Systematic selection of small molecules to promote differentiation of embryonic stem cells and experimental validation for generating cardiomyocytes.

Small molecules are being increasingly used for inducing the targeted differentiation of stem cells to different cell types. However, until now no systematic method for selecting suitable small molecules for this purpose has been presented. In this work, we propose an integrated and general bioinformatics- and cheminformatics-based approach for selecting small molecules which direct cellular differentiation in the desired way. The approach was successfully experimentally validated for differentiating stem cells into cardiomyocytes. All predicted compounds enhanced expression of cardiac progenitor (Gata4, Nkx2-5 and Mef2c) and mature cardiac markers (Actc1, myh6) significantly during and post-cardiac progenitor formation. The best-performing compound, Famotidine, increased the percentage of Myh6-positive cells from 33 to 56%, and enhanced the expression of Nkx2.5 and Tnnt2 cardiac progenitor and cardiac markers in protein level. The approach employed in the study is applicable to all other stem cell differentiation settings where gene expression data are available.YK and AB thank the European Research Council (ERC Starting Grant 2013 to AB) for funding.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/cddiscovery.2016.

A Scanning Electron Microscopic Study of the Chick Chorioallantoic Membrane: Cell Death and the Involvement of Oxygen Free Radicals

Cell death is a normal feature within the chick chorioallantoic membrane, occurring principally between days 10 and 14 of incubation. Samples of chorioallantoic membrane were obtained on days 6, 8, 10, 12 and 14 of incubation, after the creation of artificial air chambers on day 3. These were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM) after staining for acid phosphatase activity, and by light microscopy after demonstrating oxygen free radicals with nitro blue tetrazolium. On day 6, small defects in the plasmalemma, approximately 200 nm in diameter, could be seen by SEM. A sequence of events leading to complete destruction of the plasmalemma, with exposure of the nucleus and other cytoplasmic organelles, could be traced, and by day 8 the membrane was a mosaic of healthy cells and others in various stages of degeneration. TEM revealed that acid phosphatase activity was confined to the golgi apparatus and associated vesicles even in advanced stages of degeneration. By comparison, oxygen free radicals were demonstrated in individual cells from day 6 onwards. Application of superoxide dismutase and catalase to the epithelium using a nebuliser spray significantly reduced the amount of cell death seen by scanning microscopy on day 12. It is concluded that oxygen free radicals may mediate cell death in the chorioallantoic membrane

Reduction of truncated Kit Expression in Men with Abnormal Semen Parameters, Globozoospermia and History of Low or Fertilization Failure

Objective: Phospholipase C zeta 1 (PLCζ) is one of the main sperm factor involved in oocyte activation and other factors may assist this factor to induce successful fertilization. Microinjection of recombinant tr-kit, a truncated form of c-kit receptor, into metaphase II-arrested mouse oocytes initiate egg activation. Considering the potential roles of tr-KIT during spermiogenesis and fertilization, we aimed to assess expression of tr-KIT in sperm of men with normal and abnormal parameters and also in infertile men with previous failed fertilization and globozoospermia. Materials and Methods: This experimental study was conducted from September 2015 to July 2016 on 30 normozoospermic and 20 abnormozoospermic samples for experiment one, and also was carried out on 10 globozoospermic men, 10 men with a history low or failed fertilization and 13 fertile men for experiment two. Semen parameters and sperm DNA fragmentation were assessed according to WHO protocol, and TUNEL assay. Sperm tr-KIT was evaluated by flow cytometry, immunostaining and western blot. Results: The results show that tr-KIT mainly was detected in post-acrosomal, equatorial and tail regions. Percentage of tr-KIT-positive spermatozoa in abnormozoospermic men was significantly lower than normozoospermic men. Also significant correlations were observed between sperm tr-KIT with sperm count (r=0.8, P<0.001), motility (r=0.31, P=0.03) and abnormal morphology (r=-0.6, P<0.001). Expression of tr-KIT protein was significantly lower in infertile men with low/ failed fertilization and globozoospermia compared to fertile men. The significant correlation was also observed between tr-KIT protein with fertilization rate (r=-0.46, P=0.04). In addition, significant correlations were observed between sperm DNA fragmentation with fertilization rate (r=-0.56, P=0.019) and tr-KIT protein (r=-0.38, P=0.04). Conclusion: tr-KIT may play a direct or indirect role in fertilization. Therefore, to increase our insight regarding the role of tr-KIT in fertilization further research is warranted. © 2019 Royan Institute (ACECR). All rights reserved

Identification of Pithomyces chartarum causing leaf spot of cabbage in Malaysia

A leaf spot disease was observed on cabbage (Brassica oleracea L. var. capitata) affecting 80% of plants growing in greenhouses and fields in Serdang, Selangor, Malaysia. Symptomatic leaf samples were collected from infected plants and isolations made on agar medium. Single-spore isolates from resulting colonies were identified based on cultural and morphological characteristics as Pithomyces chartarum. Morphological identification was confirmed by sequence analysis of the internal transcribed spacer (ITS) regions 1 and 2, including 5.8S rDNA (ITS1-5.8S-ITS2). Pathogenicity tests indicated that P. chartarum causes leaf spot on cabbage. This is the first report of leaf spot caused by P. chartarum on cabbage in Malaysia

Spin Fidelity for Three-qubit Greenberger-Horne-Zeilinger and W States Under Lorentz Transformations

Constructing the reduced density matrix for a system of three massive spin$-\frac{1}{2}$ particles described by a wave packet with Gaussian momentum distribution and a spin part in the form of GHZ or W state, the fidelity for the spin part of the system is investigated from the viewpoint of moving observers in the jargon of special relativity. Using a numerical approach, it turns out that by increasing the boost speed, the spin fidelity decreases and reaches to a non-zero asymptotic value that depends on the momentum distribution and the amount of momentum entanglement.Comment: 12pages, 2 figure

Acoustic Actuation of Integrin‐Bound Microbubbles for Mechanical Phenotyping during Differentiation and Morphogenesis of Human Embryonic Stem Cells

Early human embryogenesis is a dynamic developmental process, involving continuous and concomitant changes in gene expression, structural reorganization, and cellular mechanics. However, the lack of investigation methods has limited the understanding of how cellular mechanical properties change during early human embryogenesis. In this study, ultrasound actuation of functionalized microbubbles targeted to integrin (acoustic tweezing cytometry, ATC) is employed for in situ measurement of cell stiffness during human embryonic stem cell (hESC) differentiation and morphogenesis. Cell stiffness, which is regulated by cytoskeleton structure, remains unchanged in undifferentiated hESCs, but significantly increases during neural differentiation. Further, using the recently established in vitro 3D embryogenesis models, ATC measurements reveal that cells continue to stiffen while maintaining pluripotency during epiblast cyst formation. In contrast, during amniotic cyst formation, cells first become stiffer during luminal cavity formation, but softens significantly when cells differentiate to form amniotic cysts. These results suggest that cell stiffness changes not only due to 3D spatial organization, but also with cell fate change. ATC therefore provides a versatile platform for in situ measurement of cellular mechanical property, and cell stiffness may be used as a mechanical biomarker for cell lineage diversification and cell fate specification during embryogenesis.Ultrasound actuation of functionalized microbubbles targeted to integrin (acoustic tweezing cytometry) is employed for in situ measurement of cell stiffness during human embryonic stem cell neural differentiation and morphogenesis in 3D embryogenesis model. The results suggest that cell stiffness changes not only due to 3D spatial organization, but also with cell fate change.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146940/1/smll201803137.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146940/2/smll201803137_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146940/3/smll201803137-sup-0001-S1.pd

ZIF-8 Modified Polypropylene Membrane: A Biomimetic Cell Culture Platform with a View to the Improvement of Guided Bone Regeneration.

PurposeDespite the significant advances in modeling of biomechanical aspects of cell microenvironment, it remains a major challenge to precisely mimic the physiological condition of the particular cell niche. Here, the metal-organic frameworks (MOFs) have been introduced as a feasible platform for multifactorial control of cell-substrate interaction, given the wide range of physical and mechanical properties of MOF materials and their structural flexibility.ResultsIn situ crystallization of zeolitic imidazolate framework-8 (ZIF-8) on the polydopamine (PDA)-modified membrane significantly raised surface energy, wettability, roughness, and stiffness of the substrate. This modulation led to an almost twofold increment in the primary attachment of dental pulp stem cells (DPSCs) compare to conventional plastic culture dishes. The findings indicate that polypropylene (PP) membrane modified by PDA/ZIF-8 coating effectively supports the growth and proliferation of DPSCs at a substantial rate. Further analysis also displayed the exaggerated multilineage differentiation of DPSCs with amplified level of autocrine cell fate determination signals, like BSP1, BMP2, PPARG, FABP4, ACAN, and COL2A. Notably, osteogenic markers were dramatically overexpressed (more than 100-folds rather than tissue culture plate) in response to biomechanical characteristics of the ZIF-8 layer.ConclusionHence, surface modification of cell culture platforms with MOF nanostructures proposed as a powerful nanomedical approach for selectively guiding stem cells for tissue regeneration. In particular, PP/PDA/ZIF-8 membrane presented ideal characteristics for using as a barrier membrane for guided bone regeneration (GBR) in periodontal tissue engineering

The effect of miRNA mimic hsa-miR-7704 on in-vitro replication of herpes simplex virus type 1

Background: Herpes simplex virus type 1 (HSV-1) infections are of the most common diseases in human population. HSV-1 can cause subclinical to severe diseases, especially in immunocompromised patients. There are few anti-herpes drugs for treatment of HSV-1 infection. Acyclovir is one of the most important drugs. The extensive use of this drug has led to the development of resistant strains. Therefore, development of new anti-herpes drugs with different mechanisms is noticeable. This study aimed to use microRNAs as a novel method for inhibiting HSV-1 infection. Methods: Synthesized miRNA mimics hsa-miR-7704 (miR-SX1) were transfected into Hela cells, and then infected with HSV-1. Cellular morphological changes were observed 24 hours post-infection by inverted microscope, and photographed. Viral titers were measured using 50 tissue culture infective dose (TCID 50 ) method. Findings: miR-SX-1-transfected cells produced low-titer HSV-1, without affecting cell viability. Conclusion: The data suggest that miR-SX1 inhibits HSV-1 replication, and may provide an alternative mechanism to prevent HSV-1 infection. © 2018, Isfahan University of Medical Sciences(IUMS). All rights reserved

HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages

BACKGROUND: Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1) differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2) control of HIV-1 alternative splicing, which is essential for optimal viral replication. RESULTS: Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins) and inhibitory factors (members of the hnRNP family). While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. CONCLUSION: While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative viral pre-mRNA splicing were observed. These changes correlated with changes in Tat expression and virus production and could play an important role in viral persistence in macrophages. This mechanism could provide a novel target for control of infection in this critical cell type, which would be necessary for eventual eradication of the virus from infected individuals

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

<p>Abstract</p> <p>Background</p> <p>The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).</p> <p>Methods</p> <p>A total of 16592 prepared spermatozoa were selected and classified into two groups: Group I, spermatozoa which presented their head attached to an HA substance (HA-bound sperm), and Group II, those spermatozoa that did not attach to the HA substance (HA-unbound sperm). HA-bound and HA-unbound spermatozoa were evaluated according to the following sperm forms: 1-Normal morphology: normal nucleus (smooth, symmetric and oval configuration, length: 4.75+/-2.8 μm and width: 3.28+/-0.20 μm, no extrusion or invagination and no vacuoles occupied more than 4% of the nuclear area) as well as acrosome, post-acrosomal lamina, neck, tail, besides not presenting a cytoplasmic droplet or cytoplasm around the head; 2-Abnormalities of nuclear form (a-Large/small; b-Wide/narrow; c-Regional disorder); 3-Abnormalities of nuclear chromatin content (a-Vacuoles: occupy >4% to 50% of the nuclear area and b-Large vacuoles: occupy >50% of the nuclear area) using a high magnification (8400x) microscopy system.</p> <p>Results</p> <p>No significant differences were obtained with respect to sperm morphological forms and the groups HA-bound and HA-unbound. 1-Normal morphology: HA-bound 2.7% and HA-unbound 2.5% (P = 0.56). 2-Abnormalities of nuclear form: a-Large/small: HA-bound 1.6% vs. HA-unbound 1.6% (P = 0.63); b-Wide/narrow: HA-bound 3.1% vs. HA-unbound 2.7% (P = 0.13); c-Regional disorders: HA-bound 4.7% vs. HA-unbound 4.4% (P = 0.34). 3. Abnormalities of nuclear chromatin content: a-Vacuoles >4% to 50%: HA-bound 72.2% vs. HA-unbound 72.5% (P = 0.74); b-Large vacuoles: HA-bound 15.7% vs. HA-unbound 16.3% (P = 0.36).</p> <p>Conclusions</p> <p>The findings suggest that HA binding assay has limited efficacy in selecting motile spermatozoa with normal morphology at high magnification.</p