Visfatin is regulated by rosiglitazone in type 2 diabetes mellitus and influenced by NFκB and JNK in human abdominal subcutaneous adipocytes

Visfatin has been proposed as an insulin-mimicking adipocytokine, predominantly secreted from adipose tissue and correlated with obesity. However, recent studies suggest visfatin may act as a proinflammatory cytokine. Our studies sought to determine the significance of this adipocytokine and its potential role in the pathogenesis of T2DM. Firstly, we examined the effects of diabetic status on circulating visfatin levels, and several other adipocytokines, demonstrating that diabetic status increased visfatin*, TNF-α*** and IL-6*** compared with non-diabetic subjects (*p<0.05, **p<0.01, ***p<0.001, respectively). We then assessed the effects of an insulin sensitizer, rosiglitazone (RSG), in treatment naïve T2DM subjects, on circulating visfatin levels. Our findings showed that visfatin was reduced post-RSG treatment [vs. pre-treatment (*p<0.05)] accompanied by a reduction in HOMA-IR**, thus implicating a role for insulin in visfatin regulation. Further studies addressed the intracellular mechanisms by which visfatin may be regulated, and may exert pro-inflammatory effects, in human abdominal subcutaneous (Abd Sc) adipocytes. Following insulin (Ins) and RSG treatment, our in vitro findings highlighted that insulin (100 nM), alone, upregulated visfatin protein expression whereas, in combination with RSG (10 nM), it reduced visfatin*, IKKβ** and p-JNK1/2*. Furthermore, inhibition of JNK protein exacted a significant reduction in visfatin expression (**p<0.01), whilst NF-κB blockade increased visfatin (*p<0.05), thus identifying JNK as the more influential factor in visfatin regulation. Additional in vitro analysis on adipokines regulating visfatin showed that only Abd Sc adipocytes treated with recombinant human (rh)IL-6 increased visfatin protein (*p<0.05), whilst rh visfatin treatment, itself, had no influence on TNF-α, IL-6 or resistin secretion from Sc adipocytes. These data highlight visfatin''s regulation by insulin and RSG, potentially acting through NF-κB and JNK mechanisms, with only rh IL-6 modestly affecting visfatin regulation. Taken together, these findings suggest that visfatin may represent a pro-inflammatory cytokine that is influenced by insulin/insulin sensitivity via the NF-κB and JNK pathways.     

EpCAM Knockdown Alters MicroRNA Expression in Retinoblastoma- Functional Implication of EpCAM Regulated MiRNA in Tumor Progression

The co-ordinated regulation of oncogenes along with miRNAs play crucial role in carcinogenesis. In retinoblastoma (RB), several miRNAs are known to be differentially expressed. Epithelial cell adhesion molecule (EpCAM) gene is involved in many epithelial cancers including, retinoblastoma (RB) tumorigenesis. EpCAM silencing effectively reduces the oncogenic miR-17-92 cluster. In order to investigate whether EpCAM has wider effect as an inducer or silencer of miRNAs, we performed a global microRNA expression profile in EpCAM siRNA knockdown Y79 cells. MicroRNA profiling in EpCAM silenced Y79 cells showed seventy-three significantly up regulated and thirty-six down regulated miRNAs. A subset of these miRNAs was also validated in tumors. Functional studies on Y79 and WERI-Rb-1 cells transfected with antagomirs against two miRNAs of miR-181c and miR-130b showed striking changes in tumor cell properties in RB cells. Treatment with anti-miR-181c and miR-130b showed significant decrease in cell viability and cell invasion. Increase in caspase-3 level was noticed in antagomir transfected cell lines indicating the induction of apoptosis. Possible genes altered by EpCAM influenced microRNAs were predicted by bioinformatic tools. Many of these belong to pathways implicated in cancer. The study shows significant influence of EpCAM on global microRNA expression. EpCAM regulated miR-181c and miR-130b may play significant roles in RB progression. EpCAM based targeted therapies may reduce carcinogenesis through several miRNAs and target genes.     

Microbial Colonization of Beech and Spruce Litter—Influence of Decomposition Site and Plant Litter Species on the Diversity of Microbial Community

The present study was conducted to investigate the effect of decomposition site and plant litter species on the colonizing microbial communities. For this, litter bag technique using beech and spruce litter was combined with RNA-based fingerprinting and cloning. Litter bags were incubated for 2 and 8 weeks in the A_h horizon of beech and beech–spruce mixed forest sites. Although sugars and starch were rapidly lost, lignin content increased by more than 40% for beech and more than doubled for spruce litter at both soil sites at the end of the experiment. Denaturing gradient gel electrophoresis analysis of 16S and 18S rRNA RT–PCR products was used for screening of differences between bacterial and fungal communities colonizing the two litter types. Development of the microbial community over time was observed to be specific for each litter type and decomposition site. RT–PCR products from both litter types incubated in beech–spruce mixed forest site were also cloned to identify the bacterial and fungal colonizers. The 16S rRNA clone libraries of beech litter were dominated by γ-proteobacterial members, whereas spruce libraries were mainly composed of α-, β-, and γ-proteobacterial members. Ascomycota members dominated the 18S rRNA clone libraries. Clones similar to Zygomycota were absent from spruce, whereas those similar to Basidiomycota and Glomeromycota were absent from beech libraries. Selective effects of litter quality were observed after 8 weeks. The study provides an insight into the bacterial and fungal communities colonizing beech and spruce litter, and the importance of litter quality and decomposition site as key factors in their development and succession.     

Spatio-temporal analysis of Egyptian flower mantis Blepharopsis mendica (order: mantodea), with notes of its future status under climate change

Egyptian flower mantis Blepharopsis mendica (Order: Mantodea) is a widespread mantis species throughout the southwest Palearctic region. The ecological and geographical distribution of such interesting species is rarely known. So, through this work, habitat suitability models for its distribution through Egyptian territory were created using MaxEnt software from 90 occurrence records. One topographic (altitude) and eleven bioclimatic variables influencing the species distribution were selected to generate the models. The predicted distribution in Egypt was focused on the Delta, South Sinai, the north-eastern part of the country, and some areas in the west including Siwa Oasis. Temporal analysis between the two periods (1900–1961) and (1961–2017) show current reduction of this species distribution through Delta and its surrounding areas, may be due to urbanization. On the other hand, it increases in newly protected areas of South Sinai. Under the future climate change scenario, the MaxEnt model predicted the habitat gains for B. mendica in RCP 2.6 for 2070 and loss of habitat in RCP 8.5 for the same year. Our results can be used as a basis for conserving this species not only in Egypt, but also throughout the whole of its range, also, it show how the using of geo-information could help in studying animal ecology.     

Natriuretic Peptide Receptor-C Agonist Attenuates the Expression of Cell Cycle Proteins and Proliferation of Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats: Role of Gi Proteins and MAPkinase/PI3kinase Signaling

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit hyperproliferation and overexpression of cell cycle proteins. We earlier showed that small peptide fragments of cytoplasmic domain of natriuretic receptor-C (NPR-C) attenuate vasoactive peptide-induced hyperproliferation of VSMC. The present study investigated if C-ANP_4–23, a specific agonist of NPR-C, could attanuate the hyperproliferation of VSMC from SHR by inhibiting the overexpression of cell cycle proteins and examine the underlying signaling pathways contributing to this inhibition. The proliferation of VSMC was determined by [³H] thymidine incorporation and the expression of proteins was determined by Western blotting. The hyperproliferation of VSMC from SHR and overexpression of cyclin D1,cyclin A, cyclin E, cyclin-dependent kinase 2 (cdk2), phosphorylated retinoblastoma protein (pRb), Giα proteins and enhanced phosphorylation of ERK1/2 and AKT exhibited by VSMC from SHR were attenuated by C-ANP_4–23 to control levels. In addition, in vivo treatment of SHR with C-ANP_4–23 also attenuated the enhanced proliferation of VSMC. Furthemore, PD98059, wortmannin and pertussis toxin, the inhibitors of MAP kinase, PI3kinase and Giα proteins respectively, also attenuated the hyperproliferation of VSMC from SHR and overexpression of cell cycle proteins to control levels. These results indicate that NPR-C activation by C-ANP_4–23 attenuates the enhanced levels of cell cycle proteins through the inhibition of enhanced expression of Giα proteins and enhanced activation of MAPkinase/PI3kinase and results in the attenuation of hyperproliferation of VSMC from SHR. It may be suggested that C-ANP_4–23 could be used as a therapeutic agent in the treatment of vascular complications associated with hypertension, atherosclerosis and restenosis.     

Cholesterol Diet Withdrawal Leads to an Initial Plaque Instability and Subsequent Regression of Accelerated Iliac Artery Atherosclerosis in Rabbits

Effect of long term cholesterol diet withdrawal on accelerated atherosclerosis in iliac artery of New Zealand White (NZW) rabbits has not been explored so far. Atherosclerosis was thus induced in rabbits by a combination of balloon injury and atherogenic diet (AD) (1% cholesterol and 6% peanut oil) feeding for 8 weeks (baseline) followed by chow diet (CD) feeding for 4, 8, 16, 32, 50 and 64 weeks. The plaque characterization was done using histology, real time RT-PCR and vasoreactivity studies. Significant elevation in plasma lipids with AD feeding was normalized following 16 weeks of CD feeding. However, baseline comparison showed advanced plaque features even after 8 weeks of CD period with significant elevation in intima/media thickness ratio and plaque area later showing reduction at 50 and 64 weeks CD periods. Lesion lipid accumulation and CD68 positivity was maintained till 16 weeks of CD feeding which significantly reduced from 32 to 64 weeks CD periods. Baseline comparison showed significant increase in ground substance, MMP-9 and significant decrease in α-actin and collagen content at 8 weeks CD period indicating features of unstable plaque. These features regressed up to 64 weeks of CD. Partial restoration of functional vasoconstriction and vasorelaxation was seen after 64 weeks of CD feeding. mRNA expression of MCP-1, VCAM-1, collagen type I and III, MMP-9, TIMP-1, IFN-γ, TNF-α, IL-10 and eNOS supported the above findings. The study thus reveals insights into initial plaque instability and subsequent regression on AD withdrawal in this model. These results are suggestive of an appropriate window for drug intervention for plaque stability/regression and restenosis as well as improves understanding of plaque regression phenomenon in this model.     

Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice

Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoE^null) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoE^null mice.     

Non-Healing Ulcer Due to Trichosporon loubieri in an Immunocompetent Host and Review of Published Reports

We report here a case of non-healing ulcer due to Trichosporon loubieri in an apparently immunocompetent female. The identity of isolate was confirmed by DNA sequencing of D1/D2 region of 26S rDNA. The minimum inhibitory concentrations of the isolate were amphotericin B—0.5 μg/ml; fluconazole—4 μg/ml; posaconazole—0.25 μg/ml; voriconazole—0.06 μg/ml. The patient was managed by extensive debridement and oral fluconazole 150 mg daily for 6 weeks. She responded to therapy. To the best of our knowledge, till date, this is the fourth report of human infection due to T. loubieri and the first of its kind in an immunocompetent host. A review of published literature on infections due to T. loubieri is also included.