Hepatitis B Virus x Protein Increases Cellular OCT3/4 and MYC and Facilitates Cellular Reprogramming.

Hepatitis B virus x (HBx) is a multifunctional protein coded by the Hepatitis B virus that is involved in various cellular processes such as proliferation, cell survival/apoptosis, and histone methylation. HBx was reported to be associated with liver "cancer stem cells." The stemness inducing properties of HBx could also facilitate the generation of pluripotent stem cells from somatic cells. It is well established that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using a cocktail of transcription factors called Yamanaka's factors (YFs) (OCT4, SOX2, KLF4, and MYC). The reprogramming process proceeds step-by-step with reprogramming factor chromatin interactions, transcription, and chromatin states changing during transitions. HBx is a "broad spectrum trans-activator" and therefore could facilitate these transitions. We electroporated low passage and high passage (difficult to reprogram) fibroblasts using YFs with and without HBx and evaluated the reprogramming efficiency. We also investigated the tri-lineage and terminal differentiation potential of iPSC derived using HBx. We found that the addition of HBx to YF improves iPSC derivation, and it increases the efficiency of iPSC generation from "difficult or hard-to-reprogram samples" such as high passage/senescent fibroblasts. Further, we show that HBx can substitute the key transcription factor MYC in the YF cocktail to generate iPSC. The cellular levels of OCT3/4 and MYC were increased in HBx expressing cells. Our results have practical value in improving the efficiency of pluripotent stem cell derivation from "difficult to reprogram" somatic cells, in addition to providing some insights into the mechanisms of liver carcinogenesis in chronic hepatitis B. To conclude, HBx improves the reprogramming efficiency of YFs. HBx increases the cellular levels of OCT3/4 and MYC.

Investigating the potential role of swertiamarin on insulin resistant and non-insulin resistant granulosa cells of poly cystic ovarian syndrome patients.

BACKGROUND AND AIM: Conventional drugs have limitations due to prevalence of contraindications in PCOS patients. To explore the potential effects of swertiamarin, on abrupted insulin and steroidogenic signaling in human luteinized granulosa cells from PCOS patients with or without insulin resistance. EXPERIMENTAL PROCEDURE: hLGCs from 8 controls and 16 PCOS patients were classified for insulin resistance based on down regulation of protein expression of insulin receptor-ß (INSR- ß) as shown in our previous paper. Cells were grouped as control, PCOS-IR and PCOS-NIR, treated with swertiamarin (66 µM) and metformin (1 mM). Expression of key molecules involved in insulin signaling, fat metabolism, IGF system and steroidogenesis were compared between groups. RESULTS: Swertiamarin significantly (P < 0.05) reversed the expression of INSR-ß, PI(3)K, p-Akt, PKC-ζ, PPARγ, (P < 0.01) IRS (Ser 307) and IGF system in PCOS-IR group and was equally potent to metformin. In the same group, candidate genes viz SREBP1c, FAS, ACC-1 and CPT-1 were down regulated by swertiamarin (P < 0.001) and metformin (P < 0.001). Significant upregulation was demonstrated in expression of StAR, CYP19A1, 17ß-HSD and 3ß-HSD when treated with swertiamarin (P < 0.01) and metformin (P < 0.01) in PCOS-IR followed by increase in 17ß-HSD and 3ß-HSD enzyme activity along with estradiol and progesterone secretions. However, swertiamarin did not reveal any effect on PCOS-NIR group as compared to metformin that significantly (P < 0.01) reversed all the parameters related to steroidogenesis and down regulated basal expression of insulin signaling genes. CONCLUSION: Swertiamarin, presents itself as a potential fertility drug in hLGCs from PCOS-IR patients.

Role of poly(ADP-ribose) polymerase-1 in regulating human islet cell differentiation.

Poly(ADP-ribose) polymerase-1 (PARP1), a fundamental DNA repair enzyme, is known to regulate ß cell death, replication, and insulin secretion. PARP1 knockout (KO) mice are resistant to diabetes, while PARP1 overactivation contributes to ß cell death. Additionally, PARP1 inhibition (PARPi) improves diabetes complications in patients with type-2 diabetes. Despite these beneficial effects, the use of PARP1 modulating agents in diabetes treatment is largely neglected, primarily due to the poorly studied mechanistic action of PARP1 catalytic function in human ß cell development. In the present study, we evaluated PARP1 regulatory action in human ß cell differentiation using the human pancreatic progenitor cell line, PANC-1. We surveyed islet census and histology from PARP1 wild-type versus KO mice pancreas in a head-to-head comparison with PARP1 regulatory action for in-vitro ß cell differentiation following either PARP1 depletion or its pharmacological inhibition in PANC-1-differentiated islet cells. shRNA mediated PARP1 depleted (SiP) and shRNA control (U6) PANC-1 cells were differentiated into islet-like clusters using established protocols. We observed complete abrogation of new ß cell formation with absolute PARP1 depletion while its inhibition using the potent inhibitor, PJ34, promoted the endocrine ß cell differentiation and maturation. Immunohistochemistry and immunoblotting for key endocrine differentiation players along with ß cell maturation markers highlighted the potential regulatory action of PARP1 and augmented ß cell differentiation due to direct interaction of unmodified PARP1 protein elicited p38 MAPK phosphorylation and Neurogenin-3 (Ngn3) re-activation. In summary, our study suggests that PARP1 is required for the proper development and differentiation of human islets. Selective inhibition with PARPi can be an advantage in pushing more insulin-producing cells under pathological conditions and delivers a potential for pilot clinical testing for ß cell replacement cell therapies for diabetes.

Clinico-Epidemiologic Attributes of 515 Persons Living With Human Immunodeficiency Virus Infection/Acquired Immunodeficiency Syndrome on Antiretroviral Therapy: A Cross-Sectional Study from a Tertiary Care Centre of North India.

Objectives: To study the clinico-epidemiologic attributes of persons living with HIV/AIDS on highly active antiretroviral therapy (HAART). Methods: Clinico-epidemiological details, CD4 counts, previous illness and mucocutaneous diseases were studied in 515 persons living with HIV/AIDS on HAART. Results: The study comprised 250 (48.5%) males and 265 (51.5%) females aged between 10 and 79 (mean 38.9) years. The 196 (38%) males were drivers, staying-alone laborers/self-employed, and 253 (49.1%) females were homemakers. All were on HAART for one month to 9 years. Heterosexual transmission was noted in 478 (92.8%) individuals. The 274 (53.5%) individuals had 200-350 CD4 cells/mm3 counts, whereas it was <200 cells/mm3 in 88 (17.2%) individuals. Candidiasis (in 48), dermatophytoses (n = 23), herpes labialis (n = 13), herpes zoster (n = 12), seborrheic dermatitis (n = 29), generalized pruritus (n = 22), and xerosis in 20 individuals were the most common dermatoses. Most dermatoses occurred with 200-350 CD4 cells/mm3. Adverse drug reactions from antiretroviral therapy (ART) and concurrent therapies also occurred. Conclusions: Although most of our patients had mild HIV-associated dermatoses while on HAART, adverse drug reactions from HAART or concurrent therapies themselves remain a potential risk. Nevertheless, knowledge of these aspects will help planning for comprehensive health care envisaged in the National AIDS Control Program phase IV.

Femoral nerve neuromonitoring for lateral lumbar interbody fusion surgery.

BACKGROUND CONTEXT: The transpsoas lateral lumbar interbody fusion (LLIF) technique is an effective alternative to traditional anterior and posterior approaches to the lumbar spine; however, nerve injuries are the most reported postoperative complication. Commonly used strategies to avoid nerve injury (eg, limiting retraction duration) have not been effective in detecting or preventing femoral nerve injuries. PURPOSE: To evaluate the efficacy of emerging intraoperative femoral nerve monitoring techniques and the importance of employing prompt surgical countermeasures when degraded femoral nerve function is detected. STUDY DESIGN/SETTING: We present the results from a retrospective analysis of a multi-center study conducted over the course of 3 years. PATIENT SAMPLE: One hundred and seventy-two lateral lumbar interbody fusion procedures were reviewed. OUTCOME MEASURES: Intraoperative femoral nerve monitoring data was correlated to immediate postoperative neurologic examinations. METHODS: Femoral nerve evoked potentials (FNEP) including saphenous nerve somatosensory evoked potentials (snSSEP) and motor evoked potentials with quadriceps recordings were used to detect evidence of degraded femoral nerve function during the time of surgical retraction. RESULTS: In 89% (n=153) of the surgeries, there were no surgeon alerts as the FNEP response amplitudes remained relatively unchanged throughout the surgery (negative group). The positive group included 11% of the cases (n=19) where the surgeon was alerted to a deterioration of the FNEP amplitudes during surgical retraction. Prompt surgical countermeasures to an FNEP alert included loosening, adjusting, or removing surgical retraction, and/or requesting an increase in blood pressure from the anesthesiologist. All the cases where prompt surgical countermeasures were employed resulted in recovery of the degraded FNEP amplitudes and no postoperative femoral nerve injuries. In two cases, the surgeons were given verbal alerts of degraded FNEPs but did not employ prompt surgical countermeasures. In both cases, the degraded FNEP amplitudes did not recover by the time of surgical closure, and both patients exhibited postoperative signs of sensorimotor femoral nerve injury including anterior thigh numbness and weakened knee extension. CONCLUSIONS: Multimodal femoral nerve monitoring can provide surgeons with a timely alert to hyperacute femoral nerve conduction failure, enabling prompt surgical countermeasures to be employed that can mitigate or avoid femoral nerve injury. Our data also suggests that the common strategy of limiting retraction duration may not be effective in preventing iatrogenic femoral nerve injuries.

Influence of metabolically compromised Adipose derived stem cell secretome on islet differentiation and functionality.

Islet integrity plays a major role in maintaining glucose homeostasis and thus replenishment of damaged islets by differentiation of resident endocrine progenitors into neo islets regulates the islet functionality. Islet differentiation is affected by many factors including crosstalk with various organs by secretome. Adipose derived stem cells (ADSC) secrete a large array of factors in the extracellular milieu that exhibit regulatory effects on other tissues including pancreatic islets. The microenvironment of metabolically compromised human ADSCs (hADSCs) has a detrimental impact on islet functionality. In the present study, the role of secretome was studied on the differentiation of islets. Expression of key transcription factors like HNF-3B, NGN-3, NeuroD, PDX- 1, Maf-A, and GLUT-2 involved in development were differentially regulated in obese hADSC secretome as compared to control hADSC secretome. Islet like cell clusters (ILCCs) functionality and viability were critically hampered under obese hADSC secretome with compromised yield, morphometry, lower expression of C-peptide and Glucagon as well as higher ROS activity and cell death parameters. This study provides considerable insights on two major findings which are (i) exploring the use of hADSC secretome in islet differentiation and (ii) understanding the regulating effect of altered hADSC secretome under a metabolically compromised condition.

To Compare and Evaluate Laryngeal Changes in Patients with Dysphonia in Laryngopharyngeal Reflux (LPR) before and after Treatment with Proton Pump Inhibitors (PPI) and Prokinetic Drugs.

To evaluate and compare pre and post treatment results using the following parameters by (a) Dual probe pH monitoring. (b) Laryngeal mucosal changes as assessed by direct video laryngoscopy/stroboscopy using Belafsky scores. (c) Voice changes by using GRBAS and Dr Speech software for speech analysis. In our study we have evaluated and compared voice and laryngeal changes in patients with dysphonia and RSI > 10 (which is suggestive of LPR) before treatment and after 6 months of treatment with Tab. Pantoprazole and Tab. Mosapride. This prospective study was carried out on 50 patients attending the ENT OPD of a tertiary care referral centre over a period of 18 months i.e. from Nov 2008 to Apr 2010. The study showed that prolonged therapy (> 6 months) is required to treat LPR effectively and 24 h ambulatory dual probe pH metry and videolaryngoscopy to assess RFS are the most preferred diagnostic tools in LPR. Dr Speech software for voice analysis can give an objective assessment of voice changes in LPR before and after treatment. The treatment consisting of PPI and prokinetic drugs proved to be effective in laryngopharyngeal reflux disease as improvement was seen in all the parameters including reflux findings score, subjective and objective voice assessment. According to results of our study, 24 h ambulatory dual probe pH metry, Reflux Finding Score (RFS), subjective and objective acoustic parameters can be used as indicators of efficacy of treatment.

Swertisin, a novel SGLT2 inhibitor, with improved glucose homeostasis for effective diabetes therapy.

Failing pancreas and subsequent loss of pancreatic ß cells worsen diabetic conditions which are further alleviated by the mounting up of glucose levels. Inhibition of sodium glucose cotransporter 2 (SGLT2) in the kidney responsible for glucose reabsorption strikingly reduces blood glucose levels. Bioactive swertisin showed a promising glucose-lowering effect. Hence, we aimed to mechanistically dissect the glucose lowering property of swertisin. A systematic in silico, in vitro, and in vivo approach was directed for target analysis of swertisin. Molecular docking was performed with Swertisn-hSGLT2 complex. Glucose uptake assay and protein expression for SGLT2 and regulatory proteins were performed under swertisin effect. Various physiological and metabolic parameters were evaluated in STZ induced BALB/c mice using swertisin treatment. SGLT2 expression was evaluated in the kidney tissue of mice. Swertisn-hSGLT2 molecularly docked complex showed similar binding energy compared to the Canagliflozin-hSGLT2 complex. Swertisin inhibited glucose uptake and decreased expression of SGLT2 in HEK293 cells. Swertisin does not affect GLUT mediated glucose transport. Swertisin treated diabetic mice demonstrated remarkable improvement in overall glucose homeostasis. Reduced expression of SGLT2 was found in kidney tissue along with reduced PKC expression which is one of the key regulators of SGLT2. Our study explored SGLT2 as a selective target of swertisin for its swift glucose-lowering action which not only inhibits SGLT2 but also reduces its expression in diabetic condition. Thus, the potential property of swertisin as a glucose-lowering agent is remarkable which points towards the likelihood of a wider avenue of diabetes therapy.

Resistin mitigates stemness and metabolic profile of human adipose-derived mesenchymal stem cells via insulin resistance.

During obesity adipose tissue abundantly secrete pro-inflammatory adipokines like Tumour Necrosis factor-alpha (TNFα), resistin, leptin, etc. but reduced anti-inflammatory adipokines like adiponectin, interleukin (IL)-10, and IL-4. In our recent clinical study, it was observed that both gene expressions and stored levels of resistin were elevated in adipose tissue of metabolically obese Indians. Resistin profoundly increases obesity, mitigates lipid metabolism, and causes peripheral insulin resistance. It dysregulates the metabolism of human adipocytes but, its effects on human adipose-derived mesenchymal stem cells (hADSC) are sparsely explored. Therefore, the present study was designed to explore the repercussion of resistin on stemness and metabolic profile of hADSC. hADSC were isolated from a healthy individual followed by immunophenotyping. Purified cells were treated with resistin and proliferation was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Cell Cycle experiments. Gene expressions of pluripotent markers, inflammatory mediators, and lipogenic genes were scrutinized. Insulin sensitivity was examined by western blot and glucose uptake assay. Further, consequences of resistin on differentiation potentials of hADSC were examined by temporal expressions of phospho (p)SMAD1/5/8 protein complex, non-phosphorylated beta (ß) catenin, and their dependent adipogenic transcription factors (ATF) and osteogenic transcription factors (OTF). MTT and cell cycle analysis revealed that resistin hampered proliferation of hADSC. Expressions of inflammatory markers and lipogenic genes were elevated. Resistin impaired insulin sensitivity and thus embarked insulin resistance in hADSC. Resistin increased adipogenesis and osteogenesis by altering expressions of activated pSMAD1/5/8 complex, activated ß catenin, ATF and OTF temporally. Downregulation of CCAAT/enhancer-binding proteins (C/EBP)α and adiponectin in adipocytes and Sirtuin (SIRT)1 in osteocytes denote that resistin induces immaturity and insulin resistance in adipocytes and osteocytes. This is the first study which, reports that resistin mitigates the stemness of hADSC by reducing proliferation, inducing insulin resistance, and hampering maturation of adipocyte and osteocyte which could lead to metabolic disorders.

Phenotypic and genotypic characterization of biofilm forming, antimicrobial resistant, pathogenic Escherichia coli isolated from Indian dairy and meat products.

Escherichia coli are commensal gastrointestinal microflora of humans, but few strains may cause food-borne diseases. Present study aimed to identify antimicrobial resistant (AMR), biofilm-forming E. coli from Indian dairy and meat products. A total of 32 E. coli isolates were identified and evaluated for biofilm-formation. EMC17, an E. coli isolate was established as a powerful biofilm-former that attained maximum biofilm-formation within 96 h on glass and stainless-steel surfaces. Presence and expression of virulence-associated genes (adhesins, invasins and polysaccharides) and ability to adhere and invade human liver carcinoma HepG2 cell lines implicates EMC17 to be pathotype belonging to Extra-intestinal Pathogenic E. coli (ExPEC). Antibiotic profiling of EMC17 identified it as multi-drug resistant (MDR) strain, possessing extended spectrum ß-lactamases (ESBL's) and biofilm phenotype. Early production of quorum sensing molecules (AHLs) alongside EPS production facilitated early onset of biofilm formation by EMC17. Furthermore, the biofilm-forming genes of EMC17 were significantly upregulated 3-27 folds in the biofilm-state. This study showed prevalence of MDR, biofilm-forming, pathogenic E. coli in Indian dairy and meat products that potentially serve as reservoirs for transmission of antimicrobial-resistant (AMR) genes of bacteria from food to humans and pose serious food safety threat.

Oncogenic transformation of human benign prostate hyperplasia with chronic cadmium exposure.

Experimentally, it has been proved that cadmium served as an effective carcinogen and able to induce tumors in rodents in a dose-specific manner. However, systemic evaluation of cadmium exposure for the transformation of prostatic hyperplasia into prostate cancer (PCa) is still unclear. In the present study, an attempt has been made to establish cadmium-induced human prostate carcinogenesis using an in vitro model of BPH cells. Wide range of cadmium concentrations, i.e., 1 nM, 10 nM, 100 nM and 1µM, were chronically exposed to the human BPH cells for transformation into PCa and monitored using cell and molecular biology approaches. After eight weeks of exposure, the cells showed subtle morphological changes and shifts of cell cycle in the G2M phase. Significant increase in expression of prostatic genes AR, PSA, ER-ß, and 5αR with increased nuclear localization of AR and pluripotency markers Cmyc, Klf4 indicated the carcinogenic effect of Cd. Further, the BPH cells exposed to Cd showed a substantial increase in the secretion of MMP-2 and MMP-9, influencing migratory potential of the cells along with decreased expression of the p63 protein which further strengthen the progression towards carcinogenesis and aggressive tumor studies. Data from the present study state that Cd exhibited marked invasiveness in BPH cells. These observations established a connecting link of BPH towards PCa pathogenesis. Further, the study will also help in investigating the intricate pathways involved in cancer progression.

Direct lineage tracing reveals Activin-a potential for improved pancreatic homing of bone marrow mesenchymal stem cells and efficient ß-cell regeneration in vivo.

BACKGROUND: Despite the potential, bone marrow-derived mesenchymal stem cells (BMSCs) show limitations for beta (ß)-cell replacement therapy due to inefficient methods to deliver BMSCs into pancreatic lineage. In this study, we report TGF-ß family member protein, Activin-a potential to stimulate efficient pancreatic migration, enhanced homing and accelerated ß-cell differentiation. METHODS: Lineage tracing of permanent green fluorescent protein (GFP)- tagged donor murine BMSCs transplanted either alone or in combination with Activin-a in diabetic mice displayed potential ß-cell regeneration and reversed diabetes. RESULTS: Pancreatic histology of Activin-a treated recipient mice reflected high GFP+BMSC infiltration into damaged pancreas with normalized fasting blood glucose and elevated serum insulin. Whole pancreas FACS profiling of GFP+ cells displayed significant homing of GFP+BMSC with Activin-a treatment (6%) compared to BMSCs alone transplanted controls (0.5%). Within islets, approximately 5% GFP+ cells attain ß-cell signature (GFP+ Ins+) with Activin-a treatment versus controls. Further, double immunostaining for mesenchymal stem cell markers CD44+/GFP+ in infiltrated GFP+BMSC deciphers substantial endocrine reprogramming and ß-cell differentiation (6.4% Ins+/GFP+) within 15 days. CONCLUSION: Our investigation thus presents a novel pharmacological approach for stimulating direct migration and homing of therapeutic BMSCs that re-validates BMSC potential for autologous stem cell transplantation therapy in diabetes.

Stromal-AR influences the growth of epithelial cells in the development of benign prostate hyperplasia.

Activation of epithelial-AR signaling is identified as the major cause of hyperproliferation of the cells during benign and malignant prostate conditions. However, the contribution of stromal-AR is also precarious due to its secretory actions that contribute to the progression of benign and malignant tumors. The present study was aimed to understand the influence of stromal-AR mediated actions on epithelial cells during BPH condition. The secretome (conditioned media-CM) was collected from AR agonist (testosterone-propionate-TP) and antagonist (Nilutamide-Nil) treated BPH patient-derived stromal cells and exposed to BPH epithelial cells. Epithelial cells exhibited increased cell proliferation with the treatment of CM derived from TP-treated stromal cells (TP-CM) but did not support the clonogenic growth of BPH epithelial cells. However, CM derived from Nil-treated stromal cells (Nil-CM) depicted delayed and aggressive BPH epithelial cell proliferation with increased clonogenicity of BPH epithelial cells. Further, decreased AR levels with increased cMyc transcripts and pAkt levels also validated the clonogenic transformation under the paracrine influence of inhibition of stromal-AR. Moreover, the CM of stromal-AR activation imparted positive regulation of basal/progenitor pool through LGR4, ß-Catenin, and ΔNP63α expression. Hence, the present study highlighted the restricted disease progression and retains the basal/progenitor state of BPH epithelial cells through the activation of stromal-AR. On the contrary, AR-independent aggressive BPH epithelial cell growth due to paracrine action of loss stromal-AR directs us to reform AR pertaining treatment regimes for better clinical outcomes.

Influence of obese phenotype on metabolic profile, inflammatory mediators and stemness of hADSC in adipose tissue.

BACKGROUND: Unhealthy dietary practices, sedentary life style and lack of physical exercise in developing countries like India are major contributors of metabolic syndrome like obesity and diabetes. Obesity in Indians is defined at Body Mass Index (BMI, kg/m2) >25 and characterized as metabolically obese. OBJECTIVE: A preliminary study performed to explore ramification of obesity on metabolic profile of adipose tissue and adipose derived stem cells (ADSC) from control and obese Indians. METHODS: Adipose tissue/lipoaspirates from both control (BMI ≤ 23) subjects, and non-diabetic obese Indians subjects (BMI ≥ 25), were scrutinized for expressions of lipogenic genes, inflammatory mediators, stored adipokine levels, and insulin signaling proteins. Further, hADSC were isolated and immune-phenotyped from both the subject groups. Comparative assessments between chADSC and ohADSC were carried out for growth kinetics, expressions of pluripotent genes, adipogenic transcriptional factors, RUNX2, inflammatory mediators (IM), insulin signaling proteins, adipogenic and osteogenic differentiation. RESULTS: Adipose tissue of obese subjects depicted high leptin and resistin levels with reduced adiponectin levels. Expressions of IM and insulin signaling proteins were elevated compared to those of control subjects. hADSC of obese subjects demonstrated diminished proliferation, altered pluripotent genes, aggravated inflammation, adipogenesis with reduced osteogenesis. hADSC of obese had established insulin resistance compared to those of control subjects. CONCLUSION: This is the first study that describes hADSC of metabolically obese Indians have insulin resistance at lower BMI compared to Caucasians exemplifying plausible role in diminishing stemness of hADSC. Study alarms Indians to restore healthy dietary habits and assess quality of hADSC in regenerative therapy.

Strengthening antioxidant defense & cardio protection by Piper betle: An in-vitro study.

INTRODUCTION: The purpose of this research work was to evaluate Piper betle ethyl acetate extract (PBEA) for its free radical scavenging, antioxidant, anti-apoptotic activities and its role in protecting against oxidative cardiac cell injury. METHODS: The Free radical scavenging activity and antioxidant potential of PBEA were evaluated using various non-cellular methods (1,1-Diphenyl-2-picrylhydrazyl, ß-carotene bleaching, superoxide anion, hydroxyl radical, hydrogen peroxide, Reducing power, Total phenolics and Total flavonoids). PBEA was standardized with Eugenol by GC-FID analysis. Furthermore, PBEA was also assessed for its cytotoprotective effect against 100µM H2O2 in H9c2 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Intracellular reactive oxygen species scavenging and anti-apoptoic activity of PBEA was assessed by using 2', 7'-Dichlorofluorescein diacetate and Annexin- Propidium Iodide, respectively. RESULTS: PBEA exhibited radical scavenging and antioxidant defense response at different magnitudes of potency. Eugenol, a cardiac protective bioactive molecule in PBEA was found to be 43.43 ± 1.46 mg/g of PBEA extract. Further, pre-incubation of H9c2 cells with 10 µg/ml PBEA for 24 h exhibited remarkable cytoprotective effect against H2O2 induced oxidative stress. PBEA at 10 µg/ml dose with 24 h contact with H9c2 cells significantly enhanced the activity of cellular defense system and significantly decreased intracellular ROS (P < 0.001) and apoptosis (P < 0.01) thereby protecting against the cytotoxic effects of H2O2. CONCLUSION: These outcomes indicated that PBEA could shield against oxidative and apoptotic cardiac cell injury in invitro studies. Thus, PBEA might be a desirable antioxidant of natural origin that has future clinical implications in both health care and food industry.

IGF-II-Conjugated Nanocarrier for Brain-Targeted Delivery of p11 Gene for Depression.

Gene therapy involving p11 cDNA has been thought to be a futuristic approach for the effective management of depression as the existing treatment regimen presents many issues regarding late onset of action, patient withdrawal and their side effects. For the effective transfection of p11 gene intracellularly, two cationic lipids based on phospholipid DOPE conjugated to basic amino acids histidine and arginine were synthesised, used for liposome formulation and evaluated for their ability as gene delivery vectors. They were further converted using IGF-II mAb into immunoliposomes for CNS targeting and mAb conjugation to liposomes were characterised by SDS-PAGE. They were further analysed by in vitro characterisation studies that include erythrocyte aggregation study, electrolyte-induced study, heparin compatibility study and serum stability studies. SHSY5Y cells were used for conducting cytotoxicity of synthesised lipids and live imaging of cell uptake for 25 min. Finally, the brain distribution studies and western blot were carried out in animals to evaluate them for their BBB permeation ability and effects on p11 protein which is believed to be a culprit. These formulated liposomes from synthesised lipids offer a promising approach for the treatment of depression.

Pancreatic resident endocrine progenitors demonstrate high islet neogenic fidelity and committed homing towards diabetic mice pancreas.

Pancreatic progenitors have been explored for their profound characteristics and unique commitment to generate new functional islets in regenerative medicine. Pancreatic resident endocrine progenitors (PREPs) with mesenchymal stem cell (MSC) phenotype were purified from BALB/c mice pancreas and characterized. PREPs were differentiated into mature islet clusters in vitro by activin-A and swertisin and functionally characterized. A temporal gene and protein profiling was performed during differentiation. Furthermore, PREPs were labeled with green fluorescent protein (GFP) and transplanted intravenously into streptozotocin (STZ) diabetic mice while monitoring their homing and differentiation leading to amelioration in the diabetic condition. PREPs were positive for unique progenitor markers and transcription factors essential for endocrine pancreatic homeostasis along with having the multipotent MSC phenotype. These cells demonstrated high fidelity for islet neogenesis in minimum time (4 days) to generate mature functional islet clusters (shortest reported period for any isolated stem/progenitor). Furthermore, GFP-labeled PREPs transplanted in STZ diabetic mice migrated and localized within the injured pancreas without trapping in any other major organ and differentiated rapidly into insulin-producing cells without an external stimulus. A rapid decrease in fasting blood glucose levels toward normoglycemia along with significant increase in fasting serum insulin levels was observed, which ameliorated the diabetic condition. This study highlights the unique potential of PREPs to generate mature islets within the shortest period and their robust homing toward the damaged pancreas, which ameliorated the diabetic condition suggesting PREPs affinity toward their niche, which can be exploited and extended to other stem cell sources in diabetic therapeutics.

Evaluation of Serum Vitamin D Levels in Patients with Systemic Sclerosis and Healthy Controls: Results of a Pilot Study.

BACKGROUND: The anti-inflammatory, immunomodulatory, and anti-proliferative effects of vitamin D in pathogenesis of autoimmune diseases have been highlighted in recent years but implications of vitamin D deficiency in systemic sclerosis (SSc) remain understudied. OBJECTIVES: To evaluate serum vitamin D levels in SSc patients and matched controls. MATERIALS AND METHODS: Serum vitamin D levels were estimated in 38 (M:F 5:33) patients aged 23-70 years of untreated SSc and age and gender matched healthy controls. Clinical and investigative evaluation for skin sclerosis by modified Rodnan skin score (mRSS), presence of digital ulcers, Raynaud's phenomenon, type of auto-antibodies, systemic involvement, and serum vitamin D levels were performed. Serum vitamin D levels were defined as normal (30-100 ng/ml), insufficient (10-30 ng/ml), and deficient (<10 ng/ml). RESULTS: Serum vitamin D levels (median ± IQR) were 19.5 ± 77.8 ng/ml in 38 patients and 100 ± 31.3 ng/ml in controls each. Vitamin D deficiency in 13 (34.2%) and insufficiency in 10 (26.3%) patients were identified. Only 2 (5.3%) controls had vitamin D insufficiency and the difference was statistically significant (P = 0.001). An inverse relationship was observed between mRSS and serum vitamin D levels. CONCLUSIONS: Patients with SSc have significantly lower serum vitamin D levels than healthy controls. Serum vitamin D levels do not correlate well with age, gender, disease duration or its variants, type of auto antibodies, presence of digital ulceration, or systemic involvement but has inverse correlation with skin sclerosis. Better-designed studies will perhaps resolve issues of potential benefits of vitamin D supplementation in modification of disease activity or severity in SSc.

Swertisin ameliorates diabetes by triggering pancreatic progenitors for islet neogenesis in Streptozotocin treated BALB/c mice.

In the present study, Swertisin's role in triggering resident pancreatic progenitors for islet neogenesis in Streptozotocin (STZ) diabetic mice was explored. STZ diabetic mice when treated with Swertisin demonstrated reversion to normoglycemia and significant elevation of fasting serum insulin levels. On screening the pancreatic tissue post Swertisin treatment in the STZ diabetic mice, we observed significant up-regulation of key transcription factors viz. Pdx1, Neurog3, MafA and Nkx6.1 required for islet neogenesis and beta cell homeostasis. We further observed increase in expression of Nestin and Neurog3 positive population; Nestin and Glut2 positive population and increase in c-peptide and Glucagon positive population within the Islets of Langerhans indicating increased pancreatic progenitor activity and their differentiation into Insulin producing beta cells in Swertisin treated STZ diabetic mice. Thus, this short study highlights pancreatic innate capability to regenerate and recover using its own resident progenitors upon appropriate stimulus, which could culminate into an effective diabetic therapy.

Differential insulin and steroidogenic signaling in insulin resistant and non-insulin resistant human luteinized granulosa cells-A study in PCOS patients.

Insulin resistance (IR) is one of the significant aberrations in polycystic ovarian syndrome (PCOS), however is only observed in 70%-80% of obese PCOS and 20%-25% of lean PCOS. Hyperinsulinemia accompanies PCOS-IR along with hyperandrogenemia against normal insulin and androgen levels in PCOS-non insulin resistance (NIR). This could possibly be due to defects in the downstream signaling pathways. The study thus aims to unravel insulin and steroidogenic signaling pathways in luteinized granulosa cells isolated from PCOS-IR and NIR vs matched controls. Luteinized granulosa cells from 30 controls and 39 PCOS were classified for IR based on a novel method of down regulation of protein expression of insulin receptor-ß (INSR- ß) as shown in our previous paper. We evaluated expression of molecules involved in insulin, steroidogenic signaling and lipid metabolism in luteinized granulosa cells followed by analysis of estradiol, progesterone and testosterone in follicular fluid. Protein expression of INSR- ß, pIRS (ser 307), PI(3)K, PKC-ζ, pAkt, ERK1/2, pP38MAPK and gene expression of IGF showed differential expression in the two groups. Increased protein expression of PPAR-γ was accompanied by up regulation in SREBP1c, FAS, CPT-1 and ACC-1 genes in PCOS-IR group. Expression of StAR, CYP19A1, 17 ß- HSD and 3 ß- HSD demonstrated significant decrease along with increase in CYP11A1, FSH-R and LH-R in both the groups. Follicular fluid testosterone increased and progesterone decreased in PCOS-IR group. This study shows how candidate molecules that were differentially expressed, aid in designing targeted therapy against the two phenotypes of PCOS.