Vascular Calcification in Rat Cultured Smooth Muscle Cells: a Role for Nitric Oxide

The underlying inflammatory storm in renal or diabetic disease may induce expression of inducible nitric oxide synthase (iNOS). Similarly, expression of iNOS or nitric oxide (NO) production in vascular smooth muscle cells (VSMCs) in a calcifying environment, may promote vascular calcification (VC) (Zaragoza et al., 2006). However, emerging data suggests that NO generated by either endothelial nitric oxide synthase (eNOS) or iNOS may protect VSMCs from VC (Kanno et al., 2008). Thus, the role of NO and its associated enzymes in the development of VC is unclear. The aim of this study was to identify whether NO produced by iNOS regulates calcification in VSMCs, and to further understanding of potential mechanisms that may mediate the actions of NO/iNOS. A significant and sustained production of NO by iNOS, which peaked at day 3 and declined thereafter was found in rat aortic smooth muscle cells (RASMCs) that were preactivated with lipopolysaccharide (LPS; 100μg ml-1) and interferon gamma (IFN-γ;100U ml-1) in the presence of calcification buffer (CB) containing calcium chloride (CaCl2; 7mM) and β-glycerophosphate (β-GP; 7mM). This was associated with formation of hydroxyapatite crystals (HA) or calcification plaques, observed via alizarin red staining (ARS) and/or fourier transform infrared (FT-IR) analysis. However, when RASMCs were incubated with the iNOS inhibitor GW274150 at 10 μM, together with LPS + IFN-γ + CB, HA crystal formation was abolished. When RASMCs were pretreated with diethylenetriamine/nitric oxide adduct (NOC 18) at either 30 or 50 μM for an hour prior to addition of CB, to generate NO; calcium levels were elevated leading to form HA crystals. However, the elevation of calcium caused by the presence of NO generated via iNOS, did not result in phosphorylation of mitogen activated protein kinases (p38 MAPK), extracellular signal-regulated kinases (Erks), and protein kinase B. Furthermore, there was a reduction of Runx2 levels (pro-calcific factor) which could be another pro-calcific factor involved in this mechanism. These findings suggest that NO may indeed play a fundamental role in calcification, enhancing mineralisation of smooth muscle cells. Furthermore, the expression of iNOS/ NO appears to be enhanced under conditions that favour calcification and these together may contribute to enhanced calcification with potential detrimental consequences in vivo

Harmful versus beneficial effects of using short-term combined oral antidiabetic therapy: ‎An open label comparative clinical trial ‎

Background: Although oral antidiabetic drugs have many beneficial pleiotropic effects, they are ‎not free from adverse reactions that may interfere with glucose homeostasis. This study aimed ‎to assess the effects of oral antidiabetic drugs as add- on-therapy to metformin, on the metabolic, ‎cardiac, and renal determinants. ‎ Material and methods: A total number of seventy-eight type 2 diabetes (T2D) patients who ‎were treated with metformin were allocated to add-on-therapy for 12 weeks, with glimepiride ‎‎(4mg/day, n=26), sitagliptin (100mg/day, n=28), and canagliflozin (300mg/day, n=24). ‎Anthropometric measurements, glycemic indices, lipid, and renal markers, were determined ‎before initiation and after the treatment. ‎ Results: All of the three treatments significantly decreased the glycemic indices, triglyceride-to-glucose index, and non-significantly altered the serum uric acid-to creatinine. Glimepiride ‎significantly increased the waist-to-height ratio (0.630±0.057 versus 0.640±0.057, p=0.040), ‎while sitagliptin and canagliflozin significantly decreased it (0.650±0.058 versus 0.640±0.054, ‎p=0.009, and 0.650±0.041 versus 0.630±0.044, p<0.001). Estimated glomerular filtration index- ‎epidemiology collaboration (ml/min/1.73m2) significantly declined by using glimepiride ‎‎(109.0±10.4 versus 103.6±10.9, p=0.001), and sitagliptin (106.1±12.4 versus 103.3±15.0, ‎p=0.013). ‎ Conclusion: Careful selection of using oral antidiabetic agents can protect T2D patients from ‎harmful events, particularly those related to cardiovascular events and renal function

Feasibility and acceptability of a technology-based, rural weight management int ervention in older adults with obesity

Background Older adults with obesity residing in rural areas have reduced access to weight management programs. We determined the feasibility, acceptability and preliminary outcomes of an integrated technology-based health promotion intervention in rural-living, older adults using remote monitoring and synchronous video-based technology. Methods A 6-month, non-randomized, non-blinded, single-arm study was conducted from October 2018 to May 2020 at a community-based aging center of adults aged ≥65 years with a body mass index (BMI) ≥30 kg/m2. Weekly dietitian visits focusing on behavior therapy and caloric restriction and twice-weekly physical therapist-led group strength, flexibility and balance training classes were delivered using video-conferencing to participants in their homes. Participants used a Fitbit Alta HR for remote monitoring with data feedback provided by the interventionists. An aerobic activity prescription was provided and monitored. Results Mean age was 72.9±3.9 years (82% female). Baseline anthropometric measures of weight, BMI, and waist circumference were 97.8±16.3 kg, 36.5±5.2 kg/m2, and 115.5±13.0 cm, respectively. A total of 142 participants were screened (n=27 ineligible), and 53 consented. There were nine dropouts (17%). Overall satisfaction with the trial (4.7+ 0.6, scale: 1 (low) to 5 (high)) and with Fitbit (4.2+ 0.9) were high. Fitbit was worn an average of 81.7±19.3% of intervention days. In completers, mean weight loss was 4.6±3.5 kg or 4.7±3.5% (p\u3c 0.001). Physical function measures of 30-s sit-to-stand repetitions increased from 13.5±5.7 to 16.7±5.9 (p\u3c 0.001), 6-min walk improved by 42.0±77.3 m (p=0.005) but no differences were observed in gait speed or grip strength. Subjective measures of late-life function improved (3.4±4.7 points, p\u3c 0.001). Conclusions A technology-based obesity intervention is feasible and acceptable to older adults with obesity and may lead to weight loss and improved physical function. Clinical trial registration Registered on Clinicaltrials.gov #NCT03104205. Registered on April 7, 2017. First participant enrolled on October 1st, 2018

Transplantation in Remission Improves the Disease-Free Survival of Patients with Advanced Myelodysplastic Syndromes Treated with Myeloablative T Cell-Depleted Stem Cell Transplants from HLA-Identical Siblings

AbstractFrom 1985 to 2004, 49 patients with advanced myelodysplastic syndromes (MDS) (≥5% blasts) or acute myeloid leukemia (AML) transformed from MDS underwent T cell depleted bone marrow or peripheral blood hematopoietic stem cell transplantation (HSCT) from HLA-identical siblings following conditioning with a myeloablative regimen that included total body irradiation (44 patients) or busulfan (5 patients). Thirty-six patients received chemotherapy (3 low dose and 33 induction doses) before conditioning, and 13 patients did not receive any chemotherapy. Prior to transplantation, 22 of the 36 treated patients were in hematologic remission; 4 were in a second refractory cytopenia phase (26 responders); 8 had failed to achieve remission; and 2 of the responders had progression or relapse of their MDS (10 failures). No post-transplantation pharmacologic prophylaxis for graft-versus-host disease (GVHD) was given. The median age was 48 yrs (range 13-61). Forty-five of the 49 patients engrafted; 2 had primary graft failure; and 2 died before engraftment. Only 3 patients developed acute GVHD (aGVHD) (grades I and III) and 1 chronic GVHD (cGVHD). At 3 yrs post-transplantation, the overall survival (OS) was 54% in the responders; 31% in the untreated group; and 0% in the failure group (P=.0004). The disease free survival (DFS) was 50%, 15% and 0% in each group respectively (P=.0008). In multivariate analysis, disease status before cytoreduction remained highly correlated with DFS (P<.001). The cumulative incidence (CI) of relapse at 2-yrs post-transplantation for the responders was 23%; for the untreated group was 38%; and for the failures was 50%. The CI of non-relapse mortality at 2-yrs post-transplantation, for the responders was 23%; for the untreated group was 38%; and for the failures was 40%. All survivors achieved a Karnofsky Performance Status (KPS) of ≥90. These results indicate that patients with advanced MDS who achieve and remain in remission or a second refractory cytopenia phase with chemotherapy before conditioning can achieve successful long-term remissions following a myeloablative T cell depleted allogeneic HSCT

Markedly Divergent Tree Assemblage Responses to Tropical Forest Loss and Fragmentation across a Strong Seasonality Gradient

We examine the effects of forest fragmentation on the structure and composition of tree assemblages within three seasonal and aseasonal forest types of southern Brazil, including evergreen, Araucaria, and deciduous forests. We sampled three southernmost Atlantic Forest landscapes, including the largest continuous forest protected areas within each forest type. Tree assemblages in each forest type were sampled within 10 plots of 0.1 ha in both continuous forests and 10 adjacent forest fragments. All trees within each plot were assigned to trait categories describing their regeneration strategy, vertical stratification, seed-dispersal mode, seed size, and wood density. We detected differences among both forest types and landscape contexts in terms of overall tree species richness, and the density and species richness of different functional groups in terms of regeneration strategy, seed dispersal mode and woody density. Overall, evergreen forest fragments exhibited the largest deviations from continuous forest plots in assemblage structure. Evergreen, Araucaria and deciduous forests diverge in the functional composition of tree floras, particularly in relation to regeneration strategy and stress tolerance. By supporting a more diversified light-demanding and stress-tolerant flora with reduced richness and abundance of shade-tolerant, old-growth species, both deciduous and Araucaria forest tree assemblages are more intrinsically resilient to contemporary human-disturbances, including fragmentation-induced edge effects, in terms of species erosion and functional shifts. We suggest that these intrinsic differences in the direction and magnitude of responses to changes in landscape structure between forest types should guide a wide range of conservation strategies in restoring fragmented tropical forest landscapes worldwide

Protocol, rationale and design of PEOPLE (Post ExpOsure Prophylaxis for LEprosy in the Comoros and Madagascar): A cluster randomized trial on effectiveness of different modalities of implementation of post-exposure prophylaxis of leprosy contacts

Background: Leprosy is an ancient infectious disease with a global annual incidence that has plateaued above 200,000 new cases since over a decade. New strategies are required to overcome this stalemate. Post-exposure prophylaxis (PEP) with a single dose of Rifampicin (SDR) has conditionally been recommended by the World Health Organization (WHO), based on a randomized-controlled-Trial in Bangladesh. More evidence is required. The Post ExpOsure Prophylaxis for Leprosy (PEOPLE) trial will assess effectiveness of different modalities of PEP on the Comoros and Madagascar. Methods: PEOPLE is a cluster-randomized trial with villages selected on previous leprosy-incidence and randomly allocated to four arms. Four annual door-To-door surveys will be performed in all arms. All consenting permanent residents will be screened for leprosy. Leprosy patients will be treated according to international guidelines and eligible contacts will be provided with SDR-PEP. Arm-1 is the comparator in which no PEP will be provided. In arms 2, 3 and 4, SDR-PEP will be provided at double the regular dose (20 mg/kg) to eligible contacts aged two years and above. In arm 2 all household-members of incident leprosy patients are eligible. In arm 3 not only household-members but also neighbourhood contacts living within 100-m of an incident case are eligible. In arm 4 such neighbourhood contacts are only eligible if they test positive to anti-PGL-I, a serological marker. Incidence rate ratios calculated between the comparator arm 1 and each of the intervention arms will constitute the primary outcome. Discussion: Different trials on PEP have yielded varying results. The pivotal COLEP trial in Bangladesh showed a 57% reduction in incidence over a two-year period post-intervention without any rebound in the following years. A study in a high-incidence setting in Indonesia showed no effect of PEP provided to close contacts but a major effect of PEP provided as a blanket measure to an entire island population. High background incidence could be the reason of the lack of effect of PEP provided to individual contacts. The PEOPLE trial will assess effectiveness of PEP in a high incidence setting and will compare three different approaches, to identify who benefits most from PEP. Trial registration: Clinicaltrials.Gov. NCT03662022. Initial Protocol Version 1.2, 27-Aug-2018

Biomarker discovery in galactosemia: Metabolomics with UPLC/HRMS in dried blood spots

Introduction:Galactosemia (GAL) is a genetic disorder that results in disturbances in galactose metabolism and can lead to life-threatening complications. However, the underlying pathophysiology of long-term complications in GAL remains poorly understood.Methods: In this study, a metabolomics approach using ultra-performance liquid chromatography coupled with high-resolution mass spectrometry was used to investigate metabolomic changes in dried blood spots of 15 patients with GAL and 39 healthy individuals.Results: The study found that 2,819 metabolites underwent significant changes in patients with GAL compared to the control group. 480 human endogenous metabolites were identified, of which 209 and 271 were upregulated and downregulated, respectively. PA (8:0/LTE4) and ganglioside GT1c (d18:0/20:0) metabolites showed the most significant difference between GAL and the healthy group, with an area under the curve of 1 and 0.995, respectively. Additionally, the study identified potential biomarkers for GAL, such as 17-alpha-estradiol-3-glucuronide and 16-alpha-hydroxy DHEA 3-sulfatediphosphate.Conclusion: This metabolomics study deepened the understanding of the pathophysiology of GAL and presented potential biomarkers that might serve as prognostic biomarkers to monitor the progression or support the clinical diagnosis of GAL

Loss of RhoB Expression Enhances the Myelodysplastic Phenotype of Mammalian Diaphanous-Related Formin mDia1 Knockout Mice

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis and hyperplastic bone marrow. Complete loss or interstitial deletions of the long arm of chromosome 5 occur frequently in MDS. One candidate tumor suppressor on 5q is the mammalian Diaphanous (mDia)-related formin mDia1, encoded by DIAPH1 (5q31.3). mDia-family formins act as effectors for Rho-family small GTP-binding proteins including RhoB, which has also been shown to possess tumor suppressor activity. Mice lacking the Drf1 gene that encodes mDia1 develop age-dependent myelodysplastic features. We crossed mDia1 and RhoB knockout mice to test whether the additional loss of RhoB expression would compound the myelodysplastic phenotype. Drf1−/−RhoB−/− mice are fertile and develop normally. Relative to age-matched Drf1−/−RhoB+/− mice, the age of myelodysplasia onset was earlier in Drf1−/−RhoB−/− animals—including abnormally shaped erythrocytes, splenomegaly, and extramedullary hematopoiesis. In addition, we observed a statistically significant increase in the number of activated monocytes/macrophages in both the spleen and bone marrow of Drf1−/−RhoB−/− mice relative to Drf1−/−RhoB+/− mice. These data suggest a role for RhoB-regulated mDia1 in the regulation of hematopoietic progenitor cells

Genomic analysis of functional haloacid-degrading gene of bacillus megaterium strain bhs1 isolated from blue lake (mavi göl, turkey)

Purpose: Bacillus megaterium strain BHS1, isolated from an alkaline water sample taken from Mavi Golu (Blue Lake, Turkey), can grow on minimal medium containing 2,2-dichloropropionic acid. We characterized this bacterium at the genomic level. Methods: The HiSeq platform was used to carry out genome sequencing, de novo assembly, and scaffolding with strain BHS1. Next, genome data were analyzed to demarcate DNA regions containing protein-coding genes and determine the function of certain BHS1 genes. Finally, results from a colorimetric chloride ion-release assay demonstrated that strain BHS1 produces dehalogenase. Results: De novo assembly of the BHS1 genomic sequence revealed a genome size of similar to 5.37 Mb with an average G+C content of 38%. The predicted nuclear genome harbors 5509 protein-coding genes, 1353 tRNA genes, 67 rRNA genes, and 6 non-coding (mRNA) genes. Genomic mapping of strain BHS1 revealed its amenability to synthesize two families of dehalogenases (Cof-type haloacid dehalogenase IIB family hydrolase and haloacid dehalogenase type II), suggesting that these enzymes can participate in the catabolism of halogenated organic acids. The mapping identified seven Na+/H+ antiporter subunits that are vital for adaptation of the bacterium to an alkaline environment. Apart from a pairwise analysis to the well-established L-2-haloacid dehalogenases, whole-cell analysis strongly suggested that the haloacid dehalogenase type II might act stereospecifically on L-2-chloropropionic acid, D,L-2-chloropropionic acid, and 2,2-dichloropropionic acid. Whole-cell studies confirmed the utilization of these three substrates and the gene's role in dehalogenation. Conclusions: To our knowledge, this is the first report of the full genome sequence for strain BHS1, which enabled the characterization of selected genes having specific metabolic activities and their roles in the biodegradation of halogenated compounds

SeqGene: a comprehensive software solution for mining exome- and transcriptome- sequencing data

Abstract Background The popularity of massively parallel exome and transcriptome sequencing projects demands new data mining tools with a comprehensive set of features to support a wide range of analysis tasks. Results SeqGene, a new data mining tool, supports mutation detection and annotation, dbSNP and 1000 Genome data integration, RNA-Seq expression quantification, mutation and coverage visualization, allele specific expression (ASE), differentially expressed genes (DEGs) identification, copy number variation (CNV) analysis, and gene expression quantitative trait loci (eQTLs) detection. We also developed novel methods for testing the association between SNP and expression and identifying genotype-controlled DEGs. We showed that the results generated from SeqGene compares favourably to other existing methods in our case studies. Conclusion SeqGene is designed as a general-purpose software package. It supports both paired-end reads and single reads generated on most sequencing platforms; it runs on all major types of computers; it supports arbitrary genome assemblies for arbitrary organisms; and it scales well to support both large and small scale sequencing projects. The software homepage is http://seqgene.sourceforge.net.</p