BVT.2733, a Selective 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor, Attenuates Obesity and Inflammation in Diet-Induced Obese Mice

BACKGROUND: Inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is being pursued as a new therapeutic approach for the treatment of obesity and metabolic syndrome. Therefore, there is an urgent need to determine the effect of 11β-HSD1 inhibitor, which suppresses glucocorticoid action, on adipose tissue inflammation. The purpose of the present study was to examine the effect of BVT.2733, a selective 11β-HSD1 inhibitor, on expression of pro-inflammatory mediators and macrophage infiltration in adipose tissue in C57BL/6J mice. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6J mice were fed with a normal chow diet (NC) or high fat diet (HFD). HFD treated mice were then administrated with BVT.2733 (HFD+BVT) or vehicle (HFD) for four weeks. Mice receiving BVT.2733 treatment exhibited decreased body weight and enhanced glucose tolerance and insulin sensitivity compared to control mice. BVT.2733 also down-regulated the expression of inflammation-related genes including monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α) and the number of infiltrated macrophages within the adipose tissue in vivo. Pharmacological inhibition of 11β-HSD1 and RNA interference against 11β-HSD1 reduced the mRNA levels of MCP-1 and interleukin-6 (IL-6) in cultured J774A.1 macrophages and 3T3-L1 preadipocyte in vitro. CONCLUSIONS/SIGNIFICANCE: These results suggest that BVT.2733 treatment could not only decrease body weight and improve metabolic homeostasis, but also suppress the inflammation of adipose tissue in diet-induced obese mice. 11β-HSD1 may be a very promising therapeutic target for obesity and associated disease

Targeted apoptosis in ovarian cancer cells through mitochondrial dysfunction in response to Sambucus nigra agglutinin

Ovarian carcinoma (OC) patients encounter the severe challenge of clinical management owing to lack of screening measures, chemoresistance and finally dearth of non-toxic therapeutics. Cancer cells deploy various defense strategies to sustain the tumor microenvironment, among which deregulated apoptosis remains a versatile promoter of cancer progression. Although recent research has focused on identifying agents capable of inducing apoptosis in cancer cells, yet molecules efficiently breaching their survival advantage are yet to be classified. Here we identify lectin, Sambucus nigra agglutinin (SNA) to exhibit selectivity towards identifying OC by virtue of its specific recognition of α-2, 6-linked sialic acids. Superficial binding of SNA to the OC cells confirm the hyper-sialylated status of the disease. Further, SNA activates the signaling pathways of AKT and ERK1/2, which eventually promotes de-phosphorylation of dynamin-related protein-1 (Drp-1). Upon its translocation to the mitochondrial fission loci Drp-1 mediates the central role of switch in the mitochondrial phenotype to attain fragmented morphology. We confirmed mitochondrial outer membrane permeabilization resulting in ROS generation and cytochrome-c release into the cytosol. SNA response resulted in an allied shift of the bioenergetics profile from Warburg phenotype to elevated mitochondrial oxidative phosphorylation, altogether highlighting the involvement of mitochondrial dysfunction in restraining cancer progression. Inability to replenish the SNA-induced energy crunch of the proliferating cancer cells on the event of perturbed respiratory outcome resulted in cell cycle arrest before G2/M phase. Our findings position SNA at a crucial juncture where it proves to be a promising candidate for impeding progression of OC. Altogether we unveil the novel aspect of identifying natural molecules harboring the inherent capability of targeting mitochondrial structural dynamics, to hold the future for developing non-toxic therapeutics for treating OC

Application of isothermal titration calorimetry in evaluation of protein–nanoparticle interactions

Nanoparticles (NPs) offer a number of advantages over small organic molecules for controlling protein behaviour inside the cell. Protein binding to the surface of NPs depends on their surface characteristics, composition and method of preparation (Mandal et al. in J Hazard Mater 248–249:238–245, 2013). It is important to understand the binding affinities, stoichiometries and thermodynamical parameters of NP–protein interactions in order to see which interaction will have toxic and hazardous consequences and thus to prevent it. On the other side, because proteins are on the brink of stability, they may experience interactions with some types of NPs that are strong enough to cause denaturation or significantly change their conformations with concomitant loss of their biological function. Structural changes in the protein may cause exposure of new antigenic sites, “cryptic” peptide epitopes, potentially triggering an immune response which can promote autoimmune disease (Treuel et al. in ACS Nano 8(1):503–513, 2014). Mechanistic details of protein structural changes at NP surface have still remained elusive. Understanding the formation and persistence of the protein corona is critical issue; however, there are no many analytical methods which could provide detailed information about the NP–protein interaction characteristics and about protein structural changes caused by interactions with nanoparticles. The article reviews recent studies in NP–protein interactions research and application of isothermal titration calorimetry (ITC) in this research. The study of protein structural changes upon adsorption on nanoparticle surface and application of ITC in these studies is emphasized. The data illustrate that ITC is a versatile tool for evaluation of interactions between NPs and proteins. When coupled with other analytical methods, it is important analytical tool for monitoring conformational changes in proteins

KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumour markers.

Background:Mutations in the Kirsten Ras (KRAS) oncogene are common in colorectal cancer (CRC). The role of KRAS-mutation status as a prognostic factor, however, is unclear. We evaluated the relationship between KRAS-mutation status and CRC survival, considering heterogeneity in this association by tumour and patient characteristics.Methods:The population-based study included individuals diagnosed with CRC between 1998-2007 in Western Washington State. Tumour specimens were tested for KRAS exon 2 mutations, the BRAF p.V600E mutation, and microsatellite instability (MSI). We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between KRAS-mutation status and disease-specific and overall survival. Stratified analyses were conducted by age, sex, tumour site, stage, and MSI. We conducted additional analyses combining KRAS-mutation, BRAF-mutation, and MSI status.Results:Among 1989 cases, 31% had KRAS-mutated CRC. Kirsten Ras (KRAS)-mutated CRC was associated with poorer disease-specific survival (HR=1.37, 95% CI: 1.13-1.66). This association was not evident in cases who presented with distant-stage CRC. Cases with KRAS-wild-type/BRAF-wild-type/MSI-high CRC had the most favourable prognosis; those with CRC exhibiting a KRAS- or BRAF-mutation and no MSI had the poorest prognosis. Patterns were similar for overall survival.Conclusion:Kirsten Ras (KRAS)-mutated CRC was associated with statistically significantly poorer survival after diagnosis than KRAS-wild-type CRC

Sarcomeric Pattern Formation by Actin Cluster Coalescence

Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells

Digital IAPT: the effectiveness & cost-effectiveness of internet-delivered interventions for depression and anxiety disorders in the Improving Access to Psychological Therapies programme: study protocol for a randomised control trial

BACKGROUND: Depression and anxiety are common mental health disorders worldwide. The UK's Improving Access to Psychological Therapies (IAPT) programme is part of the National Health Service (NHS) designed to provide a stepped care approach to treating people with anxiety and depressive disorders. Cognitive Behavioural Therapy (CBT) is widely used, with computerised and internet-delivered cognitive behavioural therapy (cCBT and iCBT, respectively) being a suitable IAPT approved treatment alternative for step 2, low- intensity treatment. iCBT has accumulated a large empirical base for treating depression and anxiety disorders. However, the cost-effectiveness and impact of these interventions in the longer-term is not routinely assessed by IAPT services. The current study aims to evaluate the clinical and cost-effectiveness of internet-delivered interventions for symptoms of depression and anxiety disorders in IAPT. METHODS: The study is a parallel-groups, randomised controlled trial examining the effectiveness and cost-effectiveness of iCBT interventions for depression and anxiety disorders, against a waitlist control group. The iCBT treatments are of 8 weeks duration and will be supported by regular post-session feedback by Psychological Wellbeing Practitioners. Assessments will be conducted at baseline, during, and at the end of the 8-week treatment and at 3, 6, 9, and 12-month follow-up. A diagnostic interview will be employed at baseline and 3-month follow-up. Participants in the waitlist control group will complete measures at baseline and week 8, at which point they will receive access to the treatment. All adult users of the Berkshire NHS Trust IAPT Talking Therapies Step 2 services will be approached to participate and measured against set eligibility criteria. Primary outcome measures will assess anxiety and depressive symptoms using the GAD-7 and PHQ-9, respectively. Secondary outcome measures will allow for the evaluation of long-term outcomes, mediators and moderators of outcome, and cost-effectiveness of treatment. Analysis will be conducted on a per protocol and intention-to-treat basis. DISCUSSION: This study seeks to evaluate the immediate and longer-term impact, as well as the cost effectiveness of internet-delivered interventions for depression and anxiety. This study will contribute to the already established literature on internet-delivered interventions worldwide. The study has the potential to show how iCBT can enhance service provision, and the findings will likely be generalisable to other health services. TRIAL REGISTRATION: Current Controlled Trials ISRCTN ISRCTN91967124. DOI: https://doi.org/10.1186/ISRCTN91967124 . Web: http://www.isrctn.com/ISRCTN91967124 . Clinicaltrials.gov : NCT03188575. Trial registration date: June 8, 2017 (prospectively registered)

Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state

DNA primase acts as a molecular brake in DNA replication

A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization. Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis. Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand