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.     

Prevalence of Dyslipidemia in Urban and Rural India: The ICMR–INDIAB Study

Aim

To study the pattern and prevalence of dyslipidemia in a large representative sample of four selected regions in India.

Methods

Phase I of the Indian Council of Medical Research–India Diabetes (ICMR-INDIAB) study was conducted in a representative population of three states of India [Tamil Nadu, Maharashtra and Jharkhand] and one Union Territory [Chandigarh], and covered a population of 213 million people using stratified multistage sampling design to recruit individuals ≥20 years of age. All the study subjects (n = 16,607) underwent anthropometric measurements and oral glucose tolerance tests were done using capillary blood (except in self-reported diabetes). In addition, in every 5th subject (n = 2042), a fasting venous sample was collected and assayed for lipids. Dyslipidemia was diagnosed using National Cholesterol Education Programme (NCEP) guidelines.

Results

Of the subjects studied, 13.9% had hypercholesterolemia, 29.5% had hypertriglyceridemia, 72.3% had low HDL-C, 11.8% had high LDL-C levels and 79% had abnormalities in one of the lipid parameters. Regional disparity exists with the highest rates of hypercholesterolemia observed in Tamilnadu (18.3%), highest rates of hypertriglyceridemia in Chandigarh (38.6%), highest rates of low HDL-C in Jharkhand (76.8%) and highest rates of high LDL-C in Tamilnadu (15.8%). Except for low HDL-C and in the state of Maharashtra, in all other states, urban residents had the highest prevalence of lipid abnormalities compared to rural residents. Low HDL-C was the most common lipid abnormality (72.3%) in all the four regions studied; in 44.9% of subjects, it was present as an isolated abnormality. Common significant risk factors for dyslipidemia included obesity, diabetes, and dysglycemia.

Conclusion

The prevalence of dyslipidemia is very high in India, which calls for urgent lifestyle intervention strategies to prevent and manage this important cardiovascular risk factor.     

Role of Ca2+ in induction and secretion of dengue virus-induced cytokines

The present study was undertaken to investigate the role of calcium ions (Ca₂₊) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca₂₊ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.     

Wheat Stripe Rust Resistance Protein WKS1 Reduces the Ability of the Thylakoid-Associated Ascorbate Peroxidase to Detoxify Reactive Oxygen Species

Stripe rust is a devastating fungal disease of wheat caused by Puccinia striiformis f. sp tritici (Pst). The WHEAT KINASE START1 (WKS1) resistance gene has an unusual combination of serine/threonine kinase and START lipid binding domains and confers partial resistance to Pst. Here, we show that wheat (Triticum aestivum) plants transformed with the complete WKS1 (variant WKS1.1) are resistant to Pst, whereas those transformed with an alternative splice variant with a truncated START domain (WKS1.2) are susceptible. WKS1.1 and WKS1.2 preferentially bind to the same lipids (phosphatidic acid and phosphatidylinositol phosphates) but differ in their protein-protein interactions. WKS1.1 is targeted to the chloroplast where it phosphorylates the thylakoid-associated ascorbate peroxidase (tAPX) and reduces its ability to detoxify peroxides. Increased expression of WKS1.1 in transgenic wheat accelerates leaf senescence in the absence of Pst. Based on these results, we propose that the phosphorylation of tAPX by WKS1.1 reduces the ability of the cells to detoxify reactive oxygen species and contributes to cell death. This response takes several days longer than typical hypersensitive cell death responses, thus allowing the limited pathogen growth and restricted sporulation that is characteristic of the WKS1 partial resistance response to Pst.     

A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane

Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.     

PPE65 of M. tuberculosis regulate pro-inflammatory signalling through LRR domains of Toll like receptor-2

Our understanding of the PE/PPE family of proteins in M. tuberculosis (Mtb) pathogenesis is still evolving and their critical roles in the host immunomodulation are still in the discovery process. Earlier studies from our group have shown that TLR2-LRR domain plays an important role in regulating cytokine signalling by PPE proteins. The importance of TLR2-LRR domain 16–20 in the regulation of PPE17-induced pro-inflammatory signalling has been established recently. However, it is yet to find whether other PPE protein also targets the TLR2-LRR 16–20 domain for induction of pro-inflammatory responses. In the current study, we have explored the structural parameters and possible role of PPE65 in generating pro-inflammatory signalling molecules mediated through IRAK3 downstream of TLR2-LRR domain 16–20. This study conceptualizes the functional characteristics of PPE65 in infection condition and might possibly provide valuable information in exploring this protein as an immunomodulator in Mtb infection.     

Nerve Growth Factor Administration Attenuates Cognitive but Not Neurobehavioral Motor Dysfunction or Hippocampal Cell Loss Following Fluid-Percussion Brain Injury in Rats

Abstract: Lateral fluid-percussion brain injury in rats results in cognitive deficits, motor dysfunction, and selective hippocampal cell loss. Neurotrophic factors have been shown to have potential therapeutic applications in neurodegenerative diseases, and nerve growth factor (NGF) has been shown to be neuroprotective in models of excitotoxicity. This study evaluated the neuroprotective efficacy of intracerebral NGF infusion after traumatic brain injury. Male Sprague-Dawley rats received lateral fluid-percussion brain injury of moderate severity (2.1–2.3 atm). A miniosmotic pump was implanted 24 h after injury to infuse NGF (n = 34) or vehicle (n = 16) directly into the region of maximal cortical injury. Infusions of NGF continued until the animal was killed at 72 h, 1 week, or 2 weeks after injury. Animals were evaluated for cognitive dysfunction (Morris Water Maze) and regional neuronal cell loss (Nissl staining) at each of the three time points. Animals surviving for 1 or 2 weeks were also evaluated for neurobehavioral motor function. Although an improvement in memory scores was not observed at 72 h after injury, animals receiving NGF infusions showed significantly improved memory scores when tested at 1 or 2 weeks after injury compared with injured animals receiving vehicle infusions ( p < 0.05). Motor scores and CA3 hippocampal cell loss were not significantly different in any group of NGF-treated animals when compared with controls. These data suggest that NGF administration, in the acute, posttraumatic period following fluid-percussion brain injury, may have potential in improving post-traumatic cognitive deficits.     

Discovery of a vorapaxar analog with increased aqueous solubility

An analog of the thrombin receptor antagonist vorapaxar (SCH 530348) with increased aqueous solubility, compound 9c (SCH 602539), was discovered through incorporation of polar substituents on the pyridine ring of the himbacine-derived lead series. This analog retained the excellent potency, pharmacokinetic and safety properties of vorapaxar while increasing the aqueous solubility by 20-fold. Also presented are in vivo evaluations of this compound in a cynomolgus monkey platelet aggregation assay and in a Folts model of thrombosis in anesthetized monkeys.     

Association of genetic polymorphisms in glutathione S-transferases and susceptibility to head and neck cancer

Polymorphism in glutathione S-transferase (GST) genes (GSTM1, GSTT1 and GSTP1) and interaction with environmental factors such as tobacco (smoking or chewing) and alcohol on susceptibility to head and neck squamous cell carcinoma (HNSCC) was studied in a case-control study. The study group consisted of 175 patients suffering from HNSCC and 200 age matched healthy controls. Statistical analysis showed an increase in risk to HNSCC in the patients with null genotype of GSTM1 (OR: 2.02; 95% CI: 1.32-3.10; P=0.001) or GSTT1 (OR: 1.66; 95% CI: 1.02-2.69; P=0.04), though the risk was not found to be significant when adjusted for age, sex, smoking, tobacco chewing or alcohol use by multivariate logistic regression model. Our data further showed that combination of deletion genotypes of GST (GSTM1 and GSTT1) confer an even higher risk of HNSCC. Interestingly, GSTP1 wild type genotype in combination with GSTM1 null or GSTT1 null genotype increased susceptibility for HNSCC (OR: 2.49 and 2.75, respectively). Likewise a much greater risk for HNSCC was observed in the patients carrying a genotype combination of GSTM1 null, GSTT1 null and GSTP1 (Ile/Ile) (OR: 4.47; 95% CI: 1.62-12.31; P=0.002). Our data have further provided evidence that tobacco chewing and alcohol consumption are the important risk factors for HNSCC. The interaction between tobacco chewing and null genotype of GSTM1 or GSTT1 resulted in about 3.5- and 2.2-fold increase in the risk respectively in the patients when compared to those not chewing tobacco. Alcohol use resulted in more than 4-fold increase in the risk in the patients with null genotype of GSTM1 as compared to those who are non-drinkers. Alcohol consumption also increased the risk (approx. 3-fold) in the cases with null genotype of GSTT1, though the association was not found to be significant when compared to non-drinkers. Our data have provided evidence that GST polymorphism modifies the susceptibility to HNSCC and have further demonstrated importance of gene-environment interaction in modulating the risk to HNSCC.     

Inactivation of Cyanobacterial Nitrogenase After Exposure to Ultraviolet-B Radiation

Exposure of the N₂-fixing cyanobacterium Anabaena BT2 to ultraviolet-B radiation (2.5 W m⁻²) for 30 min resulted in complete loss of nitrogenase activity but 100% cell killing occurred only after a 90-min exposure. Inactivation of nitrogenase activity was not specific to Anabaena BT2; other species also showed a similar effect. The time required for 100% killing and inactivation of nitrogenase activity differed in various species, and this difference may be ascribed to the presence of different levels of UV-B protection mechanisms in individual species. Inhibition of nitrogenase activity was immediate, since exposure of cultures to UV-B for as little as 5 min elicited some inhibition of activity. The activity of UV-B-inhibited nitrogenase did not recover upon transfer of exposed cells to fluorescent light, suggesting that the inhibition may be due to specific inactivation of the enzyme. By employment of inhibitors of protein synthesis and PS-II activity, it was demonstrated that restoration of nitrogenase activity in a UV-B-treated culture occurred by fresh synthesis of nitrogenase polypeptide. Our findings suggest that estimation of nitrogenase activity in diazotrophic species may be used as a marker enzyme for assessing the impact of UV-B radiation. Received: 13 June 2002 / Accepted: 22 July 2002