RELi protocol: Optimization for protein extraction from white, brown and beige adipose tissues.

Global obesity rates have reached pandemic proportions, increasing the risk of metabolic complications for hundreds of millions of individuals worldwide. Gaining insight on adipose tissue biology and understanding how fat pads behave during obesity is critical to investigate metabolic syndromes. Elucidation of cellular signaling pathways engaged by adipose tissue both in health and disease requires standardized protocols for protein extraction that yield consistently pure samples. A recurrent problem of currently available protocols is lipid or detergent contamination in extracted protein samples, which renders protein quantification inaccurate and, as a consequence, consistency and reproducibility of protein loading become unreliable. To overcome this problem, we improved the process of adipose tissue protein extraction by improving tissue lysis and decreasing lipid contamination. Here we describe the Removal of Excess Lipids (RELi) protocol to obtain increased yields of total proteins extracted from adipose tissue. The RELi protocol allows accurate and reproducible adipose tissue sample preparation for Western blot analysis and other investigative techniques requiring adipose tissue-derived proteins.

Circulating miRNAs as sensitive and specific biomarkers for the diagnosis and monitoring of human diseases: promises and challenges.

The regulation and modulation of gene expression has been a central focus of modern biomedical research ever since the first molecular elucidation of DNA. The cellular mechanisms by which genes are expressed and repressed hold valuable insight for maintaining tissue homeostasis or conversely provide mechanistic understanding of disease progression. Hence, the discovery of the first miRNA in humans roughly a decade ago profoundly shook the previously established dogmas of gene regulation. Since, these small RNAs of around 20 nucleotides have unquestionably influenced almost every area of medical research. This momentum has now spread to the clinical arena. Hundreds of papers have already been published shedding light on the mechanisms of action of miRNAs, their profound stability in almost every bodily fluid and relating their presence to disease state and severity of disease progression. In this review, we explore the diagnostic potential of miRNAs in the clinical laboratory with a focus on studies reporting the detection of miRNAs in blood and urine for investigation of human disease. Sensitivities, specificities, areas under the curve, group descriptions and miRNAs of interest for 69 studies covering a broad range of diseases are provided. We discuss the practicality of miRNAs in the screening, diagnosis and prognosis of a range of pathologies. Characteristics and pitfalls of miRNA detection in blood are also discussed. The topics covered here are pertinent in the design of future miRNA-based detection strategies for use in clinical biochemistry laboratory settings.

Omega-3 polyunsaturated fatty acids preserve retinal function in type 2 diabetic mice.

OBJECTIVE: Diabetic retinopathy (DR) is associated with hyperglycemia-driven microvascular pathology and neuronal compromise in the retina. However, DR is also linked to dyslipidemia. As omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are protective in proliferative retinopathy, we investigated the capacity of ω-3PUFAs to preserve retinal function in a mouse model of type 2 diabetes mellitus (T2DM). DESIGN: Male leptin-receptor-deficient (db/db) mice were maintained for 22 weeks (4 weeks-26 weeks of life) on calorically and compositionally matched diets, except for 2% enrichment in either ω-3 or ω-6PUFAs. Visual function was assessed at 9, 14 and 26 weeks by electroretinography. Retinal capillary and neuronal integrity, as well as glucose challenge responses, were assessed on each diet. RESULTS: The ω-3PUFA diet significantly preserved retinal function in the mouse model of T2DM to levels similar to those observed in nondiabetic control mice on normal chow. Conversely, retinal function gradually deteriorated in db/db mice on a ω-6PUFA-rich diet. There was also an enhanced ability of ω-3PUFA-fed mice to respond to glucose challenge. The protection of visual function appeared to be independent of cytoprotective or anti-inflammatory effects of ω-3PUFAs. CONCLUSION: This study identifies beneficial effects of dietary ω-3PUFAs on visual function in T2DM. The data are consistent with dyslipidemia negatively impacting retinal function. As ω-3PUFA lipid dietary interventions are readily available, safe and inexpensive, increasing ω-3PUFA intake in diabetic patients may slow the progression of vision loss in T2DM.

Computer-aided quantification of retinal neovascularization.

Rodent models of retinal angiogenesis play a pivotal role in angiogenesis research. These models are a window to developmental angiogenesis, to pathological retinopathy, and are also in vivo tools for anti-angiogenic drug screening in cancer and ophthalmic research. The mouse model of oxygen-induced retinopathy (OIR) has emerged as one of the leading in vivo models for these purposes. Many of the animal studies that laid the foundation for the recent breakthrough of anti-angiogenic treatments into clinical practice were performed in the OIR model. However, readouts from the OIR model have been time-consuming and can vary depending on user experience. Here, we present a computer-aided quantification method that is characterized by (i) significantly improved efficiency, (ii) high correlation with the established hand-measurement protocols, and (iii) high intra- and inter-individual reproducibility of results. This method greatly facilitates quantification of retinal angiogenesis while at the same time increasing lab-to-lab reproducibility of one of the most widely used in vivo models in angiogenesis research.

VRQ397 (CRAVKY): a novel noncompetitive V2 receptor antagonist.

Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC(50) = 0.69 +/- 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-L-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI(2) generation but not that of cAMP or recruitment of beta-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.

Down-regulation of human DNA-(cytosine-5) methyltransferase induces cell cycle regulators p16(ink4A) and p21(WAF/Cip1) by distinct mechanisms.

A common event in the development of human neoplasia is the loss of growth regulatory tumor suppressor functions. Methylation of 5' CpG islands of tumor suppressor genes and elevated levels of the DNA-(cytosine-5)-methyltransferase enzyme (DNA MeTase) are also prevalent features of human neoplasia. However, direct evidence that elevated DNA MeTase levels alter gene expression and influence oncogenesis has been difficult to obtain, in part due to the lack of specific DNA MeTase inhibitors. Here we show that specific reduction of cellular DNA MeTase levels in human cancer cells with potent antisense inhibitors: 1) causes demethylation of the p16(ink4A) gene promoter; 2) causes re-expression of the p16(ink4A) protein; 3) leads to accumulation of the hypophosphorylated form of the retinoblastoma protein (pRb); and 4) inhibits cell proliferation. Stepwise reduction of cellular DNA MeTase protein levels also induced a corresponding rapid increase in the cell cycle regulator p21(WAF/Cip1) protein demonstrating a regulatory link between DNA MeTase and the growth regulator p21(WAF/Cip1) that is independent of methylation of DNA. These results suggest that the elevated levels of DNA MeTase seen in cancer cells can inhibit tumor suppressors by distinct mechanisms involving either transcriptional inactivation through DNA methylation or by a methylation independent regulation.