wells). When cell-growth was chemically inhibited, high cohesive MCF10A cells formed a core surrounded by low cohesive MDA-MB-231 cells on the periphery, consistent with the differential adhesion hypothesis (DAH). Interestingly, this aggregate morphology was completely inverted when the cells were free to grow. At an initial seeding ratio of 1 : 1 (MDA-MB-231 : MCF10A), the fast growing MCF10A cells segregated in the periphery while the slow growing MDA-MB-231 cells stayed in the core. Another morphology developed at an inequal seeding ratio (4 : 1), that is, the cell mixtures developed a side-by-side aggregate morphology. We conclude that the cell self-organization depends not only on the cell cohesive properties but also on the cell seeding ratio and proliferation. Furthermore, by taking advantage of the cell self-organization, we purified human embryonic stem cells-derived pancreatic progenitors (hESCs-PPs) from co-cultured feeder cells without using any additional tools or labels.

An Aggregate IRT Procedure for Exploratory Factor Analysis

ERIC Educational Resources Information Center

Camilli, Gregory; Fox, Jean-Paul

2015-01-01

An aggregation strategy is proposed to potentially address practical limitation related to computing resources for two-level multidimensional item response theory (MIRT) models with large data sets. The aggregate model is derived by integration of the normal ogive model, and an adaptation of the stochastic approximation expectation maximization…

Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture

PubMed Central

Modulevsky, Daniel J.; Lefebvre, Cory; Haase, Kristina; Al-Rekabi, Zeinab; Pelling, Andrew E.

2014-01-01

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment. PMID:24842603

Apple derived cellulose scaffolds for 3D mammalian cell culture.

PubMed

Modulevsky, Daniel J; Lefebvre, Cory; Haase, Kristina; Al-Rekabi, Zeinab; Pelling, Andrew E

2014-01-01

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

Evaluation of self-combustion risk in tire derived aggregate fills.

PubMed

Arroyo, Marcos; San Martin, Ignacio; Olivella, Sebastian; Saaltink, Maarten W

2011-01-01

Lightweight tire derived aggregate (TDA) fills are a proven recycling outlet for waste tires, requiring relatively low cost waste processing and being competitively priced against other lightweight fill alternatives. However its value has been marred as several TDA fills have self-combusted during the early applications of this technique. An empirical review of these cases led to prescriptive guidelines from the ASTM aimed at avoiding this problem. This approach has been successful in avoiding further incidents of self-combustion. However, at present there remains no rational method available to quantify self-combustion risk in TDA fills. This means that it is not clear which aspects of the ASTM guidelines are essential and which are accessory. This hinders the practical use of TDA fills despite their inherent advantages as lightweight fill. Here a quantitative approach to self-combustion risk evaluation is developed and illustrated with a parametric analysis of an embankment case. This is later particularized to model a reported field self-combustion case. The approach is based on the available experimental observations and incorporates well-tested methodological (ISO corrosion evaluation) and theoretical tools (finite element analysis of coupled heat and mass flow). The results obtained offer clear insights into the critical aspects of the problem, allowing already some meaningful recommendations for guideline revision. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of benzothiazoles as potential polyglutamine aggregation inhibitors of Huntington's disease by using an automated filter retardation assay

PubMed Central

Heiser, Volker; Engemann, Sabine; Bröcker, Wolfgang; Dunkel, Ilona; Boeddrich, Annett; Waelter, Stephanie; Nordhoff, Eddi; Lurz, Rudi; Schugardt, Nancy; Rautenberg, Susanne; Herhaus, Christian; Barnickel, Gerhard; Böttcher, Henning; Lehrach, Hans; Wanker, Erich E.

2002-01-01

Preventing the formation of insoluble polyglutamine containing protein aggregates in neurons may represent an attractive therapeutic strategy to ameliorate Huntington's disease (HD). Therefore, the ability to screen for small molecules that suppress the self-assembly of huntingtin would have potential clinical and significant research applications. We have developed an automated filter retardation assay for the rapid identification of chemical compounds that prevent HD exon 1 protein aggregation in vitro. Using this method, a total of 25 benzothiazole derivatives that inhibit huntingtin fibrillogenesis in a dose-dependent manner were discovered from a library of ≈184,000 small molecules. The results obtained by the filter assay were confirmed by immunoblotting, electron microscopy, and mass spectrometry. Furthermore, cell culture studies revealed that 2-amino-4,7-dimethyl-benzothiazol-6-ol, a chemical compound similar to riluzole, significantly inhibits HD exon 1 aggregation in vivo. These findings may provide the basis for a new therapeutic approach to prevent the accumulation of insoluble protein aggregates in Huntington's disease and related glutamine repeat disorders. PMID:12200548

Biomechanical properties of jaw periosteum-derived mineralized culture on different titanium topography.

PubMed

Att, Wael; Kubo, Katsutoshi; Yamada, Masahiro; Maeda, Hatsuhiko; Ogawa, Takahiro

2009-01-01

This study evaluated the biomechanical properties of periosteum-derived mineralized culture on different surface topographies of titanium. Titanium surfaces modified by machining or by acid etching were analyzed using scanning electron microscopy (SEM). Rat mandibular periosteum-derived cells were cultured on either of the titanium surfaces. Cell proliferation was evaluated by cell counts, and gene expression was analyzed using a reverse-transcriptase polymerase chain reaction. Alkaline phosphatase (ALP) stain assay was employed to evaluate osteoblastic activity. Matrix mineralization was examined via von Kossa stain assay, total calcium deposition, and SEM. The hardness and elastic modulus of mineralized cultures were measured using a nano-indenter. The machined surface demonstrated a flat topographic configuration, while the acid-etched surface revealed a uniform micron-scale roughness. Both cell density and ALP activity were significantly higher on the machined surface than on the acid-etched surface. The expression of bone-related genes was up-regulated or enhanced on the acid-etched surface compared to the machined surface. Von Kossa stain showed significantly greater positive areas for the machined surface compared to the acid-etched surface, while total calcium deposition was statistically similar. Mineralized culture on the acid-etched surface was characterized by denser calcium deposition, more mature collagen deposition on the superficial layer, and larger and denser globular matrices inside the matrix than the culture on the machined surface. The mineralized matrix on the acid-etched surface was two times harder than on the machined surface, whereas the elastic modulus was comparable between the two surfaces. The design of this study can be used as a model to evaluate the effect of implant surface topography on the biomechanical properties of periosteum-derived mineralized culture. The results suggest that mandibular periosteal cells respond to different

Spin-coated Films of Squarylium Dye J-Aggregates Exhibiting Ultrafast Optical Responses

NASA Astrophysics Data System (ADS)

Tatsuura, Satoshi; Tian, Minquan; Furuki, Makoto; Sato, Yasuhiro; Pu, Lyong Sun; Wada, Osamu

2000-08-01

The formation of J-aggregates of squarylium dye derivatives in spin-coated films is reported. Squarylium dye derivatives with dipropylamino bases are found to spontaneously aggregate in a spin-coated film. Aggregation is promoted when dye molecules are dispersed in a poly(vinyl alcohol) film, and when a spin-coated film of dye molecules is heated in the presence of acid vapor. In particular, J-aggregates formed by exposure to acetic acid vapor show the narrowest spectral width. J-aggregates formed by the acid treatment method are stable at room temperature and the spectral full-width at half maximum of the J-band is 20 nm. Optical response of the acid-treated film is confirmed to exhibit a short relaxation time of bleached absorption of 300 fs.

An optimal serum-free defined condition for in vitro culture of kidney organoids.

PubMed

Nishikawa, Masaki; Kimura, Hiroshi; Yanagawa, Naomi; Hamon, Morgan; Hauser, Peter; Zhao, Lifu; Jo, Oak D; Yanagawa, Norimoto

2018-07-02

Kidney organoid is an emerging topic of importance for research in kidney development and regeneration. Conventional culture systems for kidney organoids reported thus far use culture media containing serum, which may compromise our understanding and the potential clinical applicability of the organoid system. In our present study, we tested two serum-free culture conditions and compared their suitability for the maintenance and growth of kidney organoids in culture. One of the serum-free culture conditions was the combination of keratinocytes serum free medium (KSFM) with knockout serum replacement (KSR) (KSFM + KSR), and the other was the combination of knockout DMEM/F12 (KD/F12) and KSR (KD/F12 + KSR). With cell aggregates derived from E12.5 mouse embryonic kidneys, we found that KD/F12 + KSR was superior to KSFM + KSR in promoting the growth of the aggregate with expansion of Six2 + nephron progenitor cells (NPC) and elaborated ureteric branching morphogenesis. With KD/F12 + KSR, we found that lower concentrations of KSR at 5-10% were superior to a higher concentration (20%) in promoting the growth of aggregates without affecting the expression levels of NPC marker genes. We also found that NPC in aggregates retained their differentiation potential to develop nephron tubules through mesenchyme-to-epithelial transition (MET), after being maintained in culture under these conditions for up to 7 days. In conclusion, we have identified a defined serum-free culture condition suitable for the maintenance and growth of kidney organoids that retain the differentiation potential to develop nephron structures. This defined serum-free culture condition may serve as a useful platform for further investigation of kidney organoids in vitro. Copyright © 2018 Elsevier Inc. All rights reserved.

Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

PubMed Central

Lam, Alan Tin-Lun; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul

2014-01-01

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment. PMID:24641164

Curcumin inhibits aggregation of alpha-synuclein.

PubMed

Pandey, Neeraj; Strider, Jeffrey; Nolan, William C; Yan, Sherry X; Galvin, James E

2008-04-01

Aggregation of amyloid-beta protein (Abeta) is a key pathogenic event in Alzheimer's disease (AD). Curcumin, a constituent of the Indian spice Turmeric is structurally similar to Congo Red and has been demonstrated to bind Abeta amyloid and prevent further oligomerization of Abeta monomers onto growing amyloid beta-sheets. Reasoning that oligomerization kinetics and mechanism of amyloid formation are similar in Parkinson's disease (PD) and AD, we investigated the effect of curcumin on alpha-synuclein (AS) protein aggregation. In vitro model of AS aggregation was developed by treatment of purified AS protein (wild-type) with 1 mM Fe3+ (Fenton reaction). It was observed that the addition of curcumin inhibited aggregation in a dose-dependent manner and increased AS solubility. The aggregation-inhibiting effect of curcumin was next investigated in cell culture utilizing catecholaminergic SH-SY5Y cell line. A model system was developed in which the red fluorescent protein (DsRed2) was fused with A53T mutant of AS and its aggregation examined under different concentrations of curcumin. To estimate aggregation in an unbiased manner, a protocol was developed in which the images were captured automatically through a high-throughput cell-based screening microscope. The obtained images were processed automatically for aggregates within a defined dimension of 1-6 microm. Greater than 32% decrease in mutant alpha-synuclein aggregation was observed within 48 h subsequent to curcumin addition. Our data suggest that curcumin inhibits AS oligomerization into higher molecular weight aggregates and therefore should be further explored as a potential therapeutic compound for PD and related disorders.

Aggregation of Sea Urchin Phagocytes Is Augmented In Vitro by Lipopolysaccharide

PubMed Central

Majeske, Audrey J.; Bayne, Christopher J.; Smith, L. Courtney

2013-01-01

Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ∼10% of the cells were positive for Sp185/333 proteins. At 24 hr, ∼90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the

Aggregation of sea urchin phagocytes is augmented in vitro by lipopolysaccharide.

PubMed

Majeske, Audrey J; Bayne, Christopher J; Smith, L Courtney

2013-01-01

Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ~10% of the cells were positive for Sp185/333 proteins. At 24 hr, ~90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the

Theoretical insights into aggregation-induced helicity modulation of a perylene bisimide derivative.

PubMed

Liang, Lijun; Li, Xin

2018-02-12

Formation of helical chiroptical self-assemblies via noncovalent interaction is a widely observed phenomenon in nature, the mechanism of which remains insufficiently understood. Employing an amphiphilic perylene-sugar dyad molecule (PBI-HAG) as an example, we report that the modulatable supramolecular helicity may emerge from an aggregating process that is dominated by competition between two types of noncovalent interaction: hydrogen bonding and π-π stacking. The interplay between these two driving forces, which is greatly affected by the solvent environment, determines the morphology the supramolecular assembly of PBI-HAGs. In particular, a non-layered supramolecular structure was formed in octane owing to stabilization effects of intermolecular hydrogen bonds, whereas a layered supramolecular structure was formed in water because of energetically favorable π-π stacking of aromatic rings. The formation of distinct supramolecular architectures in different solvents was reinforced by simulated circular dichroism spectra, which show opposite signals consistent with experimental observations. The results of this study could help us understand aggregation-induced supramolecular chirality of noncovalent self-assemblies. Graphical abstract Left Typical structures of amphiphilic perylene-sugar dyad (PBI-HAG) aggregates in different octane and water. Right Simulated CD and UV-Vis spectra of core PBIs aggregates in octane and water.

Cholinesterases and cell proliferation in "nonstratified" and "stratified" cell aggregates from chicken retina and tectum.

PubMed

Vollmer, G; Layer, P G

1987-12-01

Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cell-to-cell relationships ("sorting out"), but these systems never form highly laminated aggregates ("nonstratified" R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly "stratified" aggregates ("RPE-aggregates", see Vollmer et al. 1984). The present comparative study of "stratified" and "nonstratified" aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates. During early aggregate formation, in both "stratified" and "nonstratified" aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2-3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE- and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization

PubMed Central

Raju, Murugesan; Mooney, Brian P.; Thakkar, Kavi M.; Giblin, Frank J.; Schey, Kevin L.; Sharma, K. Krishna

2015-01-01

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide–mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways. PMID:25639202

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization.

PubMed

Raju, Murugesan; Mooney, Brian P; Thakkar, Kavi M; Giblin, Frank J; Schey, Kevin L; Sharma, K Krishna

2015-03-01

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide-mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protein catabolism in cultures of hepatocytes derived from mice of various ages.

PubMed

Burrows, R B; Davison, P F

1982-05-01

The degradation of pulse-labeled protein was measured in cultures of hepatocytes derived from mice of 3--4, 15--16, and 28 months of age. The rates of protein degradation were determined in culture media with varying amino acid, insulin, and glucagon concentrations. No differences with age were seen. Also no difference with age was detected in the lysosomal degradation of 125I-labeled asialofetuin.

Prions on the run: How extracellular vesicles serve as delivery vehicles for self-templating protein aggregates.

PubMed

Liu, Shu; Hossinger, André; Göbbels, Sarah; Vorberg, Ina M

2017-03-04

Extracellular vesicles (EVs) are actively secreted, membrane-bound communication vehicles that exchange biomolecules between cells. EVs also serve as dissemination vehicles for pathogens, including prions, proteinaceous infectious agents that cause transmissible spongiform encephalopathies (TSEs) in mammals. Increasing evidence accumulates that diverse protein aggregates associated with common neurodegenerative diseases are packaged into EVs as well. Vesicle-mediated intercellular transmission of protein aggregates can induce aggregation of homotypic proteins in acceptor cells and might thereby contribute to disease progression. Our knowledge of how protein aggregates are sorted into EVs and how these vesicles adhere to and fuse with target cells is limited. Here we review how TSE prions exploit EVs for intercellular transmission and compare this to the transmission behavior of self-templating cytosolic protein aggregates derived from the yeast prion domain Sup 35 NM. Artificial NM prions are non-toxic to mammalian cell cultures and do not cause loss-of-function phenotypes. Importantly, NM particles are also secreted in association with exosomes that horizontally transmit the prion phenotype to naive bystander cells, a process that can be monitored with high accuracy by automated high throughput confocal microscopy. The high abundance of mammalian proteins with amino acid stretches compositionally similar to yeast prion domains makes the NM cell model an attractive model to study self-templating and dissemination properties of proteins with prion-like domains in the mammalian context.

Pterostilbene-O-acetamidoalkylbenzylamines derivatives as novel dual inhibitors of cholinesterase with anti-β-amyloid aggregation and antioxidant properties for the treatment of Alzheimer's disease.

PubMed

Li, Yuxing; Qiang, Xiaoming; Li, Yan; Yang, Xia; Luo, Li; Xiao, Ganyuan; Cao, Zhongcheng; Tan, Zhenghuai; Deng, Yong

2016-04-15

A series of pterostilbene-O-acetamidoalkylbenzylamines were designed, synthesized and evaluated as dual inhibitors of AChE and BuChE. To further explore the multifunctional properties of the new derivatives, their antioxidant activities and inhibitory effects on self-induced Aβ1-42 aggregation and HuAChE-induced Aβ1-40 aggregation were also tested. The results showed that most of these compounds could effectively inhibit AChE and BuChE. Particularly, compound 21d exhibited the best AChE inhibitory activity (IC50=0.06 μM) and good inhibition of BuChE (IC50=28.04 μM). Both the inhibition kinetic analysis and molecular modeling study revealed that these compounds showed mixed-type inhibition, binding simultaneously to the CAS and PAS of AChE. In addition to cholinesterase inhibitory activities, these compounds showed different levels of antioxidant activity. However, the inhibitory activities against self-induced and HuAChE-induced Aβ aggregation of these new derivatives were unsatisfied. Taking into account the results of the biological evaluation, further modifications will be designed in order to increase the potency on the different targets. The results displayed in this Letter can be a new starting point for further development of multifunctional agents for Alzheimer's disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Attempt to develop taste bud models in three-dimensional culture.

PubMed

Nishiyama, Miyako; Yuki, Saori; Fukano, Chiharu; Sako, Hideyuki; Miyamoto, Takenori; Tomooka, Yasuhiro

2011-09-01

Taste buds are the end organs of taste located in the gustatory papillae, which occur on the surface of the oral cavity. The goal of the present study was to establish a culture model mimicking the lingual taste bud of the mouse. To this end, three cell lines were employed: taste bud-derived cell lines (TBD cell lines), a lingual epithelial cell-derived cell line (20A cell line), and a mesenchymal cell-derived cell line (TMD cell line). TBD cells embedded in collagen gel formed three-dimensional clusters, which had an internal cavity equipped with a tight junction-like structure, a microvilluslike structure, and a laminin-positive layer surrounding the cluster. The cells with this epitheliumlike morphology expressed marker proteins of taste cells: gustducin and NCAM. TBD cells formed a monolayer on collagen gel when they were co-cultured with TMD cells. TBD, 20A, and TMD cell lines were maintained in a triple cell co-culture, in which TBD cells were pre-seeded as aggregates or in suspension on the collagen gel containing TMD cells, and 20A cells were laid over the TBD cells. TBD cells in the triple cell co-culture expressed NCAM. This result suggests that co-cultured TBD cells exhibited a characteristic of Type III taste cells. The culture model would be useful to study morphogenesis and functions of the gustatory organ.

Establishment of Canine-Derived Giardia duodenalis Isolates in Culture.

PubMed

Tysnes, Kristoffer R; Robertson, Lucy J

2016-06-01

Researchers continue to rely on axenic cultivation of Giardia duodenalis trophozoites in vitro to study the life cycle and host-parasite interactions of G. duodenalis and to develop vaccines and drugs to prevent and treat giardiasis. The majority of in vitro studies of G. duodenalis have used a small subset of isolates, mostly of assemblage A, and these isolates are usually originally isolated from humans. The most commonly used isolate for lab studies is known as WB. Canine giardiasis is a disease of veterinary importance, but it may also be of relevance in zoonotic transmission. Few G. duodenalis isolates from dogs have been adapted to in vitro culture, probably because the methods used are not suitable for the canine-specific genotypes that tend to dominate in most dog populations. In the current study, an experimental approach to cultivating canine-derived isolates of G. duodenalis was attempted by modification of the standard protocol based on physiological differences between the human and canine digestive system. An adapted method is described for improving the rate of in vitro excystation of cysts isolated from dogs by chemically weakening the cyst wall. A new canine-derived assemblage A G. duodenalis isolate was successfully adapted to axenic culture by using this method; the dog apparently had a mixed infection of assemblages A and D, but the assemblage A successfully outcompeted the assemblage D under conditions of in vitro culture. Based on the results, reasons regarding why humans do not seem to be suitable hosts for G. duodenalis in assemblages C and D are discussed.

Compartmental culture of embryonic stem cell-derived neurons in microfluidic devices for use in axonal biology.

PubMed

Shin, Hwa Sung; Kim, Hyung Joon; Min, Seul Ki; Kim, Sung Hoon; Lee, Byung Man; Jeon, Noo Li

2010-08-01

Axonal pathology has been clearly implicated in neurodegenerative diseases making the compartmental culture of neurons a useful research tool. Primary neurons have already been cultured in compartmental microfluidic devices but their derivation from an animal is a time-consuming and difficult work and has a limit in their sources. Embryonic stem cell (ESC)-derived neurons (ESC_Ns) overcome this limit, since ESCs can be renewed without limit and can be differentiated into ESC_Ns by robust and reproducible protocols. In this research, ESC_Ns were derived from mouse ESCs in compartmental microfluidic devices, and their axons were isolated from the somal cell bodies. Once embryoid bodies (EBs) were localized in the microfluidic culture chamber, ESC_Ns spread out from the EBs and occupied the cell culture chamber. Their axons traversed the microchannels and finally were isolated from the somata, providing an arrangement comparable to dissociated primary neurons. This ESC_N compartmental microfluidic culture system not only offers a substitute for the primary neuron counterpart system but also makes it possible to make comparisons between the two systems.

Reprogramming retinal neurons and standardized quantification of their differentiation in 3-dimensional retinal cultures

PubMed Central

Hiler, Daniel J.; Barabas, Marie E.; Griffiths, Lyra M.; Dyer, Michael A.

2017-01-01

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. Other protocols to reprogram postmitotic neurons have required the inactivation of the p53 tumor suppressor. We describe a method that does not require p53 inactivation and induces reprogramming in cells purified from the retinae of reprogrammable mice in aggregates with wild-type retinal cells. After the first 10 days of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantitated using this protocol. Reprogramming retinal neurons with this protocol will take 56 days, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 days. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantitates eye field specification, optic cup formation, and retinal differentiation in 3-dimensional cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol. PMID:27658012

Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters.

PubMed

Hunt, Megan M; Meng, Guoliang; Rancourt, Derrick E; Gates, Ian D; Kallos, Michael S

2014-01-01

Traditional optimization of culture parameters for the large-scale culture of human embryonic stem cells (ESCs) as aggregates is carried out in a stepwise manner whereby the effect of varying each culture parameter is investigated individually. However, as evidenced by the wide range of published protocols and culture performance indicators (growth rates, pluripotency marker expression, etc.), there is a lack of systematic investigation into the true effect of varying culture parameters especially with respect to potential interactions between culture variables. Here we describe the design and execution of a two-parameter, three-level (3(2)) factorial experiment resulting in nine conditions that were run in duplicate 125-mL stirred suspension bioreactors. The two parameters investigated here were inoculation density and agitation rate, which are easily controlled, but currently, poorly characterized. Cell readouts analyzed included fold expansion, maximum density, and exponential growth rate. Our results reveal that the choice of best case culture parameters was dependent on which cell property was chosen as the primary output variable. Subsequent statistical analyses via two-way analysis of variance indicated significant interaction effects between inoculation density and agitation rate specifically in the case of exponential growth rates. Results indicate that stepwise optimization has the potential to miss out on the true optimal case. In addition, choosing an optimum condition for a culture output of interest from the factorial design yielded similar results when repeated with the same cell line indicating reproducibility. We finally validated that human ESCs remain pluripotent in suspension culture as aggregates under our optimal conditions and maintain their differentiation capabilities as well as a stable karyotype and strong expression levels of specific human ESC markers over several passages in suspension bioreactors.

Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves

PubMed Central

Karim, Md. Ziaul; Uesugi, Daisuke; Nakayama, Noriyuki; Hossain, M. Monzur; Ishihara, Kohji; Hamada, Hiroki

2015-01-01

Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world. PMID:28008268

Settling of dilbit-derived oil-mineral aggregates (OMAs) & transport parameters for oil spill modelling.

PubMed

O'Laughlin, Casey M; Law, Brent A; Zions, Vanessa S; King, Thomas L; Robinson, Brian; Wu, Yongsheng

2017-11-15

The size and settling velocity of oil-mineral aggregates (OMAs) derived from diluted bitumen are primary constituents in predictive models for evaluating the potential fate of oil spilled in the aquatic environment. A series of low sediment concentration (15mg·L -1 ), colder water (<10°C) wave tank experiments designed to measure variability in these parameters in naturally-formed OMAs in response the presence or absence of chemical dispersant are discussed. Corresponding lab experiments revealed settling velocities of artificially formed OMAs on the order of 0.1-0.4mm·s -1 . High-resolution imagery of settling particles were analyzed for particle size, density and settling velocity. In situ formation of OMAs in the wave tank was unsuccessful. Possible effects of chemical dispersant on natural sediment flocculation, the size of suspended oil droplets and clearance rates of suspended particles are discussed. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses

PubMed Central

Åslund, Andreas; Sigurdson, Christina J.; Klingstedt, Therése; Grathwohl, Stefan; Bolmont, Tristan; Dickstein, Dara L.; Glimsdal, Eirik; Prokop, Stefan; Lindgren, Mikael; Konradsson, Peter; Holtzman, David M.; Hof, Patrick R.; Heppner, Frank L.; Gandy, Samuel; Jucker, Mathias; Aguzzi, Adriano; Hammarström, Per; Nilsson, K. Peter R.

2010-01-01

Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying cerebral amyloidoses. Here we report the chemical design of pentameric thiophene derivatives, denoted luminescent conjugated oligothiophenes (LCOs), which could be used for real-time visualization of cerebral protein aggregates in transgenic mouse models of neurodegenerative diseases by multiphoton microscopy. One of the LCOs, p-FTAA, showed conformation-dependent optical properties and could be utilized for ex vivo spectral assignment of distinct prion deposits from two mouse-adapted prion strains. p-FTAA also revealed staining of transient soluble pre-fibrillar non-thioflavinophilic Aβ- assemblies during in vitro fibrillation of Aβ peptides. In brain tissue samples, Aβ deposits and neurofibrillary tangles (NFTs) were readily identified by a strong fluorescence from p-FTAA and the LCO staining showed complete co-localization with conventional antibodies (6E10 and AT8), indicating that p-FTAA detects all the immuno-positive aggregated proteinaceous species in Alzheimer disease, but with significantly shorter imaging time (100 fold) compared to immunofluorescence. In addition, a patchy islet-like staining of individual Aβ plaque was unveiled by the anti-oligomer A11 antibody during co-staining with p-FTAA, suggesting that pre-fibrillar species are likely an intrinsic component of Aβ plaques in human brain. The major hallmarks of Alzheimer’s disease, namely Aβ aggregates versus NFTs could also be distinguished due to distinct emission spectra from p-FTAA. Overall, we demonstrate that LCOs can be utilized as powerful practical research tools for studying protein aggregation diseases and facilitate the study of amyloid origin, evolution and maturation, Aβ−tau interactions and pathogenesis both ex vivo and in vivo. PMID:19624097

The Mechanisms of Aberrant Protein Aggregation

NASA Astrophysics Data System (ADS)

Cohen, Samuel; Vendruscolo, Michele; Dobson, Chris; Knowles, Tuomas

2012-02-01

We discuss the development of a kinetic theory for understanding the aberrant loss of solubility of proteins. The failure to maintain protein solubility results often in the assembly of organized linear structures, commonly known as amyloid fibrils, the formation of which is associated with over 50 clinical disorders including Alzheimer's and Parkinson's diseases. A true microscopic understanding of the mechanisms that drive these aggregation processes has proved difficult to achieve. To address this challenge, we apply the methodologies of chemical kinetics to the biomolecular self-assembly pathways related to protein aggregation. We discuss the relevant master equation and analytical approaches to studying it. In particular, we derive the underlying rate laws in closed-form using a self-consistent solution scheme; the solutions that we obtain reveal scaling behaviors that are very generally present in systems of growing linear aggregates, and, moreover, provide a general route through which to relate experimental measurements to mechanistic information. We conclude by outlining a study of the aggregation of the Alzheimer's amyloid-beta peptide. The study identifies the dominant microscopic mechanism of aggregation and reveals previously unidentified therapeutic strategies.

Size-dependent enrichment of waste slag aggregate fragments abraded from asphalt concrete.

PubMed

Takahashi, Fumitake; Shimaoka, Takayuki; Gardner, Kevin; Kida, Akiko

2011-10-30

Authors consider the environmental prospects of using melted waste slag as the aggregate for asphalt pavement. In particular, the enrichment of slag-derived fragments in fine abrasion dust particles originated from slag asphalt concrete and its size dependency were concerned. A series of surface abrasion tests for asphalt concrete specimens, containing only natural aggregates as reference or 30 wt% of substituted slag aggregates, were performed. Although two of three slag-asphalt concretes generated 1.5-3.0 times larger amount of abrasion dust than the reference asphalt concrete did, it could not be explained only by abrasion resistance of slag. The enrichment of slag-derived fragments in abrasion dust, estimated on the basis of the peak intensity of quartz and heavy metal concentrations, had size dependency for all slag-asphalt concretes. Slag-derived fragments were enriched in abrasion dust particles with diameters of 150-1000 μm. Enrichment factors were 1.4-2.1. In contrast, there was no enrichment in abrasion dust particles with diameter less than 75 μm. This suggests that prior airborne-size fragmentation of substituted slag aggregates does not need to be considered for tested slag aggregates when environmental risks of abrasion dust of slag-asphalt pavement are assessed. Copyright © 2011 Elsevier B.V. All rights reserved.

Hot moments in spawning aggregations: implications for ecosystem-scale nutrient cycling

NASA Astrophysics Data System (ADS)

Archer, Stephanie K.; Allgeier, Jacob E.; Semmens, Brice X.; Heppell, Scott A.; Pattengill-Semmens, Christy V.; Rosemond, Amy D.; Bush, Phillippe G.; McCoy, Croy M.; Johnson, Bradley C.; Layman, Craig A.

2015-03-01

Biogeochemical hot moments occur when a temporary increase in availability of one or more limiting reactants results in elevated rates of biogeochemical reactions. Many marine fish form transient spawning aggregations, temporarily increasing their local abundance and thus nutrients supplied via excretion at the aggregation site. In this way, nutrients released by aggregating fish could create a biogeochemical hot moment. Using a combination of empirical and modeling approaches, we estimate nitrogen and phosphorus supplied by aggregating Nassau grouper ( Epinephelus striatus). Data suggest aggregating grouper supply up to an order-of-magnitude more nitrogen and phosphorus than daily consumer-derived nutrient supply on coral reefs without aggregating fish. Comparing current and historic aggregation-level excretion estimates shows that overfishing reduced nutrients supplied by aggregating fish by up to 87 %. Our study illustrates a previously unrecognized ecosystem viewpoint regarding fish spawning aggregations and provides an additional perspective on the repercussions of their overexploitation.

Experimental and Computational Investigation of the Effect of Hydrophobicity on Aggregation and Osteoinductive Potential of BMP-2-Derived Peptide in a Hydrogel Matrix

PubMed Central

Moeinzadeh, Seyedsina; Barati, Danial; Sarvestani, Samaneh K.; Karimi, Tahereh

2015-01-01

An attractive approach to reduce the undesired side effects of bone morphogenetic proteins (BMPs) in regenerative medicine is to use osteoinductive peptide sequences derived from BMPs. Although the structure and function of BMPs have been studied extensively, there is limited data on structure and activity of BMP-derived peptides immobilized in hydrogels. The objective of this work was to investigate the effect of concentration and hydrophobicity of the BMP-2 peptide, corresponding to residues 73–92 of the knuckle epitope of BMP-2 protein, on peptide aggregation and osteogenic differentiation of human mesenchymal stem cells encapsulated in a polyethylene glycol (PEG) hydrogel. The peptide hydrophobicity was varied by capping PEG chain ends with short lactide segments. The BMP-2 peptide with a positive index of hydrophobicity had a critical micelle concentration (CMC) and formed aggregates in aqueous solution. Based on simulation results, there was a slight increase in the concentration of free peptide in solution with 1000-fold increase in peptide concentration. The dose-osteogenic response curve of the BMP-2 peptide was in the 0.0005–0.005 mM range, and osteoinductive potential of the BMP-2 peptide was significantly less than that of BMP-2 protein even at 1000-fold higher concentrations, which was attributed to peptide aggregation. Further, the peptide or PEG-peptide aggregates had significantly higher interaction energy with the cell membrane compared with the free peptide, which led to a higher nonspecific interaction with the cell membrane and loss of osteoinductive potential. Conjugation of the BMP-2 peptide to PEG increased CMC and osteoinductive potential of the peptide whereas conjugation to lactide-capped PEG reduced CMC and osteoinductive potential of the peptide. Experimental and simulation results revealed that osteoinductive potential of the BMP-2 peptide is correlated with its CMC and the free peptide concentration in aqueous medium and not the

Aggregation and fusion of modified low density lipoprotein.

PubMed

Pentikäinen, M O; Lehtonen, E M; Kovanen, P T

1996-12-01

In atherogenesis, low density lipoprotein (LDL, diameter 22 nm) accumulates in the extracellular space of the arterial intima in the form of aggregates of lipid droplets (droplet diameter up to 400 nm). Here we studied the effects of various established in vitro LDL modifications on LDL aggregation and fusion. LDL was subjected to vortexing, oxidation by copper ions, proteolysis by alpha-chymotrypsin, lipolysis by sphingomyelinase, and nonenzymatic glycosylation, and was induced to form adducts with malondialdehyde or complexes with anti-apoB-100 antibodies. To assess the amount of enlarged LDL-derived structures formed (due to aggregation or fusion), we measured the turbidity of solutions containing modified LDL, and quantified the proportion of modified LDL that 1) sedimented at low-speed centrifugation (14,000 g), 2) floated at an increased rate at high-speed centrifugation (rate zonal flotation at 285,000 gmax), 3) were excluded in size-exclusion column chromatography (exclusion limit 40 MDa), or 4) failed to enter into 0.5%. Fast Lane agarose gel during electrophoresis. To detect whether particle fusion had contributed to the formation of the enlarged LDL-derived structures, particle morphology was examined using negative staining and thin-section transmission electron microscopy. We found that 1) aggregation was induced by the formation of LDL-antibody complexes, malondialdehyde treatment, and glycosylation of LDL; 2) fusion of LDL was induced by proteolysis of LDL by alpha-chymotrypsin; and 3) aggregation and fusion of LDL were induced by vortexing, oxidation by copper ions, and lipolysis by sphingomyclinase of LDL. The various modifications of LDL differed in their ability to induce aggregation and fusion.

Damage of photoreceptor-derived cells in culture induced by light emitting diode-derived blue light

PubMed Central

Kuse, Yoshiki; Ogawa, Kenjiro; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Hara, Hideaki

2014-01-01

Our eyes are increasingly exposed to light from the emitting diode (LED) light of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smart phones. The present study aims to clarify the mechanism underlying blue LED light-induced photoreceptor cell damage. Murine cone photoreceptor-derived cells (661 W) were exposed to blue, white, or green LED light (0.38 mW/cm2). In the present study, blue LED light increased reactive oxygen species (ROS) production, altered the protein expression level, induced the aggregation of short-wavelength opsins (S-opsin), resulting in severe cell damage. While, blue LED light damaged the primary retinal cells and the damage was photoreceptor specific. N-Acetylcysteine (NAC), an antioxidant, protected against the cellular damage induced by blue LED light. Overall, the LED light induced cell damage was wavelength-, but not energy-dependent and may cause more severe retinal photoreceptor cell damage than the other LED light. PMID:24909301

Amyloid β-Derived Diffusible Ligands (ADDLs) Induce Abnormal Autophagy Associated with Aβ Aggregation Degree.

PubMed

Wen, Jie; Fang, Fang; Guo, Shu-Han; Zhang, Ying; Peng, Xiang-Lei; Sun, Wei-Min; Wei, Xiao-Ran; He, Jin-Sheng; Hung, Tao

2018-02-01

Autophagy is disturbed in Alzheimer's disease (AD) and maintaining normal autophagy homeostasis is a new therapeutic strategy for AD treatment. Amyloid β-derived diffusible ligands (ADDLs), the most toxic species of which are oligomeric forms of amyloid β peptide (Aβ) that originate from amyloid β precursor protein (APP) via autophagy; however, whether ADDLs are involved in autophagy-related AD pathogenesis remains unclear. In this study, we primarily defined the specific subsets of ADDLs, A-0, A-12, A-24, and A-48, which were generated from ADDL aggregation mixtures at different time courses of assembly. The secondary structures of ADDL subsets were detected by circular dichroism (CD). Neuronal or non-neuronal cells were exposed to the subsets of ADDLs in vitro, and then, autophagic markers were detected. Our results first showed that exogenous or endogenous LC3 puncta (autophagosomes) were induced in the cytoplasm of cells exposed to ADDLs and that the LC3 puncta were the strongest with A-24 exposure. Then, the CD spectroscopy data also indicated that the proportion of α-helices decreased, whereas the proportion of β-strands and β-turns increased during ADDL assembly from 0 to 24 h. In addition, the quantitative Western blot data demonstrated that the ratio of LC3B-II/I was significantly increased, and SQSTM1/p62 decreased over time. Finally, our results indicated that the level of phosphorylated p70 S6 kinase (p-p70 S6 kinase), which is a substrate protein in the MTOR pathway, and the ratio of p-p70 S6 kinase/p70 S6 kinase significantly decreased following A-24 exposure. Taken together, our data suggest that ADDL-induced abnormal autophagy is correlated with Aβ aggregation degree and the MTOR pathway, which might contribute to ADDL-induced AD pathogenesis.

Impact of fluidic agitation on human pluripotent stem cells in stirred suspension culture.

PubMed

Nampe, Daniel; Joshi, Ronak; Keller, Kevin; Zur Nieden, Nicole I; Tsutsui, Hideaki

2017-09-01

The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters. In particular, detailed characterizations of different agitation rates and their influence on the propagation of hPSCs are often not reported in the literature. In the current study, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and the maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Overall, our results showed that moderate agitation maximized the propagation of hPSCs to approximately 38-fold over 7 days by keeping the cell aggregates below the critical size, beyond which the cells are impacted by the diffusion limit, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. This indicates a new possibility to guide stem cell fate determination by fluidic agitation in stirred suspension cultures. Biotechnol. Bioeng. 2017;114: 2109-2120. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Temperature dependent rapid annealing effect induces amorphous aggregation of human serum albumin.

PubMed

Ishtikhar, Mohd; Ali, Mohd Sajid; Atta, Ayman M; Al-Lohedan, Hammad; Badr, Gamal; Khan, Rizwan Hasan

2016-01-01

This study represents an analysis of the thermal aggregation of human serum albumin (HSA) induced by novel rosin modified compounds. The aggregation process causes conformational alterations in the secondary and tertiary structures of the proteins. The conversion of globular protein to amorphous aggregates was carried out by spectroscopic, calorimetric and microscopic techniques to investigate the factors that are responsible for the structural, conformational and morphological alteration in the protein. Our outcome results show that the aggregation of HSA was dependent on the hydrophobicity, charge and temperature, because the formation of amorphous aggregates occurs in the presence of a novel cationic rosin compound, quaternary amine of rosin diethylaminoethyl ester (QRMAE), at 40°C and pH 7.4 (but at 25°C on similar pH value, there was no evidence of aggregate formation). In addition, the parent compound of QRMAE, i.e., abietic acid, and other derivatives such as nonionic rosin compounds [(RMPEG-750) and (RMA-MPEG-750)] do not shows the aggregating property. This work provides precise and necessary information that aid in the understanding the effects of rosin derivative compounds on HSA. This study also restrains important information for athletes, health providers, pharmaceutical companies, industries, and soft drink-processing companies. Copyright © 2015 Elsevier B.V. All rights reserved.

SENSITIVITY ANALYSIS OF AGGREGATED ENVIRONMENTAL INDICES WITH A CASE-STUDY OF THE MID-ATLANTIC REGION

EPA Science Inventory

Environmental indicators are often aggregated into a single index for various purposes in environmental studies. Aggregated indices derived from the same data set can differ, usually because the aggregated indices' sensitivities are not thoroughly analyzed. Furthermore, if a sens...

Passive participation of fixed platelets in aggregation facilitated by covalently bound fibrinogen.

PubMed

Agam, G; Livne, A

1983-01-01

The role of fibrinogen in interplatelet recognition during aggregation was examined by combining two cell types: fresh platelets (in limiting density) activated by thrombin or A23187, and formaldehyde-fixed platelets, bearing cross-linked fibrinogen. The fixed platelets did not aggregate by themselves, nor with resting platelets, but were capable of interacting with activated platelets and of participating passively in aggregation. The participation, expressed by enhanced aggregation, was assayed by the conventional turbidometric traces and by cosedimentation of fixed 3H-platelets with aggregates of fresh platelets. Platelet suspensions, prepared without special means to avert spontaneous activation, retained plasma fibrinogen to the extent of 50 micrograms/ml of a suspension containing 10(8) platelets, and the derived fixed platelets participated in aggregation, independently of added fibrinogen. The capability of such fixed platelets to participate in aggregation was sensitive to proteolytic digestion and to massive acetylation. When platelet separation was aided by apyrase or aspirin, PGE1 and gel filtration, the residual plasma fibrinogen was limited to 0.4 micrograms/ml of 10(8) platelet suspension. The derived fixed platelets were incapable of participating in aggregation unless fibrinogen was added prior to fixation. The affixed fibrinogen could not be replaced by soluble fibrinogen or affixed albumin. It is concluded that fibrinogen, which binds to platelets upon activation or is linked to them covalently, is a recognition site for platelet-platelet interaction during aggregation.

Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

PubMed Central

Kean, Thomas J.; Dennis, James E.

2015-01-01

Background Current tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential. Methods Porcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically. Results Human chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions. Conclusions

Effects of in vitro endochondral priming and pre-vascularisation of human MSC cellular aggregates in vivo.

PubMed

Freeman, Fiona E; Allen, Ashley B; Stevens, Hazel Y; Guldberg, Robert E; McNamara, Laoise M

2015-11-05

During endochondral ossification, both the production of a cartilage template and the subsequent vascularisation of that template are essential precursors to bone tissue formation. Recent studies have found the application of both chondrogenic and vascular priming of mesenchymal stem cells (MSCs) enhanced the mineralisation potential of MSCs in vitro whilst also allowing for immature vessel formation. However, the in vivo viability, vascularisation and mineralisation potential of MSC aggregates that have been pre-conditioned in vitro by a combination of chondrogenic and vascular priming, has yet to be established. In this study, we test the hypothesis that a tissue regeneration approach that incorporates both chondrogenic priming of MSCs, to first form a cartilage template, and subsequent pre-vascularisation of the cartilage constructs, by co-culture with human umbilical vein endothelial cells (HUVECs) in vitro, will improve vessel infiltration and thus mineral formation once implanted in vivo. Human MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days. These aggregates were then implanted subcutaneously in nude rats for 4 weeks. We used a combination of bioluminescent imaging, microcomputed tomography, histology (Masson's trichrome and Alizarin Red) and immunohistochemistry (CD31, CD146, and α-smooth actin) to assess the vascularisation and mineralisation potential of these MSC aggregates in vivo. Pre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation. In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days. Interestingly, the

Inhibitory effects of magnolol and honokiol on human calcitonin aggregation

PubMed Central

Guo, Caiao; Ma, Liang; Zhao, Yudan; Peng, Anlin; Cheng, Biao; Zhou, Qiaoqiao; Zheng, Ling; Huang, Kun

2015-01-01

Amyloid formation is associated with multiple amyloidosis diseases. Human calcitonin (hCT) is a typical amyloidogenic peptide, its aggregation is associated with medullary carcinoma of the thyroid (MTC), and also limits its clinical application. Magnolia officinalis is a traditional Chinese herbal medicine; its two major polyphenol components, magnolol (Mag) and honokiol (Hon), have displayed multiple functions. Polyphenols like flavonoids and their derivatives have been extensively studied as amyloid inhibitors. However, the anti-amyloidogenic property of a biphenyl backbone containing polyphenols such as Mag and Hon has not been reported. In this study, these two compounds were tested for their effects on hCT aggregation. We found that Mag and Hon both inhibited the amyloid formation of hCT, whereas Mag showed a stronger inhibitory effect; moreover, they both dose-dependently disassembled preformed hCT aggregates. Further immuno-dot blot and dynamic light scattering studies suggested Mag and Hon suppressed the aggregation of hCT both at the oligomerization and the fibrillation stages, while MTT-based and dye-leakage assays demonstrated that Mag and Hon effectively reduced cytotoxicity caused by hCT aggregates. Furthermore, isothermal titration calorimetry indicated Mag and Hon both interact with hCT. Together, our study suggested a potential anti-amyloidogenic property of these two compounds and their structure related derivatives. PMID:26324190

Arginine-glycine-aspartic acid-polyethylene glycol-polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture.

PubMed

Chen, Zhanfei; Lian, Fen; Wang, Xiaoqian; Chen, Yanling; Tang, Nanhong

The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine-glycine-aspartic acid (RGD) peptide. Our studies demonstrate that RGD-polyethylene glycol (PEG)-PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT-MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system.

Development of multifunctional, heterodimeric isoindoline-1,3-dione derivatives as cholinesterase and β-amyloid aggregation inhibitors with neuroprotective properties.

PubMed

Guzior, Natalia; Bajda, Marek; Skrok, Mirosław; Kurpiewska, Katarzyna; Lewiński, Krzysztof; Brus, Boris; Pišlar, Anja; Kos, Janko; Gobec, Stanislav; Malawska, Barbara

2015-03-06

The presented study describes the synthesis, pharmacological evaluation (AChE and BuChE inhibition, beta amyloid anti-aggregation effect and neuroprotective effect), molecular modeling and crystallographic studies of a novel series of isoindoline-1,3-dione derivatives. The target compounds were designed as dual binding site acetylcholinesterase inhibitors with an arylalkylamine moiety binding at the catalytic site of the enzyme and connected via an alkyl chain to a heterocyclic fragment, capable of binding at the peripheral anionic site of AChE. Among these molecules, compound 15b was found to be the most potent and selective AChE inhibitor (IC50EeAChE = 0.034 μM). Moreover, compound 13b in addition to AChE inhibition (IC50 EeAChE = 0.219 μM) possesses additional properties, such as the ability to inhibit Aβ aggregation (65.96% at 10 μM) and a neuroprotective effect against Aβ toxicity at 1 and 3 μM. Compound 13b emerges as a promising multi-target ligand for the further development of the therapy for age-related neurodegenerative disorders. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Conversion of Synthetic Aβ to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.

PubMed

Novotny, Renata; Langer, Franziska; Mahler, Jasmin; Skodras, Angelos; Vlachos, Andreas; Wegenast-Braun, Bettina M; Kaeser, Stephan A; Neher, Jonas J; Eisele, Yvonne S; Pietrowski, Marie J; Nilsson, K Peter R; Deller, Thomas; Staufenbiel, Matthias; Heimrich, Bernd; Jucker, Mathias

2016-05-04

The aggregation of amyloid-β peptide (Aβ) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral β-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aβ deposition was noted in untreated HSCs of postnatal Aβ precursor protein transgenic (APP tg) mice, Aβ deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aβ. Seeded Aβ deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aβ species determined the conformational characteristics of HSC Aβ deposition. HSC Aβ deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aβ, homogenates of Aβ deposits containing HSCs induced cerebral β-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aβ into a potent in vivo seeding-active form. In this study, we report the seeded induction of Aβ aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aβ aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aβ aggregates can be obtained by seeded conversion of synthetic Aβ in a living (wild-type) cellular environment. Copyright © 2016 the authors 0270-6474/16/365084-10$15.00/0.

Synthesis of new N-benzylpiperidine derivatives as cholinesterase inhibitors with β-amyloid anti-aggregation properties and beneficial effects on memory in vivo.

PubMed

Więckowska, Anna; Więckowski, Krzysztof; Bajda, Marek; Brus, Boris; Sałat, Kinga; Czerwińska, Paulina; Gobec, Stanislav; Filipek, Barbara; Malawska, Barbara

2015-05-15

Due to the complex nature of Alzheimer's disease, multi-target-directed ligand approaches are one of the most promising strategies in the search for effective treatments. Acetylcholinesterase, butyrylcholinesterase and β-amyloid are the predominant biological targets in the search for new anti-Alzheimer's agents. Our aim was to combine both anticholinesterase and β-amyloid anti-aggregation activities in one molecule, and to determine the therapeutic potential in vivo. We designed and synthesized 28 new compounds as derivatives of donepezil that contain the N-benzylpiperidine moiety combined with the phthalimide or indole moieties. Most of these test compounds showed micromolar activities against cholinesterases and aggregation of β-amyloid, combined with positive results in blood-brain barrier permeability assays. The most promising compound 23 (2-(8-(1-(3-chlorobenzyl)piperidin-4-ylamino)octyl)isoindoline-1,3-dione) is an inhibitor of butyrylcholinesterase (IC50=0.72 μM) that has β-amyloid anti-aggregation activity (72.5% inhibition at 10 μM) and can cross the blood-brain barrier. Moreover, in an animal model of memory impairment induced by scopolamine, the activity of 23 was comparable to that of donepezil. The selected compound 23 is an excellent lead structure in the further search for new anti-Alzheimer's agents. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gut bacteria mediate aggregation in the German cockroach

PubMed Central

Wada-Katsumata, Ayako; Zurek, Ludek; Nalyanya, Godfrey; Roelofs, Wendell L.; Zhang, Aijun; Schal, Coby

2015-01-01

Aggregation of the German cockroach, Blattella germanica, is regulated by fecal aggregation agents (pheromones), including volatile carboxylic acids (VCAs). We demonstrate that the gut microbial community contributes to production of these semiochemicals. Chemical analysis of the fecal extract of B. germanica revealed 40 VCAs. Feces from axenic cockroaches (no microorganisms in the alimentary tract) lacked 12 major fecal VCAs, and 24 of the remaining compounds were represented at extremely low amounts. Olfactory and aggregation bioassays demonstrated that nymphs strongly preferred the extract of control feces over the fecal extract of axenic cockroaches. Additionally, nymphs preferred a synthetic blend of 6 fecal VCAs over a solvent control or a previously identified VCA blend. To test whether gut bacteria contribute to the production of fecal aggregation agents, fecal aerobic bacteria were cultured, isolated, and identified. Inoculation of axenic cockroaches with individual bacterial taxa significantly rescued the aggregation response to the fecal extract, and inoculation with a mix of six bacterial isolates was more effective than with single isolates. The results indicate that the commensal gut microbiota contributes to production of VCAs that act as fecal aggregation agents and that cockroaches discriminate among the complex odors that emanate from a diverse microbial community. Our results highlight the pivotal role of gut bacteria in mediating insect–insect communication. Moreover, because the gut microbial community reflects the local environment, local plasticity in fecal aggregation pheromones enables colony-specific odors and fidelity to persistent aggregation sites. PMID:26644557

Aggregate formation affects ultrasonic disruption of microalgal cells.

PubMed

Wang, Wei; Lee, Duu-Jong; Lai, Juin-Yih

2015-12-01

Ultrasonication is a cell disruption process of low energy efficiency. This study dosed K(+), Ca(2+) and Al(3+) to Chlorella vulgaris cultured in Bold's Basal Medium at 25°C and measured the degree of cell disruption under ultrasonication. Adding these metal ions yielded less negatively charged surfaces of cells, while with the latter two ions large and compact cell aggregates were formed. The degree of cell disruption followed: control=K(+)>Ca(2+)>Al(3+) samples. Surface charges of cells and microbubbles have minimal effects on the microbubble number in the proximity of the microalgal cells. Conversely, cell aggregates with large size and compact interior resist cell disruption under ultrasonication. Staining tests revealed high diffusional resistance of stains over the aggregate interior. Microbubbles may not be effective generated and collapsed inside the compact aggregates, hence leading to low cell disruption efficiencies. Effective coagulation/flocculation in cell harvesting may lead to adverse effect on subsequent cell disruption efficiency. Copyright © 2015 Elsevier Ltd. All rights reserved.

Internalization of aggregated photosensitizers by tumor cells: subcellular time-resolved fluorescence spectroscopy on derivatives of pyropheophorbide-a ethers and chlorin e6 under femtosecond one- and two-photon excitations.

PubMed

Kelbauskas, L; Dietel, W

2002-12-01

Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20-30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (< 50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds.

Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations.

PubMed

Zheng, Hewen; Korendovych, Ivan V; Luk, Yan-Yeung

2016-01-01

For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa. Copyright © 2015 Elsevier Inc. All rights reserved.

Modification of Lightweight Aggregates' Microstructure by Used Motor Oil Addition.

PubMed

Franus, Małgorzata; Jozefaciuk, Grzegorz; Bandura, Lidia; Lamorski, Krzysztof; Hajnos, Mieczysław; Franus, Wojciech

2016-10-18

An admixture of lightweight aggregate substrates (beidellitic clay containing 10 wt % of natural clinoptilolite or Na-P1 zeolite) with used motor oil (1 wt %-8 wt %) caused marked changes in the aggregates' microstructure, measured by a combination of mercury porosimetry (MIP), microtomography (MT), and scanning electron microscopy. Maximum porosity was produced at low (1%-2%) oil concentrations and it dropped at higher concentrations, opposite to the aggregates' bulk density. Average pore radii, measured by MIP, decreased with an increasing oil concentration, whereas larger (MT) pore sizes tended to increase. Fractal dimension, derived from MIP data, changed similarly to the MIP pore radius, while that derived from MT remained unaltered. Solid phase density, measured by helium pycnometry, initially dropped slightly and then increased with the amount of oil added, which was most probably connected to changes in the formation of extremely small closed pores that were not available for He atoms.

The effects of an RGD-PAMAM dendrimer conjugate in 3D spheroid culture on cell proliferation, expression and aggregation.

PubMed

Jiang, Li-Yang; Lv, Bing; Luo, Ying

2013-04-01

By presenting biomolecular ligands on the surface in high density, ligand-decorated dendrimers are capable of binding to membrane receptors and cells with specificity and avidity. Despite the various uses, fundamental investigations on ligand-dendrimer conjugates have mainly focused on their binding behavior with cells, whereas their potential bioactivity and applications in multicellular systems, especially in three-dimensional (3D) culture systems, remains untapped. In this study, a typical adhesive peptide ligand - RGD - was modified to generation 4 polyamidoamine (PAMAM), and the bioactivity of suspended RGD-PAMAM conjugates was investigated on cells cultured as multicellular spheroids. Our results demonstrate that the RGD-PAMAM conjugates, after being incorporated into the 3D spheroids, were able to promote cellular proliferation and aggregation, and affect the mRNA expression of extracellular factors by NIH 3T3 cells. These bioactive functions were multivalency-dependent, as none of similar effects was observed for monovalent RGD ligand. Our study suggests that multivalent ligand-dendrimer conjugates may act as a unique type of artificial factors to mediate the cellular microenvironment in 3D culture, a property attributable to the spatial organization of the ligands and possible "cell-gluing" function of multivalent conjugates. This new finding opens the door for further exploring multivalent ligand-dendrimer conjugates for applications in 3D cell culture and tissue engineering. Copyright © 2013 Elsevier Ltd. All rights reserved.

Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: implications for therapy of Alzheimer disease.

PubMed

Bana, Laura; Minniti, Stefania; Salvati, Elisa; Sesana, Silvia; Zambelli, Vanessa; Cagnotto, Alfredo; Orlando, Antonina; Cazzaniga, Emanuela; Zwart, Rob; Scheper, Wiep; Masserini, Massimo; Re, Francesca

2014-10-01

Targeting amyloid-β peptide (Aβ) within the brain is a strategy actively sought for therapy of Alzheimer's disease (AD). We investigated the ability of liposomes bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) to affect Aβ aggregation/disaggregation features and to cross in vitro and in vivo the blood-brain barrier (BBB). Surface plasmon resonance showed that bi-functionalized liposomes strongly bind Aβ (kD=0.6 μM), while Thioflavin-T and SDS-PAGE/WB assays show that liposomes inhibit peptide aggregation (70% inhibition after 72 h) and trigger the disaggregation of preformed aggregates (60% decrease after 120 h incubation). Moreover, experiments with dually radiolabelled LIP suggest that bi-functionalization enhances the passage of radioactivity across the BBB either in vitro (permeability=2.5×10(-5) cm/min, 5-fold higher with respect to mono-functionalized liposomes) or in vivo in healthy mice. Taken together, our results suggest that mApoE-PA-LIP are valuable nanodevices with a potential applicability in vivo for the treatment of AD. From the clinical editor: Bi-functionalized liposomes with phosphatidic acid and a modified ApoE-derived peptide were demonstrated to influence Aβ aggregation/disaggregation as a potential treatment in an Alzheimer's model. The liposomes were able to cross the blood-brain barrier in vitro and in vivo. Similar liposomes may become clinically valuable nanodevices with a potential applicability for the treatment of Alzheimer's disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells

PubMed Central

van den Brink, Susanne C.; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A.; Martinez Arias, Alfonso

2014-01-01

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call ‘gastruloids’. PMID:25371360

Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells.

PubMed

van den Brink, Susanne C; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A; Martinez Arias, Alfonso

2014-11-01

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'. © 2014. Published by The Company of Biologists Ltd.

Cyclosporin A promotes mineralization by human cementoblastoma-derived cells in culture.

PubMed

Arzate, Higinio; Alvarez, Marco A; Narayanan, A Sampath

2005-06-01

The immunosuppressive drug cyclosporin A has been shown to induce cementum deposition in vivo in experimental animals. Using cementoblastoma-derived cells, we have studied whether this drug will be useful to study cementum mineralization and differentiation in vitro. Human cementoblastoma cells and gingival fibroblasts (controls) were cultured and treated with 0.5, 1.0 and 5.0 microg/ml of cyclosporin A. Cell proliferation was evaluated by MTT (tetrazolium) assay and cell number, and cell viability was assessed by trypan blue dye exclusion. Induction of mineralization was evaluated by alizarin red S staining to detect mineralized nodules and by reverse transcription-polymerase chain reaction (RT-PCR) to assess the expression of bone differentiation markers alkaline phosphatase, osteocalcin, bone sialoprotein and core-binding factor a1 (Cbfa1). Cyclosporin A at 5.0 microg/ml concentration reduced significantly the increase in the number of cementoblastoma cells. A dose-dependent increase in the number of mineralized nodules occurred in cultures of cementoblastoma-derived cells treated with cyclosporin A, and RT-PCR analyses showed significantly higher levels of expression of alkaline phosphatase, bone sialoprotein, type I collagen, matrix metalloproteinase-1, osteocalcin, osteopontin, and Cbfa1. Human gingival fibroblast proliferation and cell number were not affected. Mineralized nodules were not detected in gingival fibroblasts and bone specific proteins were not expressed. Presence of cyclosporin A during 14-day culture period appears to suppress the proliferation of cementoblastoma cells and induce the formation mineralized-like tissue by these cells.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

PubMed

Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

2016-01-01

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

PubMed

Hindié, M; Vayssade, M; Dufresne, M; Quéant, S; Warocquier-Clérout, R; Legeay, G; Vigneron, P; Olivier, V; Duval, J-L; Nagel, M-D

2006-09-01

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Microbial community dynamics in soil aggregates shape biogeochemical gas fluxes from soil profiles - upscaling an aggregate biophysical model.

PubMed

Ebrahimi, Ali; Or, Dani

2016-09-01

Microbial communities inhabiting soil aggregates dynamically adjust their activity and composition in response to variations in hydration and other external conditions. These rapid dynamics shape signatures of biogeochemical activity and gas fluxes emitted from soil profiles. Recent mechanistic models of microbial processes in unsaturated aggregate-like pore networks revealed a highly dynamic interplay between oxic and anoxic microsites jointly shaped by hydration conditions and by aerobic and anaerobic microbial community abundance and self-organization. The spatial extent of anoxic niches (hotspots) flicker in time (hot moments) and support substantial anaerobic microbial activity even in aerated soil profiles. We employed an individual-based model for microbial community life in soil aggregate assemblies represented by 3D angular pore networks. Model aggregates of different sizes were subjected to variable water, carbon and oxygen contents that varied with soil depth as boundary conditions. The study integrates microbial activity within aggregates of different sizes and soil depth to obtain estimates of biogeochemical fluxes from the soil profile. The results quantify impacts of dynamic shifts in microbial community composition on CO2 and N2 O production rates in soil profiles in good agreement with experimental data. Aggregate size distribution and the shape of resource profiles in a soil determine how hydration dynamics shape denitrification and carbon utilization rates. Results from the mechanistic model for microbial activity in aggregates of different sizes were used to derive parameters for analytical representation of soil biogeochemical processes across large scales of practical interest for hydrological and climate models. © 2016 John Wiley & Sons Ltd.

Tracking hypoxic signaling within encapsulated cell aggregates.

PubMed

Skiles, Matthew L; Sahai, Suchit; Blanchette, James O

2011-12-16

, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact β-cells whose insulin secretory function is highly dependent on oxygen. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously. The virus is stored in 10% glycerol at -80° C as many 150 μL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 10(10) pfu/mL. Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion. Western blot analysis of encapsulated cells in 20% and 1% oxygen also

Stirred tank bioreactor culture combined with serum-/xenogeneic-free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal stem/stromal cells.

PubMed

Mizukami, Amanda; Fernandes-Platzgummer, Ana; Carmelo, Joana G; Swiech, Kamilla; Covas, Dimas T; Cabral, Joaquim M S; da Silva, Cláudia L

2016-08-01

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell-based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non-invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)-free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin-based Cultispher(®) S microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. This system enabled the production of 2.4 (±1.1) x10(5) cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier-based stirred culture system, using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of the effects of several zoanthamine-type alkaloids on the aggregation of human platelets.

PubMed

Villar, Rosa M; Gil-Longo, José; Daranas, Antonio H; Souto, María L; Fernández, José J; Peixinho, Solange; Barral, Miguel A; Santafé, Gilmar; Rodríguez, Jaime; Jiménez, Carlos

2003-05-15

Ten zoanthamine-type alkaloids from two marine zoanthids belonging to the Zoanthus genus (Zoanthus nymphaeus and Zoanthus sp.) along with one semisynthetic derivative were evaluated for their antiplatelet activities on human platelet aggregation induced by several stimulating agents. 11-Hydroxyzoanthamine (11) and a synthetic derivative of norzoanthamine (16) showed strong inhibition against thrombin-, collagen- and arachidonic acid-induced aggregation, zoanthenol (15) displayed a selective inhibitory activity induced by collagen, while zoanthaminone (10) behaved as a potent aggregant agent. These evaluations allowed us to deduce several structure-activity relationships and suggest some mechanisms of action for this type of compounds.

Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

PubMed

Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

2016-01-01

As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

Marrow-derived mesenchymal stem cells: role in epithelial tumor cell determination.

PubMed

Fierro, Fernando A; Sierralta, Walter D; Epuñan, Maria J; Minguell, José J

2004-01-01

Marrow stroma represents an advantageous environment for development of micrometastatic cells. Within the cellular structure of marrow stroma, mesenchymal stem cells (MSC) have been postulated as an interacting target for disseminated cancer cells. The studies reported here were performed to gain more information on the interaction of the human breast cancer cell line MCF-7 with human bone marrow-derived MSC cells and to investigate whether this interaction affects tumor cell properties. The results showed that after co-culture with MSC, changes were detected in the morphology, proliferative capacity and aggregation pattern of MCF-7 cells, but these parameters were not affected after the co-culture of MSC cells with a non-tumorigenic breast epithelial cell line, MCF-10. Since the indirect culture of MCF-7 with MSC or its products also resulted in functional changes in the tumor cells, we evaluated whether these effects could be attributed to growth factors produced by MSC cells. It was found that VEGF and IL-6 mimic the effects produced by MSC or its products on the proliferation and aggregation properties of MCF-7, cells, respectively. Thus, it seems that after entry of disseminated tumor cells into the marrow space, their proliferative and morphogenetic organization patterns are modified after interaction with distinct stromal cells and/or with specific signals from the marrow microenvironment.

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

PubMed Central

Nakagawa, Yusuke; Muneta, Takeshi; Otabe, Koji; Ozeki, Nobutake; Mizuno, Mitsuru; Udo, Mio; Saito, Ryusuke; Yanagisawa, Katsuaki; Ichinose, Shizuko; Koga, Hideyuki; Tsuji, Kunikazu; Sekiya, Ichiro

2016-01-01

Objective Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats. Methods For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages. Results In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo.

PubMed

Nakagawa, Yusuke; Muneta, Takeshi; Otabe, Koji; Ozeki, Nobutake; Mizuno, Mitsuru; Udo, Mio; Saito, Ryusuke; Yanagisawa, Katsuaki; Ichinose, Shizuko; Koga, Hideyuki; Tsuji, Kunikazu; Sekiya, Ichiro

2016-01-01

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats. For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages. In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the

α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells

PubMed Central

Hansen, Christian; Angot, Elodie; Bergström, Ann-Louise; Steiner, Jennifer A.; Pieri, Laura; Paul, Gesine; Outeiro, Tiago F.; Melki, Ronald; Kallunki, Pekka; Fog, Karina; Li, Jia-Yi; Brundin, Patrik

2011-01-01

Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that α-synuclein–containing (α-syn–containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of α-syn from host to graft, followed by seeding of α-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed α-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP– or DsRed-tagged α-syn resulted in a gradual increase in double-labeled cells. Importantly, α-syn–GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed–α-syn, suggesting a seeding effect of transmitted α-syn. Extracellular α-syn was taken up by cells through endocytosis and interacted with intracellular α-syn. Next, following intracortical injection of recombinant α-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of α-syn between host cells and grafted dopaminergic neurons in mice overexpressing human α-syn. In summary, intercellularly transferred α-syn interacts with cytoplasmic α-syn and can propagate α-syn pathology. These results suggest that α-syn propagation is a key element in the progression of Parkinson disease pathology. PMID:21245577

Adherent culture conditions enrich the side population obtained from the cochlear modiolus-derived stem/progenitor cells.

PubMed

Chao, Ting-Ting; Wang, Chih-Hung; Chen, Hsin-Chien; Shih, Cheng-Ping; Sytwu, Huey-Kang; Huang, Kun-Lun; Chen, Shao-Yuan

2013-05-01

Previously, our group reported that sphere-forming cells derived from the organ of Corti represent the stem/progenitor cells (SPCs) of the cochlea due to their properties of self-renewal and multipotency. However, long-term propagation of sphere-forming cells under suspension culture conditions may fail to maintain the characteristic stemness of these cells. Therefore, this study investigated whether an adherent culture system would be beneficial in terms of preserving more stem-like cells for long-term manipulations in vitro. Isolated modiolus-derived SPCs were placed on poly-d-lysine-coated petri dishes to form the so-called "adherent" culture system. Modiolus SPCs cultured under adherent conditions exhibited a significantly increased percentage of cells with the side population (SP) phenotype (18.6%) compared with cells cultured under conventional suspension culture conditions (0.8%). Even after repeated passages, modiolus SPCs cultured under adherent culture conditions preserved more SP phenotype cells. In comparison with the non-SP phenotype cells, the sorted SP cells exhibited more stem-like but less differentiated properties, with an upregulated expression of the ATP-binding cassette subfamily G member 2 (ABCG2), Nestin, Sox2, and Nanog proteins. Furthermore, Retinoic acid (RA) treatment confirmed the expression of the multipotent differentiation markers in the SP cells, including TUJ1, pancytokeratin, glial fibrillary acidic protein (GFAP), and p27(Kip1). Employment of an adherent culture system, instead of a suspension culture system, resulted in the enrichment of the SP cells from SPCs while retaining their stemness and multipotency. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Phenotypic feature quantification of patient derived 3D cancer spheroids in fluorescence microscopy image

NASA Astrophysics Data System (ADS)

Kang, Mi-Sun; Rhee, Seon-Min; Seo, Ji-Hyun; Kim, Myoung-Hee

2017-03-01

Patients' responses to a drug differ at the cellular level. Here, we present an image-based cell phenotypic feature quantification method for predicting the responses of patient-derived glioblastoma cells to a particular drug. We used high-content imaging to understand the features of patient-derived cancer cells. A 3D spheroid culture formation resembles the in vivo environment more closely than 2D adherent cultures do, and it allows for the observation of cellular aggregate characteristics. However, cell analysis at the individual level is more challenging. In this paper, we demonstrate image-based phenotypic screening of the nuclei of patient-derived cancer cells. We first stitched the images of each well of the 384-well plate with the same state. We then used intensity information to detect the colonies. The nuclear intensity and morphological characteristics were used for the segmentation of individual nuclei. Next, we calculated the position of each nucleus that is appeal of the spatial pattern of cells in the well environment. Finally, we compared the results obtained using 3D spheroid culture cells with those obtained using 2D adherent culture cells from the same patient being treated with the same drugs. This technique could be applied for image-based phenotypic screening of cells to determine the patient's response to the drug.

Natural aggregates of the conterminous United States

USGS Publications Warehouse

Langer, William H.

1988-01-01

Crushed stone and sand and gravel are the two main sources of natural aggregates. These materials are commonly used construction materials and frequently can be interchanged with one another. They are widely used throughout the United States, with every State except two producing crushed stone. Together they amount to about half the mining volume in the United States. Approximately 96 percent of sand and gravel and 77 percent of the crushed stone produced in the United States are used in the construction industry. Natural aggregates are widely distributed throughout the United States in a variety of geologic environments. Sand and gravel deposits commonly are the results of the weathering of bedrock and subsequent transportation and deposition of the material by water or ice (glaciers). As such, they commonly occur as river or stream deposits or in glaciated areas as glaciofluvial and other deposits. Crushed stone aggregates are derived from a wide variety of parent bedrock materials. Limestone and other carbonates account for approximately three quarters of the rocks used for crushed stone, with granite and other igneous rocks making up the bulk of the remainder. Limestone deposits are widespread throughout the Central and Eastern United States and are scattered in the West. Granites are widely distributed in the Eastern and Western United States, with few exposures in the Midwest. Igneous rocks (excluding granites) are largely concentrated in the Western United States and in a few isolated localities in the East. Even though natural aggregates are widely distributed throughout the United States, they are not universally available for consumptive use. Some areas are devoid of sand and gravel, and potential sources of crushed stone may be covered with sufficient unconsolidated material to make surface mining impractical. In some areas many aggregates do not meet the physical property requirements for certain uses, or they may contain mineral constituents that react

Aggregation Kinetics of Diesel Soot Nanoparticles in Wet Environments.

PubMed

Chen, Chengyu; Huang, Weilin

2017-02-21

Soot produced during incomplete combustion consists mainly of carbonaceous nanoparticles (NPs) with severe adverse environmental and health effects, and its environmental fate and transport are largely controlled by aggregation. In this study, we examined the aggregation behavior for diesel soot NPs under aqueous condition in an effort to elucidate the fundamental processes that govern soot particle-particle interactions in wet environments such as rain droplets or surface aquatic systems. The influence of electrolytes and aqueous pH on colloidal stability of these NPs was investigated by measuring their aggregation kinetics in different aqueous solution chemistries. The results showed that the NPs had negatively charged surfaces and exhibited both reaction- and diffusion-limited aggregation regimes with rates depended upon solution chemistry. The aggregation kinetics data were in good agreement with the classic Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The critical coagulation concentrations (CCC) were quantified and the Hamaker constant was derived for the soot (1.4 × 10 -20 J) using the colloidal chemistry approach. The study indicated that, depending upon local aqueous chemistry, single soot NPs could remain stable against self-aggregation in typical freshwater environments and in neutral cloud droplets but are likely to aggregate under salty (e.g., estuaries) or acidic (e.g., acid rain droplets) aquatic conditions or both.

Safety and immunogenicity of a trivalent, inactivated, mammalian cell culture-derived influenza vaccine in healthy adults, seniors, and children.

PubMed

Halperin, Scott A; Smith, Bruce; Mabrouk, Taoufik; Germain, Marc; Trépanier, Pierre; Hassell, Thomas; Treanor, John; Gauthier, Richard; Mills, Elaine L

2002-01-15

We performed randomized, double-blind, controlled trials to assess the safety and immunogenicity of an inactivated, Madin Darby Canine Kidney (MDCK)-derived cell line produced influenza vaccine in healthy adults (19-50 years), children (3-12 years) and the elderly (> or =65 years). We studied three lots of cell culture-derived vaccine and one lot of licensed egg-derived vaccine in healthy adults (n=462), two lots of cell culture-derived vaccine and one lot of egg-derived vaccine in seniors (n=269), and one lot of each vaccine in children (n=209). Adverse events were collected during the first 3 days post-immunization; serum was collected before and 1 month after immunization. Rates of local and systemic adverse reactions were similar with both vaccines. An injection site adverse event rated at least moderate severity was reported by 21.9% of children who received the egg-derived vaccine and 25.0% of those who received the cell culture-derived vaccine. In healthy adults the proportions were 12.1 and 15.3%, respectively and 6.7 and 6.3%, respectively in seniors. Systemic events of at least moderate severity were 12.4 and 12.5% in children, 19.8 and 13.6% in healthy adults, and 14.1 and 9.7% in seniors; none of these differences were statistically significant. The antibody response against all three viruses was similar between the two vaccines. From 83 to 100% of children, healthy adults and seniors achieved hemagglutination inhibition titers in excess of 40 post-immunization. We conclude that the cell culture-derived vaccine was safe and immunogenic in children, healthy adults and seniors.

Fish and fisher behaviour influence the vulnerability of groupers (Epinephelidae) to fishing at a multispecies spawning aggregation site

NASA Astrophysics Data System (ADS)

Robinson, J.; Graham, N. A. J.; Cinner, J. E.; Almany, G. R.; Waldie, P.

2015-06-01

Targeted fishing of spawning aggregations is a major contributor to extinction risk in numerous species of grouper (Epinephelidae). Marine reserves are often used to protect spawning aggregation sites, including multispecies sites shared by several species of grouper. However, marine reserves may be biologically, socioeconomically or culturally unviable in some fisheries, and alternative management actions must be explored. Implementing effective management actions that control rather than prohibit fishing requires an improved understanding of how species vary in their vulnerability to fishing gears and respond to changes in fishing effort. To estimate sources of variability in vulnerability to fishing (i.e. catchability), catch-per-unit-effort (CPUE) and other fisheries data were collected in parallel with underwater visual census-derived estimates of aggregation size at a multispecies spawning site of Epinephelus fuscoguttatus and E. polyphekadion. Despite having similar abundances, E. polyphekadion was eightfold more vulnerable to capture by hook-and-line gear, clearly outcompeting its congener for bait. Contrasting with the common assumption of a proportional relationship, the CPUE of both species was unrelated to the size of their respective aggregations. Moreover, the CPUE of each species was unrelated to hook size and depth fished. However, E. polyphekadion CPUE declined as the density of fishing effort increased at the site, with gear saturation identified as the likely mechanism for this effect. E. fuscoguttatus CPUE was negatively related to the size of aggregations formed by its congener, stemming from the superior competitiveness and therefore higher selectivity of the gear for E. polyphekadion. Our findings demonstrate that CPUE is an unreliable indicator of spawning aggregation status. The other sources of variation in CPUE that we identify have implications for gear-based management, which must be based on understanding of gear selectivity for

Sizing aerosolized fractal nanoparticle aggregates through Bayesian analysis of wide-angle light scattering (WALS) data

NASA Astrophysics Data System (ADS)

Huber, Franz J. T.; Will, Stefan; Daun, Kyle J.

2016-11-01

Inferring the size distribution of aerosolized fractal aggregates from the angular distribution of elastically scattered light is a mathematically ill-posed problem. This paper presents a procedure for analyzing Wide-Angle Light Scattering (WALS) data using Bayesian inference. The outcome is probability densities for the recovered size distribution and aggregate morphology parameters. This technique is applied to both synthetic data and experimental data collected on soot-laden aerosols, using a measurement equation derived from Rayleigh-Debye-Gans fractal aggregate (RDG-FA) theory. In the case of experimental data, the recovered aggregate size distribution parameters are generally consistent with TEM-derived values, but the accuracy is impaired by the well-known limited accuracy of RDG-FA theory. Finally, we show how this bias could potentially be avoided using the approximation error technique.

SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI).

PubMed

Chen, Xueping; Guan, Teng; Li, Chen; Shang, Huifang; Cui, Liying; Li, Xin-Min; Kong, Jiming

2012-10-12

Ubiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration. To study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI) and copper-zinc superoxide dismutase (SOD1) were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining. Our data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the formation of ubiquitinated-protein aggregates in cultured

Aggregates from natural and recycled sources; economic assessments for construction applications; a materials flow study

USGS Publications Warehouse

Wilburn, David R.; Goonan, Thomas G.

1998-01-01

Increased amounts of recycled materials are being used to supplement natural aggregates (derived from crushed stone, sand and gravel) in road construction. An understanding of the economics and factors affecting the level of aggregates recycling is useful in estimating the potential for recycling and in assessing the total supply picture of aggregates. This investigation includes a descriptive analysis of the supply sources, technology, costs, incentives, deterrents, and market relationships associated with the production of aggregates.

';Best' Practices for Aggregating Subset Results from Archived Datasets

NASA Astrophysics Data System (ADS)

Baskin, W. E.; Perez, J.

2013-12-01

In response to the exponential growth in science data analysis and visualization capabilities Data Centers have been developing new delivery mechanisms to package and deliver large volumes of aggregated subsets of archived data. New standards are evolving to help data providers and application programmers deal with growing needs of the science community. These standards evolve from the best practices gleaned from new products and capabilities. The NASA Atmospheric Sciences Data Center (ASDC) has developed and deployed production provider-specific search and subset web applications for the CALIPSO, CERES, TES, and MOPITT missions. This presentation explores several use cases that leverage aggregated subset results and examines the standards and formats ASDC developers applied to the delivered files as well as the implementation strategies for subsetting and processing the aggregated products. The following topics will be addressed: - Applications of NetCDF CF conventions to aggregated level 2 satellite subsets - Data-Provider-Specific format requirements vs. generalized standards - Organization of the file structure of aggregated NetCDF subset output - Global Attributes of individual subsetted files vs. aggregated results - Specific applications and framework used for subsetting and delivering derivative data files

Dielectric spectroscopy platform to measure MCF10A epithelial cell aggregation as a model for spheroidal cell cluster analysis.

PubMed

Heileman, K L; Tabrizian, M

2017-05-02

3-Dimensional cell cultures are more representative of the native environment than traditional cell cultures on flat substrates. As a result, 3-dimensional cell cultures have emerged as a very valuable model environment to study tumorigenesis, organogenesis and tissue regeneration. Many of these models encompass the formation of cell aggregates, which mimic the architecture of tumor and organ tissue. Dielectric impedance spectroscopy is a non-invasive, label free and real time technique, overcoming the drawbacks of established techniques to monitor cell aggregates. Here we introduce a platform to monitor cell aggregation in a 3-dimensional extracellular matrix using dielectric spectroscopy. The MCF10A breast epithelial cell line serves as a model for cell aggregation. The platform maintains sterile conditions during the multi-day assay while allowing continuous dielectric spectroscopy measurements. The platform geometry optimizes dielectric measurements by concentrating cells within the electrode sensing region. The cells show a characteristic dielectric response to aggregation which corroborates with finite element analysis computer simulations. By fitting the experimental dielectric spectra to the Cole-Cole equation, we demonstrated that the dispersion intensity Δε and the characteristic frequency f c are related to cell aggregate growth. In addition, microscopy can be performed directly on the platform providing information about cell position, density and morphology. This platform could yield many applications for studying the electrophysiological activity of cell aggregates.

Freezing Responses in DMSO-Based Cryopreservation of Human iPS Cells: Aggregates Versus Single Cells.

PubMed

Li, Rui; Yu, Guanglin; Azarin, Samira M; Hubel, Allison

2018-05-01

Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.

Design, synthesis, and evaluation of resveratrol derivatives as Aß(₁-₄₂) aggregation inhibitors, antioxidants, and neuroprotective agents.

PubMed

Lu, Chuanjun; Guo, Yueyan; Li, Jianheng; Yao, Meicun; Liao, Qiongfeng; Xie, Zhiyong; Li, Xingshu

2012-12-15

A series of novel resveratrol derivatives were designed, synthesised and evaluated as potential therapeutic agents for the treatment of Alzheimer's disease. Among these compounds, compound 7l, (E)-5-(4-(isopropylamino)styryl)benzene-1,3-diol, exhibited potent ß-amyloid aggregation inhibition activity, which was confirmed by a ThT fluorescence assay (71.65% at 20 μM) and transmission electron microscopy (TEM). Compound 7l also exhibited good antioxidant activity (4.12 Trolox equivalents in an oxygen radical absorbance capacity assay and a 37% reduction in reactive oxygen species in cells at 10 μM). The cytotoxicity analysis of compounds 7f, 7i, 7j and 7l indicated that these compounds have lower toxicities than resveratrol at 60 μM. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthetic aggregate compositions derived from spent bed materials from fluidized bed combustion and fly ash

DOEpatents

Boyle, Michael J.

1994-01-01

Cementitious compositions useful as lightweight aggregates are formed from a blend of spent bed material from fluidized bed combustion and fly ash. The proportions of the blend are chosen so that ensuing reactions eliminate undesirable constituents. The blend is then mixed with water and formed into a shaped article. The shaped article is preferably either a pellet or a "brick" shape that is later crushed. The shaped articles are cured at ambient temperature while saturated with water. It has been found that if used sufficiently, the resulting aggregate will exhibit minimal dimensional change over time. The aggregate can be certified by also forming standardized test shapes, e.g., cylinders while forming the shaped articles and measuring the properties of the test shapes using standardized techniques including X-ray diffraction.

ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2

PubMed Central

Winiewska, Maria; Bugajska, Ewa

2017-01-01

The binding of four bromobenzotriazoles to the catalytic subunit of human protein kinase CK2 was assessed by two complementary methods: Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC). New algorithm proposed for the global analysis of MST pseudo-titration data enabled reliable determination of binding affinities for two distinct sites, a relatively strong one with the Kd of the order of 100 nM and a substantially weaker one (Kd > 1 μM). The affinities for the strong binding site determined for the same protein-ligand systems using ITC were in most cases approximately 10-fold underestimated. The discrepancy was assigned directly to the kinetics of ligand nano-aggregates decay occurring upon injection of the concentrated ligand solution to the protein sample. The binding affinities determined in the reverse ITC experiment, in which ligands were titrated with a concentrated protein solution, agreed with the MST-derived data. Our analysis suggests that some ITC-derived Kd values, routinely reported together with PDB structures of protein-ligand complexes, may be biased due to the uncontrolled ligand (nano)-aggregation, which may occur even substantially below the solubility limit. PMID:28273138

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

PubMed Central

Muraglia, Anita; Nguyen, Van Thi; Nardini, Marta; Mogni, Massimo; Coviello, Domenico; Dozin, Beatrice; Strada, Paolo; Baldelli, Ilaria; Formica, Matteo; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-01-01

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype. PMID:29209609

Cell culture-derived influenza vaccines from Vero cells: a new horizon for vaccine production.

PubMed

Montomoli, Emanuele; Khadang, Baharak; Piccirella, Simona; Trombetta, Claudia; Mennitto, Elisa; Manini, Ilaria; Stanzani, Valerio; Lapini, Giulia

2012-05-01

In the 20th century, three influenza pandemics killed approximately 100 million people. The traditional method of influenza vaccine manufacturing is based on using chicken eggs. However, the necessity of the availability of millions of fertile eggs in the event of a pandemic has led research to focus on the development of cell culture-derived vaccines, which offer shorter lead-in times and greater flexibility of production. So far, the cell substrates being evaluated and in use include Vero, Madin-Darby canine kidney, PER.C6 and insect cells. However, Vero cells are the most widely accepted among others. This review introduces briefly the concepts of advanced cell culture-derived influenza vaccine production and highlights the advantages of these vaccines in terms of efficiency, speed and immunogenicity based on the clinical data obtained from different studies.

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

PubMed

Díez, José M; Bauman, Ewa; Gajardo, Rodrigo; Jorquera, Juan I

2015-03-13

Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Cryopreservation of pluripotent stem cell aggregates in defined protein-free formulation.

PubMed

Sart, Sébastien; Ma, Teng; Li, Yan

2013-01-01

Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as an efficient culture configuration, enabling rapid and controlled large scale expansion. Aggregate-based PSC cryopreservation facilitates the integrated process of cell expansion and cryopreservation, but its feasibility has not been demonstrated. The goals of current study are to assess the suitability of cryopreserving intact mouse embryonic stem cell (mESC) aggregates and investigate the effects of aggregate size and the formulation of cryopreservation solution on mESC survival and recovery. The results demonstrated the size-dependent cell survival and recovery of intact aggregates. In particular, the generation of reactive oxygen species (ROS) and caspase activation were reduced for small aggregates (109 ± 55 μm) compared to medium (245 ± 77 μm) and large (365 ± 141 μm) ones, leading to the improved cell recovery. In addition, a defined protein-free formulation was tested and found to promote the aggregate survival, eliminating the cell exposure to animal serum. The cryopreserved aggregates also maintained the pluripotent markers and the differentiation capacity into three-germ layers after thawing. In summary, the cryopreservation of small PSC aggregates in a defined protein-free formulation was shown to be a suitable approach toward a fully integrated expansion and cryopreservation process at large scale. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

In vitro immunotoxicity assessment of culture-derived extracellular vesicles in human monocytes

PubMed Central

Rosas, Lucia E.; Elgamal, Ola A.; Mo, Xiaokui; Phelps, Mitch A.; Schmittgen, Thomas D.; Papenfuss, Tracey L.

2016-01-01

The potential to engineer extracellular vesicles (EV) that target specific cells and deliver a therapeutic payload has propelled a growing interest in their development as promising therapeutics. These EV are often produced from cultured cells. Very little is known about the interaction of cell culture-derived EV with cells of the immune system and their potential immunomodulatory effects. The present study evaluated potential immunotoxic effects of HEK293T-derived EV on the human monocytic cell lines THP-1 and U937. Incubation of cells with different doses of EV for 16–24 h was followed by assessment of cytotoxicity and cell function by flow cytometry. Changes in cell functionality were evaluated by the capacity of cells to phagocytize fluorescent microspheres. In addition, the internalization of labeled EV in THP-1 and U937 cells was evaluated. Exposure to EV did not affect the viability of THP-1 or U937 cells. Although lower doses of the EV increased phagocytic capacity in both cell lines, phagocytic efficiency of individual cells was not affected by EV exposure at any of the doses evaluated. This study also demonstrated that THP-1 and U937 monocytic cells are highly permissive to EV entry in a dose-response manner. These results suggest that, although HEK293T-derived EV are efficiently internalized by human monocytic cells, they do not exert a cytotoxic effect or alter phagocytic efficiency on the cell lines evaluated. PMID:27075513

Role of prion protein aggregation in neurotoxicity.

PubMed

Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Nizzari, Mario; Florio, Tullio

2012-01-01

In several neurodegenerative diseases, such as Parkinson, Alzheimer's, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.

Photodynamic toxicity of hematoporphyrin derivatives to human keratinocytes in culture.

PubMed

Kappus, H; Reinhold, C; Artuc, M

Human keratinocytes in culture were able to take up hematoporphyrin derivatives (HPDs) used during photodynamic chemotherapy of tumors. In the absence of light, HPDs showed no cytotoxic effects to keratinocytes. However, after irradiation with visible light, HPDs induced immediate cytotoxicity as measured by the neutral red uptake assay. On the other hand, cell attachment as measured by protein estimation was not affected. When the cells treated with HPDs and irradiated with light were cultured for a further 72 h, they partially lost their ability to attach to the collagen surface. Most of the cells remaining attached after 72 h were no longer viable following treatment with HPDs and light. All parameters measured depended on the intracellular concentration of HPDs used (7-50 ng/10(5) cells) and the time of irradiation (0-30 min). These results suggest that human keratinocytes are a good model to study cytotoxic effects of photodynamically active drugs. Further, keratinocytes were unable to recover after damage caused by HPDs and light.

Probabilistic prediction models for aggregate quarry siting

USGS Publications Warehouse

Robinson, G.R.; Larkins, P.M.

2007-01-01

Weights-of-evidence (WofE) and logistic regression techniques were used in a GIS framework to predict the spatial likelihood (prospectivity) of crushed-stone aggregate quarry development. The joint conditional probability models, based on geology, transportation network, and population density variables, were defined using quarry location and time of development data for the New England States, North Carolina, and South Carolina, USA. The Quarry Operation models describe the distribution of active aggregate quarries, independent of the date of opening. The New Quarry models describe the distribution of aggregate quarries when they open. Because of the small number of new quarries developed in the study areas during the last decade, independent New Quarry models have low parameter estimate reliability. The performance of parameter estimates derived for Quarry Operation models, defined by a larger number of active quarries in the study areas, were tested and evaluated to predict the spatial likelihood of new quarry development. Population density conditions at the time of new quarry development were used to modify the population density variable in the Quarry Operation models to apply to new quarry development sites. The Quarry Operation parameters derived for the New England study area, Carolina study area, and the combined New England and Carolina study areas were all similar in magnitude and relative strength. The Quarry Operation model parameters, using the modified population density variables, were found to be a good predictor of new quarry locations. Both the aggregate industry and the land management community can use the model approach to target areas for more detailed site evaluation for quarry location. The models can be revised easily to reflect actual or anticipated changes in transportation and population features. ?? International Association for Mathematical Geology 2007.

Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture

PubMed Central

Pleumeekers, M. M.; Nimeskern, L.; Koevoet, J. L. M.; Karperien, M.; Stok, K. S.; van Osch, G. J. V. M.

2018-01-01

Aims Combining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (hAMSCs) and bone-marrow-derived MSCs (hBMSCs) combined with bovine articular chondrocytes (bACs) was compared. Methods hAMSCs or hBMSCs were combined with bACs in alginate and cultured in vitro or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between bACs and hMSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted. Results The presence of hMSCs–either hAMSCs or hBMSCs—increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in hBMSC/bACs. This effect was similar when hMSCs and bAC were combined in pellet culture, in alginate culture or when conditioned media of hMSCs were used on bAC. Species-specific gene-expression analyses demonstrated that aggrecan was expressed by bACs only, indicating a predominantly trophic role for hMSCs. Collagen-10-gene expression of bACs was not affected by hBMSCs, but slightly enhanced by hAMSCs. After in-vivo implantation, hAMSC/bACs and hBMSC/bACs had similar cartilage matrix production, both appeared stable and did not calcify. Conclusions This study demonstrates that replacing 80% of bACs by either hAMSCs or hBMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by hMSCs. PMID:29489829

Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture.

PubMed

Pleumeekers, M M; Nimeskern, L; Koevoet, J L M; Karperien, M; Stok, K S; van Osch, G J V M

2018-01-01

Combining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (hAMSCs) and bone-marrow-derived MSCs (hBMSCs) combined with bovine articular chondrocytes (bACs) was compared. hAMSCs or hBMSCs were combined with bACs in alginate and cultured in vitro or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between bACs and hMSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted. The presence of hMSCs-either hAMSCs or hBMSCs-increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in hBMSC/bACs. This effect was similar when hMSCs and bAC were combined in pellet culture, in alginate culture or when conditioned media of hMSCs were used on bAC. Species-specific gene-expression analyses demonstrated that aggrecan was expressed by bACs only, indicating a predominantly trophic role for hMSCs. Collagen-10-gene expression of bACs was not affected by hBMSCs, but slightly enhanced by hAMSCs. After in-vivo implantation, hAMSC/bACs and hBMSC/bACs had similar cartilage matrix production, both appeared stable and did not calcify. This study demonstrates that replacing 80% of bACs by either hAMSCs or hBMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by hMSCs.

Biphasic cultivation strategy to avoid Epo-Fc aggregation and optimize protein expression.

PubMed

Kaisermayer, Christian; Reinhart, David; Gili, Andreas; Chang, Martina; Aberg, Per-Mikael; Castan, Andreas; Kunert, Renate

2016-06-10

In biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. In the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. The influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and pH were shifted. Applying a DoE (Design of Experiments) approach, a fractional factorial design was used to systematically evaluate the influence of cultivation temperature and pH as well as their synergistic effect on cell growth as well as on recombinant protein production and aggregation. All three responses were influenced by the cultivation temperature. Additionally, an interaction between pH and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37°C and pH 7.05, a parameter shift to low temperature and acidic pH resulted in a decrease in the aggregate fraction from 75% to less than 1%. Furthermore, the synergistic effect of temperature and pH substantially lowered the cell-specific rates of glucose and glutamine consumption as well as lactate and ammonium production. The optimized culture conditions also led to an increase of the cell-specific rates of recombinant Epo-Fc production, thus resulting in a more economic bioprocess. Copyright © 2016. Published by Elsevier B.V.

Cell culture media impact on drug product solution stability.

PubMed

Purdie, Jennifer L; Kowle, Ronald L; Langland, Amie L; Patel, Chetan N; Ouyang, Anli; Olson, Donald J

2016-07-08

To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016. © 2016 American Institute of Chemical Engineers.

Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly.

PubMed

Pham, Tu M; Tran, Si C; Lim, Yun-Sook; Hwang, Soon B

2017-02-01

Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were upregulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core protein and deposited core protein into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region, which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly. Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at the transcriptional level and altered the balance between GDP- and GTP-bound Rab32 toward production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core protein and enriched core in the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea.

PubMed

Prabhudesai, V; Bhaskaran, S

1993-03-01

An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL(-1) BA and 0.1 mgL(-1) NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL(-1)), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro.

PubMed

Baillie-Johnson, Peter; van den Brink, Susanne Carina; Balayo, Tina; Turner, David Andrew; Martinez Arias, Alfonso

2015-11-24

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/β-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

PubMed

Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa

2009-04-01

The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

Aggregator: a machine learning approach to identifying MEDLINE articles that derive from the same underlying clinical trial.

PubMed

Shao, Weixiang; Adams, Clive E; Cohen, Aaron M; Davis, John M; McDonagh, Marian S; Thakurta, Sujata; Yu, Philip S; Smalheiser, Neil R

2015-03-01

It is important to identify separate publications that report outcomes from the same underlying clinical trial, in order to avoid over-counting these as independent pieces of evidence. We created positive and negative training sets (comprised of pairs of articles reporting on the same condition and intervention) that were, or were not, linked to the same clinicaltrials.gov trial registry number. Features were extracted from MEDLINE and PubMed metadata; pairwise similarity scores were modeled using logistic regression. Article pairs from the same trial were identified with high accuracy (F1 score=0.843). We also created a clustering tool, Aggregator, that takes as input a PubMed user query for RCTs on a given topic, and returns article clusters predicted to arise from the same clinical trial. Although painstaking examination of full-text may be needed to be conclusive, metadata are surprisingly accurate in predicting when two articles derive from the same underlying clinical trial. Copyright © 2014 Elsevier Inc. All rights reserved.

Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture

PubMed Central

Eildermann, K.; Gromoll, J.; Behr, R.

2012-01-01

BACKGROUND Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro. METHODS AND RESULTS We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4. CONCLUSIONS Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells. PMID:22442249

Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

PubMed

Betzer, Cristine; Lassen, Louise Berkhoudt; Olsen, Anders; Kofoed, Rikke Hahn; Reimer, Lasse; Gregersen, Emil; Zheng, Jin; Calì, Tito; Gai, Wei-Ping; Chen, Tong; Moeller, Arne; Brini, Marisa; Fu, Yuhong; Halliday, Glenda; Brudek, Tomasz; Aznar, Susana; Pakkenberg, Bente; Andersen, Jens Peter; Jensen, Poul Henning

2018-05-01

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca 2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca 2+ levels followed by a later Ca 2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro , and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca 2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Universal timescales in the rheology of spheroid cell aggregates

NASA Astrophysics Data System (ADS)

Yu, Miao; Mahtabfar, Aria; Beleen, Paul; Foty, Ramsey; Zahn, Jeffrey; Shreiber, David; Liu, Liping; Lin, Hao

2017-11-01

The rheological properties of tissue play important roles in key biological processes including embryogenesis, cancer metastasis, and wound healing. Spheroid cell aggregate is a particularly interesting model system for the study of these phenomena. In the long time, they behave like drops with a surface tension. In the short, viscoelasticity also needs to be considered. In this work, we discover two coupled and universal timescales for spheroid aggregates. A total of 12 aggregate types (total aggregate number n =290) derived from L and GBM (glioblastoma multiforme) cells are studied with microtensiometer to obtain their surface tension. They are also allowed to relax upon release of the compression forces. The two timescales are observed during the relaxation process; their values do not depend on compression time nor the degree of deformation, and are consistent among all 12 types. Following prior work (Yu et al., Phys. Rev. Lett., 115:128303; Liu et al., J. Mech. Phys. Solids, 98:309-329) we use a rigorous mathematical theory to interpret the results, which reveals intriguing properties of the aggregates on both tissue and cellular levels. The mechanics of multicellular organization reflects both complexity and regularity due to strong active regulation.

Sulfur mustard induces the formation of keratin aggregates in human epidermal keratinocytes.

PubMed

Dillman, James F; McGary, Kriston L; Schlager, John J

2003-12-01

The vesicant sulfur mustard is an alkylating agent that has the capacity to cross-link biological molecules. We are interested in identifying specific proteins that are altered upon sulfur mustard exposure. Keratins are particularly important for the structural integrity of skin, and several genetically inherited blistering diseases have been linked to mutations in keratin 5 and keratin 14. We examined whether sulfur mustard exposure alters keratin biochemistry in cultured human epidermal keratinocytes. Western blotting with specific monoclonal antibodies revealed the formation of stable high-molecular-weight "aggregates" containing keratin 14 and/or keratin 5. These aggregates begin to form within 15 min after sulfur mustard exposure. These aggregates display a complex gel electrophoresis pattern between approximately 100 and approximately 200 kDa. Purification and analysis of these aggregates by one- and two-dimensional gel electrophoresis and mass spectrometry confirmed the presence of keratin 14 and keratin 5 and indicate that at least some of the aggregates are composed of keratin 14-keratin 14, keratin 14-keratin 5, or keratin 5-keratin 5 dimers. These studies demonstrate that sulfur mustard induces keratin aggregation in keratinocytes and support further investigation into the role of keratin aggregation in sulfur mustard-induced vesication.

HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms.

PubMed

Liste-Calleja, Leticia; Lecina, Martí; Cairó, Jordi Joan

2014-04-01

The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among those strategies, cell culture media development is of major interest. In the present work, several commercially available culture media for Human Embryonic Kidney cells (HEK293) were evaluated in terms of maximal specific growth rate and maximal viable cell concentration supported. The main objective was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained. Performing simple media supplementations with and without animal derived components, an enhancement of cell concentration from 2 × 10(6) cell/mL to 17 × 10(6) cell/mL was achieved in batch mode operation. Additionally, the media were evaluated for adenovirus production as a specific application case of HEK293 cells. None of the supplements interfered significantly with the adenovirus infection although some differences were encountered in viral productivity. To the best of our knowledge, the high cell density achieved in the work presented has never been reported before in HEK293 batch cell cultures and thus, our results are greatly promising to further study cell culture strategies in bioreactor towards bioprocess optimization. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Changes on aggregation in mine waste amended with biochar and marble mud

NASA Astrophysics Data System (ADS)

Ángeles Muñoz, María; Guzmán, Jose; Zornoza, Raúl; Moreno-Barriga, Fabián; Faz, Ángel; Lal, Rattan

2016-04-01

Mining activities have produced large amounts of wastes over centuries accumulated in tailing ponds in Southeast Spain. Applications of biochar may have a high potential for reclamation of degraded soils. Distribution, size and stability of aggregates are important indices of soil physical quality. However, research data on aggregation processes at amended mining tailings with biochar are scanty. Therefore, the aim of this study was to determine the effects of seven different treatments involving biochar and marble mud (MM) on the aggregation in mine waste (MW). Seven different treatments were tested after 90 days of incubation in the laboratory. These treatments were the mix of MW and: biochar from solid pig manure (PM), biochar from cotton crop residues (CR), biochar from municipal solid waste (MSW), marble mud (MM), PM+MM, CR+MM, MSW+MM and control without amendment. High sand percentages were identified in the MW. The biochars made from wastes (PM, CR, MSW) were obtained through pyrolysis of feedstocks. The water stability of soil aggregates was studied. The data on total aggregation were corrected for the primary particles considering the sandy texture of the MW. Moreover, partial aggregation was determined for each fraction and the mean weight diameter (MWD) of aggregates was computed. Soil bulk density and total porosity were also determined. No significant differences were observed in total aggregation and MWD among treatments including the control. For the size range of >4.75 mm, there were significant differences in aggregates > 4.75 mm between CR+MM in comparison with that for CT. There were also significant differences between MSW and PM+MM for the 1-0.425 mm fraction, and between CT and MM and CR for 0.425-0.162 mm aggregate size fractions. Therefore, CR-derived biochar applied with MM enhanced stability of macro-aggregates. Furthermore, soil bulk density was also the lowest bulk density and total porosity the highest for the CR-derived biochar

Cultured normal mammalian tissue and process

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor)

1993-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cell aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

PubMed

Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-Hara, Tomoko; Fujita, Naoya

2014-08-01

The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Extraction and Assembly of Tissue-Derived Gels for Cell Culture and Tissue Engineering

PubMed Central

Uriel, Shiri; Labay, Edwardine; Francis-Sedlak, Megan; Moya, Monica L.; Weichselbaum, Ralph R.; Ervin, Natalia; Cankova, Zdravka

2009-01-01

Interactions with the extracellular matrix (ECM) play an important role in regulating cell function. Cells cultured in, or on, three-dimensional ECM recapitulate similar features to those found in vivo that are not present in traditional two-dimensional culture. In addition, both natural and synthetic materials containing ECM components have shown promise in a number of tissue engineering applications. Current materials available for cell culture and tissue engineering do not adequately reflect the diversity of ECM composition between tissues. In this paper, a method is presented for extracting solutions of proteins and glycoproteins from soft tissues and inducing assembly of these proteins into gels. The extracts contain ECM proteins specific to the tissue source with low levels of intracellular molecules. Gels formed from the tissue-derived extracts have nanostructure similar to ECM in vivo and can be used to culture cells as both a thin substrate coating and a thick gel. This technique could be used to assemble hydrogels with varying composition depending upon the tissue source, hydrogels for three-dimensional culture, as scaffolds for tissue engineering therapies, and to study cell–matrix interactions. PMID:19115821

Diffusional creep of fine-grained olivine aggregates: Chemical and melt effects

NASA Astrophysics Data System (ADS)

Yabe, K.; Hiraga, T.

2017-12-01

still harder than olivine aggregates synthesized from naturally derived olivine crystals. Trace elements other than Ca and Al, which segregate at grain boundaries in naturally-derived olivine aggregates, is likely to induce further weakening of olivine aggregates.

Characterization of a Cartilage-Like Engineered Biomass Using a Self-Aggregating Suspension Culture Model: Molecular Composition Using FT-IRIS

PubMed Central

Kim, Minwook; Kraft, Jeffrey J.; Volk, Andrew C.; Pugarelli, Joan; Pleshko, Nancy; Dodge, George R.

2011-01-01

Maintenance of chondrocyte phenotype and robust expression and organization of macromolecular components with suitable cartilaginous properties is an ultimate goal in cartilage tissue engineering. We used a self-aggregating suspension culture (SASC) method to produce an engineered cartilage, “cartilage tissue analog” (CTA). With an objective of understanding the stability of phenotype of the CTA over long periods, we cultured chondrocytes up to 4 years and analyzed the matrix. Both early (eCTAs) (6 months) and aged (aCTAs) (4 years) showed type II collagen throughout with higher concentrations near the edge. Using Fourier transform-infrared imaging spectroscopy (FT-IRIS), proteoglycan/collagen ratio of eCTA was 2.8 times greater than native cartilage at 1 week, but the ratio was balanced to native level (p = 0.017) by 36 weeks. Surprisingly, aCTAs maintained the hyaline characteristics, but there was evidence of calcification within the tissue with a distinct range of intensities. Mineral/matrix ratio of those aCTA with “intensive” calcification was significantly higher (p = 0.017) than the “partial,” but when compared to native bone the ratio of “intensive” aCTAs was 2.4 times lower. In this study we utilized the imaging approach of FT-IRIS and have shown that a biomaterial formed is compositionally closely related to natural cartilage for long periods in culture. We show that this culture platform can maintain a CTA for extended periods of time (4 years) and under those conditions signs of mineralization can be found. This method of cartilage tissue engineering is a promising method to generate cartilaginous biomaterial and may have potential to be utilized in both cartilage and boney repairs. PMID:21630329

Which has more stem-cell characteristics: Müller cells or Müller cells derived from in vivo culture in neurospheres?

PubMed

Ji, Hong-Pei; Xiong, Yu; Zhang, En-Dong; Song, Wei-Tao; Gao, Zhao-Lin; Yao, Fei; Sun, Hong; Zhou, Rong-Rong; Xia, Xiao-Bo

2017-01-01

Müller cells can be acquired from in vitro culture or a neurosphere culture system. Both culture methods yield cells with progenitor-cell characteristics that can differentiate into mature nervous cells. We compared the progenitor-cell traits of Müller cells acquired from each method. Primary murine Müller cells were isolated in serum culture media and used to generate Müller cells derived from neurospheres in serum-free culture conditions. Gene expression of neural progenitor cell markers was examined by Q-PCR in the two groups. Expression of rhodopsin and the cone-rod homeobox protein CRX were assessed after induction with 1 μM all-trans retinoic acid (RA) for 7 days. After more than four passages, many cells were large, flattened, and difficult to passage. A spontaneously immortalized Müller cell line was not established. Three-passage neurospheres yielded few new spheres. Genes coding for Nestin, Sox2, Chx10, and Vimentin were downregulated in cells derived from neurospheres compared to the cells from standard culture, while Pax6 was upregulated. Müller cells from both culture methods were induced into rod photoreceptors, but expression of rhodopsin and CRX was greater in the Müller cells from the standard culture. Both culture methods yielded cells with stem-cell characteristics that can be induced into rod photoreceptor neurons by RA. Serum had no influence on the "stemness" of the cells. Cells from standard culture had greater "stemness" than cells derived from neurospheres. The standard Müller cells would seem to be the best choice for transplantation in cell replacement therapy for photoreceptor degeneration.

Calcium oxalate monohydrate aggregation induced by aggregation of desialylated Tamm-Horsfall protein

PubMed Central

Viswanathan, Pragasam; Rimer, Jeffrey D.; Kolbach, Ann M.; Kleinman, Jack G.

2011-01-01

Tamm-Horsfall protein (THP) is thought to protect against calcium oxalate monohydrate (COM) stone formation by inhibiting COM aggregation. Several studies reported that stone formers produce THP with reduced levels of glycosylation, particularly sialic acid levels, which leads to reduced negative charge. In this study, normal THP was treated with neuraminidase to remove sialic acid residues, confirmed by an isoelectric point shift to higher pH. COM aggregation assays revealed that desialylated THP (ds-THP) promoted COM aggregation, while normal THP inhibited aggregation. The appearance of protein aggregates in solutions at ds-THP concentrations ≥1 µg/mL in 150 mM NaCl correlated with COM aggregation promotion, implying that ds-THP aggregation induced COM aggregation. The aggregation-promoting effect of the ds-THP was independent of pH above its isoelectric point, but was substantially reduced at low ionic strength, where protein aggregation was much reduced. COM aggregation promotion was maximized at a ds-THP to COM mass ratio of ~0.025, which can be explained by a model wherein partial COM surface coverage by ds-THP aggregates promotes crystal aggregation by bridging opposing COM surfaces, whereas higher surface coverage leads to repulsion between adsorbed ds-THP aggregates. Thus, desialylation of THP apparently abrogates a normal defensive action of THP by inducing protein aggregation, and subsequently COM aggregation, a condition that favors kidney stone formation. PMID:21229239

A three-dimensional human neural cell culture model of Alzheimer's disease.

PubMed

Choi, Se Hoon; Kim, Young Hye; Hebisch, Matthias; Sliwinski, Christopher; Lee, Seungkyu; D'Avanzo, Carla; Chen, Hechao; Hooli, Basavaraj; Asselin, Caroline; Muffat, Julien; Klee, Justin B; Zhang, Can; Wainger, Brian J; Peitz, Michael; Kovacs, Dora M; Woolf, Clifford J; Wagner, Steven L; Tanzi, Rudolph E; Kim, Doo Yeon

2014-11-13

Alzheimer's disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer's disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer's disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer's disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer's disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer's disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell

What You Know, What You Do, and How You Feel: Cultural Competence, Cultural Consonance, and Psychological Distress

PubMed Central

Dressler, William W.; Balieiro, Mauro C.; dos Santos, José E.

2018-01-01

Describing the link between culture (as a phenomenon pertaining to social aggregates) and the beliefs and behaviors of individuals has eluded satisfactory resolution; however, contemporary cognitive culture theory offers hope. In this theory, culture is conceptualized as cognitive models describing specific domains of life that are shared by members of a social group. It is sharing that gives culture its aggregate properties. There are two aspects to these cultural models at the level of the individual. Persons have their own representations of the world that correspond incompletely to the shared model—this is their ‘cultural competence.’ Persons are also variable in the degree to which they can put cultural models into practice in their own lives—this is their ‘cultural consonance.’ Low cultural consonance is a stressful experience and has been linked to higher psychological distress. The relationship of cultural competence per se and psychological distress is less clear. In the research reported here, cultural competence and cultural consonance are measured on the same sample and their associations with psychological distress are examined using multiple regression analysis. Results indicate that, with respect to psychological distress, while it is good to know the cultural model, it is better to put it into practice. PMID:29379460

Measuring Aggregation of Events about a Mass Using Spatial Point Pattern Methods

PubMed Central

Smith, Michael O.; Ball, Jackson; Holloway, Benjamin B.; Erdelyi, Ferenc; Szabo, Gabor; Stone, Emily; Graham, Jonathan; Lawrence, J. Josh

2017-01-01

We present a methodology that detects event aggregation about a mass surface using 3-dimensional study regions with a point pattern and a mass present. The Aggregation about a Mass function determines aggregation, randomness, or repulsion of events with respect to the mass surface. Our method closely resembles Ripley’s K function but is modified to discern the pattern about the mass surface. We briefly state the definition and derivation of Ripley’s K function and explain how the Aggregation about a Mass function is different. We develop the novel function according to the definition: the Aggregation about a Mass function times the intensity is the expected number of events within a distance h of a mass. Special consideration of edge effects is taken in order to make the function invariant to the location of the mass within the study region. Significance of aggregation or repulsion is determined using simulation envelopes. A simulation study is performed to inform researchers how the Aggregation about a Mass function performs under different types of aggregation. Finally, we apply the Aggregation about a Mass function to neuroscience as a novel analysis tool by examining the spatial pattern of neurotransmitter release sites as events about a neuron. PMID:29046865

A novel ruthenium (II)-derived organometallic compound, TQ-6, potently inhibits platelet aggregation: Ex vivo and in vivo studies.

PubMed

Hsia, Chih-Hsuan; Velusamy, Marappan; Sheu, Joen-Rong; Khamrang, Themmila; Jayakumar, Thanasekaran; Lu, Wan-Jung; Lin, Kuan-Hung; Chang, Chao-Chien

2017-08-25

Arterial thrombosis plays a key role in cardiovascular diseases. Hence, developing more effective antithrombotic agents is necessary. We designed a ruthenium (II)-derived complex, [Ru(η 6 -cymene)2-(1H-benzoimidazol-2-yl)-quinoline Cl]BF 4 (TQ-6), as a new antiplatelet drug. TQ-6 (0.3 µM) exhibited extremely strong inhibitory activity against platelet aggregation, Src, and Syk phosphorylation stimulated by agonists in human platelets. In collagen-activated platelets, TQ-6 also inhibited ATP-release, [Ca +2 ]i, P-selectin expression, FITC-PAC-1 binding, and hydroxyl radical formation, as well as the phosphorylation of phospholipase Cγ2, protein kinase C, mitogen-activated protein kinases, and Akt. Neither FITC-JAQ1 nor FITC-triflavin binding or integrin β 3 phosphorylation stimulated by immobilized fibrinogen were diminished by TQ-6. Furthermore, TQ-6 had no effects in cyclic nucleotide formation. Moreover, TQ-6 substantially prolonged the closure time in whole blood, increased the occlusion time of thrombotic platelet plug formation and bleeding time in mice. In conclusion, TQ-6 has a novel role in inhibiting platelet activation through the inhibition of the agonist receptors-mediated inside-out signaling such as Src-Syk-PLCγ2 cascade and subsequent suppression of granule secretion, leading to disturb integrin α IIb β 3 -mediated outside-in signaling, and ultimately inhibiting platelet aggregation. Therefore, TQ-6 has potential to develop as a therapeutic agent for preventing or treating thromboembolic disorders.

Rydberg aggregates

NASA Astrophysics Data System (ADS)

Wüster, S.; Rost, J.-M.

2018-02-01

We review Rydberg aggregates, assemblies of a few Rydberg atoms exhibiting energy transport through collective eigenstates, considering isolated atoms or assemblies embedded within clouds of cold ground-state atoms. We classify Rydberg aggregates, and provide an overview of their possible applications as quantum simulators for phenomena from chemical or biological physics. Our main focus is on flexible Rydberg aggregates, in which atomic motion is an essential feature. In these, simultaneous control over Rydberg-Rydberg interactions, external trapping and electronic energies, allows Born-Oppenheimer surfaces for the motion of the entire aggregate to be tailored as desired. This is illustrated with theory proposals towards the demonstration of joint motion and excitation transport, conical intersections and non-adiabatic effects. Additional flexibility for quantum simulations is enabled by the use of dressed dipole-dipole interactions or the embedding of the aggregate in a cold gas or Bose-Einstein condensate environment. Finally we provide some guidance regarding the parameter regimes that are most suitable for the realization of either static or flexible Rydberg aggregates based on Li or Rb atoms. The current status of experimental progress towards enabling Rydberg aggregates is also reviewed.

Potential for direct interspecies electron transfer in methanogenic wastewater digester aggregates.

PubMed

Morita, Masahiko; Malvankar, Nikhil S; Franks, Ashley E; Summers, Zarath M; Giloteaux, Ludovic; Rotaru, Amelia E; Rotaru, Camelia; Lovley, Derek R

2011-01-01

Mechanisms for electron transfer within microbial aggregates derived from an upflow anaerobic sludge blanket reactor converting brewery waste to methane were investigated in order to better understand the function of methanogenic consortia. The aggregates were electrically conductive, with conductivities 3-fold higher than the conductivities previously reported for dual-species aggregates of Geobacter species in which the two species appeared to exchange electrons via interspecies electron transfer. The temperature dependence response of the aggregate conductance was characteristic of the organic metallic-like conductance previously described for the conductive pili of Geobacter sulfurreducens and was inconsistent with electron conduction through minerals. Studies in which aggregates were incubated with high concentrations of potential electron donors demonstrated that the aggregates had no significant capacity for conversion of hydrogen to methane. The aggregates converted formate to methane but at rates too low to account for the rates at which that the aggregates syntrophically metabolized ethanol, an important component of the reactor influent. Geobacter species comprised 25% of 16S rRNA gene sequences recovered from the aggregates, suggesting that Geobacter species may have contributed to some but probably not all of the aggregate conductivity. Microorganisms most closely related to the acetate-utilizing Methanosaeta concilii accounted for more than 90% of the sequences that could be assigned to methane producers, consistent with the poor capacity for hydrogen and formate utilization. These results demonstrate for the first time that methanogenic wastewater aggregates can be electrically conductive and suggest that direct interspecies electron transfer could be an important mechanism for electron exchange in some methanogenic systems.

Maintaining Elastogenicity of Mesenchymal Stem Cell-Derived Smooth Muscle Cells in Two-Dimensional Culture.

PubMed

Dahal, Shataakshi; Broekelman, Thomas; Mecham, Robert P; Ramamurthi, Anand

2018-06-01

Abdominal aortic aneurysms (AAAs) are localized expansions of the abdominal aorta that grow slowly to rupture. AAA growth is driven by irreversible elastic matrix breakdown in the aorta wall by chronically upregulated matrix metalloproteases (MMPs). Since adult vascular smooth muscle cells (SMCs) poorly regenerate elastic matrix, we previously explored utility of bone marrow mesenchymal stem cells and SMCs derived therefrom (BM-SMCs) for this purpose. One specific differentiated phenotype (cBM-SMCs) generated on a fibronectin substrate in presence of exogenous transforming growth factor-β and platelet-derived growth factor exhibited superior elastogenicity versus other phenotypes, and usefully provided proelastogenic and antiproteolytic stimuli to aneurysmal SMCs. Since in vivo cell therapy demands large cell inoculates, these derived SMCs must be propagated in vitro while maintaining their superior elastogenic, proelastogenic, and antiproteolytic characteristics. In this work, we thus investigated the culture conditions that must be provided to this propagation phase, which ensure that the differentiated SMCs maintain their phenotype and matrix regenerative benefits. Our results indicate that our BM-SMCs retain their phenotype in long-term culture even in the absence of differentiation growth factors and fibronectin substrate, but these conditions must be continued to be provided during postdifferentiation propagation if they are to maintain their superior elastic matrix deposition, crosslinking, and fiber formation properties. Our study, however, showed that cells propagated under these conditions exhibit higher expression of MMP-2, but favorably, no expression of elastolytic MMP-9. Hence, the study outcomes provide crucial guidelines to maintain phenotypic stability of cBM-SMCs during their propagation in two-dimensional culture before their delivery to the AAA wall for therapy.

A local PDE model of aggregation formation in bacterial colonies

NASA Astrophysics Data System (ADS)

Chavy-Waddy, Paul-Christopher; Kolokolnikov, Theodore

2016-10-01

We study pattern formation in a model of cyanobacteria motion recently proposed by Galante, Wisen, Bhaya and Levy. By taking a continuum limit of their model, we derive a novel fourth-order nonlinear parabolic PDE equation that governs the behaviour of the model. This PDE is {{u}t}=-{{u}xx}-{{u}xxxx}+α {{≤ft(\\frac{{{u}x}{{u}xx}}{u}\\right)}x} . We then derive the instability thresholds for the onset of pattern formation. We also compute analytically the spatial profiles of the steady state aggregation density. These profiles are shown to be of the form \\text{sec}{{\\text{h}}p} where the exponent p is related to the parameters of the model. Full numerical simulations give a favorable comparison between the continuum and the underlying discrete system, and show that the aggregation profiles are stable above the critical threshold.

Role of Prion Protein Aggregation in Neurotoxicity

PubMed Central

Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Nizzari, Mario; Florio, Tullio

2012-01-01

In several neurodegenerative diseases, such as Parkinson, Alzheimer’s, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death. PMID:22942726

Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.

PubMed

Yamazaki, Taisuke; Wakai, Mariko; Enosawa, Shin; Tokiwa, Takayoshi

2017-06-01

Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.

Genipin-crosslinked microcarriers mediating hepatocellular aggregates formation and functionalities.

PubMed

Lau, Ting Ting; Wang, Chunming; Png, Sze Wei; Su, Kai; Wang, Dong-An

2011-01-01

In engineered regenerative medicine, various types of scaffolds have been customized to pursue the optimal environment for different types of therapeutic cells. In liver therapeutic research, hepatocytes require attachment to solid anchors for survival and proliferation before they could grow into cellular aggregates with enhanced functionalities. Among the various biomaterials scaffolds and vehicles, microspherical cell carriers are suited to these requirements. Individual spheres may provide two-dimensional (2D) cell-affinitive surfaces for cell adhesion and spreading; whereas multiple microcarriers may form three-dimensional (3D) matrices with inter-spherical space for cell expansion and multicellular aggregation. In this study, we culture human liver carcinoma cell line (HepG2) cells on genipin-crosslinked gelatin microspheres of two different sizes. Results suggest that both microcarriers support cell adhesion, proliferation, and spontaneous formation of hepatocellular aggregates, among which the spheres with bigger size (200-300 μm) seem more favorable than the smaller ones in terms of aggregate formation and liver specific functionalities. These findings suggest that the genipin-crosslinked microcarrier is a competent vehicle for liver cell delivery. Copyright © 2010 Wiley Periodicals, Inc.

Hair Follicle and Sebaceous Gland De Novo Regeneration With Cultured Epidermal Stem Cells and Skin-Derived Precursors.

PubMed

Wang, Xiaoxiao; Wang, Xusheng; Liu, Jianjun; Cai, Ting; Guo, Ling; Wang, Shujuan; Wang, Jinmei; Cao, Yanpei; Ge, Jianfeng; Jiang, Yuyang; Tredget, Edward E; Cao, Mengjun; Wu, Yaojiong

2016-12-01

: Stem cell-based organ regeneration is purported to enable the replacement of impaired organs in the foreseeable future. Here, we demonstrated that a combination of cultured epidermal stem cells (Epi-SCs) derived from the epidermis and skin-derived precursors (SKPs) was capable of reconstituting functional hair follicles and sebaceous glands (SG). When Epi-SCs and SKPs were mixed in a hydrogel and implanted into an excisional wound in nude mice, the Epi-SCs formed de novo epidermis along with hair follicles, and SKPs contributed to dermal papilla in the neogenic hair follicles. Notably, a combination of culture-expanded Epi-SCs and SKPs derived from the adult human scalp were sufficient to generate hair follicles and hair. Bone morphogenetic protein 4, but not Wnts, sustained the expression of alkaline phosphatase in SKPs in vitro and the hair follicle-inductive property in vivo when SKPs were engrafted with neonatal epidermal cells into excisional wounds. In addition, Epi-SCs were capable of differentiating into sebocytes and formed de novo SGs, which excreted lipids as do normal SGs. Thus our results indicate that cultured Epi-SCs and SKPs are sufficient to generate de novo hair follicles and SGs, implying great potential to develop novel bioengineered skin substitutes with appendage genesis capacity. In postpartum humans, skin appendages lost in injury are not regenerated, despite the considerable achievement made in skin bioengineering. In this study, transplantation of a combination of culture-expanded epidermal stem cells and skin-derived progenitors from mice and adult humans led to de novo regeneration of functional hair follicles and sebaceous glands. The data provide transferable knowledge for the development of novel bioengineered skin substitutes with epidermal appendage regeneration capacity. ©AlphaMed Press.

Hair Follicle and Sebaceous Gland De Novo Regeneration With Cultured Epidermal Stem Cells and Skin-Derived Precursors

PubMed Central

Wang, Xiaoxiao; Wang, Xusheng; Liu, Jianjun; Cai, Ting; Guo, Ling; Wang, Shujuan; Wang, Jinmei; Cao, Yanpei; Ge, Jianfeng; Jiang, Yuyang; Tredget, Edward E.; Cao, Mengjun

2016-01-01

Stem cell-based organ regeneration is purported to enable the replacement of impaired organs in the foreseeable future. Here, we demonstrated that a combination of cultured epidermal stem cells (Epi-SCs) derived from the epidermis and skin-derived precursors (SKPs) was capable of reconstituting functional hair follicles and sebaceous glands (SG). When Epi-SCs and SKPs were mixed in a hydrogel and implanted into an excisional wound in nude mice, the Epi-SCs formed de novo epidermis along with hair follicles, and SKPs contributed to dermal papilla in the neogenic hair follicles. Notably, a combination of culture-expanded Epi-SCs and SKPs derived from the adult human scalp were sufficient to generate hair follicles and hair. Bone morphogenetic protein 4, but not Wnts, sustained the expression of alkaline phosphatase in SKPs in vitro and the hair follicle-inductive property in vivo when SKPs were engrafted with neonatal epidermal cells into excisional wounds. In addition, Epi-SCs were capable of differentiating into sebocytes and formed de novo SGs, which excreted lipids as do normal SGs. Thus our results indicate that cultured Epi-SCs and SKPs are sufficient to generate de novo hair follicles and SGs, implying great potential to develop novel bioengineered skin substitutes with appendage genesis capacity. Significance In postpartum humans, skin appendages lost in injury are not regenerated, despite the considerable achievement made in skin bioengineering. In this study, transplantation of a combination of culture-expanded epidermal stem cells and skin-derived progenitors from mice and adult humans led to de novo regeneration of functional hair follicles and sebaceous glands. The data provide transferable knowledge for the development of novel bioengineered skin substitutes with epidermal appendage regeneration capacity. PMID:27458264

Large χ(3) of squarylium dye J aggregates measured using the Z-scan technique

NASA Astrophysics Data System (ADS)

Tatsuura, Satoshi; Wada, Osamu; Tian, Minquan; Furuki, Makoto; Sato, Yasuhiro; Iwasa, Izumi; Pu, Lyong Sun; Kawashima, Hitoshi

2001-10-01

Third-order nonlinear optical coefficients χ(3) were measured for the J aggregates of two types of squarylium dye derivatives at resonant and near-resonant wavelengths by using the Z-scan technique. The maximum χ(3) value evaluated at one-photon resonance was 2.9×10-6 e.s.u., which was greater than that of phthalocyanines by 4 orders of magnitude. χ(3) for one squarylium derivative was approximately two times as large as that of the other. This can be attributed to the difference of the number of molecules contributing to a coherent state in each J aggregate.

Elastic cartilage reconstruction by transplantation of cultured hyaline cartilage-derived chondrocytes.

PubMed

Mizuno, M; Takebe, T; Kobayashi, S; Kimura, S; Masutani, M; Lee, S; Jo, Y H; Lee, J I; Taniguchi, H

2014-05-01

Current surgical intervention of craniofacial defects caused by injuries or abnormalities uses reconstructive materials, such as autologous cartilage grafts. Transplantation of autologous tissues, however, places a significant invasiveness on patients, and many efforts have been made for establishing an alternative graft. Recently, we and others have shown the potential use of reconstructed elastic cartilage from ear-derived chondrocytes or progenitors with the unique elastic properties. Here, we examined the differentiation potential of canine joint cartilage-derived chondrocytes into elastic cartilage for expanding the cell sources, such as hyaline cartilage. Articular chondrocytes are isolated from canine joint, cultivated, and compared regarding characteristic differences with auricular chondrocytes, including proliferation rates, gene expression, extracellular matrix production, and cartilage reconstruction capability after transplantation. Canine articular chondrocytes proliferated less robustly than auricular chondrocytes, but there was no significant difference in the amount of sulfated glycosaminoglycan produced from redifferentiated chondrocytes. Furthermore, in vitro expanded and redifferentiated articular chondrocytes have been shown to reconstruct elastic cartilage on transplantation that has histologic characteristics distinct from hyaline cartilage. Taken together, cultured hyaline cartilage-derived chondrocytes are a possible cell source for elastic cartilage reconstruction. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Non-pathogenic protein aggregates in skeletal muscle in MLF1 transgenic mice.

PubMed

Li, Zhi-Fang; Wu, Xiaohua; Jiang, Yun; Liu, Jianxiang; Wu, Chun; Inagaki, Masaki; Izawa, Ichiro; Mizisin, Andrew P; Engvall, Eva; Shelton, G Diane

2008-01-15

Protein aggregate formation in muscle is thought to be pathogenic and associated with clinical weakness. Over-expression of either wild type or a mutant form of myeloid leukemia factor 1 (MLF1) in transgenic mouse skeletal muscle and in cultured cells resulted in aggregate formation. Aggregates were detected in MLF1 transgenic mice at 6 weeks of age, and increased in size with age. However, histological examination of skeletal muscles of MLF1 transgenic mice revealed no pathological changes other than the aggregates, and RotaRod testing did not detect functional deficits. MLF1 has recently been identified as a protein that could neutralize the toxicity of intracellular protein aggregates in a Drosophila model of Huntington's disease (HD). We also demonstrate that MLF1 interacts with MRJ, a heat shock protein, which can independently neutralize the toxicity of intracellular protein aggregates in the Drosophila HD model. Our data suggest that over-expression of MLF1 has no significant impact on skeletal muscle function in mice; that progressive formation of protein aggregates in muscle are not necessarily pathogenic; and that MLF1 and MRJ may function together to ameliorate the toxic effects of polyglutamine or mutant proteins in myodegenerative diseases such as inclusion body myositis and oculopharyngeal muscular dystrophy, as well as neurodegenerative disease.

Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions.

PubMed

Fukuda, Takayuki; Takayama, Kazuo; Hirata, Mitsuhi; Liu, Yu-Jung; Yanagihara, Kana; Suga, Mika; Mizuguchi, Hiroyuki; Furue, Miho K

2017-03-15

Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, labor-intensive, cost-intensive, and unstable. In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells. We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC-derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Co-production of GroELS discriminates between intrinsic and thermally-induced recombinant protein aggregation during substrate quality control

PubMed Central

2011-01-01

Background The effects and effectiveness of the chaperone pair GroELS on the yield and quality of recombinant polypeptides produced in Escherichia coli are matter of controversy, as the reported activities of this complex are not always consistent and eventually indicate undesired side effects. The divergence in the reported data could be due, at least partially, to different experimental conditions in independent research approaches. Results We have then selected two structurally different model proteins (namely GFP and E. coli β-galactosidase) and two derived aggregation-prone fusions to explore, in a systematic way, the eventual effects of GroELS co-production on yield, solubility and conformational quality. Host cells were cultured at two alternative temperatures below the threshold at which thermal stress is expected to be triggered, to minimize the involvement of independent stress factors. Conclusions From the analysis of protein yield, solubility and biological activity of the four model proteins produced alone or along the chaperones, we conclude that GroELS impacts on yield and quality of aggregation-prone proteins with intrinsic determinants but not on thermally induced protein aggregation. No effective modifications of protein solubility have been observed, but significant stabilization of small (encapsulable) substrates and moderate chaperone-induced degradation of larger (excluded) polypeptides. These findings indicate that the activities of this chaperone pair in the context of actively producing recombinant bacteria discriminate between intrinsic and thermally-induced protein aggregation, and that the side effects of GroELS overproduction might be determined by substrate size. PMID:21992454

Aggregate nanostructures of organic molecular materials.

PubMed

Liu, Huibiao; Xu, Jialiang; Li, Yongjun; Li, Yuliang

2010-12-21

Conjugated organic molecules are interesting materials because of their structures and their electronic, electrical, magnetic, optical, biological, and chemical properties. However, researchers continue to face great challenges in the construction of well-defined organic compounds that aggregate into larger molecular materials such as nanowires, tubes, rods, particles, walls, films, and other structural arrays. Such nanoscale materials could serve as direct device components. In this Account, we describe our recent progress in the construction of nanostructures formed through the aggregation of organic conjugated molecules and in the investigation of the optical, electrical, and electronic properties that depend on the size or morphology of these nanostructures. We have designed and synthesized functional conjugated organic molecules with structural features that favor assembly into aggregate nanostructures via weak intermolecular interactions. These large-area ordered molecular aggregate nanostructures are based on a variety of simpler structures such as fullerenes, perylenes, anthracenes, porphyrins, polydiacetylenes, and their derivatives. We have developed new methods to construct these larger structures including organic vapor-solid phase reaction, natural growth, association via self-polymerization and self-organization, and a combination of self-assembly and electrochemical growth. These methods are both facile and reliable, allowing us to produce ordered and aligned aggregate nanostructures, such as large-area arrays of nanowires, nanorods, and nanotubes. In addition, we can synthesize nanoscale materials with controlled properties. Large-area ordered aggregate nanostructures exhibit interesting electrical, optical, and optoelectronic properties. We also describe the preparation of large-area aggregate nanostructures of charge transfer (CT) complexes using an organic solid-phase reaction technique. By this process, we can finely control the morphologies and

Characterization of a cartilage-like engineered biomass using a self-aggregating suspension culture model: molecular composition using FT-IRIS.

PubMed

Kim, Minwook; Kraft, Jeffrey J; Volk, Andrew C; Pugarelli, Joan; Pleshko, Nancy; Dodge, George R

2011-12-01

Maintenance of chondrocyte phenotype and robust expression and organization of macromolecular components with suitable cartilaginous properties is an ultimate goal in cartilage tissue engineering. We used a self-aggregating suspension culture (SASC) method to produce an engineered cartilage, "cartilage tissue analog" (CTA). With an objective of understanding the stability of phenotype of the CTA over long periods, we cultured chondrocytes up to 4 years and analyzed the matrix. Both early (eCTAs) (6 months) and aged (aCTAs) (4 years) showed type II collagen throughout with higher concentrations near the edge. Using Fourier transform-infrared imaging spectroscopy (FT-IRIS), proteoglycan/collagen ratio of eCTA was 2.8 times greater than native cartilage at 1 week, but the ratio was balanced to native level (p = 0.017) by 36 weeks. Surprisingly, aCTAs maintained the hyaline characteristics, but there was evidence of calcification within the tissue with a distinct range of intensities. Mineral/matrix ratio of those aCTA with "intensive" calcification was significantly higher (p = 0.017) than the "partial," but when compared to native bone the ratio of "intensive" aCTAs was 2.4 times lower. In this study we utilized the imaging approach of FT-IRIS and have shown that a biomaterial formed is compositionally closely related to natural cartilage for long periods in culture. We show that this culture platform can maintain a CTA for extended periods of time (4 years) and under those conditions signs of mineralization can be found. This method of cartilage tissue engineering is a promising method to generate cartilaginous biomaterial and may have potential to be utilized in both cartilage and boney repairs. Copyright © 2011 Orthopaedic Research Society.

Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway

PubMed Central

Zhang, Wei; Wang, Hanning; Zhang, Shaopeng; Zhong, Liang; Wang, Yanliang; Pei, Yangli; Cao, Suying

2018-01-01

Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs), are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs) under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX). These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM) of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal–epithelial transition (MET) through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation. PMID:29419748

Expression of organophosphorus-degradation gene ( opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria

NASA Astrophysics Data System (ADS)

Li, Qiong; Tang, Qing; Xu, Xudong; Gao, Hong

2010-11-01

Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene ( opd) into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, P psbA . From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25°C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.

A microfabricated platform to form three-dimensional toroidal multicellular aggregate.

PubMed

Masuda, Taisuke; Takei, Natsuki; Nakano, Takuma; Anada, Takahisa; Suzuki, Osamu; Arai, Fumihito

2012-12-01

Techniques that allow cells to self-assemble into three-dimensional (3D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular in fields such as stem cell research, tissue engineering, and cancer biology. Appropriate simulation of the 3D environment in which tissues normally develop and function is crucial for the engineering of in vitro models that can be used for the formation of complex tissues. We have developed a unique multicellular aggregate formation platform that utilizes a maskless gray-scale photolithography. The cellular aggregate formed using this platform has a toroidal-like geometry and includes a micro lumen that facilitates the supply of oxygen and growth factors and the expulsion of waste products. As a result, this platform was capable of rapidly producing hundreds of multicellular aggregates at a time, and of regulating the diameter of aggregates with complex design. These toroidal multicellular aggregates can grow as long-term culture. In addition, the micro lumen can be used as a continuous channel and for the insertion of a vascular system or a nerve system into the assembled tissue. These platform characteristics highlight its potential to be used in a wide variety of applications, e.g. as a bioactuator, as a micro-machine component or in drug screening and tissue engineering.

Attogram detection of picric acid by hexa-peri-hexabenzocoronene-based chemosensors by controlled aggregation-induced emission enhancement.

PubMed

Vij, Varun; Bhalla, Vandana; Kumar, Manoj

2013-06-12

Hexa-peri-hexabenzocoronene (HBC) based molecules 5 and 6 have been designed and synthesized. These planar coronenes are appended with rotors to invoke aggregation induced emission enhancement (AIEE) phenomenon by controlling the ratio of H2O in solutions of aggregates. These aggregates of HBC derivatives serve as highly selective chemosensors for picric acid (PA) in mixed aqueous solution. These aggregates are also able to detect PA in vapor phase. In addition, fluorescent test strips have been prepared by dip-coating the Whatman paper with aggregates of both compounds for trace detection of PA in contact mode with detection limits in attograms.

Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

PubMed

Selheim, F; Holmsen, H; Vassbotn, F S

1999-08-15

We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.

Lewy Body-like α-Synuclein Aggregates Resist Degradation and Impair Macroautophagy*♦

PubMed Central

Tanik, Selcuk A.; Schultheiss, Christine E.; Volpicelli-Daley, Laura A.; Brunden, Kurt R.; Lee, Virginia M. Y.

2013-01-01

Cytoplasmic α-synuclein (α-syn) aggregates, referred to as Lewy bodies, are pathological hallmarks of a number of neurodegenerative diseases, most notably Parkinson disease. Activation of macroautophagy is suggested to facilitate degradation of certain proteinaceous inclusions, but it is unclear if this pathway is capable of degrading α-syn aggregates. Here, we examined this issue by utilizing cellular models in which intracellular Lewy body-like α-syn inclusions accumulate after internalization of pre-formed α-syn fibrils into α-syn-expressing HEK293 cells or cultured primary neurons. We demonstrate that α-syn inclusions cannot be effectively degraded, even though they co-localize with essential components of both the autophagic and proteasomal protein degradation pathways. The α-syn aggregates persist even after soluble α-syn levels have been substantially reduced, suggesting that once formed, the α-syn inclusions are refractory to clearance. Importantly, we also find that α-syn aggregates impair overall macroautophagy by reducing autophagosome clearance, which may contribute to the increased cell death that is observed in aggregate-bearing cells. PMID:23532841

Decellularized adipose tissue microcarriers as a dynamic culture platform for human adipose-derived stem/stromal cell expansion.

PubMed

Yu, Claire; Kornmuller, Anna; Brown, Cody; Hoare, Todd; Flynn, Lauren E

2017-03-01

With the goal of designing a clinically-relevant expansion strategy for human adipose-derived stem/stromal cells (ASCs), methods were developed to synthesize porous microcarriers derived purely from human decellularized adipose tissue (DAT). An electrospraying approach was applied to generate spherical DAT microcarriers with an average diameter of 428 ± 41 μm, which were soft, compliant, and stable in long-term culture without chemical crosslinking. Human ASCs demonstrated enhanced proliferation on the DAT microcarriers relative to commercially-sourced Cultispher-S microcarriers within a spinner culture system over 1 month. ASC immunophenotype was maintained post expansion, with a trend for reduced expression of the cell adhesion receptors CD73, CD105, and CD29 under dynamic conditions. Upregulation of the early lineage-specific genes PPARγ, LPL, and COMP was observed in the ASCs expanded on the DAT microcarriers, but the cells retained their multilineage differentiation capacity. Comparison of adipogenic and osteogenic differentiation in 2-D cultures prepared with ASCs pre-expanded on the DAT microcarriers or Cultispher-S microcarriers revealed similar adipogenic and enhanced osteogenic marker expression in the DAT microcarrier group, which had undergone a higher population fold change. Further, histological staining results suggested a more homogeneous differentiation response in the ASCs expanded on the DAT microcarriers as compared to either Cultispher-S microcarriers or tissue culture polystyrene. A pilot chondrogenesis study revealed higher levels of chondrogenic gene and protein expression in the ASCs expanded on the DAT microcarriers relative to all other groups, including the baseline controls. Overall, this study demonstrates the promise of applying dynamic culture with tissue-specific DAT microcarriers as a means of deriving regenerative cell populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

NASA Technical Reports Server (NTRS)

Schatten, H.; Hedrick, J.; Chakrabarti, A.

1998-01-01

Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid.

PubMed

Makoolati, Zohreh; Movahedin, Mansoureh; Forouzandeh-Moghadam, Mehdi

2016-12-01

An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0-5  μM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh),  α6 integrin,  β1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3  μM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 μM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs. © 2016 The Author(s).

Directing Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Impact of Medium Composition and Culture Condition.

PubMed

Petry, L; Kippenberger, S; Meissner, M; Kleemann, J; Kaufmann, R; Rieger, U M; Wellenbrock, S; Reichenbach, G; Zöller, N; Valesky, E

2018-04-28

Adipose-derived stem cells (ASC) are known to transdifferentiate into a wide range of different cell species in vitro including along the epidermal lineage. This property makes them a promising tool for regenerative medicine in order to restore the epidermal barrier. The present study is dedicated to identify in vitro conditions enabling transdifferentiation to a keratinocyte-like phenotype. Especially, the impact of different culture conditions (media compositions, 2D-, 3D-cultures) and extracellular matrix (ECM) molecules was evaluated. ASC derived from subcutaneous abdominal fat were characterized by stemness associated markers and subjected to different media. Epithelial differentiation in 2D cultures was monitored by pan-cytokeratin expression using flow cytometry and immunocytochemistry. In order to evaluate the impact of different ECM molecules on epidermal stratification, 3D cultures were produced, lifted to the air-liquid-interface (ALI) and examined by histological analysis and quantitative real-time RT-PCR. We identified a medium composition containing retinoic acid, hydrocortisone, ascorbic acid and BMP-4 enabling maximum pan-cytokeratin expression in 2D cultures. Moreover, adhesion to type IV collagen further promotes the pan-cytokeratin expression. When cultures were lifted to the ALI, significant stratification was observed, particularly in supports coated with type IV collagen or fibronectin. Moreover, epidermal differentiation markers (involucrin, cytokeratin 1 and 14) become induced. Conditions with hampered wound healing such as non-healing ulcers demand new treatment regimes. The here introduced optimized protocols for transdifferentiation of ASC into keratinocyte-like cells may help to establish more effective treatment procedures. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Dichloromethane Fermentation by a Dehalobacter sp. in an Enrichment Culture Derived from Pristine River Sediment

PubMed Central

Justicia-Leon, Shandra D.; Ritalahti, Kirsti M.; Mack, E. Erin

2012-01-01

Dichloromethane (DCM) as the sole substrate supported growth of a Dehalobacter sp. in an enrichment culture derived from noncontaminated river sediment. DCM was not reductively dechlorinated, and acetate was produced, indicating DCM fermentation and further suggesting Dehalobacter growth is not limited to organohalide respiration. PMID:22179245

Random access with adaptive packet aggregation in LTE/LTE-A.

PubMed

Zhou, Kaijie; Nikaein, Navid

While random access presents a promising solution for efficient uplink channel access, the preamble collision rate can significantly increase when massive number of devices simultaneously access the channel. To address this issue and improve the reliability of the random access, an adaptive packet aggregation method is proposed. With the proposed method, a device does not trigger a random access for every single packet. Instead, it starts a random access when the number of aggregated packets reaches a given threshold. This method reduces the packet collision rate at the expense of an extra latency, which is used to accumulate multiple packets into a single transmission unit. Therefore, the tradeoff between packet loss rate and channel access latency has to be carefully selected. We use semi-Markov model to derive the packet loss rate and channel access latency as functions of packet aggregation number. Hence, the optimal amount of aggregated packets can be found, which keeps the loss rate below the desired value while minimizing the access latency. We also apply for the idea of packet aggregation for power saving, where a device aggregates as many packets as possible until the latency constraint is reached. Simulations are carried out to evaluate our methods. We find that the packet loss rate and/or power consumption are significantly reduced with the proposed method.

ROCK and PRK-2 Mediate the Inhibitory Effect of Y-27632 on Polyglutamine Aggregation

PubMed Central

Shao, Jieya; Welch, William J.; Diamond, Marc I.

2009-01-01

Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, H-1077, HA-1152), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal. PMID:18423405

One-step generation of murine embryonic stem cell-derived mesoderm progenitors and chondrocytes in a serum-free monolayer differentiation system.

PubMed

Waese, Elaine Y L; Stanford, William L

2011-01-01

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high-throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. We present a novel, single-step method of generating murine ESC-derived chondrocytes in monolayer cultures under chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, activin A, or Wnt3a. Prolonged culture with sustained activin A, TGFβ3, or BMP4 supplementation led to robust chondrogenic induction. A short pulse of activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with activin A or with activin A followed by TGFβ3 promoted articular cartilage formation. Thus, we devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. Copyright © 2010 Elsevier B.V. All rights reserved.

Can the common brain parasite, Toxoplasma gondii, influence human culture?

PubMed Central

Lafferty, Kevin D

2006-01-01

The latent prevalence of a long-lived and common brain parasite, Toxoplasma gondii, explains a statistically significant portion of the variance in aggregate neuroticism among populations, as well as in the ‘neurotic’ cultural dimensions of sex roles and uncertainty avoidance. Spurious or non-causal correlations between aggregate personality and aspects of climate and culture that influence T. gondii transmission could also drive these patterns. A link between culture and T. gondii hypothetically results from a behavioural manipulation that the parasite uses to increase its transmission to the next host in the life cycle: a cat. While latent toxoplasmosis is usually benign, the parasite's subtle effect on individual personality appears to alter the aggregate personality at the population level. Drivers of the geographical variation in the prevalence of this parasite include the effects of climate on the persistence of infectious stages in soil, the cultural practices of food preparation and cats as pets. Some variation in culture, therefore, may ultimately be related to how climate affects the distribution of T. gondii, though the results only explain a fraction of the variation in two of the four cultural dimensions, suggesting that if T. gondii does influence human culture, it is only one among many factors. PMID:17015323

Maintenance of human adipose derived stem cell (hASC) differentiation capabilities using a 3D culture.

PubMed

Lin, Ching-Yu; Huang, Chi-Hui; Wu, Yuan-Kun; Cheng, Nai-Chen; Yu, Jiashing

2014-07-01

In this study, 3D culture system for human adipose-derived stem cell (hASC) using a BioLevitator as the bioreactor for microcarrier-based cultures was established. During the culturing period, hASCs preferred to grow in crevices between microcarriers and a high viability was maintained even when reaching confluency. Adipogenic or osteogenic differential medium was used to induce hASCs and differential potentials of these cells were compared between 2D and 3D environments via RT-PCR and staining quantifications. CEBP/α gene expression was significant higher in 3D condition at day 21 (P < 0.05). Staining quantification indicates that cells cultured in 3D condition have significant better differentiation potential from day 14 to 21 for both adipogenic and osteogenic lineages (P < 0.01).

Embryonic mescencephalon derived neurospheres contain progenitors as well as differentiated neurons and glia.

PubMed

Khaing, Zin Z; Roberts, James L

2009-01-01

Stem cells and progenitor cells in the central nervous system may have potential for therapeutic use in patients with degenerative diseases or after injury. Neural precursor cells can be grown in culture in the presence of mitogens as aggregates termed neurospheres (NSs), as a source of proliferating progenitor cells. Withdrawal of mitogen and allowing the NSs to adhere to a substrate is the conventional way to study the differentiation potential of the progenitor cells propagated in NSs form. Here we asked if differentiation occurs within NSs cultured in the normal manner, in the presence of mitogen. We used non-passaged NSs derived from E13.5 mouse ventral mesencephalon. The NSs contained not only progenitor cells but also phenotypically-differentiated neurons and glia, in the presence of mitogen. Extracellular matrix molecules (fibronectin, laminin and collagen type IV) were also detected within these NSs, which may aid in the differentiation of progenitors inside the NSs. The cell types within NSs were also organized in a way that the differentiated cells were found in the inner cell mass while progenitors were found in the outer region. Additionally, the proportion of differentiated cell types within the NSs was also affected by exposure to different mitogens. Moreover, when placed together in to co-culture, dissociated embryonic striatal and mesencephalic cells aggregated spontaneously to form mixed NSs, enhancing the eventual differentiation into dopaminergic neurons from progenitors within these NSs. Therefore, the NSs contained progenitor cells and differentiated neurons and glial cells. In addition, NS culture system can be used to study cellular differentiation in vitro in non-adherent conditions.

Human immunodeficiency virus-infected macrophages produce soluble factors that cause histological and neurochemical alterations in cultured human brains.

PubMed Central

Pulliam, L; Herndier, B G; Tang, N M; McGrath, M S

1991-01-01

We wanted to establish an in vitro human model for AIDS-associated dementia and pursue the hypothesis that this disease process may be a result of soluble factors produced by HIV-infected macrophages. Human brain aggregates were prepared from nine different brain specimens, and were treated with supernatants from in vitro HIV-infected macrophages (SI), uninfected macrophages (SU), infected T cells, or macrophage-conditioned media from four AIDS patients. Seven of nine treated brains exposed to SI showed peripheral rarefaction after 1 wk of incubation that by ultrastructural analysis showed cytoplasmic vacuolation. Aggregates from two of three brain cultures treated with SI for 3 wk became smaller, an approximately 50% decrease in size. The degree of apparent toxicity in brains exposed to patient-derived macrophage supernatants paralleled the proportion of macrophages found to be expressing HIV p24. Ultrastructural abnormalities were not observed in brains treated with supernatants from HIV-infected T cells, uninfected macrophages, or LPS-activated macrophages. Levels of five neurotransmitter amino acids were decreased in comparison to the structural amino acid leucine. These findings suggest that HIV-infected macrophages, infected both in vitro as well as derived from AIDS patients' peripheral blood, produce factors that cause reproducible histochemical, ultrastructural, and functional abnormalities in human brain aggregates. Images PMID:1671392

Interference between Coulombic and CT-mediated couplings in molecular aggregates: H- to J-aggregate transformation in perylene-based π-stacks

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

Hestand, Nicholas J.; Spano, Frank C.

2015-12-28

The spectroscopic differences between J and H-aggregates are traditionally attributed to the spatial dependence of the Coulombic coupling, as originally proposed by Kasha. However, in tightly packed molecular aggregates wave functions on neighboring molecules overlap, leading to an additional charge transfer (CT) mediated exciton coupling with a vastly different spatial dependence. The latter is governed by the nodal patterns of the molecular LUMOs and HOMOs from which the electron (t{sub e}) and hole (t{sub h}) transfer integrals derive. The sign of the CT-mediated coupling depends on the sign of the product t{sub e}t{sub h} and is therefore highly sensitive tomore » small (sub-Angstrom) transverse displacements or slips. Given that Coulombic and CT-mediated couplings exist simultaneously in tightly packed molecular systems, the interference between the two must be considered when defining J and H-aggregates. Generally, such π-stacked aggregates do not abide by the traditional classification scheme of Kasha: for example, even when the Coulomb coupling is strong the presence of a similarly strong but destructively interfering CT-mediated coupling results in “null-aggregates” which spectroscopically resemble uncoupled molecules. Based on a Frenkel/CT Holstein Hamiltonian that takes into account both sources of electronic coupling as well as intramolecular vibrations, vibronic spectral signatures are developed for integrated Frenkel/CT systems in both the perturbative and resonance regimes. In the perturbative regime, the sign of the lowest exciton band curvature, which rigorously defines J and H-aggregation, is directly tracked by the ratio of the first two vibronic peak intensities. Even in the resonance regime, the vibronic ratio remains a useful tool to evaluate the J or H nature of the system. The theory developed is applied to the reversible H to J-aggregate transformations recently observed in several perylene bisimide systems.« less

Characterization of cell cultures derived from Lutzomyia spinicrassa (Diptera: Psychodidae) and their susceptibility to infection with Leishmania (Viannia) braziliensis.

PubMed

Zapata Lesmes, Angela Cristina; Cárdenas Castro, Estrella; Bello, Felio

2005-12-01

The sand fly Lutzomyia spinicrassa (Morales, Osorno-Mesa, Osorno & de Hoyos, 1969) is a vector of Leishmania (Viannia) braziliensis, an etiological agent of cutaneous leishmaniasis in Colombia. The present article describes, for the first time, the morphological, karyotypical, and isozymatic characteristics of cell cultures derived from L. Spinicrassa embryonic tissues as well as the interaction of L. Braziliensis with these cell cultures. L. Spinicrassa embryonated eggs and neonate larvae were taken for tissue explants. These were seeded in Grace, L-15, Grace/L-15, MM/VP12, and MK/VP12 culture media. The pH range in these media was 6.7 to 6.9 and the cultures were incubated at 28 degrees C. The MHOM/CO/86/CL250 strain of L. Braziliensis was used for experimental infection of cell cultures of L. Spinicrassa. Cell growth was achieved in L-15 medium and a confluent monolayer was obtained 180 days after the embryonated eggs were explanted. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer of these cell cultures and in the subcultures the predominant cell types were later fibroblast-like and epithelial-like. Cultured cells were predominantly diploid (2n=8); however, significant percentages of aneuploids were also recorded. The cell culture isozyme patterns of L. Spinicrassa coincided with pupae samples from the same species. Promastigote forms of L. Braziliensis could invade cells and transform into amastigote-like forms inside them. The characteristics of cell cultures derived from L. Spinicrassa embryonic tissues were determined. These cultures emerge as a new model to study the life-cycle of L. Braziliensis.

Activated stress response pathways within multicellular aggregates utilize an autocrine component.

PubMed

Jack, Graham D; Cabrera, M Carla; Manning, Michael L; Slaughter, Stephen M; Potts, Malcolm; Helm, Richard F

2007-04-01

Multicellular aggregates (spheroids) of primary human foreskin fibroblasts (HFF-2) and a glioblastoma cell line (T98G) entered and exited from long term (2 weeks) metabolic arrest utilizing an autocrine response. Cytokine production (specifically IFN-gamma) activated a Gadd45alpha/p38 pathway that led to increased AP-1 (c-jun and ATF3) transcription factor levels, augmenting cytokine production in an autocrine fashion. Whereas HFF-2 aggregates were capable of surviving long term arrest and recovery during NF-kappaB inhibition independent of JNK activation, T98G aggregates were not. Such endogenous processes are not easily observed with adherent monolayer cell culturing systems, strongly suggesting that more emphasis needs to be placed on determining the operational signal transduction cascades within multicellular aggregates. Extracellular inputs such as spheroid formation, arrest, and regrowth as monolayers invoke intracellular signaling responses converging at the AP-1 transcription factor level. Variations in responses are both cell type and transformation state dependent and require an autocrine cytokine component. The data are discussed in relation to the wounding response and avascular tumor growth mechanisms.

Interactions between polystyrene microplastics and marine phytoplankton lead to species-specific hetero-aggregation.

PubMed

Long, Marc; Paul-Pont, Ika; Hégaret, Hélène; Moriceau, Brivaela; Lambert, Christophe; Huvet, Arnaud; Soudant, Philippe

2017-09-01

To understand the fate and impacts of microplastics (MP) in the marine ecosystems, it is essential to investigate their interactions with phytoplankton as these may affect MP bioavailability to marine organisms as well as their fate in the water column. However, the behaviour of MP with marine phytoplanktonic cells remains little studied and thus unpredictable. The present study assessed the potential for phytoplankton cells to form hetero-aggregates with small micro-polystyrene (micro-PS) particles depending on microalgal species and physiological status. A prymnesiophycea, Tisochrysis lutea, a dinoflagellate, Heterocapsa triquetra, and a diatom, Chaetoceros neogracile, were exposed to micro-PS (2 μm diameter; 3.96 μg L -1 ) during their growth culture cycles. Micro-PS were quantified using an innovative flow-cytometry approach, which allowed the monitoring of the micro-PS repartition in microalgal cultures and the distinction between free suspended micro-PS and hetero-aggregates of micro-PS and microalgae. Hetero-aggregation was observed for C. neogracile during the stationary growth phase. The highest levels of micro-PS were "lost" from solution, sticking to flasks, with T. lutea and H. triquetra cultures. This loss of micro-PS sticking to the flask walls increased with the age of the culture for both species. No effects of micro-PS were observed on microalgal physiology in terms of growth and chlorophyll fluorescence. Overall, these results highlight the potential for single phytoplankton cells and residual organic matter to interact with microplastics, and thus potentially influence their distribution and bioavailability in experimental systems and the water column. Copyright © 2017 Elsevier Ltd. All rights reserved.

Glycoprotein Ibα receptor instability is associated with loss of quality in platelets produced in culture.

PubMed

Robert, Amélie; Boyer, Lucie; Pineault, Nicolas

2011-03-01

The development of culture processes for hematopoietic progenitors could lead to the development of a complementary source of platelets for therapeutic purposes. However, functional characterization of culture-derived platelets remains limited, which raises some uncertainties about the quality of platelets produced in vitro. The aim of this study was to define the proportion of functional platelets produced in cord blood CD34+ cell cultures. Toward this, the morphological and functional properties of culture-derived platelet-like particles (PLPs) were critically compared to that of blood platelets. Flow cytometry combined with transmission electron microscopy analyses revealed that PLPs formed a more heterogeneous population of platelets at a different stage of maturation than blood platelets. The majority of PLPs harbored the fibrinogen receptor αIIbβ3, but a significant proportion failed to maintain glycoprotein (GP)Ibα surface expression, a component of the vWF receptor essential for platelet functions. Importantly, GPIbα extracellular expression correlated closely with platelet function, as the GPIIb+ GPIbα+ PLP subfraction responded normally to agonist stimulation as evidenced by α-granule release, adhesion, spreading, and aggregation. In contrast, the GPIIb+ GPIbα⁻ subfraction was unresponsive in most functional assays and appeared to be metabolically inactive. The present study confirms that functional platelets can be generated in cord blood CD34+ cell cultures, though these are highly susceptible to ectodomain shedding of receptors associated with loss of function. Optimization of culture conditions to prevent these deleterious effects and to homogenize PLPs is necessary to improve the quality and yields of culture-derived platelets before they can be recognized as a suitable complementary source for therapeutic purposes.

Properties of lightweight aggregate concrete prepared with PVC granules derived from scraped PVC pipes.

PubMed

Kou, S C; Lee, G; Poon, C S; Lai, W L

2009-02-01

This paper aims to investigate the fresh and hardened properties of lightweight aggregate concretes that are prepared with the use of recycled plastic waste sourced from scraped PVC pipes to replace river sand as fine aggregates. A number of laboratory prepared concrete mixes were tested, in which river sand was partially replaced by PVC plastic waste granules in percentages of 0%, 5%, 15%, 30% and 45% by volume. Two major findings are identified. The positive side shows that the concrete prepared with a partial replacement by PVC was lighter (lower density), was more ductile (greater Poisson's ratios and reduced modulus of elasticity), and had lower drying shrinkage and higher resistance to chloride ion penetration. The negative side reveals that the workability, compressive strength and tensile splitting strength of the concretes were reduced. The results gathered would form a part of useful information for recycling PVC plastic waste in lightweight concrete mixes.

Synthesis, aggregation and spectroscopic studies of novel water soluble metal free, zinc, copper and magnesium phthalocyanines and investigation of their anti-bacterial properties

NASA Astrophysics Data System (ADS)

Bayrak, Rıza; Akçay, Hakkı Türker; Beriş, Fatih Şaban; Şahin, Ertan; Bayrak, Hacer; Demirbaş, Ümit

2014-12-01

In this study, novel phthalonitrile derivative (3) was synthesized by the reaction between 4-nitrophthalonitrile (2) and a triazole derivative (1) containing pyridine moiety. Crystal structure of compound (3) was characterized by X-ray diffraction. New metal free and metallo-phthalocyanine complexes (Zn, Cu, and Mg) were synthesized using the phthalonitrile derivative (3). Cationic derivatives of these phthalocyanines (5, 7, 9, and 11) were prepared from the non-ionic phthalocyanines (4, 6, 8, and 10). All proposed structures were supported by instrumental methods. The aggregation behaviors of the phthalocyanines (4-11) were investigated in different solvents such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), chloroform and water. Water soluble cationic Pcs (5, 7, 9, and 11) aggregated in water and sodium dodecyl sulfate was used to prevent the aggregation. The second derivatives of the UV-Vis spectra of aggregated Pcs were used for analyzing the Q and B bands of aggregated species. Thermal behaviors of the phthalocyanines were also studied. In addition, anti-bacterial properties of the phthalocyanines were investigated. We used four gram negative and two gram positive bacteria to determine antibacterial activity of these compounds. Compound 7 has the best activity against the all bacteria with 125 μg/mL of MIC value. Compounds 4, 6, and 10 have the similar effect on the bacteria with 250 μg/mL of MIC value.

Three Dimensional Primary Hepatocyte Culture

NASA Technical Reports Server (NTRS)

Yoffe, Boris

1998-01-01

Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

Properties of concrete blocks prepared with low grade recycled aggregates.

PubMed

Poon, Chi-Sun; Kou, Shi-cong; Wan, Hui-wen; Etxeberria, Miren

2009-08-01

Low grade recycled aggregates obtained from a construction waste sorting facility were tested to assess the feasibility of using these in the production of concrete blocks. The characteristics of the sorted construction waste are significantly different from that of crushed concrete rubbles that are mostly derived from demolition waste streams. This is due to the presence of higher percentages of non-concrete components (e.g. >10% soil, brick, tiles etc.) in the sorted construction waste. In the study reported in this paper, three series of concrete block mixtures were prepared by using the low grade recycled aggregates to replace (i) natural coarse granite (10mm), and (ii) 0, 25, 50, 75 and 100% replacement levels of crushed stone fine (crushed natural granite <5mm) in the concrete blocks. Test results on properties such as density, compressive strength, transverse strength and drying shrinkage as well as strength reduction after exposure to 800 degrees C are presented below. The results show that the soil content in the recycled fine aggregate was an important factor in affecting the properties of the blocks produced and the mechanical strength deceased with increasing low grade recycled fine aggregate content. But the higher soil content in the recycled aggregates reduced the reduction of compressive strength of the blocks after exposure to high temperature due probably to the formation of a new crystalline phase. The results show that the low grade recycled aggregates obtained from the construction waste sorting facility has potential to be used as aggregates for making non-structural pre-cast concrete blocks.

Web-based access, aggregation, and visualization of future climate projections with emphasis on agricultural assessments

NASA Astrophysics Data System (ADS)

Villoria, Nelson B.; Elliott, Joshua; Müller, Christoph; Shin, Jaewoo; Zhao, Lan; Song, Carol

2018-01-01

Access to climate and spatial datasets by non-specialists is restricted by technical barriers involving hardware, software and data formats. We discuss an open-source online tool that facilitates downloading the climate data from the global circulation models used by the Inter-Sectoral Impacts Model Intercomparison Project. The tool also offers temporal and spatial aggregation capabilities for incorporating future climate scenarios in applications where spatial aggregation is important. We hope that streamlined access to these data facilitates analysis of climate related issues while considering the uncertainties derived from future climate projections and temporal aggregation choices.

Measurement of Average Aggregate Density by Sedimentation and Brownian Motion Analysis.

PubMed

Cavicchi, Richard E; King, Jason; Ripple, Dean C

2018-05-01

The spatially averaged density of protein aggregates is an important parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of protein in aggregate form in a sample. We obtained a series of images of protein aggregates exhibiting Brownian diffusion while settling under the influence of gravity in a sealed capillary. The aggregates were formed by stir-stressing a monoclonal antibody (NISTmAb). Image processing yielded particle tracks, which were then examined to determine settling velocity and hydrodynamic diameter down to 1 μm based on mean square displacement analysis. Measurements on polystyrene calibration microspheres ranging in size from 1 to 5 μm showed that the mean square displacement diameter had improved accuracy over the diameter derived from imaged particle area, suggesting a future method for correcting size distributions based on imaging. Stokes' law was used to estimate the density of each particle. It was found that the aggregates were highly porous with density decreasing from 1.080 to 1.028 g/cm 3 as the size increased from 1.37 to 4.9 μm. Published by Elsevier Inc.

A cGMP-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue.

PubMed

Shelley, Brandon C; Gowing, Geneviève; Svendsen, Clive N

2014-06-15

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as "chopping" that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

An online detection system for aggregate sizes and shapes based on digital image processing

NASA Astrophysics Data System (ADS)

Yang, Jianhong; Chen, Sijia

2017-02-01

Traditional aggregate size measuring methods are time-consuming, taxing, and do not deliver online measurements. A new online detection system for determining aggregate size and shape based on a digital camera with a charge-coupled device, and subsequent digital image processing, have been developed to overcome these problems. The system captures images of aggregates while falling and flat lying. Using these data, the particle size and shape distribution can be obtained in real time. Here, we calibrate this method using standard globules. Our experiments show that the maximum particle size distribution error was only 3 wt%, while the maximum particle shape distribution error was only 2 wt% for data derived from falling aggregates, having good dispersion. In contrast, the data for flat-lying aggregates had a maximum particle size distribution error of 12 wt%, and a maximum particle shape distribution error of 10 wt%; their accuracy was clearly lower than for falling aggregates. However, they performed well for single-graded aggregates, and did not require a dispersion device. Our system is low-cost and easy to install. It can successfully achieve online detection of aggregate size and shape with good reliability, and it has great potential for aggregate quality assurance.

Social aggregation as a cooperative game

NASA Astrophysics Data System (ADS)

Vilone, Daniele; Guazzini, Andrea

2011-07-01

A new approach for the description of phenomena of social aggregation is suggested. On the basis of psychological concepts (as for instance social norms and cultural coordinates), we deduce a general mechanism for social aggregation in which different clusters of individuals can merge according to cooperation among the agents. In their turn, the agents can cooperate or defect according to the clusters' distribution inside the system. The fitness of an individual increases with the size of its cluster, but decreases with the work the individual had to do in order to join it. In order to test the reliability of such a new approach, we introduce a couple of simple toy models with the features illustrated above. We see, from this preliminary study, how cooperation is the most convenient strategy only in the presence of very large clusters, while on the other hand it is not necessary to have one hundred percent of cooperators for reaching a totally ordered configuration with only one megacluster filling the whole system.

An assessment of adhesion, aggregation and surface charges of Lactobacillus strains derived from the human oral cavity.

PubMed

Piwat, S; Sophatha, B; Teanpaisan, R

2015-07-01

There is limited information concerning the adhesion and aggregation of human oral lactobacilli. In this study, the adhesion of 10 Lactobacillus species was investigated using H357 oral keratinocyte cells as an in vitro model for oral mucosa. Coaggregation with the representative oral pathogen, Streptococcus mutans ATCC 25175, and the physicochemical cell properties was also evaluated. The results demonstrated significant variations in adhesion (42-96%) and aggregation (autoaggregation, 14-95%; coaggregation, 19-65%). All strains showed a high affinity for chloroform, and most strains had a moderate-to-high hydrophobicity. All strains, except Lactobacillus casei and Lactobacillus gasseri, showed a moderate affinity for ethyl acetate. There was a strong association of autoaggregation with coaggregation (rs = 0·883, P < 0·001). The highest mean for autoaggregation (74%) and coaggregation (47%) belonged to the Lact. gasseri strains. Correlations between the adhesion and surface characteristics and aggregation were observed among the Lactobacillus fermentum and Lactobacillus paracasei strains; however, there was a variation in the strains properties within and between species. This study indicated that the Lact. gasseri, Lact. fermentum, and Lact. paracasei strains might be potential probiotics for the human oral cavity given their desirable properties. It should also be emphasized that a selective process for probiotic strains is required. Adhesion to host tissues and bacterial aggregation (auto- and coaggregation) are the highly important criteria for selecting strains with probiotic potential. These abilities are commonly involved with surface-charged characteristics. This is the first study to investigate the oral Lactobacillus species using an oral keratinocyte cell line. Significant results were found for the correlations between the adhesion and surface charge characteristics and for aggregation among certain strains of Lactobacillus gasseri, Lactobacillus

Sarcocystis neurona manipulation using culture-derived merozoites for bradyzoite and sporocyst production.

PubMed

Chaney, Sarah B; Marsh, Antoinette E; Lewis, Stephanie; Carman, Michelle; Howe, Daniel K; Saville, William J; Reed, Stephen M

2017-04-30

Equine protozoal myeloencephalitis (EPM) remains a significant central nervous system disease of horses in the American continents. Sarcocystis neurona is considered the primary causative agent and its intermediate life stages are carried by a wide host-range including raccoons (Procyon lotor) in North America. S. neurona sarcocysts mature in raccoon skeletal muscle and can produce central nervous system disease in raccoons, mirroring the clinical presentation in horses. The study aimed to develop laboratory tools whereby the life cycle and various life stages of S. neurona could be better studied and manipulated using in vitro and in vivo systems and compare the biology of two independent isolates. This study utilized culture-derived parasites from S. neurona strains derived from a raccoon or from a horse to initiate raccoon infections. Raccoon tissues, including fresh and cryopreserved tissues, were used to establish opossum (Didelphis virginiana) infections, which then shed sporocyts with retained biological activity to cause encephalitis in mice. These results demonstrate that sarcocysts can be generated using in vitro-derived S. neurona merozoites, including an isolate originally derived from a naturally infected horse with clinical EPM. This study indicates the life cycle can be significantly manipulated in the laboratory without affecting subsequent stage development, allowing further purification of strains and artificial maintenance of the life cycle. Published by Elsevier B.V.

Mechanisms of Soil Aggregation: a biophysical modeling framework

NASA Astrophysics Data System (ADS)

Ghezzehei, T. A.; Or, D.

2016-12-01

Soil aggregation is one of the main crosscutting concepts in all sub-disciplines and applications of soil science from agriculture to climate regulation. The concept generally refers to adhesion of primary soil particles into distinct units that remain stable when subjected to disruptive forces. It is one of the most sensitive soil qualities that readily respond to disturbances such as cultivation, fire, drought, flooding, and changes in vegetation. These changes are commonly quantified and incorporated in soil models indirectly as alterations in carbon content and type, bulk density, aeration, permeability, as well as water retention characteristics. Soil aggregation that is primarily controlled by organic matter generally exhibits hierarchical organization of soil constituents into stable units that range in size from a few microns to centimeters. However, this conceptual model of soil aggregation as the key unifying mechanism remains poorly quantified and is rarely included in predictive soil models. Here we provide a biophysical framework for quantitative and predictive modeling of soil aggregation and its attendant soil characteristics. The framework treats aggregates as hotspots of biological, chemical and physical processes centered around roots and root residue. We keep track of the life cycle of an individual aggregate from it genesis in the rhizosphere, fueled by rhizodeposition and mediated by vigorous microbial activity, until its disappearance when the root-derived resources are depleted. The framework synthesizes current understanding of microbial life in porous media; water holding and soil binding capacity of biopolymers; and environmental controls on soil organic matter dynamics. The framework paves a way for integration of processes that are presently modeled as disparate or poorly coupled processes, including storage and protection of carbon, microbial activity, greenhouse gas fluxes, movement and storage of water, resistance of soils against

GEOMETRIC CROSS SECTIONS OF DUST AGGREGATES AND A COMPRESSION MODEL FOR AGGREGATE COLLISIONS

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

Suyama, Toru; Wada, Koji; Tanaka, Hidekazu

2012-07-10

Geometric cross sections of dust aggregates determine their coupling with disk gas, which governs their motions in protoplanetary disks. Collisional outcomes also depend on geometric cross sections of initial aggregates. In a previous paper, we performed three-dimensional N-body simulations of sequential collisions of aggregates composed of a number of sub-micron-sized icy particles and examined radii of gyration (and bulk densities) of the obtained aggregates. We showed that collisional compression of aggregates is not efficient and that aggregates remain fluffy. In the present study, we examine geometric cross sections of the aggregates. Their cross sections decrease due to compression as wellmore » as to their gyration radii. It is found that a relation between the cross section and the gyration radius proposed by Okuzumi et al. is valid for the compressed aggregates. We also refine the compression model proposed in our previous paper. The refined model enables us to calculate the evolution of both gyration radii and cross sections of growing aggregates and reproduces well our numerical results of sequential aggregate collisions. The refined model can describe non-equal-mass collisions as well as equal-mass cases. Although we do not take into account oblique collisions in the present study, oblique collisions would further hinder compression of aggregates.« less

Two compounds in bed bug feces are sufficient to elicit off-host aggregation by bed bugs, Cimex lectularius.

PubMed

Olson, Joelle F; Vers, Leonard M Ver; Moon, Roger D; Kells, Stephen A

2017-01-01

After feeding, bed bugs aggregate in cracks and crevices near a host. Aggregation and arrestment are mediated by tactile and chemical stimuli associated with the bugs' feces and exuviae. Volatiles derived from fecally stained filter papers were analyzed by solid-phase microextraction (SPME) and evaluated using a multichoice behavioral assay to determine their impact on bed bug aggregation. In addition, crude fecal extracts were collected in methanol, analyzed by gas chromatography coupled with electroantennogram detection (GC-EAD) and mass spectrometry (GC-MS) and evaluated in open-air multichoice behavioral assays. The SPME method was used to detect (E)-2-hexenal and (E)-2-octenal in heated bed bug feces. The presence of these two volatile components did not affect aggregation. Analysis of the crude fecal extracts revealed several semi-volatile nitrogenous compounds, a carboxylic acid and a sulfur-based compound. Adult antennae responded to compounds eluted from three regions of the crude extract using GC-EAD. A combination of two compounds, dimethyl trisulfide and methyldiethanolamine, resulted in aggregation responses equivalent to the original crude extract. Bed bug aggregation is mediated by semi-volatile compounds derived from fecal extracts, and two compounds are sufficient to elicit aggregation. The two compounds identified here could be used to enhance the effectiveness of insecticidal applications or improve monitoring techniques. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Defining and systematic analyses of aggregation indices to evaluate degree of calcium oxalate crystal aggregation

NASA Astrophysics Data System (ADS)

Chaiyarit, Sakdithep; Thongboonkerd, Visith

2017-12-01

Crystal aggregation is one of the most crucial steps in kidney stone pathogenesis. However, previous studies of crystal aggregation were rarely done and quantitative analysis of aggregation degree was handicapped by a lack of the standard measurement. We thus performed an in vitro assay to generate aggregation of calcium oxalate monohydrate (COM) crystals with various concentrations (25-800 µg/ml) in saturated aggregation buffer. The crystal aggregates were analyzed by microscopic examination, UV-visible spectrophotometry, and GraphPad Prism6 software to define a total of 12 aggregation indices (including number of aggregates, aggregated mass index, optical density, aggregation coefficient, span, number of aggregates at plateau time-point, aggregated area index, aggregated diameter index, aggregated symmetry index, time constant, half-life, and rate constant). The data showed linear correlation between crystal concentration and almost all of these indices, except only for rate constant. Among these, number of aggregates provided the greatest regression coefficient (r=0.997; p<0.001), whereas the equally second rank included aggregated mass index and optical density (r=0.993; p<0.001 and r=‑0.993; p<0.001, respectively) and the equally forth were aggregation coefficient and span (r=0.991; p<0.001 for both). These five indices are thus recommended as the most appropriate indices for quantitative analysis of COM crystal aggregation in vitro.

Transgene delivery to endothelial cultures derived from porcine carotid artery ex vivo.

PubMed

Andoh, J; Sawyer, B; Szewczyk, K; Nortley, M; Rossetti, T; Loftus, I M; Yáñez-Muñoz, R J; Hainsworth, A H

2013-10-01

Carotid artery disease is a widespread cause of morbidity and mortality. Porcine models of vascular disease are well established in vivo, but existing endothelial systems in vitro (e.g. human umbilical vein endothelial cells, rat aortic endothelial cultures) poorly reflect carotid endothelium. A reliable in vitro assay would improve design of in vivo experiments and allow reduction and refinement of animal use. This study aimed (1) to develop ex vivo endothelial cultures from porcine carotid and (2) to test whether these were suitable for lentivector-mediated transgene delivery. Surplus carotid arteries were harvested from young adult female Large White pigs within 10 min post-mortem. Small sectors of carotid artery wall (approximately 4 mm×4 mm squares) were immobilised in a stable gel matrix. Cultures were exposed to HIV-derived lentivector (LV) encoding a reporter transgene or the equivalent integration-deficient vector (IDLV). After 7-14 days in vitro, cultures were fixed and labelled histochemically. Thread-like multicellular outgrowths were observed that were positive for endothelial cell markers (CD31, VEGFR2, von Willebrand factor). A minority of cells co-labelled for smooth muscle markers. Sensitivity to cytotoxic agents (paclitaxel, cycloheximide, staurosporine) was comparable to that in cell cultures, indicating that the gel matrix permits diffusive access of small pharmacological molecules. Transgene-expressing cells were more abundant following exposure to LV than IDLV (4.7, 0.1% of cells, respectively). In conclusion, ex vivo adult porcine carotid artery produced endothelial cell outgrowths that were effectively transduced by LV. This system will facilitate translation of novel therapies to clinical trials, with reduction and refinement of in vivo experiments.

Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

PubMed

Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

2014-09-01

This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Sustainable aggregates production : green applications for aggregate by-products.

DOT National Transportation Integrated Search

2015-06-01

Increased emphasis in the construction industry on sustainability and recycling requires production of : aggregate gradations with lower dust (cleaner aggregates) and smaller maximum sizeshence, increased : amount of quarry by-products (QBs). QBs ...

A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila

NASA Astrophysics Data System (ADS)

Apostol, Barbara L.; Kazantsev, Alexsey; Raffioni, Simona; Illes, Katalin; Pallos, Judit; Bodai, Laszlo; Slepko, Natalia; Bear, James E.; Gertler, Frank B.; Hersch, Steven; Housman, David E.; Marsh, J. Lawrence; Michels Thompson, Leslie

2003-05-01

The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds.

Aggregation effects on tritium-based mean transit times and young water fractions in spatially heterogeneous catchments and groundwater systems

NASA Astrophysics Data System (ADS)

Stewart, Michael K.; Morgenstern, Uwe; Gusyev, Maksym A.; Małoszewski, Piotr

2017-09-01

Kirchner (2016a) demonstrated that aggregation errors due to spatial heterogeneity, represented by two homogeneous subcatchments, could cause severe underestimation of the mean transit times (MTTs) of water travelling through catchments when simple lumped parameter models were applied to interpret seasonal tracer cycle data. Here we examine the effects of such errors on the MTTs and young water fractions estimated using tritium concentrations in two-part hydrological systems. We find that MTTs derived from tritium concentrations in streamflow are just as susceptible to aggregation bias as those from seasonal tracer cycles. Likewise, groundwater wells or springs fed by two or more water sources with different MTTs will also have aggregation bias. However, the transit times over which the biases are manifested are different because the two methods are applicable over different time ranges, up to 5 years for seasonal tracer cycles and up to 200 years for tritium concentrations. Our virtual experiments with two water components show that the aggregation errors are larger when the MTT differences between the components are larger and the amounts of the components are each close to 50 % of the mixture. We also find that young water fractions derived from tritium (based on a young water threshold of 18 years) are almost immune to aggregation errors as were those derived from seasonal tracer cycles with a threshold of about 2 months.

Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

PubMed Central

Tanase, Maya; Zolla, Valerio; Clement, Cristina C; Borghi, Francesco; Urbanska, Aleksandra M; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura

2016-01-01

Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. PMID:25521790

Thermodynamics of Protein Aggregation

NASA Astrophysics Data System (ADS)

Osborne, Kenneth L.; Barz, Bogdan; Bachmann, Michael; Strodel, Birgit

Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis.

PubMed

Lee, Seung Tae; Choi, Mun Hwan; Lee, Eun Ju; Gong, Seung Pyo; Jang, Mi; Park, Sang Hyun; Jee, Hyang; Kim, Dae Yong; Han, Jae Yong; Lim, Jeong Mook

2008-11-01

To evaluate whether autologous embryonic stem cells can be established without generating clone embryos. Prospective model study. Gamete and stem cell biotechnology laboratory in Seoul National University, Seoul, Korea. F1 hybrid B6D2F1 mice. Preantral follicles were cultured, and oocytes matured in the follicles were parthenogenetically activated. Preimplantation development and stem cell characterization. More intrafollicular oocytes that were retrieved from secondary follicles matured and developed into blastocysts after parthenogenesis than those that were retrieved from primary follicles. Of those 35 blastocysts derived from 193 parthenotes, one line of colony-forming cells was established from the culturing of early secondary follicles. The established cells were positive for embryonic stem cell-specific markers and had normal diploid karyotype and telomerase activity. They differentiated into embryoid bodies in vitro and teratomas in vivo. Inducible differentiation of the established cells into neuronal lineage cells also was possible. Autologous embryonic stem cells can be established by preantral follicle culture and oocyte parthenogenesis. A combined technique of follicle culture and oocyte parthenogenesis that does not use developmentally competent oocytes has the potential to replace somatic cell nuclear transfer for autologous cell therapy.

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

PubMed

Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

2018-03-22

Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

Self-Assembly of Supramolecular Aggregates Based on Sector- and Cone-Shaped Dendrons and Bolaamphiphiles

NASA Astrophysics Data System (ADS)

Shcherbina, M. A.; Chvalun, S. N.

2018-06-01

Using a number of classes of such sector-shaped macromolecules as derivatives of 2,3,4- and 3,4,5- tri(dodecyloxy)benzenesulfonic acid and dendrimers based on gallic acid as an example, the main stages in the formation of supramolecular ensembles are considered: the formation of individual supramolecular aggregates due to the weak noncovalent interactions of mesogenic groups, and the subsequent ordering within these aggregates, which lowers the free energy of a system. Supramolecular aggregates are in turn organized into two- or three-dimensional supramolecular lattices. It is shown that the shape of the supramolecular aggregates and its change along with temperature are functions of the chemical structure of the mesogenic group (resulting in the controlled design of complex self-organizing systems with a given response to external stimuli).

Derivation and characterization of gut-like structures from embryonic stem cells.

PubMed

Yamada, Takatsugu; Nakajima, Yoshiyuki

2006-01-01

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages of all three embryonic germ layers in vitro. The hanging drop culture of ES cell suspension in the absence of leukemia inhibitory factor induces aggregation and differentiation of the cells into simple or cystic embryoid bodies (EBs). After 6 d of hanging drop culture, the resulting EBs are plated onto plastic dishes for the outgrowth culture. At d 21 after outgrowth culture, cell populations of EBs can give rise to three-dimensional gut-like structures that exhibit spontaneous contraction and highly coordinated peristalsis. The gut-like structures have large lumens surrounded by three layers: epithelium, lamina propria, and muscularis. Ganglia are scattered along the periphery, and interstitial cells of Cajal are distributed among the smooth muscle cells. The fundamental process of formation of the in vitro organized gut-like structures is similar to embryonic gastrointestinal development in vivo. The EBs at the 6-d egg-cylinder stage may have the potential to regulate developmental programs associated with cell lineage commitment and provide an appropriate microenvironment to differentiate ES cells into enteric derivatives of all three embryonic germ layers and reproduce the gut organization process in vitro.

Synthesis, aggregation and spectroscopic studies of novel water soluble metal free, zinc, copper and magnesium phthalocyanines and investigation of their anti-bacterial properties.

PubMed

Bayrak, Rıza; Akçay, Hakkı Türker; Beriş, Fatih Şaban; Sahin, Ertan; Bayrak, Hacer; Demirbaş, Ümit

2014-12-10

In this study, novel phthalonitrile derivative (3) was synthesized by the reaction between 4-nitrophthalonitrile (2) and a triazole derivative (1) containing pyridine moiety. Crystal structure of compound (3) was characterized by X-ray diffraction. New metal free and metallo-phthalocyanine complexes (Zn, Cu, and Mg) were synthesized using the phthalonitrile derivative (3). Cationic derivatives of these phthalocyanines (5, 7, 9, and 11) were prepared from the non-ionic phthalocyanines (4, 6, 8, and 10). All proposed structures were supported by instrumental methods. The aggregation behaviors of the phthalocyanines (4-11) were investigated in different solvents such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), chloroform and water. Water soluble cationic Pcs (5, 7, 9, and 11) aggregated in water and sodium dodecyl sulfate was used to prevent the aggregation. The second derivatives of the UV-Vis spectra of aggregated Pcs were used for analyzing the Q and B bands of aggregated species. Thermal behaviors of the phthalocyanines were also studied. In addition, anti-bacterial properties of the phthalocyanines were investigated. We used four gram negative and two gram positive bacteria to determine antibacterial activity of these compounds. Compound 7 has the best activity against the all bacteria with 125μg/mL of MIC value. Compounds 4, 6, and 10 have the similar effect on the bacteria with 250μg/mL of MIC value. Copyright © 2014 Elsevier B.V. All rights reserved.

Neuroforecasting Aggregate Choice

PubMed Central

Knutson, Brian; Genevsky, Alexander

2018-01-01

Advances in brain-imaging design and analysis have allowed investigators to use neural activity to predict individual choice, while emerging Internet markets have opened up new opportunities for forecasting aggregate choice. Here, we review emerging research that bridges these levels of analysis by attempting to use group neural activity to forecast aggregate choice. A survey of initial findings suggests that components of group neural activity might forecast aggregate choice, in some cases even beyond traditional behavioral measures. In addition to demonstrating the plausibility of neuroforecasting, these findings raise the possibility that not all neural processes that predict individual choice forecast aggregate choice to the same degree. We propose that although integrative choice components may confer more consistency within individuals, affective choice components may generalize more broadly across individuals to forecast aggregate choice. PMID:29706726

Theoretical deposition of carcinogenic particle aggregates in the upper respiratory tract.

PubMed

Sturm, Robert

2013-10-01

Numerous particles suspended in the atmosphere are composed of smaller particular components that form aggregates with highly irregular shape. Such aggregates, among which dusts and soot are the most prominent examples, may be taken up into the respiratory tract and, in the worst case, initiate a malignant transformation of lung cells. Particle aggregates were theoretically modelled by using small spheres with equal diameters (1 nm) and arranging them randomly. This procedure resulted in the generation of various aggregate shapes (chain-like, loose, compact), for which essential parameters such as dynamic shape factors, χ, and aerodynamic diameters, dae , were computed. Deposition of aggregates consisting of 10, 50, 100, and 1,000 nano-spheres was simulated for the uppermost parts of the human respiratory system (extrathoracic region and airway generation 0 to 4), thereby distinguishing between sitting and light-work breathing as well as between nasal and oral inhalation. Based upon the modelling results, aggregate deposition in the human respiratory system can be described as a function of (I) aerodynamic diameter; (II) inhaled particle position within the airway system; and (III) breathing conditions. Therefore, highest deposition values were obtained for nano-scale aggregates (<10 nm), whereas larger aggregates exhibited slightly to significantly reduced deposition probabilities. Extrathoracic regions and uppermost bronchi (generations 0 to 1) were marked by most effective particle capture. Any increase of inhaled air volumes and reduction of breathing times resulted in an enhancement of deposition probabilities of larger particles. Based on the results derived from this study it may be concluded that small particle aggregates are accumulated in the uppermost compartments of the human respiratory tract, where they may unfold their unwholesome potential. In the case of carcinogenic particles being stored in epithelial cells for a longer time span, malignant

Calcium response and FcepsilonRI expression in bone marrow-derived mast cells co-cultured with SCG neurites.

PubMed

Suzuki, Akio; Suzuki, Ryo; Furuno, Tadahide; Teshima, Reiko; Nakanishi, Mamoru

2005-10-01

Communication between nerves and mast cells is a prototypic demonstration of neuro-immune interaction. Numerous studies have shown that the stimulation of nerves (or addition of neurotransmitters) can evoke activation of mast cells, and that mast cell-derived mediators can influence neuronal activity. However, it is still unknown whether high affinity IgE receptors (FcepsilonRI) themselves are involved directly in the communication between nerves and mast cells. In the present experiments, we used an in vitro co-culture approach comprising interaction between immune (bone marrow-derived mast cells, BMMCs) and nerve cells (superior cervical ganglia, SCG) to solve the above problem. We found that the intracellular calcium ion concentration ([Ca2+]i) increased much more in BMMCs after antigen (DNP7-BSA) stimulation when they were associated with SCG neurites in the co-culture system. But the [Ca2+]i in BMMCs was less increased when they were not associated with the neurites. Further, the in vitro co-culture approach of BMMCs with SCG neurites for 3 d showed the increases of FcepsilonRI expression occurred on the plasma membranes of BMMCs which were attached to the neurites. On the contrary, N-cadherin molecules which localized on the interface between on the plasma membrane of BMMCs and SCG neurites did not increase with the co-culture for 3 d. All of these results indicated that co-culturing BMMCs with SCG neurites for 3 d promoted not only the calcium response but also the FcepsilonRI expression in BMMCs.

The effect of nanofibrous galactosylated chitosan scaffolds on the formation of rat primary hepatocyte aggregates and the maintenance of liver function.

PubMed

Feng, Zhang-Qi; Chu, Xuehui; Huang, Ning-Ping; Wang, Tao; Wang, Yichun; Shi, Xiaolei; Ding, Yitao; Gu, Zhong-Ze

2009-05-01

Liver tissue engineering requires a perfect extracellular matrix (ECM) for primary hepatocytes culture to maintain high level of liver-specific functions and desirable mechanical stability. The aim of this study was to develop a novel natural nanofibrous scaffold with surface-galactose ligands to enhance the bioactivity and mechanical stability of primary hepatocytes in culture. The nanofibrous scaffold was fabricated by electrospinning a natural material, galactosylated chitosan (GC), into nanofibers with an average diameter of approximately 160 nm. The GC nanofibrous scaffolds displayed slow degradation and suitable mechanical properties as an ECM for hepatocytes according to the evaluation of disintegration and Young's modulus testing. The results of morphology characterization, double-staining fluorescence assay and function detection showed that hepatocytes cultured on GC nanofibrous scaffold formed stably immobilized 3D flat aggregates and exhibited superior cell bioactivity with higher levels of liver-specific function maintenance in terms of albumin secretion, urea synthesis and cytochrome P-450 enzyme than 3D spheroid aggregates formed on GC films. These spheroid aggregates could be detached easily during culture period from the flat GC films. We suggest such GC-based nanofibrous scaffolds could be useful for various applications such as bioartificial liver-assist devices and tissue engineering for liver regeneration as primary hepatocytes culture substrates.

Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer

PubMed Central

Feng, Y; Zhu, M; Dangelmajer, S; Lee, Y M; Wijesekera, O; Castellanos, C X; Denduluri, A; Chaichana, K L; Li, Q; Zhang, H; Levchenko, A; Guerrero-Cazares, H; Quiñones-Hinojosa, A

2014-01-01

Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM. PMID:25501828

Optimizing Culture Medium Composition to Improve Oligodendrocyte Progenitor Cell Yields In Vitro from Subventricular Zone-Derived Neural Progenitor Cell Neurospheres

PubMed Central

Franco, Paula G.; Pasquini, Juana M.; Silvestroff, Lucas

2015-01-01

Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC. PMID:25837625

Calcium Ions Promote Superoxide Dismutase 1 (SOD1) Aggregation into Non-fibrillar Amyloid

PubMed Central

Leal, Sónia S.; Cardoso, Isabel; Valentine, Joan S.; Gomes, Cláudio M.

2013-01-01

Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca2+ dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca2+. We show that at physiological pH, Ca2+ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca2+ boosts the onset of SOD1 aggregation. In agreement, Ca2+ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca2+ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca2+-induced aggregates, thus indicating that Ca2+ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca2+ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca2+ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions. PMID:23861388

Three-dimensional Myoblast Aggregates--Effects of Modeled Microgravity

NASA Technical Reports Server (NTRS)

Byerly, Diane; Sognier, M. A.; Marquette, M. L.

2006-01-01

The overall objective of these studies is to elucidate the molecular and cellular alterations that contribute to muscle atrophy in astronauts caused by exposure to microgravity conditions in space. To accomplish this, a three-dimensional model test system was developed using mouse myoblast cells (C2C12). Myoblast cells were grown as three-dimensional aggregates (without scaffolding or other solid support structures) in both modeled microgravity (Rotary Cell Culture System, Synthecon, Inc.) and at unit gravity in coated Petri dishes. Evaluation of H&E stained thin sections of the aggregates revealed the absence of any necrosis. Confocal microscopy evaluations of cells stained with the Live/Dead assay (Molecular Probes) confirmed that viable cells were present throughout the aggregates with an average of only three dead cells observed per aggregate. Preliminary results from gene array analysis (Affymetrix chip U74Av2) showed that approximately 14% of the genes were down regulated (decreased more than 3 fold) and 4% were upregulated in cells exposed to modeled microgravity for 12 hours compared to unit gravity controls. Additional studies using fluorescent phallacidin revealed a decrease in F-actin in the cells exposed to modeled microgravity compared to unit gravity. Myoblast cells grown as aggregates in modeled microgravity exhibited spontaneous differentiation into syncitia while no differentiation was seen in the unit gravity controls. These studies show that 1)the model test system developed is suitable for assessing cellular and molecular alterations in myoblasts; 2) gene expression alterations occur rapidly (within 12 hours) following exposure to modeled microgravity; and 3) modeled microgravity conditions stimulated myoblast cell differentiation. Achieving a greater understanding of the molecular alterations leading to muscle atrophy will eventually enable the development of cell-based countermeasures, which may be valuable for treatment of muscle diseases on

Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

PubMed Central

Tyedmers, Jens

2012-01-01

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI+] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates. PMID:22449721

Agreement between aggregate and individual-level measures of income and education: a comparison across three patient groups.

PubMed

Marra, Carlo A; Lynd, Larry D; Harvard, Stephanie S; Grubisic, Maja

2011-03-31

The association between lower socioeconomic status and poorer health outcomes has been observed using both individual-level and aggregate-level measures of income and education. While both are predictive of health outcomes, previous research indicates poor agreement between individual-level and aggregate-level measures. The purpose of this study was to determine the level of agreement between aggregate-level and individual-level measures of income and education among three distinct patient groups, specifically asthma, diabetes, and rheumatoid patients. Individual-level measures of annual household income and education were derived from three separate surveys conducted among patients with asthma (n = 359), diabetes (n = 281) and rheumatoid arthritis (n = 275). Aggregate-level measures of income and education were derived from the 2001 Canadian census, including both census tract-and dissemination area-level measures. Cross-tabulations of individual-level income by aggregate-level income were used to determine the percentage of income classifications in agreement. The kappa statistic (simple and weighted), Spearman's rank correlations, and intra-class correlation coefficient (ICC) were also calculated. Individual-level and aggregate-level education was compared using Chi-Square tests within patient groups. Point biserial correlation coefficients between individual-level and aggregate-level education were computed. Individual-level income was poorly correlated with aggregate-level measures, which provided the worst estimations of income among patients in the lowest income category at the individual-level. Both aggregate-level measures were best at approximating individual-level income in patients with diabetes, in whom aggregate-level estimates were only significantly different from individual-level measures for patients in the lowest income category. Among asthma patients, the proportion of patients classified by aggregate-level measures as having a university degree

A comparative study of the properties and self-aggregation behavior of collagens from the scales and skin of grass carp (Ctenopharyngodon idella).

PubMed

Liu, Yaowen; Ma, Donghui; Wang, Yihao; Qin, Wen

2018-01-01

Collagens were extracted from the scales and skin of Ctenopharyngodon idella (C. idella) as raw materials using an acid-enzyme hybrid method. The structural properties of the extracted collagens were compared using ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and differential scanning calorimetry. Additionally, the in vitro self-aggregation behaviors of the two types of collagens (fish skin- and scale-derived collagens) were compared using turbidimetric assays, aggregation assays, and scanning electron microscopy (SEM). The results showed that both types of extracted collagen were typical type I collagen with two α chains and intact triple-helical structures. The denaturation temperatures of the collagens from fish scales and skin were 34.99°C and 39.75°C, respectively. Both types of collagens were capable of self-aggregation in neutral salt solution at 30°C, with aggregation degrees of 28% and 27.33% for the scale and skin collagens, respectively. SEM analysis revealed that both types of collagens could self-aggregate into interwoven fibers, and the fish scale-derived collagen had a more pronounced reticular fiber structure with a striped periodic D-band pattern of collagen fibrils, whereas the collagen fibers from the self-aggregation of fish skin-derived collagen had a certain degree of disruption without any D-band pattern. Copyright © 2017 Elsevier B.V. All rights reserved.

Color-tunable, aggregation-induced emission of a butterfly-shaped molecule comprising a pyran skeleton and two cholesteryl wings.

PubMed

Tong, Hui; Hong, Yuning; Dong, Yongqiang; Ren, Yan; Häussler, Matthias; Lam, Jacky W Y; Wong, Kam Sing; Tang, Ben Zhong

2007-03-01

A chiral pyran derivative containing two cholesteryl groups (1) is synthesized, and its optical properties are investigated. Whereas the isolated molecule of 1 is virtually nonluminescent in dilute solutions, it becomes highly emissive with a 2 orders of magnitude increase in fluorescence quantum yield upon aggregation in poor solvents or in solid state, showing a novel phenomenon of aggregation-induced emission (AIE). The color and efficiency of the AIE of 1 can be tuned by varying the morphology of its aggregates: photoluminescence of its aggregates formed in a tetrahydrofuran/water mixture progressively red-shifts (green --> yellow --> red) with increasing water content of the mixture, with the crystalline aggregates emitting bluer lights in higher efficiencies than their amorphous counterparts.

Self-aggregation behavior of synthetic amphiphile derived from triazolylbenzoic acid: CMC and phase transition

NASA Astrophysics Data System (ADS)

Tajuddin, Hairul Anuar; Idris, Tarmezee; Zul, Nurul Faiezin; Sadidarto, Ahmad Bayhaki; Abdullah, Zanariah; Ahmad, Noraini

2017-12-01

An anionic surfactant with intention to mimic the natural fatty acids with highly conjugated component, has been synthesized and analyzed by using NMR, FTIR and MS. The polar head group of the structure was linked to the hydrophobic tail, through triazolyl moiety that was formed from the Cu(I) cycloaddition between an azidobenzoic acid and an acetylene. The critical micellar concentration (cmc) of the surfactant was determined at 0.1 mM by measuring its conductivity at a constant pH, consistent with the UV-Visible absorption. Phase transitions of the surfactant were then observed, by gradually decreasing the pH from 10 to 7. There were two stages of phase transitions observed at pH 8.9 and 7.6 upon titration, suggesting the transition from smaller to larger size of the aggregation structures. The results indicate that the aggregation mechanism of the synthesized surfactant w a s greatly influenced by the conversion of t h e head group from -COO- to -COOH, which is similar to the natural fatty acids in aqueous form.

Non-Arrhenius protein aggregation.

PubMed

Wang, Wei; Roberts, Christopher J

2013-07-01

Protein aggregation presents one of the key challenges in the development of protein biotherapeutics. It affects not only product quality but also potentially impacts safety, as protein aggregates have been shown to be linked with cytotoxicity and patient immunogenicity. Therefore, investigations of protein aggregation remain a major focus in pharmaceutical companies and academic institutions. Due to the complexity of the aggregation process and temperature-dependent conformational stability, temperature-induced protein aggregation is often non-Arrhenius over even relatively small temperature windows relevant for product development, and this makes low-temperature extrapolation difficult based simply on accelerated stability studies at high temperatures. This review discusses the non-Arrhenius nature of the temperature dependence of protein aggregation, explores possible causes, and considers inherent hurdles for accurately extrapolating aggregation rates from conventional industrial approaches for selecting accelerated conditions and from conventional or more advanced methods of analyzing the resulting rate data.

The breakup mechanism of biomolecular and colloidal aggregates in a shear flow

NASA Astrophysics Data System (ADS)

Ó Conchúir, Breanndán; Zaccone, Alessio

2014-03-01

The theory of self-assembly of colloidal particles in shear flow is incomplete. Previous analytical approaches have failed to capture the microscopic interplay between diffusion, shear and intermolecular interactions which controls the aggregates fate in shear. In this work we analytically solved the drift-diffusion equation for the breakup rate of a dimer in flow. Then applying rigidity percolation theory, we found that the lifetime of a generic cluster formed under shear is controlled by the typical lifetime of a single bond in its interior, which in turn depends on the efficiency of the stress transmitted from other bonds in the cluster. We showed that aggregate breakup is a thermally-activated process where the activation energy is controlled by the interplay between intermolecular forces and the shear drift, and where structural parameters determine whether cluster fragmentation or surface erosion prevails. In our latest work, we analyzed floppy modes and nonaffine deformations to derive a lower bound on the fractal dimension df below which aggregates are mechanically unstable, ie. for large aggregates df ~= 2.4. This theoretical framework is in quantitative agreement with experiments and can be used for population balance modeling of colloidal and protein aggregation.

Biotechnological production of caffeic acid derivatives from cell and organ cultures of Echinacea species.

PubMed

Murthy, Hosakatte Niranjana; Kim, Yun-Soo; Park, So-Young; Paek, Kee-Yoeup

2014-09-01

Caffeic acid derivatives (CADs) are a group of bioactive compounds which are produced in Echinacea species especially Echinacea purpurea, Echinacea angustifolia, and Echinacea pallida. Echinacea is a popular herbal medicine used in the treatment of common cold and it is also a prominent dietary supplement used throughout the world. Caffeic acid, chlorogenic acid (5-O-caffeoylquinic acid), caftaric acid (2-O-caffeoyltartaric acid), cichoric acid (2, 3-O-dicaffeoyltartaric acid), cynarin, and echinacoside are some of the important CADs which have varied pharmacological activities. The concentrations of these bioactive compounds are species specific and also they vary considerably with the cultivated Echinacea species due to geographical location, stage of development, time of harvest, and growth conditions. Due to these reasons, plant cell and organ cultures have become attractive alternative for the production of biomass and caffeic acid derivatives. Adventitious and hairy roots have been induced in E. pupurea and E. angustifolia, and suspension cultures have been established from flask to bioreactor scale for the production of biomass and CADs. Tremendous progress has been made in this area; various bioprocess methods and strategies have been developed for constant high-quality productivity of biomass and secondary products. This review is aimed to discuss biotechnological methods and approaches employed for the sustainable production of CADs.

The use of index tests to determine the mechanical properties of crushed aggregates from Precambrian basement complex rocks, Ado-Ekiti, SW Nigeria

NASA Astrophysics Data System (ADS)

Afolagboye, Lekan Olatayo; Talabi, Abel Ojo; Oyelami, Charles Adebayo

2017-05-01

This study assessed the possibility of using index tests to determine the mechanical properties of crushed aggregates. The aggregates used in this study were derived from major Precambrian basement rocks in Ado-Ekiti, Nigeria. Regression analyses were performed to determine the empirical relations that mechanical properties of the aggregates may have with the point load strength (IS(50)), Schmidt rebound hammer value (SHR) and unconfined compressive strength (UCS) of the rocks. For all the data, strong correlation coefficients were found between IS(50), SHR, UCS, and mechanical properties of the aggregates. The regression analysis conducted on the different rocks separately showed that correlations coefficients obtained between the IS(50), SHR, UCS and mechanical properties of the aggregates were stronger than those of the grouped rocks. The T-test and F-test showed that the derived models were valid. This study has shown that the mechanical properties of the aggregates can be estimated from IS(50), SHR and USC but the influence of rock type on the relationships should be taken into consideration.

Effect of aggregate graining compositions on skid resistance of Exposed Aggregate Concrete pavement

NASA Astrophysics Data System (ADS)

Wasilewska, Marta; Gardziejczyk, Wladysław; Gierasimiuk, Pawel

2018-05-01

The paper presents the evaluation of skid resistance of EAC (Exposed Aggregate Concrete) pavements which differ in aggregate graining compositions. The tests were carried out on concrete mixes with a maximum aggregate size of 8 mm. Three types of coarse aggregates were selected depending on their resistance to polishing which was determined on the basis of the PSV (Polished Stone Value). Basalt (PSV 48), gabbro (PSV 50) and trachybasalt (PSV 52) aggregates were chosen. For each type of aggregate three graining compositions were designed, which differed in the content of coarse aggregate > 4mm. Their content for each series was as follows: A - 38%, B - 50% and C - 68%. Evaluation of the skid resistance has been performed using the FAP (Friction After Polishing) test equipment also known as the Wehner/Schulze machine. Laboratory method enables to compare the skid resistance of different types of wearing course under specified conditions simulating polishing processes. In addition, macrotexture measurements were made on the surface of each specimen using the Elatexure laser profile. Analysis of variance showed that at significance level α = 0.05, aggregate graining compositions as well as the PSV have a significant influence on the obtained values of the friction coefficient μm of the tested EAC pavements. The highest values of the μm have been obtained for EAC with the lowest amount of coarse aggregates (compositions A). In these cases the resistance to polishing of the aggregate does not significantly affect the friction coefficients. This is related to the large areas of cement mortar between the exposed coarse grains. Based on the analysis of microscope images, it was observed that the coarse aggregates were not sufficiently exposed. It has been proved that PSV significantly affected the coefficient of friction in the case of compositions B and C. This is caused by large areas of exposed coarse aggregate. The best parameters were achieved for the EAC pavements

Maximal aggregation of polynomial dynamical systems

PubMed Central

Cardelli, Luca; Tschaikowski, Max

2017-01-01

Ordinary differential equations (ODEs) with polynomial derivatives are a fundamental tool for understanding the dynamics of systems across many branches of science, but our ability to gain mechanistic insight and effectively conduct numerical evaluations is critically hindered when dealing with large models. Here we propose an aggregation technique that rests on two notions of equivalence relating ODE variables whenever they have the same solution (backward criterion) or if a self-consistent system can be written for describing the evolution of sums of variables in the same equivalence class (forward criterion). A key feature of our proposal is to encode a polynomial ODE system into a finitary structure akin to a formal chemical reaction network. This enables the development of a discrete algorithm to efficiently compute the largest equivalence, building on approaches rooted in computer science to minimize basic models of computation through iterative partition refinements. The physical interpretability of the aggregation is shown on polynomial ODE systems for biochemical reaction networks, gene regulatory networks, and evolutionary game theory. PMID:28878023

Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL.

PubMed

Wang, Xia; An, Qiang; Zhao, Bin; Guo, Jin Song; Huang, Yuan Sheng; Tian, Meng

2018-02-01

Enterobacter sp. strain FL was newly isolated from activated sludge and exhibited significant capability of auto-aggregation as well as aerobic denitrification. The removal efficiencies of NO 3 - -N, total nitrogen (TN), and TOC by strain FL in batch culture reached 94.6, 63.9, and 72.5% in 24 h, respectively. The production of N 2 O and N 2 in the presence of oxygen demonstrated the occurrence of aerobic denitrification. The auto-aggregation index of strain FL reached 54.3%, suggesting a high tendency that the cells would agglomerate into aggregates. The production of extracellular polymeric substances (EPSs), which were mainly composed of proteins followed by polysaccharides, was considered to be related to the cell aggregation according to Fourier transform infrared (FT-IR) and confocal laser scanning microscopy (CLSM). The proteins in EPS were evenly and tightly combined to cells and altered the protein secondary structures of cell surface from random coils to β-sheets and three-turn helices. The alteration of protein secondary structures of cell surface caused by the proteins in EPS might play a dominant role in the auto-aggregation of strain FL. To further assess the feasibility of strain FL for synthetic wastewater treatment, a sequencing batch reactor (SBR), solely inoculated with strain FL, was conducted. During the 16 running cycles, the removal efficiency of NO 3 - -N was 90.2-99.7% and the auto-aggregation index was stabilized at 35.0-41.5%. The EPS promoted the biomass of strain FL to aggregate in the SBR.

Evaluation of Canadian unconfined aggregate freeze-thaw tests for identifying nondurable aggregates.

DOT National Transportation Integrated Search

2012-06-01

Concrete is the most widely used material in construction. Aggregates contribute 60% to 75% of the total volume : of concrete. The aggregates play a key role in concrete durability. The U.S. Midwest has many aggregates that can : show distress in the...

A new and efficient culture method for porcine bone marrow-derived M1- and M2-polarized macrophages.

PubMed

Gao, Jiye; Scheenstra, Maaike R; van Dijk, Albert; Veldhuizen, Edwin J A; Haagsman, Henk P

2018-06-01

Macrophages play an important role in the innate immune system as part of the mononuclear phagocyte system (MPS). They have a pro-inflammatory signature (M1-polarized macrophages) or anti-inflammatory signature (M2-polarized macrophages) based on expression of surface receptors and secretion of cytokines. However, very little is known about the culture of macrophages from pigs and more specific about the M1 and M2 polarization in vitro. Porcine monocytes or mononuclear bone marrow cells were used to culture M1- and M2-polarized macrophages in the presence of GM-CSF and M-CSF, respectively. Surface receptor expression was measured with flow cytometry and ELISA was used to quantify cytokine secretion in response to LPS and PAM 3 CSK 4 stimulation. Human monocyte-derived macrophages were used as control. Porcine M1- and M2-polarized macrophages were cultured best using porcine GM-CSF and murine M-CSF, respectively. Cultures from bone marrow cells resulted in a higher yield M1- and M2-polarized macrophages which were better comparable to human monocyte-derived macrophages than cultures from porcine monocytes. Porcine M1-polarized macrophages displayed the characteristic fried egg shape morphology, lower CD163 expression and low IL-10 production. Porcine M2-polarized macrophages contained the spindle-like morphology, higher CD163 expression and high IL-10 production. Porcine M1- and M2-polarized macrophages can be most efficiently cultured from mononuclear bone marrow cells using porcine GM-CSF and murine M-CSF. The new culture method facilitates more refined studies of porcine macrophages in vitro, important for both porcine and human health since pigs are increasingly used as model for translational research. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

PubMed Central

Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

2012-01-01

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths.

PubMed Central

Snyder, S W; Ladror, U S; Wade, W S; Wang, G T; Barrett, L W; Matayoshi, E D; Huffaker, H J; Krafft, G A; Holzman, T F

1994-01-01

One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and

Anti-protein aggregation is a potential target for preventing delayed neuronal death after transient ischemia.

PubMed

Ge, Pengfei; Luo, Yinan; Wang, Haifeng; Ling, Feng

2009-12-01

Brain ischemia has been an important risk factor for human being health, there is no effective medicine can be used to protect delayed neuronal injury or death secondary to blood reperfusion following ischemia. Recent discovery shows protein aggregation is an important factor resulting in ischemia-induced neuron death. Therefore, we propose the hypothesis that inhibiting protein aggregation may be an effective way to prevent delayed neuronal death after transient ischemia. At present, in vitro studies show some chemicals such as 4PBA (sodium 4-phenylbutyrate) and trehalose have the features of antagonizing protein aggregation in vitro. Moreover, polyQ-binding peptide (QBP1), geldanamycin, amino acids and amino acid derivatives have been also used in vitro to decrease aggregation and to increase protein stability. Although in vivo and systematical study should be performed to evaluate their effects of anti-protein aggregation, this enlightening us on using them to protect ischemic-induced neuronal death, and find new potential chemicals or methods which could be effective in keeping protein stable and prevent forming aggregates.

Spatial Modeling of Iron Transformations Within Artificial Soil Aggregates

NASA Astrophysics Data System (ADS)

Kausch, M.; Meile, C.; Pallud, C.

2008-12-01

Structured soils exhibit significant variations in transport characteristics at the aggregate scale. Preferential flow occurs through macropores while predominantly diffusive exchange takes place in intra-aggregate micropores. Such environments characterized by mass transfer limitations are conducive to the formation of small-scale chemical gradients and promote strong spatial variation in processes controlling the fate of redox-sensitive elements such as Fe. In this study, we present a reactive transport model used to spatially resolve iron bioreductive processes occurring within a spherical aggregate at the interface between advective and diffusive domains. The model is derived from current conceptual models of iron(hydr)oxide (HFO) transformations and constrained by literature and experimental data. Data were obtained from flow-through experiments on artificial soil aggregates inoculated with Shewanella putrefaciens strain CN32, and include the temporal evolution of the bulk solution composition, as well as spatial information on the final solid phase distribution within aggregates. With all iron initially in the form of ferrihydrite, spatially heterogeneous formation of goethite/lepidocrocite, magnetite and siderite was observed during the course of the experiments. These transformations were reproduced by the model, which ascribes a central role to divalent iron as a driver of HFO transformations and master variable in the rate laws of the considered reaction network. The predicted dissolved iron breakthrough curves also match the experimental ones closely. Thus, the computed chemical concentration fields help identify factors governing the observed trends in the solid phase distribution patterns inside the aggregate. Building on a mechanistic description of transformation reactions, fluid flow and solute transport, the model was able to describe the observations and hence illustrates the importance of small-scale gradients and dynamics of bioreductive

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity.

PubMed

Villar-Piqué, Anna; Lopes da Fonseca, Tomás; Sant'Anna, Ricardo; Szegö, Éva Mónika; Fonseca-Ornelas, Luis; Pinho, Raquel; Carija, Anita; Gerhardt, Ellen; Masaracchia, Caterina; Abad Gonzalez, Enrique; Rossetti, Giulia; Carloni, Paolo; Fernández, Claudio O; Foguel, Debora; Milosevic, Ira; Zweckstetter, Markus; Ventura, Salvador; Outeiro, Tiago Fleming

2016-10-18

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity.

A Novel Method to Quantify Soil Aggregate Stability by Measuring Aggregate Bond Energies

NASA Astrophysics Data System (ADS)

Efrat, Rachel; Rawlins, Barry G.; Quinton, John N.; Watts, Chris W.; Whitmore, Andy P.

2016-04-01

Soil aggregate stability is a key indicator of soil quality because it controls physical, biological and chemical functions important in cultivated soils. Micro-aggregates are responsible for the long term sequestration of carbon in soil, therefore determine soils role in the carbon cycle. It is thus vital that techniques to measure aggregate stability are accurate, consistent and reliable, in order to appropriately manage and monitor soil quality, and to develop our understanding and estimates of soil as a carbon store to appropriately incorporate in carbon cycle models. Practices used to assess the stability of aggregates vary in sample preparation, operational technique and unit of results. They use proxies and lack quantification. Conflicting results are therefore drawn between projects that do not provide methodological or resultant comparability. Typical modern stability tests suspend aggregates in water and monitor fragmentation upon exposure to an un-quantified amount of ultrasonic energy, utilising a laser granulometer to measure the change in mean weight diameter. In this project a novel approach has been developed based on that of Zhu et al., (2009), to accurately quantify the stability of aggregates by specifically measuring their bond energies. The bond energies are measured operating a combination of calorimetry and a high powered ultrasonic probe, with computable output function. Temperature change during sonication is monitored by an array of probes which enables calculation of the energy spent heating the system (Ph). Our novel technique suspends aggregates in heavy liquid lithium heteropolytungstate, as opposed to water, to avoid exposing aggregates to an immeasurable disruptive energy source, due to cavitation, collisions and clay swelling. Mean weight diameter is measured by a laser granulometer to monitor aggregate breakdown after successive periods of calculated ultrasonic energy input (Pi), until complete dispersion is achieved and bond

Inhibitory effects of 2'-hydroxychalcones on rat lens aldose reductase and rat platelet aggregation.

PubMed

Lim, S S; Jung, S H; Ji, J; Shin, K H; Keum, S R

2000-11-01

Inhibitory effects of synthetic 2'-hydroxychalcone derivatives on rat lens aldose reductase (RLAR) and on platelet aggregation were investigated for the prevention or the treatment of chronic diabetic complications. 5'-chloro-4,2'-dihydroxychalcone (8) and 5'-chloro-3,2'-dihydroxychalcone (27) exhibited a potent inhibitory effect on rat platelet aggregation induced by ADP (IC50=0.10 and 0.06 mg/ml, respectively) and collagen (IC50=44 and 16 microg/ml, respectively) but showed relatively weak inhibitory activities on RLAR.

The relationship between organizational culture and the health and wellbeing of hospital nurses worldwide: a mixed methods systematic review protocol.

PubMed

Whitcombe, Anne; Cooper, Kay; Palmer, Emma

2016-06-01

The objective of this mixed methods systematic review is to examine the relationship between organizational culture and the health and wellbeing of hospital nurses, and to develop an aggregated synthesis of quantitative and qualitative systematic reviews to derive recommendations for policy and practice.Organizational culture comprises factors such as leadership, management and support, a health and safety oriented workplace climate and job characteristics.The quantitative component of this review will explore the relationship between organizational culture and the following outcomes in hospital nurses which may be indicators of health and wellbeing: work-related injury such as needlestick or sharp injuries, musculoskeletal injuries and conditions such as low back pain, burnout and general wellbeing.The qualitative component of this review will explore the perceptions of hospital nurses in relation to the impact of organizational culture on their own health and wellbeing and those of their nursing colleagues.

Synthesis and Properties of Gelators Derived from Tetraphenylethylene and Gallic Acid with Aggregation-Induced Emission

NASA Astrophysics Data System (ADS)

Luo, Miao; Zhou, Xie; Chi, Zhenguo; Ma, Chunping; Zhang, Yi; Liu, Siwei; Xu, Jiarui

2013-09-01

Two novel organogelators (TEG and TAG) based on tetraphenylethylene and 3,4,5-tris(dodecyloxy) benzoic acid were synthesized through ester bond and amido bond linkages, respectively. Compounds TEG and TAG were able to induce gelation in ethanol. Aggregation-induced enhanced emission was observed in these organogelator molecules, with increased fluorescence intensity from the solutions to the gels. The completely thermoreversible gelation occurred due to the aggregation of the organogelators. In the process, a fibrous network was formed by a combination of intermolecular hydrogen bonding, π-π stacking and van der Waals interactions. These phenomena were observed in the xerogels by field-emission scanning electron microscopy and Fourier-transform infrared spectroscopy. The results of differential scanning calorimetry and polarized optical microscopy indicated that compound TAG exhibited stable liquid crystalline phases over a wide temperature range. The linking groups have severe influence on the properties of the organogelators, which was mainly attributed to the hydrogen bonding interaction in compound TAG.

Recovery of MSWI and soil washing residues as concrete aggregates.

PubMed

Sorlini, Sabrina; Abbà, Alessandro; Collivignarelli, Carlo

2011-02-01

The aim of the present work was to study if municipal solid waste incinerator (MSWI) residues and aggregates derived from contaminated soil washing could be used as alternative aggregates for concrete production. Initially, chemical, physical and geometric characteristics (according to UNI EN 12620) of municipal solid waste incineration bottom ashes and some contaminated soils were evaluated; moreover, the pollutants release was evaluated by means of leaching tests. The results showed that the reuse of pre-treated MSWI bottom ash and washed soil is possible, either from technical or environmental point of view, while it is not possible for the raw wastes. Then, the natural aggregate was partially and totally replaced with these recycled aggregates for the production of concrete mixtures that were characterized by conventional mechanical and leaching tests. Good results were obtained using the same dosage of a high resistance cement (42.5R calcareous Portland cement instead of 32.5R); the concrete mixture containing 400 kg/m(3) of washed bottom ash and high resistance cement was classified as structural concrete (C25/30 class). Regarding the pollutants leaching, all concrete mixtures respected the limit values according to the Italian regulation. Copyright © 2010 Elsevier Ltd. All rights reserved.

Curcumin Attenuates Amyloid-β Aggregate Toxicity and Modulates Amyloid-β Aggregation Pathway.

PubMed

Thapa, Arjun; Jett, Stephen D; Chi, Eva Y

2016-01-20

The abnormal misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet enriched insoluble deposits initiates a cascade of events leading to pathological processes and culminating in cognitive decline in Alzheimer's disease (AD). In particular, soluble oligomeric/prefibrillar Aβ have been shown to be potent neurotoxins. The naturally occurring polyphenol curcumin has been shown to exert a neuroprotective effect against age-related neurodegenerative diseases such as AD. However, its protective mechanism remains unclear. In this study, we investigated the effects of curcumin on the aggregation of Aβ40 as well as Aβ40 aggregate induced neurotoxicity. Our results show that the curcumin does not inhibit Aβ fibril formation, but rather enriches the population of "off-pathway" soluble oligomers and prefibrillar aggregates that were nontoxic. Curcumin also exerted a nonspecific neuroprotective effect, reducing toxicities induced by a range of Aβ conformers, including monomeric, oligomeric, prefibrillar, and fibrillar Aβ. The neuroprotective effect is possibly membrane-mediated, as curcumin reduced the extent of cell membrane permeabilization induced by Aβ aggregates. Taken together, our study shows that curcumin exerts its neuroprotective effect against Aβ induced toxicity through at least two concerted pathways, modifying the Aβ aggregation pathway toward the formation of nontoxic aggregates and ameliorating Aβ-induced toxicity possibly through a nonspecific pathway.

Ammonificins C and D, Hydroxyethylamine Chromene Derivatives from a Cultured Marine Hydrothermal Vent Bacterium, Thermovibrio ammonificans

PubMed Central

Andrianasolo, Eric H.; Haramaty, Liti; Rosario-Passapera, Richard; Vetriani, Costantino; Falkowski, Paul; White, Eileen; Lutz, Richard

2012-01-01

Chemical and biological investigation of the cultured marine hydrothermal vent bacterium, Thermovibrio ammonifican led to the isolation of two hydroxyethylamine chromene derivatives, ammonificins C and D. Their structures were elucidated using combination of NMR and mass spectrometry. Absolute stereochemistry was ascertained by comparison of experimental and calculated CD spectra. Biological evaluation and assessment were determined using the patented ApopScreen cell-based screen for apoptosis-induction. Ammonificins C and D induce apoptosis in micromolar concentrations. To our knowledge, this finding is the first report of chemical compounds that induce apoptosis from the cultured deep-sea marine organism, hydrothermal vent bacterium, Thermovibrio ammonificans. PMID:23170085

Construction of an in vitro primary lung co-culture platform derived from New Zealand white rabbits

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

Powell, Joshua D.; Hess, Becky M.; Hutchison, Janine R.

2015-05-01

We report the construction of an in vitro three dimensional (3D) co-culture platform consisting of differentiated lung epithelial cells and monocytes from New Zealand white rabbits. Rabbit lung epithelial cells were successfully grown at air-liquid interface, produced mucus, and expressed both sialic acid alpha-2,3 and alpha-2,6. Blood-derived CD14+ monocytes were deposited above the epithelial layer resulting in the differentiation of a subset of monocytes into CD11c+ cells within the co-culture. These proof-of-concept findings provide a convenient means to comparatively study in vitro versus in vivo rabbit lung responses as they relate to inhalation or lung-challenge studies.

Biotechnological approaches to enhance salidroside, rosin and its derivatives production in selected Rhodiola spp. in vitro cultures.

PubMed

Grech-Baran, Marta; Sykłowska-Baranek, Katarzyna; Pietrosiuk, Agnieszka

Rhodiola (Crassulaceae) an arctic-alpine plant, is extensively used in traditional folk medicine in Asian and European countries. A number of investigations have demonstrated that Rhodiola preparations exhibit adaptogenic, neuroprotective, anti-tumour, cardioprotective, and anti-depressant effects. The main compounds responsible for these activities are believed to be salidroside, rosin and its derivatives which became the target of biotechnological investigations. This review summarizes the results of the diverse biotechnological approaches undertaken to enhance the production of salidroside, rosin and its derivatives in callus, cell suspension and organ in vitro cultures of selected Rhodiola species.

Lysosomal membrane permeability stimulates protein aggregate formation in neurons of a lysosomal disease.

PubMed

Micsenyi, Matthew C; Sikora, Jakub; Stephney, Gloria; Dobrenis, Kostantin; Walkley, Steven U

2013-06-26

Protein aggregates are a common pathological feature of neurodegenerative diseases and several lysosomal diseases, but it is currently unclear what aggregates represent for pathogenesis. Here we report the accumulation of intraneuronal aggregates containing the macroautophagy adapter proteins p62 and NBR1 in the neurodegenerative lysosomal disease late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). CLN2 disease is caused by a deficiency in the lysosomal enzyme tripeptidyl peptidase I, which results in aberrant lysosomal storage of catabolites, including the subunit c of mitochondrial ATP synthase (SCMAS). In an effort to define the role of aggregates in CLN2, we evaluated p62 and NBR1 accumulation in the CNS of Cln2(-/-) mice. Although increases in p62 and NBR1 often suggest compromised degradative mechanisms, we found normal ubiquitin-proteasome system function and only modest inefficiency in macroautophagy late in disease. Importantly, we identified that SCMAS colocalizes with p62 in extra-lysosomal aggregates in Cln2(-/-) neurons in vivo. This finding is consistent with SCMAS being released from lysosomes, an event known as lysosomal membrane permeability (LMP). We predicted that LMP and storage release from lysosomes results in the sequestration of this material as cytosolic aggregates by p62 and NBR1. Notably, LMP induction in primary neuronal cultures generates p62-positive aggregates and promotes p62 localization to lysosomal membranes, supporting our in vivo findings. We conclude that LMP is a previously unrecognized pathogenic event in CLN2 disease that stimulates cytosolic aggregate formation. Furthermore, we offer a novel role for p62 in response to LMP that may be relevant for other diseases exhibiting p62 accumulation.

Lysosomal Membrane Permeability Stimulates Protein Aggregate Formation in Neurons of a Lysosomal Disease

PubMed Central

Micsenyi, Matthew C.; Sikora, Jakub; Stephney, Gloria; Dobrenis, Kostantin

2013-01-01

Protein aggregates are a common pathological feature of neurodegenerative diseases and several lysosomal diseases, but it is currently unclear what aggregates represent for pathogenesis. Here we report the accumulation of intraneuronal aggregates containing the macroautophagy adapter proteins p62 and NBR1 in the neurodegenerative lysosomal disease late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). CLN2 disease is caused by a deficiency in the lysosomal enzyme tripeptidyl peptidase I, which results in aberrant lysosomal storage of catabolites, including the subunit c of mitochondrial ATP synthase (SCMAS). In an effort to define the role of aggregates in CLN2, we evaluated p62 and NBR1 accumulation in the CNS of Cln2−/− mice. Although increases in p62 and NBR1 often suggest compromised degradative mechanisms, we found normal ubiquitin–proteasome system function and only modest inefficiency in macroautophagy late in disease. Importantly, we identified that SCMAS colocalizes with p62 in extra-lysosomal aggregates in Cln2−/− neurons in vivo. This finding is consistent with SCMAS being released from lysosomes, an event known as lysosomal membrane permeability (LMP). We predicted that LMP and storage release from lysosomes results in the sequestration of this material as cytosolic aggregates by p62 and NBR1. Notably, LMP induction in primary neuronal cultures generates p62-positive aggregates and promotes p62 localization to lysosomal membranes, supporting our in vivo findings. We conclude that LMP is a previously unrecognized pathogenic event in CLN2 disease that stimulates cytosolic aggregate formation. Furthermore, we offer a novel role for p62 in response to LMP that may be relevant for other diseases exhibiting p62 accumulation. PMID:23804102

Aggregation Properties and Liquid Crystal Phase of a Dye Based on Naphthalenetetracarboxylic Acid

NASA Astrophysics Data System (ADS)

Tomasik, Michelle; Collings, Peter

2007-03-01

R003 is a dye produced for thin film optical components by Optiva, Inc.^1 made from the sulfonation of the dibenzimidazole derivative of naphthalenetetracarboxylic acid. Its molecular structure is very different from the aggregating food dye previously investigated in our laboratory^2 and R003 forms a liquid crystal phase at significantly lower concentrations. We have performed polarizing microscopy, absorption spectroscopy, and x-ray diffraction experiments in order to determine the phase diagram and aggregate structure. In addition, we have included both translational and orientational entropy in the theoretical analysis of the aggregation process, and have used a more realistic lineshape in analyzing the absorption data. Our results indicate that the ``bond energy'' for molecules in an aggregate is even larger than for the previously studied dye and that the aggregate structure has a cross-sectional area equal to two or three molecular areas rather than one.^1Lazarev, P., N. Ovchinnikova, M. Paukshto, SID Int. Symp. Digest of Tech. Papers, San Jose, California, June XXXII, 571 (2001).^2V. R. Horowitz, L. A. Janowitz, A. L. Modic, P. A. Heiney, and P. J. Collings, Phys. Rev. E 72, 041710 (2005).

Cytotoxicity Testing of Temporary Luting Cements with Two- and Three-Dimensional Cultures of Bovine Dental Pulp-Derived Cells

PubMed Central

Ülker, Hayriye Esra; Ülker, Mustafa; Gümüş, Hasan Önder; Yalçın, Muhammet; Şengün, Abdulkadir

2013-01-01

This study evaluated the cytotoxicity of eugenol-containing and eugenol-free temporary luting cements. For cytotoxicity testing, bovine pulp-derived cells transfected with Simian virus 40 Large T antigen were exposed to extracts of eugenol-containing (Rely X Temp E) and eugenol-free (Provicol, PreVISION CEM, and Rely X Temp NE) temporary luting cements for 24 h. The cytotoxicity of the same materials was also evaluated in a dentin barrier test device using three-dimensional cell cultures of bovine pulp-derived cells. The results of the cytotoxicity studies with two-dimensional cultures of bovine dental pulp-derived cells revealed that cell survival with the extracts of Rely X Temp E, Provicol, PreVISION CEM, and Rely X Temp NE was 89.1%, 84.9%, 92.3%, and 66.8%, respectively. Rely X Temp NE and Provicol showed cytotoxic effects on bovine dental pulp-derived cells (P < 0.05). The results of the dentin barrier test revealed that cell survival with the above-mentioned temporary cement was 101.5%, 91.9%, 93.5%, and 90.6%, respectively. None of the temporary luting cements significantly reduced cell survival compared with the negative control in the dentin barrier test (P > 0.05). Biologically active materials released from temporary luting cements may not influence the dentine-pulp complex if the residual dentine layer is at least 0.5 mm thick. PMID:23984419

Effects of Phytoplankton Growth Phase on Delayed Settling Behavior of Marine Snow Aggregates at Sharp Density Transitions

NASA Astrophysics Data System (ADS)

Proctor, K. W.; Montgomery, Q. W.; Prairie, J. C.

2016-02-01

Marine snow aggregates play a fundamental role in the marine carbon cycle. Since marine snow aggregates are larger and thus sink faster than individual phytoplankton, aggregates often dominate carbon flux. Previous studies have shown that marine snow aggregates will significantly decrease their settling velocity when passing through sharp density transitions within the ocean, a phenomenon defined as delayed settling. Given the importance of aggregate settling to carbon export, these small-scale changes in aggregate settling dynamics may have significant impacts on the efficiency of the biological pump. However, there is still a lack of knowledge about how different physical properties of aggregates can affect this delayed settling. In this study, we investigated the effect of phytoplankton growth phase on delayed settling behavior. Using phytoplankton cultures stopped at four different growth phases, we formed marine snow aggregates in the laboratory in rotating cylindrical tanks. We then observed individual aggregates as they settled through a stratified tank. We will present data which illustrates that aggregates experience greatly reduced settling rates when passing through sharp density gradients and that the growth phase of the phytoplankton used to form these aggregates has a significant effect on this delayed settling behavior. A thorough understanding of the impact of phytoplankton growth phase on the delayed settling behavior of marine snow will offer insight into the way phytoplankton growth phase may influence the efficiency of the biological pump, carbon flux, and the carbon cycle as a whole.

3D printing human induced pluripotent stem cells with novel hydroxypropyl chitin bioink: scalable expansion and uniform aggregation.

PubMed

Li, Yang; Jiang, Xulin; Li, Ling; Chen, Zhi-Nan; Gao, Ge; Yao, Rui; Sun, Wei

2018-06-28

Human induced pluripotent stem cells (hiPSCs) are more likely to successfully avoid the immunological rejection and ethical problems that are often encountered by human embryonic stem cells in various stem cell studies and applications. To transfer hiPSCs from the laboratory to clinical applications, researchers must obtain sufficient cell numbers. In this study, 3D cell printing was used as a novel method for iPSC scalable expansion. Hydroxypropyl chitin (HPCH), utilized as a new type of bioink, and a set of optimized printing parameters were shown to achieve high cell survival (> 90%) after the printing process and high proliferation efficiency (~ 32.3 folds) during subsequent 10-day culture. After the culture, high levels of pluripotency maintenance were recognized by both qualitative and quantitative detections. Compared with static suspension (SS) culture, hiPSC aggregates formed in 3D printed constructs showed a higher uniformity in size. Using novel dual-fluorescent labelling method, hiPSC aggregates in the constructs were found more inclined to form by <i>in situ</i> proliferation rather than multicellular aggregation. This study revealed unique advantages of non-ionic crosslinking bioink material HPCH, including high gel strength and rapid temperature response in hiPSC printing, and achieved primed state hiPSC printing for the first time. Features achieved in this study, such as high cell yield, high pluripotency maintenance and uniform aggregation provide good foundations for further hiPSC studies on 3D micro-tissue differentiation and drug screening. © 2018 IOP Publishing Ltd.

Proteins aggregation and human diseases

NASA Astrophysics Data System (ADS)

Hu, Chin-Kun

2015-04-01

Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease.

Polythiophenes Inhibit Prion Propagation by Stabilizing Prion Protein (PrP) Aggregates*

PubMed Central

Margalith, Ilan; Suter, Carlo; Ballmer, Boris; Schwarz, Petra; Tiberi, Cinzia; Sonati, Tiziana; Falsig, Jeppe; Nyström, Sofie; Hammarström, Per; Åslund, Andreas; Nilsson, K. Peter R.; Yam, Alice; Whitters, Eric; Hornemann, Simone; Aguzzi, Adriano

2012-01-01

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrPC (PrPSc) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrPSc to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23–231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23–231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrPSc by stabilizing the conformation of PrPC or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrPSc deposits. PMID:22493452

Oleaginous yeast Yarrowia lipolytica culture with synthetic and food waste-derived volatile fatty acids for lipid production.

PubMed

Gao, Ruiling; Li, Zifu; Zhou, Xiaoqin; Cheng, Shikun; Zheng, Lei

2017-01-01

The sustainability of microbial lipids production from traditional carbon sources, such as glucose or glycerol, is problematic given the high price of raw materials. Considerable efforts have been directed to minimize the cost and find new alternative carbon sources. Volatile fatty acids (VFAs) are especially attractive raw materials, because they can be produced from a variety of organic wastes fermentation. Therefore, the use of volatile fatty acids as carbon sources seems to be a feasible strategy for cost-effective microbial lipid production. Lipid accumulation in Y. lipolytica using synthetic and food waste-derived VFAs as substrates was systematically compared and evaluated in batch cultures. The highest lipid content obtained with acetic, butyric, and propionic acids reached 31.62 ± 0.91, 28.36 ± 0.74, and 28.91 ± 0.66%, respectively. High concentrations of VFA inhibited cell growth in the following order: butyric acid > propionic acid > acetic acid. Within a 30-day experimental period, Y. lipolytica could adapt up to 20 g/L acetic acid, whereas the corresponding concentration of propionic acid and butyric acid were 10 and 5 g/L, respectively. Cultures on a VFA mixture showed that the utilization of different types of VFA by Y. lipolytica was not synchronized but rather performed in a step-wise manner. Although yeast fermentation is an exothermic process, and the addition of VFA will directly affect the pH of the system by increasing environmental acidity, cultures at a cultivation temperature of 38 °C and uncontrolled pH demonstrated that Y. lipolytica had high tolerance in the high temperature and acidic environment when a low concentration (2.5 g/L) of either synthetic or food waste-derived VFA was used. However, batch cultures fed with food fermentate yielded lower lipid content (18.23 ± 1.12%) and lipid productivity (0.12 ± 0.02 g/L/day). The lipid composition obtained with synthetic and food waste-derived VFA was similar to

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

PubMed Central

Pijuan-Galitó, Sara; Tamm, Christoffer; Schuster, Jens; Sobol, Maria; Forsberg, Lars; Merry, Catherine L. R.; Annerén, Cecilia

2016-01-01

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. PMID:27405751

Derivation and characterization of cell cultures from the skin of the Indo-Pacific humpback dolphin Sousa chinensis.

PubMed

Jin, Wei; Jia, Kuntong; Yang, Lili; Chen, Jialin; Wu, Yuping; Yi, Meisheng

2013-06-01

The marine mammalian Indo-Pacific humpback dolphin, once widely lived in waters of the Indian to western Pacific oceans, has become an endangered species. The individual number of this dolphin has significantly declined in recent decades, which raises the concern of extinction. Direct concentration on laboratorial conservation of the genetic and cell resources should be paid to this marine species. Here, we report the successful derivation of cell lines form the skin of Indo-Pacific humpback dolphin. The cell cultures displayed the characteristics of fibroblast in morphology and grew rapidly at early passages, but showed obvious growth arrest at higher passages. The karyotype of the cells consisted of 42 autosomes and sex chromosomes X and Y. The immortalized cell lines obtained by forced expression of the SV40 large T-antigen were capable of proliferation at high rate in long-term culture. Immortalization and long-term culture did not cause cytogenetically observable abnormality in the karyotype. The cell type of the primary cultures and immortalized cell lines were further characterized as fibroblasts by the specific expression of vimentin. Gene transfer experiments showed that exogenetic genes could be efficiently delivered into the cells by both plasmid transfection and lentivirus infection. The cells derived from the skin of the Indo-Pacific humpback dolphin may serve as a useful in vitro system for studies on the effects of environmental pollutants and pathogens in habitats on the dolphin animals. More importantly, because of their high proliferation rate and susceptibility to lentivirus, these cells are potential ideal materials for generation of induced pluripotent stem cells.

Skeletal muscle satellite cells cultured in simulated microgravity

NASA Technical Reports Server (NTRS)

Molnar, Greg; Hartzell, Charles R.; Schroedl, Nancy A.; Gonda, Steve R.

1993-01-01

Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of

Effects of maximum aggregate size on UPV of brick aggregate concrete.

PubMed

Mohammed, Tarek Uddin; Mahmood, Aziz Hasan

2016-07-01

Investigation was carried out to study the effects of maximum aggregate size (MAS) (12.5mm, 19.0mm, 25.0mm, 37.5mm, and 50.0mm) on ultrasonic pulse velocity (UPV) of concrete. For investigation, first class bricks were collected and broken to make coarse aggregate. The aggregates were tested for specific gravity, absorption capacity, unit weight, and abrasion resistance. Cylindrical concrete specimens were made with different sand to aggregate volume ratio (s/a) (0.40 and 0.45), W/C ratio (0.45, 0.50, and 0.55), and cement content (375kg/m(3) and 400kg/m(3)). The specimens were tested for compressive strength and Young's modulus. UPV through wet specimen was measured using Portable Ultrasonic Non-destructive Digital Indicating Tester (PUNDIT). Results indicate that the pulse velocity through concrete increases with an increase in MAS. Relationships between UPV and compressive strength; and UPV and Young's modulus of concrete are proposed for different maximum sizes of brick aggregate. Copyright © 2016 Elsevier B.V. All rights reserved.

Memantine inhibits β-amyloid aggregation and disassembles preformed β-amyloid aggregates.

PubMed

Takahashi-Ito, Kaori; Makino, Mitsuhiro; Okado, Keiko; Tomita, Taisuke

2017-11-04

Memantine, an uncompetitive glutamatergic N-methyl-d-aspartate (NMDA) receptor antagonist, is widely used as a medication for the treatment of Alzheimer's disease (AD). We previously reported that chronic treatment of AD with memantine reduces the amount of insoluble β-amyloid (Aβ) and soluble Aβ oligomers in animal models of AD. The mechanisms by which memantine reduces Aβ levels in the brain were evaluated by determining the effect of memantine on Aβ aggregation using thioflavin T and transmission electron microscopy. Memantine inhibited the formation of Aβ(1-42) aggregates in a concentration-dependent manner, whereas amantadine, a structurally similar compound, did not affect Aβ aggregation at the same concentrations. Furthermore, memantine inhibited the formation of different types of Aβ aggregates, including Aβs carrying familial AD mutations, and disaggregated preformed Aβ(1-42) fibrils. These results suggest that the inhibition of Aβ aggregation and induction of Aβ disaggregation may be involved in the mechanisms by which memantine reduces Aβ deposition in the brain. Copyright © 2017 Elsevier Inc. All rights reserved.

Spaceflight Enhances Cell Aggregation and Random Budding in Candida albicans

PubMed Central

Woolley, Christine M.; Barrila, Jennifer; Buchanan, Kent; McCracken, James; Inglis, Diane O.; Searles, Stephen C.; Nelman-Gonzalez, Mayra A.; Ott, C. Mark; Wilson, James W.; Pierson, Duane L.; Stefanyshyn-Piper, Heidemarie M.; Hyman, Linda E.; Nickerson, Cheryl A.

2013-01-01

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans–induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid

Diversity of Marine-Derived Fungal Cultures Exposed by DNA Barcodes: The Algorithm Matters

PubMed Central

Andreakis, Nikos; Høj, Lone; Kearns, Philip; Hall, Michael R.; Ericson, Gavin; Cobb, Rose E.; Gordon, Benjamin R.; Evans-Illidge, Elizabeth

2015-01-01

Marine fungi are an understudied group of eukaryotic microorganisms characterized by unresolved genealogies and unstable classification. Whereas DNA barcoding via the nuclear ribosomal internal transcribed spacer (ITS) provides a robust and rapid tool for fungal species delineation, accurate classification of fungi is often arduous given the large number of partial or unknown barcodes and misidentified isolates deposited in public databases. This situation is perpetuated by a paucity of cultivable fungal strains available for phylogenetic research linked to these data sets. We analyze ITS barcodes produced from a subsample (290) of 1781 cultured isolates of marine-derived fungi in the Bioresources Library located at the Australian Institute of Marine Science (AIMS). Our analysis revealed high levels of under-explored fungal diversity. The majority of isolates were ascomycetes including representatives of the subclasses Eurotiomycetidae, Hypocreomycetidae, Sordariomycetidae, Pleosporomycetidae, Dothideomycetidae, Xylariomycetidae and Saccharomycetidae. The phylum Basidiomycota was represented by isolates affiliated with the genera Tritirachium and Tilletiopsis. BLAST searches revealed 26 unknown OTUs and 50 isolates corresponding to previously uncultured, unidentified fungal clones. This study makes a significant addition to the availability of barcoded, culturable marine-derived fungi for detailed future genomic and physiological studies. We also demonstrate the influence of commonly used alignment algorithms and genetic distance measures on the accuracy and comparability of estimating Operational Taxonomic Units (OTUs) by the automatic barcode gap finder (ABGD) method. Large scale biodiversity screening programs that combine datasets using algorithmic OTU delineation pipelines need to ensure compatible algorithms have been used because the algorithm matters. PMID:26308620

Dielectric aggregation kinetics of cells in a uniform AC electric field.

PubMed

Tada, Shigeru; Natsuya, Tomoyuki; Tsukamoto, Akira

2014-01-01

Cell manipulation and separation technologies have potential biological and medical applications, including advanced clinical protocols such as tissue engineering. An aggregation model was developed for a human carcinoma (HeLa) cell suspension exposed to a uniform AC electric field, in order to explore the field-induced structure formation and kinetics of cell aggregates. The momentum equations of cells under the action of the dipole-dipole interaction were solved theoretically and the total time required to form linear string-like cluster was derived. The results were compared with those of a numerical simulation. Experiments using HeLa cells were also performed for comparison. The total time required to form linear string-like clusters was derived from a simple theoretical model of the cell cluster kinetics. The growth rates of the average string length of cell aggregates showed good agreement with those of the numerical simulation. In the experiment, cells were found to form massive clusters on the bottom of a chamber. The results imply that the string-like cluster grows rapidly by longitudinal attraction when the electric field is first applied and that this process slows at later times and is replaced by lateral coagulation of short strings. The findings presented here are expected to enable design of methods for the organization of three-dimensional (3D) cellular structures without the use of micro-fabricated substrates, such as 3D biopolymer scaffolds, to manipulate cells into spatial arrangement.

Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors.

PubMed

Surrao, Denver C; Boon, Kathryn; Borys, Breanna; Sinha, Sarthak; Kumar, Ranjan; Biernaskie, Jeff; Kallos, Michael S

2016-12-01

Human skin-derived precursor cells (hSKPs) are multipotent adult stem cells found in the dermis of human skin. Incorporation of hSKPs into split-thickness skin grafts (STSGs), the current gold standard to treat severe burns or tissue resections, has been proposed as a treatment option to enhance skin wound healing and tissue function. For this approach to be clinically viable substantial quantities of hSKPs are required, which is the rate-limiting step, as only a few thousand hSKPs can be isolated from an autologous skin biopsy without causing donor site morbidity. In order to produce sufficient quantities of clinically viable cells, we have developed a bioprocess capable of expanding hSKPs as aggregates in stirred suspension bioreactors (SSBs). In this study, we found hSKPs from adult donors to expand significantly more (P < 0.05) at 60 rpm in SSBs than in static cultures. Furthermore, the utility of the SSBs, at 60 rpm is demonstrated by serial passaging of hSKPs from a small starting population, which can be isolated from an autologous skin biopsy without causing donor site morbidity. At 60 rpm, aggregates were markedly smaller and did not experience oxygen diffusional limitations, as seen in hSKPs cultured at 40 rpm. While hSKPs also grew at 80 rpm (0.74 Pa) and 100 rpm (1 Pa), they produced smaller aggregates due to high shear stress. The pH of the media in all the SSBs was closer to biological conditions and significantly different (P < 0.05) from static cultures, which recorded acidic pH conditions. The nutrient concentrations of the media in all the SSBs and static cultures did not drop below acceptable limits. Furthermore, there was no significant build-up of waste products to limit hSKP expansion in the SSBs. In addition, hSKP markers were maintained in the 60 rpm SSB as demonstrated by immunocytochemistry. This method of growing hSKPs in a batch culture at 60 rpm in a SSB represents an important first step in developing an

Oil-Price Shocks: Beyond Standard Aggregate Demand/Aggregate Supply Analysis.

ERIC Educational Resources Information Center

Elwood, S. Kirk

2001-01-01

Explores the problems of portraying oil-price shocks using the aggregate demand/aggregate supply model. Presents a simple modification of the model that differentiates between production and absorption of goods, which enables it to better reflect the effects of oil-price shocks on open economies. (RLH)

Metal concentrations in aggregate interiors, exteriors, whole aggregates, and bulk of Costa Rican soils

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

Wilcke, W.; Kretzschmar, S.; Bundt, M.

1999-10-01

In many temperate soils the preferential weathering and leaching of aggregate surfaces and the nonaggregated material between aggregates depletes geogenic metals. It also shifts metals from strongly to more weakly bound metal forms. Deposited metals are sorbed preferentially on aggregate surfaces and between aggregates. The authors examined whether preferential desilication under tropical climate causes an enrichment in the aggregate exteriors in oxidic forms of metals. They also studied where deposited metals are bound in these soils. Aggregates (2--20 mm) were selected manually from the A horizons of eight Oxisols, six Andisols, two Mollisols, and two Inceptisols in Costa Rica. Allmore » samples were fractionated into interior and exterior portions and treated with a seven-step sequence to extract Al, Cd, Cu, Fe, Mn, Pb, and Zn. Total concentrations of all metals except Zn were higher in the aggregate exteriors than in the interiors. The average Cd and Pb concentrations in easily extractable fractions were significantly higher in the aggregate exteriors. There were no significant differences in metal partitioning between interiors and exteriors except for Pb, which had higher proportions in extractable forms with NH{sub 2}OH {center{underscore}dot} HCl {gt} NH{sub 4} - acetate, pH 6.0 {gt} EDTA in the exteriors. There were few significant differences in metal concentrations and partitioning between bulk soil and whole aggregates. The results may be explained by (i) preferential desilication of the aggregate exteriors and (ii) preferential sorption of deposited heavy metals mainly in easily extractable forms.« less

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity

PubMed Central

Villar-Piqué, Anna; Lopes da Fonseca, Tomás; Sant’Anna, Ricardo; Szegö, Éva Mónika; Fonseca-Ornelas, Luis; Pinho, Raquel; Carija, Anita; Gerhardt, Ellen; Masaracchia, Caterina; Abad Gonzalez, Enrique; Rossetti, Giulia; Carloni, Paolo; Fernández, Claudio O.; Foguel, Debora; Milosevic, Ira; Zweckstetter, Markus; Ventura, Salvador; Outeiro, Tiago Fleming

2016-01-01

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity. PMID:27708160

Low-molecular weight heparin protamine complex augmented the potential of adipose-derived stromal cells to ameliorate limb ischemia.

PubMed

Kishimoto, Satoko; Inoue, Ken-Ichi; Nakamura, Shingo; Hattori, Hidemi; Ishihara, Masayuki; Sakuma, Masashi; Toyoda, Shigeru; Iwaguro, Hideki; Taguchi, Isao; Inoue, Teruo; Yoshida, Ken-Ichiro

2016-06-01

Heparin/protamine micro/nanoparticles (LH/P-MPs) were recently developed as low-molecular weight, biodegradable carriers for adipose-derived stromal cells (ADSCs). These particles can be used for a locally delivered stem cell therapy that promotes angiogenesis. LH/P-MPs bind to the cell surface of ADSCs and promote cell-to-cell interaction and aggregation of ADSCs. Cultured ADSC/LH/P-MP aggregates remain viable. Here, we examined the ability of these aggregates to rescue limb loss in a mouse model of hindlimb ischemia. Unilateral hindlimb ischemia was induced in adult male BALB/c mice by ligation of the iliac artery and hindlimb vein. For allotransplantation of ADSCs from the same inbred strain, we injected ADSC alone or ADSC/LH/P-MP aggregates or control medium (sham-treated) directly into the ischemic muscles. Ischemic limb blood perfusion, vessel density, and vessel area were recorded. The extent of ischemic limb necrosis or limb loss was assessed on postoperative days 2, 7, and 14. Compared with the sham-treatment control, treatment with ADSCs alone showed modest effects on blood perfusion recovery and increased the number of α-SMA-positive vessels. Response to ADSC/LH/P-MP aggregates was significantly greater than ADSCs alone for every endpoint. ADSC/LH/P-MP aggregates more effectively prevented the loss of ischemic hindlimbs than ADSCs alone or the sham-treatment. The LH/P-MPs augmented the effects of ADSCs on angiogenesis and reversal of limb ischemia. Use of ADSC/LH/P-MP aggregates offers a novel and convenient treatment method and potentially represents a promising new therapeutic approach to inducing angiogenesis in ischemic diseases. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Comparison of two different culture conditions for derivation of early hiPSC.

PubMed

Hey, Caroline A B; Saltõkova, Katarina B; Bisgaard, Hanne C; Møller, Lisbeth B

2018-03-30

Different culture-systems for derivation of induced pluripotent stem cells (iPSC) in vitro from human fibroblasts have been established. Here, we compared the efficacy of two different feeder-free culture-systems; Matrigel-coated surfaces in combination with mTeSR1 medium versus Vitronectin-coated surfaces in combination with Essential 8 (E8) medium. The comparison was performed by counting the number of emerging iPSC-looking colonies of re-programmed fibroblasts. The fibroblasts were re-programmed using episomal plasmids expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28, and a p53 knock down shP53. Three different fibroblast lines, K40 and K48 from healthy controls and BBS1 from a patient with Bardet-Biedl syndrome, were used in two independent setups. The BBS1 line was used in both setups in combination with K40 and K48 respectively. In all four re-programming experiments, we observed a significantly higher number of emerging colonies with the combination Matrigel/mTeSR1 as compared to the combination Vitronectin/E8. The presence of iPSC was verified by alkaline phosphatase and Tra-1-60 staining. Furthermore, a higher expression of the pluripotency-associated markers NANOG and SOX2 in cells under Matrigel/mTeSR1 conditions compared with Vitronectin/E8 supported the higher proportion of iPSC on Matrigel/mTeSR1 plates. In conclusion, the combination Matrigel/mTeSR1 is more efficient for derivation of iPSC compared to the Vitronectin/E8 combination. © 2018 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

Role of CaCO3 and Charcoal Application on Organic Matter Retention in Silt-sized Aggregates

NASA Astrophysics Data System (ADS)

Berhe, A. A.; Kaiser, M.; Ghezzehei, T.; Myrold, D.; Kleber, M.

2011-12-01

' combination to have the largest effect on silt-size scale aggregation processes because the amount of microbial derived cementing agents, charcoal derived functional groups containing OM, and Ca2+ ions are enhanced at the same time.

Optimized suspension culture: the rotating-wall vessel

NASA Technical Reports Server (NTRS)

Hammond, T. G.; Hammond, J. M.

2001-01-01

Suspension culture remains a popular modality, which manipulates mechanical culture conditions to maintain the specialized features of cultured cells. The rotating-wall vessel is a suspension culture vessel optimized to produce laminar flow and minimize the mechanical stresses on cell aggregates in culture. This review summarizes the engineering principles, which allow optimal suspension culture conditions to be established, and the boundary conditions, which limit this process. We suggest that to minimize mechanical damage and optimize differentiation of cultured cells, suspension culture should be performed in a solid-body rotation Couette-flow, zero-headspace culture vessel such as the rotating-wall vessel. This provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis, characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. Optimization of suspension culture is achieved by applying three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Next, rotation in the rotating-wall vessel induces both Coriolis and centrifugal forces, directly dependent on terminal velocity and minimized as terminal velocity is minimized. Last, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. In the transduction of mechanical culture conditions into cellular effects, several lines of evidence support a role for multiple molecular mechanisms. These include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as protein kinase C. The discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and

Statistical Inference for Cultural Consensus Theory

DTIC Science & Technology

2014-02-24

Social Network Conference XXXII , Redondo Beach, California, March 2012. Agrawal, K. (Presenter), and Batchelder, W. H. Cultural Consensus Theory...Aggregating Complete Signed Graphs Under a Balance Constraint -- Part 2. International Sunbelt Social Network Conference XXXII , Redondo Beach

The Validity and Structure of Culture-Level Personality Scores: Data From Ratings of Young Adolescents

PubMed Central

McCrae, Robert R.; Terracciano, Antonio; De Fruyt, Filip; De Bolle, Marleen; Gelfand, Michele J.; Costa, Paul T.; Aguilar-Vafaie, Maria E.; Ahn, Chang-kyu; Ahn, Hyun-nie; Alcalay, Lidia; Allik, Jüri; Avdeyeva, Tatyana V.; Blatný, Marek; Bratko, Denis; Brunner-Sciarra, Marina; Cain, Thomas R.; Chittcharat, Niyada; Crawford, Jarret T.; de Lima, Margarida P.; Fehr, Ryan; Ficková, Emília; Gülgöz, Sami; Hřebíčková, Martina; Jussim, Lee; Klinkosz, Waldemar; Knežević, Goran; de Figueroa, Nora Leibovich; Löckenhoff, Corinna E.; Martin, Thomas A.; Marušić, Iris; Mastor, Khairul Anwar; Nakazato, Katsuharu; Nansubuga, Florence; Porrata, Jose; Purić, Danka; Realo, Anu; Reátegui, Norma; Rolland, Jean-Pierre; Schmidt, Vanina; Sekowski, Andrzej; Shakespeare-Finch, Jane; Shimonaka, Yoshiko; Simonetti, Franco; Siuta, Jerzy; Szmigielska, Barbara; Vanno, Vitanya; Wang, Lei; Yik, Michelle

2010-01-01

We examined properties of culture-level personality traits in ratings of targets (N = 5,109) aged 12 to 17 in 24 cultures. Aggregate scores were generalizable across gender, age, and relationship groups and showed convergence with culture-level scores from previous studies of self-reports and observer ratings of adults, but they were unrelated to national character stereotypes. Trait profiles also showed cross-study agreement within most cultures, eight of which had not previously been studied. Multidimensional scaling showed that Western and non-Western cultures clustered along a dimension related to Extraversion. A culture-level factor analysis replicated earlier findings of a broad Extraversion factor, but generally resembled the factor structure found in individuals. Continued analysis of aggregate personality scores is warranted. PMID:20573127

The validity and structure of culture-level personality scores: data from ratings of young adolescents.

PubMed

McCrae, Robert R; Terracciano, Antonio; De Fruyt, Filip; De Bolle, Marleen; Gelfand, Michele J; Costa, Paul T

2010-06-01

We examined properties of culture-level personality traits in ratings of targets (N=5,109) ages 12 to 17 in 24 cultures. Aggregate scores were generalizable across gender, age, and relationship groups and showed convergence with culture-level scores from previous studies of self-reports and observer ratings of adults, but they were unrelated to national character stereotypes. Trait profiles also showed cross-study agreement within most cultures, 8 of which had not previously been studied. Multidimensional scaling showed that Western and non-Western cultures clustered along a dimension related to Extraversion. A culture-level factor analysis replicated earlier findings of a broad Extraversion factor but generally resembled the factor structure found in individuals. Continued analysis of aggregate personality scores is warranted.

Projected entrainment of fish resulting from aggregate dredging.

PubMed

Drabble, Ray

2012-02-01

Previous research to assess impacts from aggregate dredging has focussed on infaunal species with few studies made of fish entrainment. Entrainment evidence from hydraulic dredging studies is reviewed to develop a sensitivity index for benthic fish. Environmental monitoring attendant with the granting of new licences in the Eastern Channel Region (ECR) in 2006 offers a unique opportunity to assess the effects of dredging upon fish. Projected theoretical fish entrainment rates are calculated based upon: abundance data from 4m beam trawl sampling of fish species over the period 2005-2008; sensitivity data; and dredging activity and footprint derived from Electronic monitoring System (EMS) data. Results have been compared with actual entrainment rates and also against summary results from independent analysis of the changes in fish population over the period 2005-2008 (Drabble, 2012). The case is made for entrainment surveys to form part of impact monitoring for marine aggregate dredging. Copyright © 2011 Elsevier Ltd. All rights reserved.

Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture

PubMed Central

Phetfong, J.; Tawonsawatruk, T.; Seenprachawong, K.; Srisarin, A.; Isarankura-Na-Ayudhya, C.

2017-01-01

Objectives Adipose-derived mesenchymal stem cells (ADMSCs) are a promising strategy for orthopaedic applications, particularly in bone repair. Ex vivo expansion of ADMSCs is required to obtain sufficient cell numbers. Xenogenic supplements should be avoided in order to minimise the risk of infections and immunological reactions. Human platelet lysate and human plasma may be an excellent material source for ADMSC expansion. In the present study, use of blood products after their recommended transfusion date to prepare human platelet lysate (HPL) and human plasma (Hplasma) was evaluated for in vitro culture expansion and osteogenesis of ADMSCs. Methods Human ADMSCs were cultured in medium supplemented with HPL, Hplasma and a combination of HPL and Hplasma (HPL+Hplasma). Characteristics of these ADMSCs, including osteogenesis, were evaluated in comparison with those cultured in fetal bovine serum (FBS). Results HPL and HPL+Hplasma had a significantly greater growth-promoting effect than FBS, while Hplasma exhibited a similar growth-promoting effect to that of FBS. ADMSCs cultured in HPL and/or Hplasma generated more colony-forming unit fibroblasts (CFU-F) than those cultured in FBS. After long-term culture, ADMSCs cultured in HPL and/or Hplasma showed reduced cellular senescence, retained typical cell phenotypes, and retained differentiation capacities into osteogenic and adipogenic lineages. Conclusion HPL and Hplasma prepared from blood products after their recommended transfusion date can be used as an alternative and effective source for large-scale ex vivo expansion of ADMSCs. Cite this article: J. Phetfong, T. Tawonsawatruk, K. Seenprachawong, A. Srisarin, C. Isarankura-Na-Ayudhya, A. Supokawej. Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture. Bone Joint Res 2017;6:414–422. DOI: 10.1302/2046-3758.67.BJR-2016-0342.R1. PMID:28720606

Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants.

PubMed

Chen, M H; Wang, P J; Maeda, E

1987-10-01

The regeneration potential of shoot tip, stem, leaf, cotyledon and root explants of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studed. Callus induction of these two cultivars of papaya showed that the shoot tips and stems are most suitable for forming callus, while leaves, cotyledons and roots are comparatively difficult to induce callus. Callus induction also varied with the varities. Somatic embryogenesis was obtained from 3-month-old root cultures. A medium containing half strength of MS inorganic salts, 160 mg/l adenine sulfate, 1.0 mg/1 NAA, 0.5 mg/1 kinetin and 1.0 mg/1 GA3 was optimal for embryogenesis. The callus maintained high regenerative capacity after two years of culture on this medium. Plants derived from somatic embryos were obtained under green-house conditions.

The aggregation paths and products of Aβ42 dimers are distinct from Aβ42 monomer

PubMed Central

O'Malley, Tiernan T.; Witbold, William M.; Linse, Sara; Walsh, Dominic M.

2017-01-01

Extracts of Alzheimer's disease (AD) brain that contain what appear to be SDS-stable amyloid β-protein (Aβ) dimers potently block LTP and impair memory consolidation. Brain-derived dimers can be physically separated from Aβ monomer, consist primarily of Aβ42 and resist denaturation by powerful chaotropic agents. In nature, covalently cross-linked Aβ dimers could be generated in only one of two different ways - either by the formation of a dityrosine (DiY) or an isopeptide ε-(γ-glutamyl)-lysine (Q-K) bond. We enzymatically cross-linked recombinant Aβ42 monomer to produce DiY and Q-K dimers and then applied a range of biophysical methods to study their aggregation. Both Q-K and DiY dimers aggregate to form soluble assemblies distinct from the fibrillar aggregates formed by Aβ monomer. These results suggest that Aβ dimers allow the formation of soluble aggregates akin to those in aqueous extracts of AD brain. Thus it seems that Aβ dimers may play an important role in determining the formation of soluble rather than insoluble aggregates. PMID:27750419

The influence of auxins on the biosynthesis of isoprene derivatives in callus cultures of Vaccinium corymbosum var. bluecrop.

PubMed

Migas, Piotr; Luczkiewicz, Maria; Cisowski, Wojciech

2006-01-01

Callus cultures of Vaccinium corymbosum var. bluecrop were optimized for their isoprene derivatives production by supplementing Schenk-Hildebrandt (SH) medium with constant concentration of kinetin (2.32 microM) and two different amounts of selected auxins. Every auxin, except for IBA, used in 10-time higher concentration (2,4D, NAA, IAA, NOA) stimulated biosynthesis of beta-sitosterol and inhibited triterpene synthesis. Quantitative analysis of isoprene derivatives in callus biomass collected on the 25th day of the experiment proved that the analyzed callus of Vaccinium corymbosum var. bluecrop synthesized the highest amount of isoprene derivatives after subculturing on SH medium modified with 22.6 microM of 2,4D and 2.32 microM of kinetin.

Protein aggregate turbidity: Simulation of turbidity profiles for mixed-aggregation reactions.

PubMed

Hall, Damien; Zhao, Ran; Dehlsen, Ian; Bloomfield, Nathaniel; Williams, Steven R; Arisaka, Fumio; Goto, Yuji; Carver, John A

2016-04-01

Due to their colloidal nature, all protein aggregates scatter light in the visible wavelength region when formed in aqueous solution. This phenomenon makes solution turbidity, a quantity proportional to the relative loss in forward intensity of scattered light, a convenient method for monitoring protein aggregation in biochemical assays. Although turbidity is often taken to be a linear descriptor of the progress of aggregation reactions, this assumption is usually made without performing the necessary checks to provide it with a firm underlying basis. In this article, we outline utilitarian methods for simulating the turbidity generated by homogeneous and mixed-protein aggregation reactions containing fibrous, amorphous, and crystalline structures. The approach is based on a combination of Rayleigh-Gans-Debye theory and approximate forms of the Mie scattering equations. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

Porphyrin Cyclodextrin Conjugates Modulate Amyloid Beta Peptide Aggregation and Cytotoxicity.

PubMed

Oliveri, Valentina; Zimbone, Stefania; Giuffrida, Maria Laura; Bellia, Francesco; Tomasello, Marianna Flora; Vecchio, Graziella

2018-04-25

Although fibrillar amyloid beta peptide (Aβ) aggregates are one of the major hallmarks of Alzheimer's disease, increasing evidence suggests that soluble Aβ oligomers are the primary toxic species. Targeting the oligomeric species could represent an effective strategy to interfere with Aβ toxicity. In this work, the biological properties of 5[4-(6-O-β-cyclodextrin)-phenyl],10,15,20-tri(4-hydroxyphenyl)-porphyrin and its zinc complex were tested, as new molecules that interact with Aβ and effectively prevent its cytotoxicity. We found that these systems can cross the cell membrane to deliver Aβ intracellularly and promote its clearance. Our results provide evidence for the use of cyclodextrin-porphyrin derivatives as a promising strategy to target amyloid aggregation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of wastes derived from earthquakes for the production of concrete masonry partition wall blocks

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

Xiao Zhao; Faculty of Architecture, Civil Engineering and Environment Engineering and Mechanics, Sichuan University; Ling, Tung-Chai

2011-08-15

Highlights: > Solved the scientific and technological challenges impeding use of waste rubble derived from earthquake, by providing an alternative solution of recycling the waste in moulded concrete block products. > Significant requirements for optimum integration on the utilization of the waste aggregates in the production of concrete blocks are investigated. > A thorough understanding of the mechanical properties of concrete blocks made with waste derived from earthquake is reported. - Abstract: Utilization of construction and demolition (C and D) wastes as recycled aggregates in the production of concrete and concrete products have attracted much attention in recent years. However,more » the presence of large quantities of crushed clay brick in some the C and D waste streams (e.g. waste derived collapsed masonry buildings after an earthquake) renders the recycled aggregates unsuitable for high grade use. One possibility is to make use of the low grade recycled aggregates for concrete block production. In this paper, we report the results of a comprehensive study to assess the feasibility of using crushed clay brick as coarse and fine aggregates in concrete masonry block production. The effects of the content of crushed coarse and fine clay brick aggregates (CBA) on the mechanical properties of non-structural concrete block were quantified. From the experimental test results, it was observed that incorporating the crushed clay brick aggregates had a significant influence on the properties of blocks. The hardened density and drying shrinkage of the block specimens decreased with an increase in CBA content. The use of CBA increased the water absorption of block specimens. The results suggested that the amount of crushed clay brick to be used in concrete masonry blocks should be controlled at less than 25% (coarse aggregate) and within 50-75% for fine aggregates.« less

Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived.

PubMed

Dame, Michael K; Jiang, Yan; Appelman, Henry D; Copley, Kelly D; McClintock, Shannon D; Aslam, Muhammad Nadeem; Attili, Durga; Elmunzer, B Joseph; Brenner, Dean E; Varani, James; Turgeon, D Kim

2014-02-01

In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca(2+) (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3-5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1-3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3-4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

PubMed

Rao, Fang; Yang, Ren-Qiang; Chen, Xiao-Shu; Xu, Jin-Song; Fu, Hui-Min; Su, Hai; Wang, Ling

2014-01-01

Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

PPARγ Ligands Decrease Hydrostatic Pressure-Induced Platelet Aggregation and Proinflammatory Activity

PubMed Central

Chen, Xiao-Shu; Xu, Jin-Song; Fu, Hui-Min; Su, Hai; Wang, Ling

2014-01-01

Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure. PMID:24586940

Propionibacterium-Produced Coproporphyrin III Induces Staphylococcus aureus Aggregation and Biofilm Formation

PubMed Central

Wollenberg, Michael S.; Claesen, Jan; Escapa, Isabel F.; Aldridge, Kelly L.; Fischbach, Michael A.

2014-01-01

ABSTRACT The majority of bacteria detected in the nostril microbiota of most healthy adults belong to three genera: Propionibacterium, Corynebacterium, and Staphylococcus. Among these staphylococci is the medically important bacterium Staphylococcus aureus. Almost nothing is known about interspecies interactions among bacteria in the nostrils. We observed that crude extracts of cell-free conditioned medium from Propionibacterium spp. induce S. aureus aggregation in culture. Bioassay-guided fractionation implicated coproporphyrin III (CIII), the most abundant extracellular porphyrin produced by human-associated Propionibacterium spp., as a cause of S. aureus aggregation. This aggregation response depended on the CIII dose and occurred during early stationary-phase growth, and a low pH (~4 to 6) was necessary but was not sufficient for its induction. Additionally, CIII induced plasma-independent S. aureus biofilm development on an abiotic surface in multiple S. aureus strains. In strain UAMS-1, CIII stimulation of biofilm depended on sarA, a key biofilm regulator. This study is one of the first demonstrations of a small-molecule-mediated interaction among medically relevant members of the nostril microbiota and the first description of a role for CIII in bacterial interspecies interactions. Our results indicate that CIII may be an important mediator of S. aureus aggregation and/or biofilm formation in the nostril or other sites inhabited by Propionibacterium spp. and S. aureus. PMID:25053784

Incorporation of Biomaterials in Multicellular Aggregates Modulates Pluripotent Stem Cell Differentiation

PubMed Central

Bratt-Leal, Andrés M.; Carpenedo, Richard L.; Ungrin, Mark; Zandstra, Peter W.; McDevitt, Todd C.

2010-01-01

Biomaterials are increasingly being used to engineer the biochemical and biophysical properties of the extracellular stem cell microenvironment in order to tailor niche characteristics and direct cell phenotype. To date, stem cell-biomaterial interactions have largely been studied by introducing stem cells into artificial environments, such as 2D cell culture on biomaterial surfaces, encapsulation of cell suspensions within hydrogel materials, or cell seeding on 3D polymeric scaffolds. In this study, microparticles fabricated from different materials, such as agarose, PLGA and gelatin, were stably integrated, in a dose-dependent manner, within aggregates of pluripotent stem cells (PSCs) prior to differentiation as a means to directly examine stem cell-biomaterial interactions in 3D. Interestingly, the presence of the materials within the stem cell aggregates differentially modulated the gene and protein expression patterns of several differentiation markers without adversely affecting cell viability. Microparticle incorporation within 3D stem cell aggregates can control the spatial presentation of extracellular environmental cues (i.e. soluble factors, extracellular matrix and intercellular adhesion molecules) as a means to direct the differentiation of stem cells for tissue engineering and regenerative medicine applications. In addition, these results suggest that the physical presence of microparticles within stem cell aggregates does not compromise PSC differentiation, but in fact the choice of biomaterials can impact the propensity of stem cells to adopt particular differentiated cell phenotypes. PMID:20864164

Aggregation and breakup of colloidal particle aggregates in shear flow, studied with video microscopy.

PubMed

Tolpekin, V A; Duits, M H G; van den Ende, D; Mellema, J

2004-03-30

We used video microscopy to study the behavior of aggregating suspensions in shear flow. Suspensions consisted of 920 nm diameter silica spheres, dispersed in a methanol/bromoform solvent, to which poly(ethylene glycol) (M = 35.000 g) was added to effect weak particle aggregation. With our solvent mixture, the refractive index of the particles could be closely matched, to allow microscopic observations up to 80 microm deep into the suspension. Also the mass density is nearly equal to that of the particles, thus allowing long observation times without problems due to aggregate sedimentation. Particles were visualized via fluorescent molecules incorporated into their cores. Using a fast confocal scanning laser microscope made it possible to characterize the (flowing) aggregates via their contour-area distributions as observed in the focal plane. The aggregation process was monitored from the initial state (just after adding the polymer), until a steady state was reached. The particle volume fraction was chosen at 0.001, to obtain a characteristic aggregation time of a few hundred seconds. On variation of polymer concentration, cP (2.2-12.0 g/L), and shear rate, gamma (3-6/s), it was observed that the volume-averaged size, Dv, in the steady state became larger with polymer concentration and smaller with shear rate. This demonstrates that the aggregate size is set by a competition between cohesive forces caused by the polymer and rupture forces caused by the flow. Also aggregate size distributions were be measured (semiquantitatively). Together with a description for the internal aggregate structure they allowed a modeling of the complete aggregation curve, from t = 0 up to the steady state. A satisfactory description could be obtained by describing the aggregates as fractal objects, with Df = 2.0, as measured from CSLM images after stopping the flow. In this modeling, the fitted characteristic breakup time was found to increase with cP.

Differentiation Potential of Human Chorion-Derived Mesenchymal Stem Cells into Motor Neuron-Like Cells in Two- and Three-Dimensional Culture Systems.

PubMed

Faghihi, Faezeh; Mirzaei, Esmaeil; Ai, Jafar; Lotfi, Abolfazl; Sayahpour, Forough Azam; Barough, Somayeh Ebrahimi; Joghataei, Mohammad Taghi

2016-04-01

Many people worldwide suffer from motor neuron-related disorders such as amyotrophic lateral sclerosis and spinal cord injuries. Recently, several attempts have been made to recruit stem cells to modulate disease progression in ALS and also regenerate spinal cord injuries. Chorion-derived mesenchymal stem cells (C-MSCs), used to be discarded as postpartum medically waste product, currently represent a class of cells with self renewal property and immunomodulatory capacity. These cells are able to differentiate into mesodermal and nonmesodermal lineages such as neural cells. On the other hand, gelatin, as a simply denatured collagen, is a suitable substrate for cell adhesion and differentiation. It has been shown that electrospinning of scaffolds into fibrous structure better resembles the physiological microenvironment in comparison with two-dimensional (2D) culture system. Since there is no report on potential of human chorion-derived MSCs to differentiate into motor neuron cells in two- and three-dimensional (3D) culture systems, we set out to determine the effect of retinoic acid (RA) and sonic hedgehog (Shh) on differentiation of human C-MSCs into motor neuron-like cells cultured on tissue culture plates (2D) and electrospun nanofibrous gelatin scaffold (3D).

Aggregate stability in mine residues after reclamation with biochar

NASA Astrophysics Data System (ADS)

Moreno-Barriga, Fabián; Díaz, Vicente; Acosta, José; Faz, Ángel; Zornoza, Raúl

2016-04-01

This study aims to assess how the addition of biochar and marble waste to acidic mine residues affected aggregate stability (AS) and contributed to the improvement of soil texture. For this purpose, a lab incubation was carried out for 90 days. Biochars derived from pig manure (PM), crop residues (CR) and municipal solid waste (MSW) were added to the soil at a rate of 20 g kg-1. The marble waste (MW) was added at a rate of 200 g kg-1, with the aim of increasing pH from 3 to 8 (pH of the native soils of the area). Biochars and MW were applied independently and combined. A control treatment was used without application of amendments. The evolution of AS was periodically monitored at 2, 4, 7, 15, 30 and 90 days by the method of artificial rainfall. Results showed, at the end of the incubation, that the addition of MW alone did not significantly increased AS with comparison to CT (30%). However, the biochar, alone or together with MW, significantly increased AS, the treatment receiving CR derived biochar being the one with the highest values (46%). Increments in AS were significant from the day 30 of incubation. AS showed a significant correlation with the total organic carbon content, but was not correlated with organic carbon fractions (soluble, labile, recalcitrant), inorganic carbon, microbial biomass carbon, enzyme activities, exchangeable fraction of heavy metals (As, Cd, Cu, Pb, Zn), pH, electrical conductivity nor greenhouse gas emissions (NO₂, CH₄). Thus, the application of biochar (alone or in combination with MW as a source of calcium carbonate) significantly increased the formation of stable aggregates in former acidic mine residues, favoring the development of soil structure, essential to create a soil from residues. It seems that the total content of organic carbon is directly controlling aggregation, rather than other labile organic sources. Moreover, pH, salinity or the presence of exchangeable metals did not seem to affect soil aggregation

Organic carbon, water repellency and soil stability to slaking at aggregate and intra-aggregate scales

NASA Astrophysics Data System (ADS)

Jordán López, Antonio; García-Moreno, Jorge; Gordillo-Rivero, Ángel J.; Zavala, Lorena M.; Cerdà, Artemi; Alanís, Nancy; Jiménez-Compán, Elizabeth

2015-04-01

Water repellency (WR) is a property of some soils that inhibits or delays water infiltration between a few seconds and days or weeks. Inhibited or delayed infiltration contributes to ponding and increases runoff flow generation, often increasing soil erosion risk. In water-repellent soils, water infiltrates preferentially through cracks or macropores, causing irregular soil wetting patterns, the development of preferential flow paths and accelerated leaching of nutrients. Although low inputs of hydrophobic organic substances and high mineralization rates lead to low degrees of WR in cropped soils, it has been reported that conservative agricultural practices may induce soil WR. Although there are many studies at catchment, slope or plot scales very few studies have been carried out at particle or aggregate scale. Intra-aggregate heterogeneity of physical, biological and chemical properties conditions the transport of substances, microbial activity and biochemical processes, including changes in the amount, distribution and chemical properties of organic matter. Some authors have reported positive relationships between soil WR and aggregate stability, since it may delay the entry of water into aggregates, increase structural stability and contribute to reduce soil erosion risk. Organic C (OC) content, aggregate stability and WR are therefore strongly related parameters. In the case of agricultural soils, where both the type of management as crops can influence all these parameters, it is important to evaluate the interactions among them and their consequences. Studies focused on the intra-aggregate distribution of OC and WR are necessary to shed light on the soil processes at a detailed scale. It is extremely important to understand how the spatial distribution of OC in soil aggregates can protect against rapid water entry and help stabilize larger structural units or lead to preferential flow. The objectives of this research are to study [i] the OC content and the

Office of Naval Research Aggregate Dynamics in the Sea Workshop Held at Pacific Grove, California on September 22-24, 1986

DTIC Science & Technology

1986-09-01

collision, etc.) originate from largely biogenically derived component particles. Local loss terms include sinking, advection and decomposition which...Some quarry or scrape away the aggregate surface, others consume entire particles. Bacterial decomposition on the particle surfaces may also weaken...major role in the degradation of aggregates. Only limited information is available regarding microbial colonization, hydrolysis , and metabolism of the

Specific growth stimulation of cultured smooth muscle cells from spontaneously hypertensive rats by platelet-derived growth factor A-chain homodimer.

PubMed Central

Resink, T J; Scott-Burden, T; Hahn, A W; Rouge, M; Hosang, M; Powell, J S; Bühler, F R

1990-01-01

Cultured vascular smooth muscle cells (VSMC)1 from spontaneously hypertensive rats (SHR) possess specific cell surface receptors for both homodimeric forms of platelet-derived growth factor (PDGF-AA and PDGF-BB), in contrast to cells from normotensive Wistar Kyoto (WKY) animals, which express receptors only for the B-chain form of PDGF. Stimulation of quiescent VSMC from SHR with PDGF-AA resulted in activation of S6-kinase and induction of phosphoinositide catabolism, as well as cellular proliferation when cultures were maintained for prolonged periods with daily supplementation of the growth factor. WKY-derived VSMC showed no response to PDGF-AA, which was consistent with their lack of specific receptors for this homodimer. The responsiveness of quiescent cells from SHR and WKY to the B-chain homodimer was similar. The enhanced growth responsiveness of SHR-derived cells to fetal calf serum, as compared with cells from their normotensive counterparts, may be accounted for in part by their expression of receptors for the AA homodimer of PDGF. PMID:1965150

Morphology-Variable Aggregates Prepared from Cholesterol-Containing Amphiphilic Glycopolymers: Their Protein Recognition/Adsorption and Drug Delivery Applications

PubMed Central

Wang, Zhao; Luo, Ting; Cao, Amin; Sun, Jingjing; Jia, Lin

2018-01-01

In this study, a series of diblock glycopolymers, poly(6-O-methacryloyl-d-galactopyranose)-b-poly(6-cholesteryloxyhexyl methacrylate) (PMAgala-b-PMAChols), with cholesterol/galactose grafts were prepared through a sequential reversible addition-fragmentation chain transfer (RAFT) polymerization and deprotection process. The glycopolymers could self-assemble into aggregates with various morphologies depending on cholesterol/galactose-containing block weight ratios, as determined by transmission electronic microscopy (TEM) and dynamic laser light scattering (DLS). In addition, the lectin (Ricinus communis agglutinin II, RCA120) recognition and bovine serum albumin (BSA) adsorption of the PMAgala-b-PMAChol aggregates were evaluated. The SK-Hep-1 tumor cell inhibition properties of the PMAgala-b-PMAChol/doxorubicin (DOX) complex aggregates were further examined in vitro. Results indicate that the PMAgala-b-PMAChol aggregates with various morphologies showed different interaction/recognition features with RCA120 and BSA. Spherical aggregates (d ≈ 92 nm) possessed the highest RCA120 recognition ability and lowest BSA protein adsorption. In addition, the DOX-loaded spherical complex aggregates exhibited a better tumor cell inhibition property than those of nanofibrous complex aggregates. The morphology-variable aggregates derived from the amphiphilic glycopolymers may serve as multifunctional biomaterials with biomolecular recognition and drug delivery features. PMID:29495614

Media Compositions for Three Dimensional Mammalian Tissue Growth Under Microgravity Culture Conditions

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor)

1998-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Media Compositions for Three-Dimensional Mammalian Tissue Growth under Microgravity Culture Conditions

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor)

1998-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue.The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

The morphology, proliferation rate, and population doubling time factor of adipose-derived mesenchymal stem cells cultured on to non-aqueous SiO2, TiO2, and hybrid sol-gel-derived oxide coatings.

PubMed

Marycz, Krzysztof; Krzak-Roś, Justyna; Donesz-Sikorska, Anna; Śmieszek, Agnieszka

2014-11-01

In recent years, much attention has been paid to the development of tissue engineering and regenerative medicine, especially when stem cells of various sources are concerned. In addition to the interest in mesenchymal stem cells isolated from bone marrow, recently more consideration has been given to stem cells isolated from adipose tissue (AdMSCs), due to their less invasive method of collection as well as their ease of isolation and culture. However, the development of regenerative medicine requires both the application of biocompatible material and the stem cells to accelerate the regeneration. In this study, we investigated the morphology, proliferation rate index (PRi), and population doubling time factor of adipose-derived mesenchymal stem cells cultured on non-aqueous sol-gel-derived SiO2, TiO2, and SiO2/TiO2 oxide coatings. The results indicated an increase in PRi of AdMSCs when cultured on to titanium dioxide, suggesting its high attractiveness for AdMSCs. In addition, the proper morphology and the shortest doubling time of AdMSCs were observed when cultured on titanium dioxide coating. © 2014 Wiley Periodicals, Inc.

Methods of predicting aggregate voids.

DOT National Transportation Integrated Search

2013-03-01

Percent voids in combined aggregates vary significantly. Simplified methods of predicting aggregate : voids were studied to determine the feasibility of a range of gradations using aggregates available in Kansas. : The 0.45 Power Curve Void Predictio...

Blended aggregate study : final report.

DOT National Transportation Integrated Search

1980-03-01

Louisiana produces no naturally occurring skid resistant aggregate and, therefore, must import these aggregates at great expense. : In an effort to extend the yield of these aggregates, a laboratory investigation was initiated to determine the feasib...

A survey of natural aggregate properties and characteristics important in remote sensing and airborne geophysics

USGS Publications Warehouse

Knepper, D.H.; Langer, W.H.; Miller, S.

1995-01-01

Natural aggregate is vital to the construction industry. Although natural aggregate is a high volume/low value commodity that is abundant, new sources are becoming increasingly difficult to find and develop because of rigid industry specifications, political considerations, development and transportation costs, and environmental concerns. There are two primary sources of natural aggregate: (1) exposed or near-surface bedrock that can be crushed, and (2) deposits of sand and gravel. Remote sensing and airborne geophysics detect surface and near-surface phenomena, and may be useful for detecting and mapping potential aggregate sources; however, before a methodology for applying these techniques can be developed, it is necessary to understand the type, distribution, physical properties, and characteristics of natural aggregate deposits. The distribution of potential aggregate sources is closely tied to local geologic history. Conventional exploration for natural aggregate deposits has been largely a ground-based operation, although aerial photographs and topographic maps have been extensively used to target possible deposits. Today, the exploration process also considers factors such as the availability of the land, space and water supply for processing, political and environmental factors, and distance from the market; exploration and planning cannot be separated. There are many physical properties and characteristics by which to judge aggregate material for specific applications; most of these properties and characteristics pertain only to individual aggregate particles. The application of remote sensing and airborne geophysical measurements to detecting and mapping potential aggregate sources, however, is based on intrinsic bulk physical properties and extrinsic characteristics of the deposits that can be directly measured, mathematically derived from measurement, or interpreted with remote sensing and geophysical data. ?? 1995 Oxford UniversityPress.

Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

PubMed

Miranda, Moysés S; Nascimento, Hamilton S; Costa, Mayra P R; Costa, Nathália N; Brito, Karynne N L; Lopes, Cinthia T A; Santos, Simone S D; Cordeiro, Marcela S; Ohashi, Otávio M

2016-10-01

Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 10 3 and 10 4 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 10 4 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 10 4 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 10 3 b-ATMSCs/mL (p = 0.051), group 10 4 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 10 4 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms.

PubMed

Kornmuller, Anna; Brown, Cody F C; Yu, Claire; Flynn, Lauren E

2017-04-11

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads.

PubMed

Nakada, Takashi; Masuda, Takeshi; Naito, Hiroyuki; Yoshida, Masao; Ashida, Shinji; Morita, Koji; Miyazaki, Hideki; Kasuya, Yuji; Ogitani, Yusuke; Yamaguchi, Junko; Abe, Yuki; Honda, Takeshi

2016-03-15

Trastuzumab conjugates consisting of exatecan derivatives were prepared and their biological activities and physicochemical properties were evaluated. The ADCs showed strong efficacy and a low aggregation rate. The exatecan derivatives were covalently connected via a peptidyl spacer (Gly-Gly-Phe-Gly), which is assumed to be stable in circulation, and were cleaved by lysosomal enzymes following ADC internalization into tumor tissue. These anti-HER2 ADCs exhibited a high potency, specifically against HER2-positive cancer cell lines in vitro. The ADCs, bearing exatecan derivatives which have more than two methylene chains, exhibited superior cytotoxicity. It was speculated that steric hindrance of the cleavable amide moiety could be involved in the drug release. The adequate alkyl lengths of exatecan derivatives (13, 14, 15) were from two to four in terms of aggregation rate. The ADC having a hydrophilic moiety showed good efficacy in a HER2-positive and Trastuzumab-resistant breast carcinoma cell model in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell culture density affects the stemness gene expression of adipose tissue-derived mesenchymal stem cells.

PubMed

Kim, Dae Seong; Lee, Myoung Woo; Lee, Tae-Hee; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

2017-03-01

The results of clinical trials using mesenchymal stem cells (MSCs) are controversial due to the heterogeneity of human MSCs and differences in culture conditions. In this regard, it is important to identify gene expression patterns according to culture conditions, and to determine how the cells are expanded and when they should be clinically used. In the current study, stemness gene expression was investigated in adipose tissue-derived MSCs (AT-MSCs) harvested following culture at different densities. AT-MSCs were plated at a density of 200 or 5,000 cells/cm 2 . After 7 days of culture, stemness gene expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The proliferation rate of AT-MSCs harvested at a low density (~50% confluent) was higher than that of AT-MSCs harvested at a high density (~90% confluent). Although there were differences in the expression levels of stemness gene, such as octamer-binding transcription factor 4, nanog homeobox ( Nanog ), SRY-box 2, Kruppel like factor 4, v-myc avian myelocytomatosis viral oncogene homolog ( c-Myc ), and lin-28 homolog A, in the AT-MSCs obtained from different donors, RT-qPCR analysis demonstrated differential gene expression patterns according to the cell culture density. Expression levels of stemness genes, particularly Nanog and c-Myc , were upregulated in AT-MSCs harvested at a low density (~50% confluent) in comparison to AT-MSCs from the same donor harvested at a high density (~90% confluent). These results imply that culture conditions, such as the cell density at harvesting, modulate the stemness gene expression and proliferation of MSCs.

A Genome-wide Analysis of Human Pluripotent Stem Cell-Derived Endothelial Cells in 2D or 3D Culture.

PubMed

Zhang, Jue; Schwartz, Michael P; Hou, Zhonggang; Bai, Yongsheng; Ardalani, Hamisha; Swanson, Scott; Steill, John; Ruotti, Victor; Elwell, Angela; Nguyen, Bao Kim; Bolin, Jennifer; Stewart, Ron; Thomson, James A; Murphy, William L

2017-04-11

A defined protocol for efficiently deriving endothelial cells from human pluripotent stem cells was established and vascular morphogenesis was used as a model system to understand how synthetic hydrogels influence global biological function compared with common 2D and 3D culture platforms. RNA sequencing demonstrated that gene expression profiles were similar for endothelial cells and pericytes cocultured in polyethylene glycol (PEG) hydrogels or Matrigel, while monoculture comparisons identified distinct vascular signatures for each cell type. Endothelial cells cultured on tissue-culture polystyrene adopted a proliferative phenotype compared with cells cultured on or encapsulated in PEG hydrogels. The proliferative phenotype correlated to increased FAK-ERK activity, and knockdown or inhibition of ERK signaling reduced proliferation and expression for cell-cycle genes while increasing expression for "3D-like" vasculature development genes. Our results provide insight into the influence of 2D and 3D culture formats on global biological processes that regulate cell function. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Soil aggregation and aggregate-associated carbon under four typical halophyte communities in an arid area.

PubMed

Yang, Haichang; Wang, Jingya; Zhang, Fenghua

2016-12-01

The soil microbial biomass carbon (MBC) is considered as a sensitive index of soil carbon ecosystem. The distribution of aggregate-associated MBC determines the capacity of the soil to store soil organic carbon (SOC). We compared soil aggregate-associated SOC and aggregate-associated MBC under four halophyte communities: Karelinia caspia (Pall.) Less. (Abbr. K. caspia), Bassia dasyphylla (Fisch. et C. A. Mey.) Kuntze. (Abbr. B. dasyphylla), Haloxylon ammodendron (C. A. Mey.) Bunge. (Abbr. H. ammodendron), and Tamarix ramosissima Lour (Abbr. T. ramosissima) on an alluvial fan in the Manasi River Basin, Xinjiang, China. The specific objectives of the study were to determine which aggregate size fraction was the most important for MBC and SOC retention in these soils of four halophyte communities. The results showed that the 0.053-0.25 mm fraction contained 47 to 75 % of the total soil mass. The amount of soil in the 0.053-0.25 mm fraction was significantly greater than that in the >0.25 and the <0.053 mm fractions. The >0.25 and the <0.053 mm fractions contained 7.8 to 43.0 % of the soil mass. Aggregate-associated SOC concentrations ranged from 1.70 to 13.68 g kg -1 , and the aggregate-associated SOC were the highest under the H. ammodendron and T. ramosissima communities. The aggregate-associated MBC ranged from 55.26 to 217.11 g kg -1 , and the aggregate-associated MBC were higher under the K. caspia and B. dasyphylla communities. The aggregate-associated SOC concentrations were significantly higher in the >0.25 and the <0.053 mm fractions than in the 0.053-0.25 mm fraction. The aggregate-associated MBC in the 20-40 cm depth was consistent with its law. However, in the 0-20 cm depth, the aggregate-associated MBC concentrations were significantly higher in the >0.25 mm fraction than the other two aggregate fractions, and there were no significant differences in 0.25-0.053 or <0.053 mm fraction. Correlation analyses showed that the aggregate

Kinetics of Aggregation with Choice

DOE PAGES

Ben-Naim, Eli; Krapivsky, Paul

2016-12-01

Here we generalize the ordinary aggregation process to allow for choice. In ordinary aggregation, two random clusters merge and form a larger aggregate. In our implementation of choice, a target cluster and two candidate clusters are randomly selected and the target cluster merges with the larger of the two candidate clusters.We study the long-time asymptotic behavior and find that as in ordinary aggregation, the size density adheres to the standard scaling form. However, aggregation with choice exhibits a number of different features. First, the density of the smallest clusters exhibits anomalous scaling. Second, both the small-size and the large-size tailsmore » of the density are overpopulated, at the expense of the density of moderate-size clusters. Finally, we also study the complementary case where the smaller candidate cluster participates in the aggregation process and find an abundance of moderate clusters at the expense of small and large clusters. Additionally, we investigate aggregation processes with choice among multiple candidate clusters and a symmetric implementation where the choice is between two pairs of clusters.« less

Sectional methods for aggregation problems: application to volcanic eruptions

NASA Astrophysics Data System (ADS)

Rossi, E.

2016-12-01

Particle aggregation is a general problem that is common to several scientific disciplines such as planetary formation, food industry and aerosol sciences. So far the ordinary approach to this class of problems relies on the solution of the Smoluchowski Coagulation Equations (SCE), a set of Ordinary Differential Equations (ODEs) derived from the Population Balance Equations (PBE), which basically describe the change in time of an initial grain-size distribution due to the interaction of "single" particles. The frequency of particles collisions and their sticking efficiencies depend on the specific problem under analysis, but the mathematical framework and the possible solutions to the ODEs seem to be somehow discipline-independent and very general. In this work we will focus on the problem of volcanic ash aggregation, since it represents an extreme case of complexity that can be relevant also to other disciplines. In fact volcanic ash aggregates observed during the fallouts are characterized by relevant porosities and they do not fit with simplified descriptions based on monomer-like structures or fractal geometries. In this work we propose a bidimensional approach to the PBEs which uses additive (mass) and non-additive (volume) internal descriptors in order to better characterize the evolution of volcanic ash aggregation. In particular we used sectional methods (fixed-pivot) to discretize the internal parameters space. This algorithm has been applied to a one dimensional volcanic plume model in order to investigate how the Total Grain Size Distribution (TGSD) changes throughout the erupted column in real scenarios (i.e. Eyjafjallajokull 2010, Sakurajima 2013 and Mt. Saint Helens 1980).

New ambuic acid derivatives from the solid culture of Pestalotiopsis neglecta and their nitric oxide inhibitory activity.

PubMed

Qi, Qiu-Yue; Li, Er-Wei; Han, Jun-Jie; Pei, Yun-Fei; Ma, Ke; Bao, Li; Huang, Ying; Zhao, Feng; Liu, Hong-Wei

2015-05-19

Four new ambuic acid derivatives (1-4), and four known derivatives (5-8), were isolated from the solid culture of a plant pathogenic fungus Pestalotiopsis neglecta. Their structures were elucidated by extensive NMR experiments. The absolute configuration of the C-16 secondary alcohol in 1 was deduced via the CD data of the in situ formed [Rh2(OCOCF3)4] complex with the acetonide derivative of 1. The absolute configuration in 3 was assigned by comparison of the experimental and simulated electronic circular dichroism (ECD) spectrum. The NMR data of compound 5 was reported for the first time. In the nitric oxide (NO) inhibition assay, compounds 4, 6 and 7 showed inhibitory activity against the NO production in the lipopolysaccharide (LPS)-induced macrophage with IC50 values of 88.66, 11.20, and 20.80 µM, respectively.

Soil aggregation and aggregating agents as affected by long term contrasting management of an Anthrosol

NASA Astrophysics Data System (ADS)

Zhang, Shulan; Wang, Renjie; Yang, Xueyun; Sun, Benhua; Li, Qinghui

2016-12-01

Soil aggregation was studied in a 21-year experiment conducted on an Anthrosol. The soil management regimes consisted of cropland abandonment, bare fallow without vegetation and cropping system. The cropping system was combined with the following nutrient management treatments: control (CONTROL, no nutrient input); nitrogen, phosphorus and potassium (NPK); straw plus NPK (SNPK); and manure (M) plus NPK (MNPK). Compared with the CONTROL treatment, the abandonment treatment significantly increased the formation of large soil macroaggregates (>2 mm) and consequently improved the stability of aggregates in the surface soil layer due to enhancement of hyphal length and of soil organic matter content. However, in response to long-term bare fallow treatment aggregate stability was low, as were the levels of aggregating agents. Long term fertilization significantly redistributed macroaggregates; this could be mainly ascribed to soil organic matter contributing to the formation of 0.5-2 mm classes of aggregates and a decrease in the formation of the >2 mm class of aggregates, especially in the MNPK treatment. Overall, hyphae represented a major aggregating agent in both of the systems tested, while soil organic compounds played significantly different roles in stabilizing aggregates in Anthrosol when the cropping system and the soil management regimes were compared.

Soil aggregation and aggregating agents as affected by long term contrasting management of an Anthrosol

PubMed Central

Zhang, Shulan; Wang, Renjie; Yang, Xueyun; Sun, Benhua; Li, Qinghui

2016-01-01

Soil aggregation was studied in a 21-year experiment conducted on an Anthrosol. The soil management regimes consisted of cropland abandonment, bare fallow without vegetation and cropping system. The cropping system was combined with the following nutrient management treatments: control (CONTROL, no nutrient input); nitrogen, phosphorus and potassium (NPK); straw plus NPK (SNPK); and manure (M) plus NPK (MNPK). Compared with the CONTROL treatment, the abandonment treatment significantly increased the formation of large soil macroaggregates (>2 mm) and consequently improved the stability of aggregates in the surface soil layer due to enhancement of hyphal length and of soil organic matter content. However, in response to long-term bare fallow treatment aggregate stability was low, as were the levels of aggregating agents. Long term fertilization significantly redistributed macroaggregates; this could be mainly ascribed to soil organic matter contributing to the formation of 0.5–2 mm classes of aggregates and a decrease in the formation of the >2 mm class of aggregates, especially in the MNPK treatment. Overall, hyphae represented a major aggregating agent in both of the systems tested, while soil organic compounds played significantly different roles in stabilizing aggregates in Anthrosol when the cropping system and the soil management regimes were compared. PMID:27958366

Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells

PubMed Central

Sánchez-Mendoza, Eduardo H.; Bellver-Landete, Victor; Arce, Carmen; Doeppner, Thorsten R.; Hermann, Dirk M.

2017-01-01

The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs. PMID:28493916

A study of the aggregation of cyclodextrins: Determination of the critical aggregation concentration, size of aggregates and thermodynamics using isodesmic and K2-K models.

PubMed

Do, Thao Thi; Van Hooghten, Rob; Van den Mooter, Guy

2017-04-15

The aggregation of three different cyclodextrins (CDs): 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) and sulfobutylether-β-cyclodextrin (SBE-β-CD) was studied. The critical aggregation concentration (cac) of these three CDs is quite similar and is situated at ca. 2% (m/v). There was only a small difference in the cac values determined by DLS and 1 H NMR. DLS measurements revealed that CDs in solution have three size populations wherein one of them is that of a single CD molecule. The size of aggregates determined by TEM appears to be similar to the size of the aggregates in the second size distribution determined by DLS. Isodesmic and K 2 -K self-assembly models were used for studying the aggregation process of HP-β-CD, HP-γ-CD and SBE-β-CD. The results showed that the aggregation process of these CDs is a cooperative one, where the first step of aggregation is less favorable than the next steps. The determined thermodynamic parameters showed that the aggregation process of all three CDs is spontaneous and exothermic and it is driven by an increase of the entropy of the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative high-throughput population dynamics in continuous-culture by automated microscopy.

PubMed

Merritt, Jason; Kuehn, Seppe

2016-09-12

We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation.

Abrogation of E-cadherin-mediated cellular aggregation allows proliferation of pluripotent mouse embryonic stem cells in shake flask bioreactors.

PubMed

Mohamet, Lisa; Lea, Michelle L; Ward, Christopher M

2010-09-23

A fundamental requirement for the exploitation of embryonic stem (ES) cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times. Here we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/-) or the neutralising antibody DECMA-1 (EcadAb), allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers. This is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are necessary to increase cell viability of hES cells in suspension.

Improving Large-Scale Image Retrieval Through Robust Aggregation of Local Descriptors.

PubMed

Husain, Syed Sameed; Bober, Miroslaw

2017-09-01

Visual search and image retrieval underpin numerous applications, however the task is still challenging predominantly due to the variability of object appearance and ever increasing size of the databases, often exceeding billions of images. Prior art methods rely on aggregation of local scale-invariant descriptors, such as SIFT, via mechanisms including Bag of Visual Words (BoW), Vector of Locally Aggregated Descriptors (VLAD) and Fisher Vectors (FV). However, their performance is still short of what is required. This paper presents a novel method for deriving a compact and distinctive representation of image content called Robust Visual Descriptor with Whitening (RVD-W). It significantly advances the state of the art and delivers world-class performance. In our approach local descriptors are rank-assigned to multiple clusters. Residual vectors are then computed in each cluster, normalized using a direction-preserving normalization function and aggregated based on the neighborhood rank. Importantly, the residual vectors are de-correlated and whitened in each cluster before aggregation, leading to a balanced energy distribution in each dimension and significantly improved performance. We also propose a new post-PCA normalization approach which improves separability between the matching and non-matching global descriptors. This new normalization benefits not only our RVD-W descriptor but also improves existing approaches based on FV and VLAD aggregation. Furthermore, we show that the aggregation framework developed using hand-crafted SIFT features also performs exceptionally well with Convolutional Neural Network (CNN) based features. The RVD-W pipeline outperforms state-of-the-art global descriptors on both the Holidays and Oxford datasets. On the large scale datasets, Holidays1M and Oxford1M, SIFT-based RVD-W representation obtains a mAP of 45.1 and 35.1 percent, while CNN-based RVD-W achieve a mAP of 63.5 and 44.8 percent, all yielding superior performance to the