A Small Non-Coding RNA Modulates Expression of Pilus-1 Type in Streptococcus pneumoniae

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, and about 30% of the pneumococcal clinical isolates show type I pili-like structures. These long proteinaceous polymers extending from the bacterial surface are encoded by pilus islet 1 and play major roles in adhesion and host colonization. Pili expression is bistable and is controlled by the transcriptional activator RlrA. In this work, we demonstrate that the previously identified small noncoding RNA srn135 also participates in pilus regulation. Our findings show that srn135 is generated upon processing of the 5′-UTR region of rrgA messenger and its deletion prevents the synthesis of RrgA, the main pili adhesin. Moreover, overexpression of srn135 increases the expression of all pili genes and rises the percentage of piliated bacteria within a clonal population. This regulation is mediated by the stabilization of rlrA mRNA since higher levels of srn135 increase its half-life to 165%. Our findings suggest that srn135 has a dual role in pilus expression acting both in cis- (on the RrgA levels) and in trans- (modulating the levels of RlrA) and contributes to the delicate balance between pili expressing and non-expressing bacteria.

Long-Term Hypoxia Maintains a State of Dedifferentiation and Enhanced Stemness in Fetal Cardiovascular Progenitor Cells

Early-stage mammalian embryos survive within a low oxygen tension environment and develop into fully functional, healthy organisms despite this hypoxic stress. This suggests that hypoxia plays a regulative role in fetal development that influences cell mobilization, differentiation, proliferation, and survival. The long-term hypoxic environment is sustained throughout gestation. Elucidation of the mechanisms by which cardiovascular stem cells survive and thrive under hypoxic conditions would benefit cell-based therapies where stem cell survival is limited in the hypoxic environment of the infarcted heart. The current study addressed the impact of long-term hypoxia on fetal Islet-1+ cardiovascular progenitor cell clones, which were isolated from sheep housed at high altitude. The cells were then cultured in vitro in 1% oxygen and compared with control Islet-1+ cardiovascular progenitor cells maintained at 21% oxygen. RT-PCR, western blotting, flow cytometry, and migration assays evaluated adaptation to long term hypoxia in terms of survival, proliferation, and signaling. Non-canonical Wnt, Notch, AKT, HIF-2α and Yap1 transcripts were induced by hypoxia. The hypoxic niche environment regulates these signaling pathways to sustain the dedifferentiation and survival of fetal cardiovascular progenitor cells.

In Vitro Assessment of the Gene Expression Level of EZH-2 and P300 During Motor Neuron Differentiation of Human Cord Blood Mesenchymal Stem Cells

Introduction: The dedication of stem cells for dissociation into a specific type of cell requires the over expression of genes related to a particular phenotype and suppression of the other genes. Through imposing corresponding alterations on the genome, the genome modulators such as transcription factors can be regulated by histone-modifying enzymes. Maintenance of the neurogenesis process depend on the function of some of these genes which can regulate shifting of cells from proliferation to differentiation such as Enhancer of zeste homolog 2 (EZH2) known as an evolutionarily conserved gene. Moreover, motor neurons (MN) in spinal cord can be regulated during neuronal differentiation via one of the histone acetyltransferase (P300). Up until now, the mechanism of epigenetic regulation and gene expression underlie transition process of human cord blood mesenchymal stem cells (hCB-MSCs) into MNs has not been clarified very well. Therefore, the aim of this study was to explore the quantitative expression of MN-related genes including ChAT, Islet-1, and Mnx-1 along with two epigenetic regulatory genes P300 and EZH2 involved in neurogenesis during differentiation of hCB-MSCs into MNs, using two morphogens including Sonic hedgehog (Shh) and Retinoic acid (RA) involved in the specification of MNs during the growth of nervous system. Methods: Flow cytometry was done to characterize the cells (hCB-MSCs). The cells were differentiated into MN-like cells according to our previous procedure using RA (0.01mM) and Shh (100ng/ ml) as the inducing morphogens. CB-MSCs with no treatment were assumed as control cells. RT-qPCR and Immunocytochemistry were performed to find the expression of interested genes in this study. Results: The expression of MN-related markers was confirmed at the level of mRNA and protein by induction of differentiation. The results was confirmed by immunocytochemistry showed that a number of cells about 55.33±15.885% and 49.67±13.796% could express Islet-1 and ChAT, respectively. The level of gene expression of Islet-1 and ChAT was significantly increased at the first and second week of exposure, respectively. After two weeks, expression level of P300 and EZH-2 genes was increased remarkably. No significant expression of Mnx-1 was detected when compared with the control sample. Conclusion: In this study MN-related markers, Islet-1 and ChAT, were detected in differentiated cells of hCB-MSCs supporting the potency of cord blood cells in regeneration of MN-related disorders. The over expression of Islet-1, as an early MN marker, in the presence of Shh and RA indicates the supportive effect of these morphogens for the onset of motor neuron generation. We could also detect significant expression of two potent epigenetic regulatory genes involved in neurogenesis, P300 and EZH2 accompanied by ChAT as the mature motor neuron marker at the second week of exposure due to the elimination of Shh and RA at later time of differentiation. To our knowledge, the evaluation of P300 and EZH2 during differentiation of hCB-MSCs into MN-like cells was performed in this study for the first time. However, the assessment of these epigenetic regulatory genes at the level of protein can be suggested to confirm their functional epigenetic modifying effects during motor neuron differentiation.

In vitro characterization of human bone marrow mesenchymal stem cell-derived motor neurons induced by epigenetic modifiers

Background Motor neurons (MNs) are distinct types of cells in the dorso-ventral axis of the spinal cord. These cells are developed in the presence of two main morphogens, including Sonic hedgehog (Shh) and retinoic acid (RA). On the other hand, human bone marrow mesenchymal stem cells (hBM-MSCs) are known as a multipotent type of cells with neural differentiation capacity. In this regard, the aim of this study was to quantitatively evaluate the expression of MN-related genes and the potent epigenetic regulatory genes involved in neurogenesis, including Enhancer of zeste homolog 2 (EZH-2) and P300, during hBM-MSC differentiation into MN-like cells, using RA and Shh. After isolating and inducing the cells with Shh and RA, the results were evaluated using immunocytochemistry and qRT-PCR. Results Our findings showed that the treated cells could express choline acetyltransferase (ChAT) and insulin gene enhancer binding protein-1 (Islet-1) antigens at the protein level, 2 weeks after induction. Moreover, at the second week after induction, the induced cells expressed MN-related genes (ChAT and ISLET-1) and epigenetic regulatory genes (EZH-2 and P300) at significant levels compared to the control (non-treated BM-MSCs) and to the induced cells at the first week (day 7). In addition, the expression of EZH-2, as a histone-modifying gene, was also significantly upregulated at the first week compared to the control. No significant upregulation was detected in the expression of motor neuron and pancreas homeobox 1 (MNX-1) in the treated groups compared to the control group. Conclusion We concluded that epigenetic modifiers, P300 and EZH-2, are important mediators for regulating the process of motor neuron differentiation induced by RA and Shh.

Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

Background Despite recent advances in diagnostic and therapeutic approaches for gastric cancer (GC), the survival of patients with advanced GC remains very low. Islet-1 (ISL1) is a LIM-homeodomain transcription factor, which is upregulated and promotes cell proliferation in GC. The exact mechanism by which ISL1 influences GC development is unclear. Methods Co-immunoprecipitation (co-IP) and glutathione S-transferase (GST)-pulldown assays were employed to evaluate the interaction of ISL1 with CDK1. Western blot and immunohistochemistry analyses were performed to evaluate the ability of CDK1 to phosphorylate ISL1 at Ser 269 in GC cell and tissue specimens. Chromatin immunoprecipitation (ChIP), ChIP re-IP, luciferase reporter, and CCK-8 assays were combined with flow cytometry cell cycle analysis to detect the transactivation potency of ISL1-S269-p and its ability to promote cell proliferation. The self-stability and interaction with CDK1 of ISL1-S269-p were also determined. Results ISL1 is phosphorylated by CDK1 at serine 269 (S269) in vivo. Phosphorylation of ISL1 by CDK1 on serine 269 strengthened its binding on the cyclin B1 and cyclin B2 promoters and increased its transcriptional activity in GC. Furthermore, CDK1-dependent phosphorylation of ISL1 correlated positively with ISL1 protein self-stability in NIH3T3 cells. Conclusions ISL1-S269-p increased ISL1 transcriptional activity and self-stability while binding to the cyclinB1 and cyclinB2 promoters promotes cell proliferation. ISL1-S269-p is therefore crucial for tumorigenesis and potentially a direct therapeutic target for GC.

The SSBP3 Co-Regulator Is a Novel Driver of Islet Cell Structure and Function

The activities of transcriptional complexes drive the proper development and function of insulin producing beta-cells, ultimately required for the regulation of glucose homeostasis. Our prior work helped to establish that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), directly interacts with the Ldb1 co-regulator in developing and adult beta-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with the Isl1:Ldb1 complex in beta-cells and primary islets to impact critical target genes MafA and Glp1R. Members of the SSBP family of co-regulators stabilize TF complexes in various tissues, ranging from brain to skin, by binding directly to Ldb1 and protecting the factors from ubiquitin-mediated turnover. Because of this, we hypothesized that SSBP3 would have similarly critical roles as Isl1 and Ldb1 for beta-cell development and function in vivo. To assess this, we first developed a novel SSBP3 floxed mouse line, where Cre-mediated recombination is predicted to impart loss of the Ldb1-interacting domain, plus an early termination. We bred this mouse into a Pax6-Cre transgenic line to recombine SSBP3 in the developing pancreatic islet, a model termed SSBP3islet. We found that SSBP3islet neonates become progressively hyperglycemic and both male and female mice are glucose intolerant as early as postnatal day (P) 21. These results are similar to previous Ldb1 and Isl1 knockouts in the embryonic islet, both of which were hyperglycemic by P10. We observed a reduction of the beta-cell maturity marker, MafA, and disruptions in islet cell architecture with an apparent increase in both glucagon+ alpha-cells and ghrelin+epsilon-cells at P10 and P28. In ongoing studies we are generating embryonic day (E)18.5 embryos to determine islet development defects and will conduct chromatin immunoprecipitation (ChIP) experiments to determine the beta-cell and islet genes directly bound by SSBP3 in vivo. These experiments will further elucidate the regulation of islet function by LIM complexes, knowledge that is central not only for our understanding of glucose homeostasis but for the development of novel diabetes therapeutics.

The Impact of Spaceflight and Microgravity on the Human Islet-1+ Cardiovascular Progenitor Cell Transcriptome

Understanding the transcriptomic impact of microgravity and the spaceflight environment is relevant for future missions in space and microgravity-based applications designed to benefit life on Earth. Here, we investigated the transcriptome of adult and neonatal cardiovascular progenitors following culture aboard the International Space Station for 30 days and compared it to the transcriptome of clonally identical cells cultured on Earth. Cardiovascular progenitors acquire a gene expression profile representative of an early-stage, dedifferentiated, stem-like state, regardless of age. Signaling pathways that support cell proliferation and survival were induced by spaceflight along with transcripts related to cell cycle re-entry, cardiovascular development, and oxidative stress. These findings contribute new insight into the multifaceted influence of reduced gravitational environments.

Distinctive Alteration of Neuropeptide Y Expression Responsible for Neuro-Proliferation Following Zebrafish Spinal Cord Injury

In strong contrast to the limited repair within the mammalian central nervous system, the spinal cord of adult zebrafish is capable of regeneration following injury. Understanding the mechanism underlying neural regeneration and functional recovery in spinal cord-injured zebrafish may lead to effective therapies for human spinal cord injury (SCI). Since neuropeptide Y (NPY) plays a protective role in the pathogenesis of several neurological diseases, in the present study, the effects of NPY on neuronal repair and subsequent recovery of motor function in adult zebrafish post-SCI were evaluated. Real-time quantitative PCR (qRT-PCR), in situ hybridization (ISH) and immunostaining of NPY revealed decreased NPY expression at 12 hours (h), 6 days (d) and 21 d after SCI. Double-immunostaining for NPY and Islet-1, a motoneuron marker, showed that NPY was expressed in spinal cord motoneurons. NPY morpholino (MO) treatment resulted in suppressed locomotor recovery and axon regrowth. PCNA and Islet-1 double-staining showed suppressed motoneuron proliferation in NPY-MO zebrafish. Similar to NYP, the mRNA level for NPY1R was also expressed within motoneurons and downregulated at 12 h and 21 d after SCI. Collectively, these data suggest that NPY expression in motoneurons promotes locomotor recovery and axon regrowth in adult zebrafish, possibly by regulating motoneuron proliferation through the activation of NPY1R.

Differentiation of Mesenchymal Stem Cells Derived From Human Adipose Tissue Into Cholinergic-like Cells; in vitro

Cholinergic associated diseases currently are major cause of neurological and neurodegenerative disabilities. As the drugs are not efficient in improving the suffered tissues, stem cell treatment is considered as an effective strategy for substituting the lost cells. In the current study, we set out to investigate the differentiation properties of human adipose-derived mensenchymal stem cells (AD-MSCs) into cholinergic-like cells by two morphogens; including retinoid acid (RA) and Sonic hedgehog (Shh) using a three- step in vitro procedure. The results were evaluated using Real-time PCR, Flowcytometry and Immunocytochemistry for two weeks. Our data showed that the cells could express cholinergic specific markers; including Islet-1, AChE, SMI-32 and Nestin at the level of mRNA and protein. We could also quantitatively evaluate the expression of Islet-1, AChE and Nestin at 14 days post- induction using flowcytometry. It is concluded that human AD-MSCs are potent type cell to differentiate into cholinergic like cells in the presence of RA and Shh through a three- step protocol; thus it could be a suitable cell candidate for regeneration of cholinergic associated diseases; however, more functional and electrophysiological analysis are needed.