Increasing evidence shows the beneficial effects of fish oil on breast cancer growth and invasion in vitro and in animal models. Expression of CSF-1 (colony stimulating factor-1) by breast cancer cells acts as potent activator of malignancy and metastasis. In this report, we used two human breast cancer cell lines, MDA-MB-231 and MCF-7, to show that the bioactive fish oil component DHA (docosahexaenoic acid) inhibits expression of CSF-1 and its secretion from these cancer cells. We found that the tumor suppressor protein PTEN regulates CSF-1 expression through PI 3 kinase/Akt signaling via a transcriptional mechanism. The enhanced abundance of microRNA-21 (miR-21) in breast cancer cells contributes to the growth and metastasis. Interestingly, DHA significantly inhibited expression of miR-21. miR-21 Sponge, which derepresses the miR-21 targets, markedly decreased expression of CSF-1 and its secretion. Furthermore, miR-21-induced upregulation of CSF-1 mRNA and its transcription were prevented by expression of PTEN mRNA lacking 3′-untranslated region (UTR) and miR-21 recognition sequence. Strikingly, miR-21 reversed DHA-forced reduction of CSF-1 expression and secretion. Finally, we found that expression of miR-21 as well as CSF-1 was significantly attenuated in breast tumors of mice receiving a diet supplemented with fish oil. Our results reveal a novel mechanism for the therapeutic function of fish oil diet that blocks miR-21, thereby increasing PTEN levels to prevent expression of CSF-1 in breast cancer.
The data derived from epidemiological and animal models confirm a beneficial effect of fish oil (rich in ω-3 polyunsaturated fatty acids) in the amelioration of tumor growth and progression, including breast cancer. The breast cancer patients often develop bone metastasis evidenced by osteolytic lesions, leading to severe pain and bone fracture. Using a mouse model of MDA-MB-231 human breast cancer cell metastasis to bone, here we show that fish oil diet enriched in DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) prevents the formation of osteolytic lesions in bone, indicating suppression of cancer cell metastasis to bone. These results are supported by our data showing both DHA and EPA significantly attenuate the migration/invasion of MDA-MB-231 breast cancer cells in culture. The mechanism that limits breast cancer cells to selective metastasis to bone remains hitherto unexplored. Aberrant increased expression of CD44 is associated with generation of cancer stem cells, which contribute to metastasis of breast cancer cells. We demonstrate that DHA and EPA significantly inhibit the expression of CD44 protein and mRNA by a transcriptional mechanism. Furthermore, we show markedly reduced levels of CD44 mRNA and protein in the tumors of mice, which were fed fish oil diet than those in control diet. Our data provide the first evidence for a salutary effect of fish oil on breast cancer metastasis to bone. Our results identify a novel function of the fish oil active components, DHA and EPA, which target the cell-intrinsic pro-metastatic molecule CD44 to inhibit migration/invasion.
Laparoscopic cholecystectomy (LC) is associated with a high risk of postoperative nausea and vomiting (PONV). Palonosetron is a newer 5HT3 receptor antagonist, which is routinely used in our institution to prevent PONV in patients scheduled for LC, under general anesthesia (GA). We formulated this study to find out whether the palonosetron and dexamethasone combination will be a better choice than palonosetron alone in the prevention of PONV.
Materials and Methods:
Sixty American Society of Anesthesiologists (ASA) physical status I and II patients, scheduled for LC under GA, were randomized to receive either palonosetron or a combination of palonosetron and dexamethasone. The number of complete responders (no emesis, no requirement of rescue anti-emetic medication) and the four-point nausea score was recorded at 2, 6, 24, 48 h postoperatively and the data was analyzed statistically.
The number of complete responders, as well as the nausea score, did not vary significantly (P=0.718) between the two groups over the 48-h postoperative period.
The palonosetron and dexamethasone combination was not more effective than palonosetron alone in the prevention of PONV, in patients undergoing LC under GA.
Complete responders; four-point nausea score; 5HT3 receptor antagonist
Sustained activation of Akt kinase acts as a focal regulator to increase cell growth and survival, which cause tumorigenesis including breast cancer. Statins, potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, display anticancer activity. The molecular mechanisms by which statins block cancer cell growth are poorly understood. We demonstrate that in the tumors derived from MDA-MB-231 human breast cancer cell xenografts, simvastatin significantly inhibited phosphorylation of Akt with concomitant attenuation of expression of the anti-apoptotic protein BclXL. In many cancer cells, BclXL is a target of NFκB. Simvastatin inhibited the DNA binding and transcriptional activities of NF κ B resulting in marked reduction in transcription of BclXL. Signals transmitted by anti-neoplastic mechanism implanted in the cancer cells serve to obstruct the initial outgrowth of tumors. One such mechanism represents the action of the tumor suppressor protein PTEN, which negatively regulates Akt kinase activity. We provide the first evidence for significantly increased levels of PTEN in the tumors of simvastatin-administered mice. Importantly, simvastatin markedly prevented binding of NFκB to the two canonical recognition elements, NFRE-1 and NFRE-2 present in the PTEN promoter. Contrary to the transcriptional suppression of BclXL, simvastatin significantly increased the transcription of PTEN. Furthermore, expression of NFκ B p65 subunit inhibited transcription of PTEN, resulting in reduced protein expression, which leads to enhanced phosphorylation of Akt. Taken together, our data present a novel bifaceted mechanism where simvastatin acts on a nodal transcription factor NFκ B, which attenuates the expression of anti-apoptotic BclXL and simultaneously derepresses the expression of anti-proliferative/proapoptotic tumor suppressor PTEN to prevent breast cancer cell growth.
Statin; Breast tumor; BclXL; Akt kinase
Up to 40% of acute ischaemic strokes in young adults are cryptogenic in nature, that is, no cause is determined. In more than half of these patients, patent foramen ovale (PFO) is seen along with an increased incidence of atrial septal aneurysm. The commonest method of investigation is echocardiography (preferably transoesophageal echocardiography). On the basis of available evidence, low risk patients are treated with antiplatelet agents and high risk patients with warfarin. There are inconclusive data on the efficacy of PFO closure to prevent stroke recurrence. However, if there is recurrent stroke or intolerance to medical therapy, percutaneous closure is carried out.
atrial septal defect; cryptogenic stroke; patent foramen ovale
Giardia lamblia is a microaerophilic human gastrointestinal parasite and considered as an early-diverged eukaryote. In vitro oxidative stress generation plays a significant role in cell cycle progression and cell death of this parasite. In the present study hydrogen peroxide, metronidazole, and a modified growth medium without cysteine and ascorbic acid have been chosen as oxidative stress-inducing agents. Cell cycle progression has been found to be regulated by different types of oxidative stresses. Apoptosis is not an established pathway in Giardia, which is devoid of ideal mitochondria, but in the present investigation, apoptosis-like programmed cell death has been found by the experiments like AnnexinV-FITC assay, DNA fragmentation pattern, etc. On the contrary, Caspase-9 assay, which confirms the caspase-mediated apoptotic pathway, has been found to be negative in all the stress conditions. Protease inhibitor assay confirmed that, even in absence of any proteases, programmed cell death does occur in this primitive eukaryote. All these results signify a novel pathway of programmed suicidal death in Giardia lamblia under oxidative stress. This is the first demonstration of protease-independent programmed cell death regulation in Giardia exclusive for its own specialties.
Giardia lamblia; oxidative stress; reactive oxygen species; programmed cell death; apoptosis; early branching eukaryotes
Using synthetic antisense RNA from the 5'-untranslated region of the beta-tubulin gene as probe in gel retardation assays, a heat stable RNA-binding factor was identified in promastigotes of the kinetoplastid protozoan Leishmania donovani. The same or similar factors interact with several small ribosomal RNA (srRNA) species and, more weakly, with tRNA, as shown by binding and competition experiments. Deletion analysis indicated involvement of repeated purine-rich motifs on the antisense RNA, in the reaction. Related, conserved motifs occur on at least two of the srRNAs. By a modified Western blot assay, the RNA-binding species was identified as a single, small polypeptide. The activity is apparently specific for the promastigote stage of the parasite, being undetectable in amastigotes. The properties of this RNA-binding factor suggest that it is a novel, previously uncharacterized protein.
Carbonic anhydrase (CA), a well-characterized metalloenzyme, is associated with oxygen-18 ( 18O)-isotopic fractionations of CO2. To investigate how CA activity links the 18O of breath CO2 to pre-diabetes (PD) and type 2 diabetes (T2D) during metabolism, we studied pre- and post-dose CA activities in erythrocytes with simultaneous monitoring of 18O/ 16O-isotope ratios of breath CO2 and thereafter elucidated potential metabolic pathways underlying CA alteration in the pathogenesis of T2D. Here we show that the post-dose CA activity in both T2D and PD was markedly enhanced, whereas the non-diabetic controls (NDC) exhibited a considerable reduction in post-dose CA activity when compared with their basal CA activities. However, T2D and PD exhibited isotopic enrichments of 18O in breath CO2, while a marked depletion of 18O in CO2 was manifested in NDC. Thus, the isotopic enrichments and depletions of 18O in breath CO2 were well correlated with the changes in CA activities for controls, PD and T2D. Our findings suggest the changes in CA activities in erythrocytes may contribute to the pathogenesis of T2D and the breath C 18O 16O regulated by the CA activity as a potential biomarker for non-invasive assessment of T2D, and thus may open a new method for treating T2D.
Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica controls glioblastoma (GBM) growth.
Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma.
We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against GBM in vitro and in vivo. Azt caused cell cycle arrest, most prominently at the G1-S stage in GBM cells expressing EGFRvIII, an oncogene present in about 20-25% of GBMs. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma (RB) protein, cell cycle arrest at G1-S and cell death. Independent of RB hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of GBM cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor induced phosphorylation of Akt, Erk1/2 and STAT3. These effects were specific since Azt did not affect mTOR or other cell cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of GBM growth.
Our preclinical findings demonstrate Nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo.
Keratoconus (KC) is characterized by progressive vision loss due to corneal thinning and structural abnormalities. It is hypothesized that KC is caused by deregulated collagen levels and collagen fibril-maturating enzyme lysyl oxidase (LOX). Further, it is currently not understood whether the gene expression deregulated by the corneal epithelium influences KC pathogenesis. We studied (i) the expressions of the LOX, collagen I (COL IA1), collagen IV (COL IVA1), MMP9, and IL6 genes in KC corneal epithelia, (ii) validated their expression levels in patient tissues, and (iii) correlated expression levels with KC disease severity. The primary goal of this study was to evaluate the importance of these genes in the progression of KC.
We analyzed the gene expression levels of the key proteins LOX, collagens (COL IA1 and COL IVA1), MMP9, and IL6 in debrided corneal epithelia from a large cohort of KC patients (90 eyes) and compared them to control patients (52 eyes) without KC. We measured the total LOX activity in the tears of KC patients compared to controls. We also correlated the protein expression levels of LOX and collagens by immunohistochemistry (IHC) in primary tissues from KC patients (27 eyes) undergoing keratoplasty compared to healthy donor corneas (15 eyes).
We observed a significant reduction in LOX transcript levels in KC corneal epithelia, and LOX activity in KC tears correlated with disease severity. Collagen transcripts were also reduced in KC while MMP9 transcript levels were upregulated and correlated with disease severity. IL6 was moderately increased in KC patients. IHC demonstrated a reduction in the protein expression levels of LOX in the epithelium and collagen IV in the basement membrane of KC patients compared to healthy donor corneas.
The data demonstrates that the structural deformity of the KC cornea may be dependent on reduced expressions of collagens and LOX, as well as on MMP9 elevated by the corneal epithelium.
MicroRNAs regulate post-transcriptomic landscape in many tumors including renal cell carcinoma. We have recently shown significantly increased expression of miR-21 in renal tumors and that this miRNA contributes to the proliferation of renal cancer cells in culture. However, the mechanism by which miR-21 regulates renal cancer cells proliferation is poorly understood. Addiction to constitutive NFκB activity is hallmark of many cancers including renal cancer. Using miR-21 Sponge in renal cancer cells to block endogenous function of miR-21, we show inhibition of phosphorylation of p65 subunit of NFκB, IKKβ and IκB, which results in attenuation of NFκB transcriptional activity. Subtle reduction in the tumor suppressor PTEN has been linked to various malignancies. We showed previously that miR-21 targeted PTEN in renal cancer cells. Inhibition of PTEN by siRNAs restored miR-21 Sponge-induced suppression of phosphorylation of p65, IKKβ, IκB and NFκB transcriptional activity along with reversal of miR-21 Sponge-reduced phosphorylation of Akt. Expression of constitutively active Akt protected against miR-21 Sponge- and PTEN-mediated decrease in p65/IKKβ/IκB phosphorylation and NFκB transcriptional activity. Furthermore, IKKβ and p65 were required for miR-21-induced renal cancer cell proliferation. Interestingly, miR-21 controlled the expression of cyclin D1 through NFκB-dependent transcription. Finally, we demonstrate that miR-21-regulated renal cancer cell proliferation is mediated by cyclin D1 and CDK4. Together, our results establish a molecular order of a phosphatase-kinase couple involving PTEN/Akt/IKKβ and NFκB-dependent cyclin D1 expression for renal carcinoma cell proliferation by increased miR-21 levels.
microRNA; Renal cancer; PTEN; NFκB
We have reported that prophylactic as well as therapeutic administration of neem leaf glycoprotein (NLGP) induces significant restriction of solid tumor growth in mice. Here, we investigate whether the effect of such pretreatment (25µg/mice; weekly, 4 times) benefits regulation of tumor angiogenesis, an obligate factor for tumor progression. We show that NLGP pretreatment results in vascular normalization in melanoma and carcinoma bearing mice along with downregulation of CD31, VEGF and VEGFR2. NLGP pretreatment facilitates profound infiltration of CD8+ T cells within tumor parenchyma, which subsequently regulates VEGF-VEGFR2 signaling in CD31+ vascular endothelial cells to prevent aberrant neovascularization. Pericyte stabilization, VEGF dependent inhibition of VEC proliferation and subsequent vascular normalization are also experienced. Studies in immune compromised mice confirmed that these vascular and intratumoral changes in angiogenic profile are dependent upon active adoptive immunity particularly those mediated by CD8+ T cells. Accumulated evidences suggest that NLGP regulated immunomodulation is active in tumor growth restriction and normalization of tumor angiogenesis as well, thereby, signifying its clinical translation.
Osteoarthritis and Osteoporosis are highly prevalent disease, so is hypovitaminosis D. We tried to find out prevalence of osteoporosis and hypovitaminosis D in patients suffering from primary knee Osteoarthritis. We also compared the prevalence of oseotoporosis between general population and patients of primary osteoarthritis.
Patients suffering from primary knee OA were taken from Rheumatology OPD of Medical College Hospital and SSKM Hospital Kolkata, India. For each patient age and sex matched friend or relative of same locality was taken in the study as controls. Hospital staffs that come from different part of state was taken in the study as controls. The control population was the representative of general population.
Total number of participants in this study was 206. Out of which there were 98 cases and 108 controls. BMD status correlates significantly with Primary OA. Serum Vitamin D3 status correlates significantly with Osteoarthritis. Age of the patients correlated significantly with both BMD Status and Knee OA but not with the vVitamin D level. There were significant correlation between the Serum Vitamin D3 status and BMD of the subjects.
Osteoporosis is prevalent both in general population and patients suffering from Knee Osteoarthritis and may increase the disability. The matter is complicated by the fact hypovitaminosis D is also prevalent in the population and positively correlated with both Osteoporosis and osteoarthrosis, though we cannot comment on further pathogenesis because of cross sectional design of the study.
Knee osteoarthitis; Osteoporosis; Hypovitaminosis D
Cerebellar ataxia is a rare manifestation of neuropsychiatric systemic lupus erythematosus (SLE). Development of vasculitic infarcts in the cerebellum is the most plausible reason of this manifestation. We report the case of a patient who presented with characteristic skin rashes of lupus along with cerebellar signs. Imaging of brain in this patient revealed prominent cerebellar atrophy. She was treated with mycophenolate mofetil and oral corticosteroid, and there was no further progression of her neurological signs after the initiation of therapy. In the clinical context of varied presentations of neurolupus, this is one of the rare sightings and our treatment protocol holds promise as first-line therapy in future.
Ataxia; immunosuppression; systemic lupus erythematosus
Enhanced TGFβ activity contributes to the accumulation of matrix proteins including collagen I (α2) by proximal tubular epithelial cells in progressive kidney disease. Although TGFβ rapidly activates its canonical Smad signaling pathway, it also recruits noncanonical pathway involving mTOR kinase to regulate renal matrix expansion. The mechanism by which chronic TGFβ treatment maintains increased mTOR activity to induce the matrix protein collagen I (α2) expression is not known. Deptor is an mTOR interacting protein that suppresses mTOR activity in both mTORC1 and mTORC2. In proximal tubular epithelial cells, TGFβ reduced deptor levels in a time-dependent manner with concomitant increase in both mTORC1 and mTORC2 activities. Expression of deptor abrogated activity of mTORC1 and mTORC2, resulting in inhibition of collagen I (α2) mRNA and protein expression via transcriptional mechanism. In contrast, neutralization of endogenous deptor by shRNAs increased activity of both mTOR complexes and expression of collagen I (α2) similar to TGFβ treatment. Importantly, downregulation of deptor by TGFβ increased the expression of Hif1α by increasing translation of its mRNA. TGFβ-induced deptor downregulation promotes Hif1α binding to its cognate hypoxia responsive element in the collagen I (α2) gene to control its protein expression via direct transcriptional mechanism. Interestingly, knockdown of raptor to specifically block mTORC1 activity significantly inhibited expression of collagen I (α2) and Hif1α while inhibition of rictor to prevent selectively mTORC2 activation did not have any effect. Critically, our data provide evidence for the requirement of TGFβ-activated mTORC1 only by deptor downregulation, which dominates upon the bystander mTORC2 activity for enhanced expression of collagen I (α2). Our results also suggest the presence of a safeguard mechanism involving deptor-mediated suppression of mTORC1 activity against developing TGFβ-induced renal fibrosis.
The contribution of chronic hepatitis B virus (HBV) infection in the pathogenesis of hepatocellular carcinoma (HCC) through progressive stages of liver fibrosis is exacerbated by the acquisition of naturally occurring mutations in its genome. This study has investigated the prevalence of single and combo mutations in the genome of HBV-genotype D from treatment naïve Indian patients of progressive liver disease stages and assessed their impact on the disease progression to HCC.
The mutation profile was determined from the sequence analysis of the full-length HBV genome and compared with the reference HBV sequences. SPSS 16.0 and R software were used to delineate their statistical significance in predicting HCC occurrence.
Age was identified as associated risk factor for HCC development in chronic hepatitis B (CHB) patients (p≤0.01). Beyond the classical mutations in basal core promoter (BCP) (A1762T/G1764A) and precore (G1862T), persistence of progressively accumulated mutations in enhancer-I, surface, HBx and core were showed significant association to liver disease progression. BCP_T1753C, core_T147C, surface_L213I had contributed significantly in the disease progression to HCC (p<0.05) in HBeAg positive patients whereas precore_T1858C, core_I116L, core_P130Q and preS1_S98T in HBeAg negative patients. Furthermore, the effect of individual mutation was magnified by the combination with A1762T/G1764A in HCC pathogenesis. Multivariate risk analysis had confirmed that core_P130Q [OR 20.71, 95% CI (1.64–261.77), p = 0.019] in B cell epitope and core_T147C [OR 14.58, 95% CI (1.17–181.76), p = 0.037] in CTL epitope were two independent predictors of HCC in HBeAg positive and negative patients respectively.
Thus distinct pattern of mutations distributed across the entire HBV genome may be useful in predicting HCC in high-risk CHB patients and pattern of mutational combinations may exert greater impact on HCC risk prediction more accurately than point mutations and hence these predictors may support the existing surveillance strategies in proper management of the patients.
The main objective of the present study is to effectively utilize the de-oiled algal biomass (DAB) to minimize the waste streams from algal biofuel by using it as an adsorbent. Methylene blue (MB) was used as a sorbate for evaluating the potential of DAB as a biosorbent. The DAB was characterized by SEM, FTIR, pHPZC, particle size, pore volume and pore diameter to understand the biosorption mechanism. The equilibrium studies were carried out by variation in different parameters, i.e., pH (2–9), temperature (293.16–323.16 K), biosorbent dosage (1–10 g L−1), contact time (0–1,440 min), agitation speed (0–150 rpm) and dye concentration (25–2,500 mg L−1). MB removal was greater than 90% in both acidic and basic pH. The optimum result of MB removal was found at 5–7 g L−1 DAB concentration. DAB removes 86% dye in 5 minutes under static conditions and nearly 100% in 24 hours when agitated at 150 rpm. The highest adsorption capacity was found 139.11 mg g−1 at 2,000 mg L−1 initial MB concentration. The process attained equilibrium in 24 hours. It is an endothermic process whose spontaneity increases with temperature. MB biosorption by DAB follows pseudo-second order kinetics. Artificial neural network (ANN) model also validates the experimental dye removal efficiency (R2 = 0.97) corresponding with theoretically predicted values. Sensitivity analysis suggests that temperature and agitation speed affect the process most with 23.62% and 21.08% influence on MB biosorption, respectively. Dye adsorption capacity of DAB in fixed bed column was 107.57 mg g−1 in preliminary study while it went up to 139.11 mg g−1 in batch studies. The probable mechanism for biosorption in this study is chemisorptions via surface active charges in the initial phase followed by physical sorption by occupying pores of DAB.
Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as Type 2 Diabetes and atherosclerosis) has shifted the focus from Western diet-induced changes in gut microbiota per se to release of gut bacteria-derived products into circulation as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. Under physiological conditions, an intact intestinal barrier prevents this release of LPS underscoring the importance of examining and modulating the direct effects of Western diet on intestinal barrier function. In the present study we evaluated two strategies, namely selective gut decontamination and supplementation with oral curcumin, to modulate Western-diet (WD) induced changes in intestinal barrier function and subsequent development of glucose intolerance and atherosclerosis. LDLR−/− mice were fed WD for 16 weeks and either received non-absorbable antibiotics (Neomycin and polymyxin) in drinking water for selective gut decontamination or gavaged daily with curcumin. WD significantly increased intestinal permeability as assessed by in vivo translocation of FITC-dextran and plasma LPS levels. Selective gut decontamination and supplementation with curcumin significantly attenuated the WD-induced increase in plasma LPS levels (3.32 vs 1.90 or 1.51 EU/ml, respectively) and improved intestinal barrier function at multiple levels (restoring intestinal alkaline phosphatase activity and expression of tight junction proteins, ZO-1 and Claudin-1). Consequently, both these interventions significantly reduced WD-induced glucose intolerance and atherosclerosis in LDLR−/− mice. Activation of macrophages by low levels of LPS (50 ng/ml) and its exacerbation by fatty acids is likely the mechanism by which release of trace amounts of LPS into circulation due to disruption of intestinal barrier function induces the development of these diseases. These studies not only establish the important role of intestinal barrier function, but also identify oral supplementation with curcumin as a potential therapeutic strategy to improve intestinal barrier function and prevent the development of metabolic diseases.
M. phaseolina, a global devastating necrotrophic fungal pathogen causes charcoal rot disease in more than 500 host plants. With the aim of understanding the plant-necrotrophic pathogen interaction associated with charcoal rot disease of jute, biochemical approach was attempted to study cellular nitric oxide production under diseased condition. This is the first report on M. phaseolina infection in Corchorus capsularis (jute) plants which resulted in elevated nitric oxide, reactive nitrogen species and S nitrosothiols production in infected tissues. Time dependent nitric oxide production was also assessed with 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate using single leaf experiment both in presence of M. phaseolina and xylanases obtained from fungal secretome. Cellular redox status and redox active enzymes were also assessed during plant fungal interaction. Interestingly, M. phaseolina was found to produce nitric oxide which was detected in vitro inside the mycelium and in the surrounding medium. Addition of mammalian nitric oxide synthase inhibitor could block the nitric oxide production in M. phaseolina. Bioinformatics analysis revealed nitric oxide synthase like sequence with conserved amino acid sequences in M. phaseolina genome sequence. In conclusion, the production of nitric oxide and reactive nitrogen species may have important physiological significance in necrotrophic host pathogen interaction.
Liver is the major organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or as bile acids. Intracellular hydrolysis of lipoprotein-derived cholesteryl esters (CE) is essential to generate the free cholesterol (FC) required for this process. We earlier demonstrated that over-expression of human cholesteryl ester hydrolase (Gene symbol CES1) increased bile acid synthesis in human hepatocytes and enhanced reverse cholesterol transport in mice. The objective of the present study was to demonstrate that liver-specific deletion of its murine ortholog, Ces3, would decrease cholesterol elimination from the body and increase atherosclerosis.
Approach and Results
Liver-specific Ces3 knockout mice (Ces3-LKO) were generated and Ces3 deficiency did not affect the expression of genes involved in cholesterol homeostasis and FC or bile acid transport. The effects of Ces3 deficiency on the development of Western diet-induced atherosclerosis were examined in LDLR-/- mice. Despite similar plasma lipoprotein profiles, there was increased lesion development in LDLR-/-Ces3-LKO mice along with a significant decrease in bile acids content of the bile. Ces3 deficiency significantly reduced the flux of cholesterol from [3H]-CE labeled HDL to feces (as FC and bile acids) and decreased total fecal sterol elimination.
Our results demonstrate that hepatic Ces3 modulates the hydrolysis of lipoprotein-delivered CE and thereby regulates FC and bile acid secretion into the feces. Its deficiency, therefore, results in reduced cholesterol elimination from the body leading to significant increase in atherosclerosis. Collectively, these data establish the anti-atherogenic role of hepatic CE hydrolysis.
Hepatic cholesteryl ester hydrolysis; atherosclerosis; liver-specific knockout mice; cholesterol elimination from the body; bile acids
The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored.
The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed.
HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC.
Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy.
To assess efficacy and safety of lornoxicam as analgesic after surgery on head and neck in comparison to tramadol. Forty five patients undergoing operations on head and neck were recruited and randomly assigned to two parallel groups—lornoxicam and tramadol, both given intramuscular on the first post-operative day followed by oral tablets for the consecutive 4 days. Treatment was given single blind. 10 cm visual analog scale (VAS) pain score and wound tenderness assessed by a 3-point ordinal scale were the primary efficacy parameters. Use of rescue medication and percentage of subjects having at least 50 % pain relief by 48 h were also compared as secondary parameters. The groups were comparable at baseline regarding age, sex and VAS score. There was steady decline in VAS pain score from baseline to study end in both the groups, indicating good analgesic efficacy with either drug. Between groups comparisons of VAS score showed no significant difference at any time point. Between groups comparisons of wound tenderness also showed no significant difference. Five patients on lornoxicam and one patient on tramadol experienced at least 50 % pain relief at 48 hours compared to baseline while five patients from the lornoxicam group and eight from the tramadol group required rescue medicine. The tolerability of lornoxicam appeared to be significantly superior to tramadol, with less number of patients experiencing adverse drug reactions. Lornoxicam is safe, effective and comparable to tramadol for relieving postoperative pain after operations on head and neck.
Lornoxicam; Tramadol; Post-operative pain; VAS score
Transforming growth factorβ (TGFβ)-induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ time-dependently stimulated eIF4E phosphorylation at Ser-209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen-activated and extracellular signal-regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFβ-induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser-209 is mediated by Mnk-1 (mitogen-activated protein kinase signal-integrating kinase-1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk-1 in response to TGFβ. Dominant negative Mnk-1 significantly inhibited TGFβ-stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E-4EBP-1 complex, decreased TGFβ-stimulated phosphorylation of eIF4E without any effect on Mnk-1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFβ. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP-1-eIF4E complex for Mnk-1-mediated phosphorylation of eIF4E. Moreover, we conclude that TGFβ-induced noncanonical signaling circuit involving PI 3 kinase-dependent Mnk-1-mediated phosphorylation of eIF4E at Ser-209 is required to facilitate mesangial cell hypertrophy.
We describe a case of anhidrotic ectodermal dysplasia (AED) with an autosomal recessive mode of inheritance, a very rare entity, in a 2-year-old female child of two asymptomatic, consanguineous parents. Their previous child also had a similar condition. Autosomal recessive AED (AR-AED) can have its full expression both in males and females and it is clinically indistinguishable from the x-linked recessive AED (XL-AED), which is the most common type of ectodermal dysplasia. Unlike the partially symptomatic carriers of XL-AED, the heterozygotes of AR-AED are phenotypically asymptomatic.
Anhidrotic; autosomal; dysplasia; ectodermal; recessive