Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

High throughput screening of hydrolytic enzymes from termites using a natural substrate derived from sugarcane bagasse

Background

Short rotation coppice willow is a potential lignocellulosic feedstock in the United Kingdom and elsewhere; however, research on optimising willow specifically for bioethanol production has started developing only recently. We have used the feedstock Salix viminalis × Salix schwerinii cultivar 'Olof' in a three-month pot experiment with the aim of modifying cell wall composition and structure within the stem to the benefit of bioethanol production. Trees were treated for 26 or 43 days with tension wood induction and/or with an application of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile that is specific to secondary cell walls. Reaction wood (tension and opposite wood) was isolated from material that had received the 43-day tension wood induction treatment.

Results

Glucan content, lignin content and enzymatically released glucose were assayed. All measured parameters were altered without loss of total stem biomass yield, indicating that enzymatic saccharification yield can be enhanced by both alterations to cell wall structure and alterations to absolute contents of either glucan or lignin.

Conclusions

Final glucose yields can be improved by the induction of tension wood without a detrimental impact on biomass yield. The increase in glucan accessibility to cell wall degrading enzymes could help contribute to reducing the energy and environmental impacts of the lignocellulosic bioethanol production process.     

Sequence uniqueness and sequence variability as modulating factors of human anti-HCV humoral immune response

We recently compared the HCV polyprotein to the human proteome in order to test whether amino acid sequences unique to the virus could represent immunodominant epitopic determinants of the human humoral immune response against HCV. We identified a relatively limited number of HCV fragments with no/low similarity to the human host that represented exclusive HCV motifs. In this study, the peptides corresponding to low/zero similarity sequences were synthesized and assayed with HCV-infected sera. With different patterns, the synthetic HCV peptides corresponding to low/zero similarity sequences were found to be immunoreactive. In particular, the HCV E1 (315-323) HRMAWDMMM, HCV E2/NS1 (547-555) NWFGCTWMN, and HCV NS5 (2638-2646) YDTRCFDST sequences were immunodominant in the HCV-infected cohort under study. These three peptides correspond to sequences that are endowed with low-similarity to the human proteome, are highly conserved among various HCV strains, and have, potentially, a scarce susceptibility to proteolytic attacks. These data may be of help in defining the multiple factors which concur in the modulation of the human immune response against HCV, eventually providing information for the design of effective anti-HCV vaccines.     

Intraesophageal chemicals enhance responsiveness of upper thoracic spinal neurons to mechanical stimulation of esophagus in rats

Esophageal hypersensitivity is one of the most common causes of noncardiac chest pain in patients. In this study, we investigated whether exposure of the esophagus to acid and other chemical irritants affected activity of thoracic spinal neurons responding to esophageal distension (ED) in rats. Extracellular potentials of single thoracic (T3) spinal neurons were recorded in pentobarbital sodium-anesthetized, -paralyzed, and -ventilated male rats. ED (0.2 or 0.4 ml, 20 s) was produced by water inflation of a latex balloon placed orally into the middle thoracic region of the esophagus. The chemicals were administered via a tube that was passed through the stomach and placed in the thoracic esophagus. To irritate the esophagus, 0.2 ml of HCl (0.01 N), bradykinin (10 microg/ml), or capsaicin (10 microg/ml) were injected for 1-2 min. Only neurons excited by ED were included in this study. Results showed that intraesophageal instillation of HCl, bradykinin, and capsaicin increased activity in 3/20 (15%), 7/25 (28%), and 9/20 (45%) neurons but enhanced excitatory responses to ED in 9/17 (53%), 8/15 (53%), and 7/11 (64%) of the remaining spinal neurons, respectively. Furthermore, intraesophageal chemicals were more likely to enhance the responsiveness of low-threshold neurons than high-threshold neurons to the esophageal mechanical stimulus. Normal saline (pH 7.4, 0.2 ml) or vehicle instilled in the esophagus did not significantly affect activity or ED responses of neurons. We conclude that enhanced responses of thoracic spinal neurons to ED by the chemically challenged esophagus may provide a possible pathophysiological basis for visceral hypersensitivity in patients with gastroesophageal reflux and/or esophagitis.     

Ninety-six haploid yeast strains with individual disruptions of open reading frames between YOR097C and YOR192C, constructed for the Saccharomyces genome deletion project, have an additional mutation in the mismatch repair gene MSH3

As part of the Saccharomyces Genome Deletion Project, sets of presumably isogenic haploid and diploid strains that differed only by single gene deletions were constructed. We found that one set of 96 strains (containing deletions of ORFs located between YOR097C and YOR192C) in the collection, which was derived from the haploid BY4741, has an additional mutation in the MSH3 mismatch repair gene.     

Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family

By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.     

Genome scans for transmission ratio distortion regions in mice

Transmission ratio distortion (TRD) is the departure from the expected genotypic frequencies under Mendelian inheritance. This departure can be due to multiple physiological mechanisms during gametogenesis, fertilization, fetal and embryonic development, and early neonatal life. Although a few TRD loci have been reported in mouse, inheritance patterns have never been evaluated for TRD. In this article, we developed a Bayesian binomial model accounting for additive and dominant deviation TRD mechanisms. Moreover, this model was used to perform genome-wide scans for TRD quantitative trait loci (QTL) on six F2 mouse crosses involving between 296 and 541 mice and between 72 and 1854 genetic markers. Statistical significance of each model was checked at each genetic marker with Bayes factors. Genome scans revealed overdominance TRD QTL located in mouse chromosomes 1, 2, 12, 13, and 14 and additive TRD QTL in mouse chromosomes 2, 3, and 15, although these results did not replicate across mouse crosses. This research contributes new statistical tools for the analysis of specific genetic patterns involved in TRD in F2 populations, our results suggesting a relevant incidence of TRD phenomena in mouse with important implications for both statistical analyses and biological research.     

ApoB-containing lipoproteins regulate angiogenesis by modulating expression of VEGF receptor 1

Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.     

Regulation of brain tumor dispersal by NKCC1 through a novel role in focal adhesion regulation

Glioblastoma (GB) is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1) can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF), which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1) through the regulation of focal adhesion dynamics and cell contractility and (2) through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms.