Differentiation of mesenchymal stem cells derived from pancreatic islets and bone marrow into islet-like cell phenotype

Background

Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself.

Methodology/Principal Findings

In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin.

Conclusions/Significance

Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.     

The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis

Background

GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation.

Aims

To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis.

Methods

Colitis was induced in wild type and GP-BAR1^−/− mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies.

Results

GP-BAR1^−/− mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn''s disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1.

Conclusions

GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn''s disease. Ciprofloxacin is a GP-BAR1 ligand.     

Probiotics modulate intestinal expression of nuclear receptor and provide counter-regulatory signals to inflammation-driven adipose tissue activation

Background

Adipocytes from mesenteric white adipose tissue amplify the inflammatory response and participate in inflammation-driven immune dysfunction in Crohn''s disease by releasing proinflammatory mediators. Peroxisome proliferator-activated receptors (PPAR)-α and -γ, pregnane x receptor (PXR), farnesoid x receptor (FXR) and liver x-receptor (LXR) are ligand-activated nuclear receptor that provide counter-regulatory signals to dysregulated immunity and modulates adipose tissue.

Aims

To investigate the expression and function of nuclear receptors in intestinal and adipose tissues in a rodent model of colitis and mesenteric fat from Crohn''s patients and to investigate their modulation by probiotics.

Methods

Colitis was induced by TNBS administration. Mice were administered vehicle or VSL#3, daily for 10 days. Abdominal fat explants obtained at surgery from five Crohn''s disease patients and five patients with colon cancer were cultured with VSL#3 medium.

Results

Probiotic administration attenuated development of signs and symptoms of colitis, reduced colonic expression of TNFα, IL-6 and IFNγ and reserved colonic downregulation of PPARγ, PXR and FXR caused by TNBS. Mesenteric fat depots isolated from TNBS-treated animals had increased expression of inflammatory mediators along with PPARγ, FXR, leptin and adiponectin. These changes were prevented by VSL#3. Creeping fat and mesenteric adipose tissue from Crohn''s patients showed a differential expression of PPARγ and FXR with both tissue expressing high levels of leptin. Exposure of these tissues to VSL#3 medium abrogates leptin release.

Conclusions

Mesenteric adipose tissue from rodent colitis and Crohn''s disease is metabolically active and shows inflammation-driven regulation of PPARγ, FXR and leptin. Probiotics correct the inflammation-driven metabolic dysfunction.     

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins

Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.     

FLO11-based model for air-liquid interfacial biofilm formation by Saccharomyces cerevisiae

Sardinian wine strains of Saccharomyces cerevisiae used to make sherry-like wines form a biofilm at the air-liquid interface at the end of ethanolic fermentation, when grape sugar is depleted and further growth becomes dependent on access to oxygen. Here, we show that FLO11, which encodes a hydrophobic cell wall glycoprotein, is required for the air-liquid interfacial biofilm and that biofilm cells have a buoyant density greater than the suspending medium. We propose a model for biofilm formation based on an increase in cell surface hydrophobicity occurring at the diauxic shift. This increase leads to formation of multicellular aggregates that effectively entrap carbon dioxide, providing buoyancy. A visible biofilm appears when a sufficient number of hydrophobic cell aggregates are carried to and grow on the liquid surface.     

Evolution of an acylase active on cephalosporin C

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ~90% conversion of cephalosporin C to 7-amino-cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.     

A bunch-oligonucleotide forming stable monomolecular quadruplex containing a T-tetrad

The chemical synthesis of bunch-ODN I and II prone to form quadruplex structures containing G-and T-tetrads has been reported. Structural studies were performed by 1H-NMR and CD melting experiments.     

Effects of acrolein on the quadruplex forming d(TTAGGG)4 telomeric repeat sequence

HPLC and ESI-MS analysis have been used to investigate the effect of acrolein exposure on d(TITAGGG)4 human telomeric repeat. Preliminary results disclosed a novel relationship between the structure assumed by oligodeoxynucleotides (ODNs) and the capability of their nucleobase residues to react with acrolein.     

Unusual monomolecular DNA quadruplex structures using bunch-oligonucleotides

The chemical synthesis of several G-rich bunch-oligonucleotides and the structural characterization of the corresponding monomolecular G-quadruplexes (I-IV) have been reported. The synthetic method allow the achievement of monomolecular DNA quadruplex structures having unusual and predeterminable oligodeoxyribonucleotide (ODN) strand orientation.     

Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists

A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a Solid-Phase Extraction column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post exercise versus pre exercise condition in cyclists (F: 136+/-93 nmol/l versus 67+/-50 nmol/l (p<0.001); E: 797+/-400 nmol/l versus 408+/-252 nmol/l (p<0.001)). The F/E ratio was 0.18+/-0.11 versus 0.16+/-0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11+/-0.07) and cyclist individuals at rest (p<0.05).