Expression of the stress-response regulators CtsR and HrcA in the uropathogen Staphylococcus saprophyticus during heat shock.

The uropathogen Staphylococcus saprophyticus is an ubiquitous bacterium but little is known about mechanisms that allow its persistence in diverse environments. Here we evaluated S. saprophyticus growth and survival during heat shock, the expression of stress response regulators ctsR and hrcA through qRT-PCR and heat shock protein synthesis through 35S-Met metabolic labeling. S. saprophyticus does not tolerate temperatures much higher than the optimal 37 °C, as its growth is greatly affected at 42 °C, though viability is maintained up to 48 °C. At 42 °C, the expression of ctsR and hrcA repressor genes approximately triple when compared to 37 °C and continue to increase together with temperature till 48 °C. Expression of hrcA peaks after 20 min of heat shock and decreases significantly after 30 min, indicating that heat stress response regulated by this gene may last 20-30 min. An increase in temperature is accompanied by the synthesis of at least eight proteins, three of which are likely the chaperones DnaK, GroEL and ClpB. In silico analysis indicate that the groEL gene may be regulated by HrcA, clpB by CtsR and dnaK by both repressors. This is the first work to discuss heat stress response in S. saprophyticus and a step forward in the understanding of mechanisms that make this a widespread and emergent pathogen.

Evidences for the involvement of cell surface glycans in stem cell pluripotency and differentiation.

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development, several issues regarding their pluripotency, differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study, we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that, upon spontaneous differentiation, iPS-MBMC present high amounts of terminal ß-galactopyranoside residues, pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation, prompting an ectoderm commitment. Exposed ß-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface, which could modulate intercellular or intracellular interactions. Together, our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and, therefore, the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation.

Modeling and rescue of the vascular phenotype of Williams-Beuren syndrome in patient induced pluripotent stem cells.

Elastin haploinsufficiency in Williams-Beuren syndrome (WBS) leads to increased vascular smooth muscle cell (SMC) proliferation and stenoses. Our objective was to generate a human induced pluripotent stem (hiPS) cell model for in vitro assessment of the WBS phenotype and to test the ability of candidate agents to rescue the phenotype. hiPS cells were reprogrammed from skin fibroblasts of a WBS patient with aortic and pulmonary stenosis and healthy control BJ fibroblasts using four-factor retrovirus reprogramming and were differentiated into SMCs. Differentiated SMCs were treated with synthetic elastin-binding protein ligand 2 (EBPL2) (20 µg/ml) or the antiproliferative drug rapamycin (100 nM) for 5 days. We generated four WBS induced pluripotent stem (iPS) cell lines that expressed pluripotency genes and differentiated into all three germ layers. Directed differentiation of BJ iPS cells yielded an 85%-92% pure SMC population that expressed differentiated SMC markers, were functionally contractile, and formed tube-like structures on three-dimensional gel assay. Unlike BJ iPS cells, WBS iPS cells generated immature SMCs that were highly proliferative, showed lower expression of differentiated SMC markers, reduced response to the vasoactive agonists, carbachol and endothelin-1, impaired vascular tube formation, and reduced calcium flux. EBPL2 partially rescued and rapamycin fully rescued the abnormal SMC phenotype by decreasing the smooth muscle proliferation rate and enhancing differentiation and tube formation. WBS iPS cell-derived SMCs demonstrate an immature proliferative phenotype with reduced functional and contractile properties, thereby recapitulating the human disease phenotype. The ability of rapamycin to rescue the phenotype provides an attractive therapeutic candidate for patients with WBS and vascular stenoses.

Human menstrual blood-derived mesenchymal cells as a cell source of rapid and efficient nuclear reprogramming.

Induced pluripotent stem cells (iPSCs) were originally generated by forced ectopic expression of four transcription factors genes-OCT4, KLF4, SOX2, and c-MYC-in fibroblasts. However, the efficiency of iPSCs obtention is extremely low, and reprogramming takes about 20 days. We reasoned that adult cells showing basal expression of core embryonic stem (ES) cell regulator genes could be a better cell source for reprogramming. Menstrual blood-derived mesenchymal cells (MBMCs) are multipotent cells that show detectable levels of some of the core ES cells regulators. The aim of this study was to determine whether reprogramming efficiency could be increased by using MBMCs as a cell source to generate iPSCs. MBMCs were transduced with recombinant retroviruses expressing the coding regions of OCT4, SOX2, and KLF4 genes. Cells with high nucleus/cytoplasm ratio can be detected about 5 days of posttransduction, and colonies of typical ES-like cells begun to appear after 7 days. At day 15, colonies were picked up and expanded for characterization. Most of the clones were morphologically identical to ES cells and positive at the mRNA and protein levels for all pluripotency markers tested. The clones are capable of forming embryoid bodies and to differentiate in vitro into cells of the three germ cell layers. Our results show that the reprogramming was faster and with efficiency around 2-5%, even in the absence of ectopic expression of c-MYC. To date, this is the first study showing MBMCs as a cell source for nuclear reprogramming.