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Research Containing: Reverse Transcriptase Polymerase Chain Reaction

Media ion composition controls regulatory and virulence response of Salmonella in spaceflight

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/19079590

Development of a Novel Three-Dimensional, Automatable and Integrated Bioprocess for the Differentiation of Embryonic Stem Cells into Pulmonary Alveolar Cells in a Rotating Vessel Bioreactor System

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Application of stem cells for cell therapy of respiratory diseases is a developing field. We have previously established several protocols for the differentiation of embryonic stem cells (ESC) into alveolar epithelial cells, which require a high degree of operator interference and result in a low yield of target cells. Herein, we have shown that, by provision of a medium conditioned using A549 cells and by integration of classic steps of ESC differentiation into a single step through encapsulation in hydrogels (three-dimensional) and culture in a rotary bioreactor, murine ESC (mESC) could be directed to differentiate into distal respiratory epithelial cells. Type I and II pneumocytes (with a yield of 50% for type II) and Clara cells were demonstrated by the expression of aquaporin 5, surfactant protein C, and Clara cell secretory protein, respectively. We identified target cells as early as day 5 of culture and stably maintained our differentiated cells in vitro for 100 days. Electron microscopy demonstrated microvilli and intracellular lamellar bodies (LB), and fluorescent staining confirmed the active process of exocytosis of these LB in differentiated type II cells. When these cells were decapsulated and cultured in static conditions in flask cultures (two-dimensional), they retained their characteristic type II phenotype and morphology. In conclusion, our protocol offers integrated bioprocessing, shorter time of differentiation, lower cost, no use of growth factors, high reproducibility, and high phenotypic and functional stability, as well as being amenable to automation and being scalable, which would move this field closer to future clinical applications.

Related URLs:
<Go to ISI>://WOS:000302136300003

Modeled Microgravity Sensitizes Osteoclast Precursors to RANKL Mediated Osteoclastogenesis by Increasing DAP12

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Mechanical forces are essential to maintain skeletal integrity, and microgravity exposure leads to bone loss. The underlying molecular mechanisms leading to the changes in osteoblasts and osteoclast differentiation and function remain to be fully elucidated. Because of the infrequency of spaceflights and payload constraints, establishing in vitro and in vivo systems that mimic microgravity conditions becomes necessary. We have established a simulated microgravity (modeled microgravity, MMG) system to study the changes induced in osteoclast precursors. We observed that MMG, on its own, was unable to induce osteoclastogenesis of osteoclast precursors; however, 24 h of MMG activates osteoclastogenesis-related signaling molecules ERK, p38, PLCgamma2, and NFATc1. Receptor activator of NFkB ligand (RANKL) (with or without M-CSF) stimulation for 3-4 days in gravity of cells that had been exposed to MMG for 24 h enhanced the formation of very large tartrate-resistant acid phosphatase (TRAP)-positive multinucleated (>30 nuclei) osteoclasts accompanied by an upregulation of the osteoclast marker genes TRAP and cathepsin K. To validate the in vitro system, we studied the hindlimb unloading (HLU) system using BALB/c mice and observed a decrease in BMD of femurs and a loss of 3D microstructure of both cortical and trabecular bone as determined by micro-CT. There was a marked stimulation of osteoclastogenesis as determined by the total number of TRAP-positive multinucleated osteoclasts formed and also an increase in RANKL-stimulated osteoclastogenesis from precursors removed from the tibias of mice after 28 days of HLU. In contrast to earlier reported findings, we did not observe any histomorphometric changes in the bone formation parameters. Thus, the foregoing observations indicate that microgravity sensitizes osteoclast precursors for increased differentiation. The in vitro model system described here is potentially a valid system for testing drugs for preventing microgravity-induced bone loss by targeting the molecular events occurring in microgravity-induced enhanced osteoclastogenesis.

Related URLs:
<Go to ISI>://WOS:000259411002251

Modulation of Pleurodeles waltl DNA polymerase mu expression by extreme conditions encountered during spaceflight

by cfynanon 9 June 2015in Biology & Biotechnology No comment

DNA polymerase micro is involved in DNA repair, V(D)J recombination and likely somatic hypermutation of immunoglobulin genes. Our previous studies demonstrated that spaceflight conditions affect immunoglobulin gene expression and somatic hypermutation frequency. Consequently, we questioned whether Polmu expression could also be affected. To address this question, we characterized Polmu of the Iberian ribbed newt Pleurodeles waltl and exposed embryos of that species to spaceflight conditions or to environmental modifications corresponding to those encountered in the International Space Station. We noted a robust expression of Polmu mRNA during early ontogenesis and in the testis, suggesting that Polmu is involved in genomic stability. Full-length Polmu transcripts are 8-9 times more abundant in P. waltl than in humans and mice, thereby providing an explanation for the somatic hypermutation predilection of G and C bases in amphibians. Polmu transcription decreases after 10 days of development in space and radiation seem primarily involved in this down-regulation. However, space radiation, alone or in combination with a perturbation of the circadian rhythm, did not affect Polmu protein levels and did not induce protein oxidation, showing the limited impact of radiation encountered during a 10-day stay in the International Space Station.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/23936065

Effects of Microgravity Modeled by Large Gradient High Magnetic Field on the Osteogenic Initiation of Human Mesenchymal Stem Cells

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Microgravity (MG) leads to a decrease in osteogenic potential of human bone marrow-derived mesenchymal stem cells (hMSCs). In the present study, we used large gradient high magnetic field (LGHMF) produced by a superconducting magnet to model MG (LGHMF-MG) and analyzed the effects of LGHMF-MG on survival, cytoskeleton and osteogenic potential of hMSCs. Results showed that the LGHMF-MG treatment for 6 h disrupted the cytoskeleton of hMSCs, and the LGHMF-MG treatment for 24 h led to cell death. LGHMF-MG treatments for 6 h in early stages of osteogenic induction (the pre-treatment before osteogenic induction, the beginning-treatment in the beginning-stage of osteogenic induction and the middle-treatment in the middle-stage of osteogenic induction) resulted in suppression on osteogenesis of hMSCs. The suppression intensity was reduced gradually as the treatment stage of LGHMF-MG was postponed. The LGHMF-MG treatment for 6 h in the ending-stage of osteogenic induction (the ending-treatment) had no obvious effect on osteogenesis of hMSCs. These results indicated that LGHMF-MG should affect the initiation of osteogenesis. Finally, the possible mechanism for the inhibition effect of LGHMF-MG on osteogenesis of hMSCs is discussed.

Related URLs:
<Go to ISI>://WOS:000282424200009

Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3 x 10(5) cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.

Related URLs:
<Go to ISI>://WOS:000293752000009

Modeled microgravity and hindlimb unloading sensitize osteoclast precursors to RANKL-mediated osteoclastogenesis

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Mechanical forces are essential to maintain skeletal integrity, and microgravity exposure leads to bone loss. The underlying molecular mechanisms leading to the changes in osteoblasts and osteoclast differentiation and function remain to be fully elucidated. Because of the infrequency of spaceflights and payload constraints, establishing in vitro and in vivo systems that mimic microgravity conditions becomes necessary. We have established a simulated microgravity (modeled microgravity, MMG) system to study the changes induced in osteoclast precursors. We observed that MMG, on its own, was unable to induce osteoclastogenesis of osteoclast precursors; however, 24 h of MMG activates osteoclastogenesis-related signaling molecules ERK, p38, PLC gamma 2, and NFATc1. Receptor activator of NFkB ligand (RANKL) (with or without M-CSF) stimulation for 3-4 days in gravity of cells that had been exposed to MMG for 24 h enhanced the formation of very large tartrate-resistant acid phosphatase (TRAP)-positive multinucleated (> 30 nuclei) osteoclasts accompanied by an upregulation of the osteoclast marker genes TRAP and cathepsin K. To validate the in vitro system, we studied the hindlimb unloading (HLU) system using BALB/c mice and observed a decrease in BMD of femurs and a loss of 3D microstructure of both cortical and trabecular bone as determined by micro-CT. There was a marked stimulation of osteoclastogenesis as determined by the total number of TRAP-positive multinucleated osteoclasts formed and also an increase in RANKL-stimulated osteoclastogenesis from precursors removed from the tibias of mice after 28 days of HLU. In contrast to earlier reported findings, we did not observe any histomorphometric changes in the bone formation parameters. Thus, the foregoing observations indicate that microgravity sensitizes osteoclast precursors for increased differentiation. The in vitro model system described here is potentially a valid system for testing drugs for preventing microgravity-induced bone loss by targeting the molecular events occurring in microgravity-induced enhanced osteoclastogenesis.

Related URLs:
<Go to ISI>://WOS:000286119000014

Microgravity promotes differentiation and meiotic entry of postnatal mouse male germ cells

by cfynanon 9 June 2015in Biology & Biotechnology No comment

A critical step of spermatogenesis is the entry of mitotic spermatogonia into meiosis. Progresses on these topics are hampered by the lack of an in vitro culture system allowing mouse spermatogonia differentiation and entry into meiosis. Previous studies have shown that mouse pachytene spermatocytes cultured in simulated microgravity (SM) undergo a spontaneous meiotic progression. Here we report that mouse mitotic spermatogonia cultured under SM with a rotary cell culture system (RCCS) enter into meiosis in the absence of any added exogenous factor or contact with somatic cells. We found that isolated Kit-positive spermatogonia under the RCCS condition enter into the prophase of the first meiotic division (leptotene stage), as monitored by chromosomal organization of the synaptonemal complex 3 protein (Scp3) and up-regulation of several pro-meiotic genes. SM was found to activate the phosphatidyl inositol 3 kinase (PI3K) pathway and to induce in Kit-positive spermatogonia the last round of DNA replication, typical of the preleptotene stage. A PI3K inhibitor abolished Scp3 induction and meiotic entry stimulated by RCCS conditions. A positive effect of SM on germ cell differentiation was also observed in undifferentiated (Kit-negative) spermatogonia, in which RCCS conditions stimulate the expression of Kit and Stra8. In conclusion, SM is an artificial environmental condition which promotes postnatal male germ cell differentiation and might provide a tool to study the molecular mechanisms underlying the switch from mitosis to meiosis in mammals.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed9&AN=2010168350
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1371%2Fjournal.pone.0009064&issn=1932-6203&isbn=&volume=5&issue=2&spage=e9064&pages=&date=2010&title=PLoS+ONE&atitle=Microgravity+promotes+differentiation+and+meiotic+entry+of+postnatal+mouse+male+germ+cells&aulast=Pellegrini&pid=%3Cauthor%3EPellegrini+M.%3C%2Fauthor%3E&%3CAN%3E2010168350%3C%2FAN%3E

The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis.In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10(-7)M and 10(-8)M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains.In testes, immunofluorescent reaction for 3beta- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1beta were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3beta and 17beta steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 beta expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. -90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules.Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/22558148

Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT-PCR, in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5-fold or greater change. When these data were averaged (n = 4), we identified 12 genes that were significantly up- or down-regulated by at least 1.5-fold after spaceflight (P < or = 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT-PCR were as follows: Rbm3 (up-regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down-regulated). QRT-PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla-4, IFN-alpha2a (up-regulated) and CD44 (down-regulated). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/20213684

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