Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn ^?/? ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn ^?/? MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn ^?/? MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn ^?/? MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn^?/? MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn ^?/? MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.     

Evaluation of chemical interactions of maleic acid with sodium hypochlorite and chlorhexidine gluconate

Introduction

The elimination of microorganisms from the root canal system necessitates the use of combination of irrigating solutions to enhance their antimicrobial property. The combination of irrigants and their interaction sometimes could be detrimental to the outcome of the root canal therapy. The purposes of this study were (1) to evaluate the interaction between 7% maleic acid (MA) and 2% chlorhexidine gluconate solution (CHX) and to find out the availability of individual irrigant and (2) to determine the free available chlorine content when 7% MA was mixed with 2.5% sodium hypochlorite (NaOCl) solution.

Methods

Interaction between MA and CHX was assessed by high-performance liquid chromatography. Available chlorine content in NaOCl was evaluated by the standard iodine/thiosulfate titration method.

Results

It was observed that more than 90% free MA and CHX were available when MA was combined with CHX. It was also observed that there was no precipitate formation when 7% MA was mixed with 2% CHX. Available chlorine content decreased significantly in the MA/NaOCl mixture.

Conclusions

There were no adverse interactions or precipitate formation observed when MA was combined with CHX, but the available chlorine content was reduced when NaOCl was mixed with MA.     

Vaccine for tuberculosis: up-regulation of IL-15 by Ag85A and not by ESAT-6

IFN-γ is the most commonly measured cytokine released by the cells to define the cellular immune responses induced by the vaccine candidates for tuberculosis. IL-15 acts as a co-stimulator in IFN-γ production by NK cells and may therefore be important in the control of Mycobacterium tuberculosis that requires IFN-γ for clearance. The aim of the study is to determine whether Ag85A can also stimulate the innate immune response through the expression of IL-15, a cytokine that bridges the innate and adaptive immune systems. The expression of IL-15 was up regulated by about 4 fold in PPD+ healthy controls as compared with TB patients. Significantly higher expression of IL-15 mRNA in the Ag85A stimulated cells not only in PPD+ healthy controls but also in TB patients substantiates the use of Ag85A as a vaccine candidate over ESAT-6.     

Chronic exposure to a high-fat diet induces hepatic steatosis, impairs nitric oxide bioavailability, and modifies the mitochondrial proteome in mice

Obesity-related pathologies, such as nonalcoholic fatty liver disease, are linked to mitochondrial dysfunction and nitric oxide (NO) deficiency. Herein, we tested the hypothesis that a high-fat diet (HFD) modifies the liver mitochondrial proteome and alters proteins involved in NO metabolism, namely arginase 1 and endothelial NO synthase. Male C57BL/6 mice were fed a control or HFD and liver mitochondria were isolated for proteomics and reactive oxygen species measurements. Steatosis and hepatocyte ballooning were present in livers of HFD mice, with no pathology observed in the controls. HFD mice had increased serum glucose and decreased adiponectin. Mitochondrial reactive oxygen species was increased after 8 weeks in the HFD mice, but decreased at 16 weeks compared with the control, which was accompanied by increased uncoupling protein 2. Using proteomics, 22 proteins were altered as a consequence of the HFD. This cohort consists of oxidative phosphorylation, lipid metabolism, sulfur amino acid metabolism, and chaperone proteins. We observed a HFD-dependent increase in arginase 1 and decrease in activated endothelial NO synthase. Serum and liver nitrate + nitrite were decreased by HFD. In summary, these data demonstrate that a HFD causes steatosis, alters NO metabolism, and modifies the liver mitochondrial proteome; thus, NO may play an important role in the processes responsible for nonalcoholic fatty liver disease.     

Thioredoxin 1 enhances neovascularization and reduces ventricular remodeling during chronic myocardial infarction: a study using thioredoxin 1 transgenic mice

Oxidative stress plays a crucial role in disruption of neovascularization by alterations in thioredoxin 1 (Trx1) expression and its interaction with other proteins after myocardial infarction (MI). We previously showed that Trx1 has angiogenic properties, but the possible therapeutic significance of overexpressing Trx1 in chronic MI has not been elucidated. Therefore, we explored the angiogenic and cardioprotective potential of Trx1 in an in vivo MI model using transgenic mice overexpressing Trx1. Wild-type (W) and Trx1 transgenic (Trx1^Tg/+) mice were randomized into W sham (WS), Trx1^Tg/+ sham (TS), WMI, and TMI. MI was induced by permanent occlusion of LAD coronary artery. Hearts from mice overexpressing Trx1 exhibited reduced fibrosis and oxidative stress and attenuated cardiomyocyte apoptosis along with increased vessel formation compared to WMI. We found significant inhibition of Trx1 regulating proteins, TXNIP and AKAP 12, and increased p-Akt, p-eNOS, p-GSK-3β, HIF-1α, β-catenin, VEGF, Bcl-2, and survivin expression in TMI compared to WMI. Echocardiography performed 30 days after MI revealed significant improvement in myocardial functions in TMI compared to WMI. Our study identifies a potential role for Trx1 overexpression and its association with its regulatory proteins TXNIP, AKAP12, and subsequent activation of Akt/GSK-3β/β-catenin/HIF-1α-mediated VEGF and eNOS expression in inducing angiogenesis and reduced ventricular remodeling. Hence, Trx1 and other proteins identified in our study may prove to be potential therapeutic targets in the treatment of ischemic heart disease.     

Evidence of bluetongue virus serotype 21 (BTV-21) divergence

Bluetongue virus serotype 21 (BTV-21) was originally isolated from Australia, but has now been reported from India, Indonesia, China and Japan. We report the isolation, and sequencing of BTV-21 from India. The complete ORF sequence of VP2 gene of this isolate showed that it is closely related to recent BTV-21 isolates from Japan (93–94% identity), and distantly related to BTV-21 reference strain (86% identity). Our results, along with the available sequences of Japanese isolates, suggest that the currently circulating BTV-21 strains from India and Japan are divergent from the original strain(s) from Australia and shed light on designing molecular diagnostics for the detection of BTV.     

Polymerase Chain Reaction and its Correlation with Clinical Features and Treatment Response in Tubercular Uveitis

Purpose: Correlation of results of polymerase chain reaction for Mycobacterium tuberculosis (MTB PCR) with clinical features and treatment response in tubercular uveitis.

Methods: Retrospective case study.

Results: Among 56 patients, 31 (55.3%) had acute and 25 (44.6%) had chronic uveitis. Uveitis was unilateral in 40 (71.4%) and bilateral in the remaining 16 (28.6%). Anatomical subtypes of uveitis were: anterior in 10 (13.9%) eyes, intermediate in 9 (12.5%), posterior in 17 (23.6%), and pan uveitis in 36 (50%) eyes. MTB PCR was positive in 24 patients. There was an 80% correlation between clinical response to antitubercular therapy (ATT) and PCR results. Twenty-two patients with positive PCR had a good clinical response. The sensitivity and specificity was 73.3% and 92.3%, respectively.

Conclusions: The diagnosis of intraocular TB requires strong clinical suspicion with corroborative laboratory and radiological evidence. A positive PCR is reliable whereas negative results should be correlated with clinical features. An adequate response to ATT supports PCR results.