The antidiabetic actions of [A14K]PGLa-AM1, an analog of peptide glycine-leucine-amide-AM1 isolated from skin secretions of the octoploid frog Xenopus amieti, were investigated in genetically diabetic-obese db/db mice. Twice daily administration of [A14K]PGLa-AM1 (75 nmol/kg body weight) for 28 days significantly (P < 0.05) decreased circulating blood glucose and HbA1c and increased plasma insulin concentrations leading to improvements in glucose tolerance. The elevated levels of triglycerides, LDL and cholesterol associated with the db/db phenotype were significantly reduced by peptide administration. Elevated plasma alanine transaminase, aspartic acid transaminase, and alkaline phosphatase activities and creatinine concentrations were also significantly decreased. Peptide treatment increased pancreatic insulin content and improved the responses of isolated islets to established insulin secretagogues. No significant changes in islet β-cell and α-cell areas were observed in [A14K]PGLa-AM1 treated mice but the loss of large and medium-size islets was prevented. Peptide administration resulted in a significant (P < 0.01) increase in islet expression of the gene encoding Pdx-1, a major transcription factor in islet cells determining β-cell survival and function, resulting in increased expression of genes involved with insulin secretion (Abcc8, Kcnj11, Slc2a2, Cacn1c) together with the genes encoding the incretin receptors Glp1r and Gipr. In addition, the elevated expression of insulin signalling genes (Slc2a4, Insr, Irs1, Akt1, Pik3ca, Ppm1b) in skeletal muscle associated with the db/db phenotype was downregulated by peptide treatment These data suggest that the anti-diabetic properties of [A14K]PGLa-AM1 are mediated by molecular changes that enhance both the secretion and action of insulin.

There is growing interest in assessing possible immunotoxicological effects in anesthetized patients. There are controversial findings concerning the effect of nitrous oxide (N2O) anesthetic gas effect on inflammatory response. We tested the hypothesis that N2O associated with desflurane (inhalational anesthetic) was likely to worsen neuro-immune-endocrine effects when compared with desflurane alone in this randomized trial. The primary endpoint of this study was to evaluate the systemic proinflammatory interleukin (IL)-6, and the secondary endpoints included other systemic (IL-1β, TNF-α, IL-8, IL-10, IL-17A and high-sensitivity C-reactive protein - hs-CRP) and genetic inflammatory markers (NF-kB, IL-6 and COX-2) as well as hormones (adrenocorticotropic hormone, cortisol and prolactin) comparing patients undergoing minor surgery with or without N2O-desflurane. As a second aim, we assessed whether there were changes in the neuro-immune-endocrine profiles within each group. Blood samples were collected before anesthesia, 90 min after anesthesia induction, and the day after surgery. We assessed serum cytokines using a cytometric bead array and hs-CRP by chemiluminescent immunoassay. Expression of three proinflammatory transcripts was assessed by real-time quantitative polymerase chain reaction, and neuroendocrine hormones were detected by chemiluminescent microparticle immunoenzymatic assay. There were no significant between-group differences for any analyzed biomarkers. However, there was a significant increase in: (a) systemic IL-6 and hs-CRP values one day after surgery in both groups and (b) prolactin levels in the intraoperative period compared to baseline and postoperative period levels for both groups. In conclusion, N2O does not impair the inflammatory profile and neuroendocrine response compared to patients who receive only desflurane anesthesia.

Regulation of gene transcription is an essential mechanism for differentiation and adaptation of organisms. A key actor in this regulation process is the repressor element 1 (RE1)-silencing transcription factor (REST), a transcriptional repressor that controls more than 2000 putative target genes, most of which are neuron-specific. With the purpose of modulating REST expression, we exploited synthetic, ad hoc designed, RNA binding proteins (RBPs) able to specifically target and dock to REST mRNA. Among the various families of RBPs, we focused on the Pumilio and FBF (PUF) proteins, present in all eukaryotic organisms and controlling a variety of cellular functions. Here, a combined experimental and computational approach was used to design and test 8- and 16-repeat PUF proteins specific for REST mRNA. We explored the conformational properties and atomic features of the PUF-RNA recognition code by Molecular Dynamics simulations. Biochemical assays revealed that the 8- and 16-repeat PUF-based variants specifically bind the endogenous REST mRNA without affecting its translational regulation. The data also indicate a key role of stacking residues in determining the binding specificity. The newly characterized REST-specific PUF-based constructs act as excellent RNA-binding modules and represent a versatile and functional platform to specifically target REST mRNA and modulate its endogenous expression.

BACKGROUND: Linking genetic risk loci identified by genome-wide association studies (GWAS) to their causal genes remains a major challenge. Disease-associated genetic variants are concentrated in regions containing regulatory DNA elements, such as promoters and enhancers. Although researchers have previously published DNA maps of these regulatory regions for kidney tubule cells and glomerular endothelial cells, maps for podocytes and mesangial cells have not been available.
METHODS: We generated regulatory DNA maps (DNase-seq) and paired gene expression profiles (RNA-seq) from primary outgrowth cultures of human glomeruli that were composed mainly of podocytes and mesangial cells. We generated similar datasets from renal cortex cultures, to compare with those of the glomerular cultures. Because regulatory DNA elements can act on target genes across large genomic distances, we also generated a chromatin conformation map from freshly isolated human glomeruli.
RESULTS: We identified thousands of unique regulatory DNA elements, many located close to transcription factor genes, which the glomerular and cortex samples expressed at different levels. We found that genetic variants associated with kidney diseases (GWAS) and kidney expression quantitative trait loci were enriched in regulatory DNA regions. By combining GWAS, epigenomic, and chromatin conformation data, we functionally annotated 46 kidney disease genes.
CONCLUSIONS: We demonstrate a powerful approach to functionally connect kidney disease-/trait-associated loci to their target genes by leveraging unique regulatory DNA maps and integrated epigenomic and genetic analysis. This process can be applied to other kidney cell types and will enhance our understanding of genome regulation and its effects on gene expression in kidney disease.

Long noncoding RNAs (lncRNAs) have been implicated in many biological processes. However, due to the unique nature of lncRNAs and the consequential difficulties associated with their characterization, there is a growing disparity between the rate at which lncRNAs are being discovered and the assignment of biological function to these transcripts. Here we present a molecular biology toolbox equipped to help dissect aspects of lncRNA biology and reveal functionality. We outline an approach that begins with a broad survey of genome-wide, high-throughput datasets to identify potential lncRNA candidates and then narrow the focus on specific methods that are well suited to interrogate the transcripts of interest more closely. This involves the use of imaging-based strategies to validate these candidates and observe the behaviors of these transcripts at single molecule resolution in individual cells. We also describe the use of gene editing tools and interactome capture techniques to interrogate functionality and infer mechanism, respectively. With the emergence of lncRNAs as important molecules in healthy and diseased cellular function, it remains crucial to deepen our understanding of their biology.

Background: Vitamin D deficiency, defined as a serum 25-hydroxyvitamin D [25(OH)D] concentration <20 ng/mL, is correlated with a more atherogenic lipid profile. However, oral vitamin D supplementation does not lower LDL-cholesterol concentrations or raise HDL-cholesterol concentrations. This uncoupling between association and causation may result from a failure of oral vitamin D to mimic the effect of dermally synthesized vitamin D in response to ultraviolet type B (UVB) light.Objective: We tested the hypothesis that, in vitamin D-deficient adults, the replenishment of vitamin D with UVB exposure would lower LDL-cholesterol concentrations compared with the effect of oral vitamin D3 supplementation.Design: We performed a randomized clinical trial in vitamin D-deficient adults and compared vitamin D replenishment between subjects who received oral vitamin D3 (n = 60) and those who received narrow-band UVB exposure (n = 58) ≤6 mo.Results: There was no difference in the change from baseline LDL-cholesterol concentrations between oral vitamin D3 and UVB groups (difference in median of oral vitamin D3 minus that of UVB: 1.5 mg/dL; 95% CI: -5.0, 7.0 mg/dL). There were also no differences within groups or between groups for changes in total or HDL cholesterol or triglycerides. Transcriptional profiling of skin and blood, however, revealed significant upregulation of immune pathway signaling with oral vitamin D3 but significant downregulation with UVB.Conclusions: Correcting vitamin D deficiency with either oral vitamin D3 or UVB does not improve the lipid profile. Beyond cholesterol, these 2 modalities of raising 25(OH)D have disparate effects on gene transcription. This trial was registered at clinicaltrials.gov as NCT01688102.