Probing protein aggregation through spectroscopic insights and multimodal approaches: A comprehensive review for counteracting neurodegenerative disorders.

Aberrant accumulation of protein misfolding can cause aggregation and fibrillation and is one of the primary characteristic features of neurodegenerative diseases. Because they are disordered, misfolded, and aggregated proteins pose a significant setback in drug designing. The structural study of intermediate steps in these kinds of aggregated proteins will allow us to determine the conformational changes as well as the probable pathways encompassing various neurodegenerative disorders. The analysis of protein aggregates involved in neurodegenerative diseases relies on a diverse toolkit of biophysical techniques, encompassing both morphological and non-morphological methods. Additionally, Thioflavin T (ThT) assays and Circular Dichroism (CD) spectroscopy facilitate investigations into aggregation kinetics and secondary structure alterations. The collective application of these biophysical techniques empowers researchers to comprehensively unravel the intricate nature of protein aggregates associated with neurodegeneration. Furthermore, the topics covered in this review have summed up a handful of well-established techniques used for the structural analysis of protein aggregation. This multifaceted approach advances our fundamental understanding of the underlying mechanisms driving neurodegenerative diseases and informs potential therapeutic strategies.

Amyloid-induced neurodegeneration: A comprehensive review through aggregomics perception of proteins in health and pathology.

Amyloidosis of protein caused by fibrillation and aggregation are some of the most exciting new edges not only in protein sciences but also in molecular medicines. The present review discusses recent advancements in the field of neurodegenerative diseases and therapeutic applications with ongoing clinical trials, featuring new areas of protein misfolding resulting in aggregation. The endogenous accretion of protein fibrils having fibrillar morphology symbolizes the beginning of neuro-disorders. Prognostic amyloidosis is prominent in numerous degenerative infections such as Alzheimer's and Parkinson's disease, Amyotrophic lateral sclerosis (ALS), etc. However, the molecular basis determining the intracellular or extracellular evidence of aggregates, playing a significant role as a causative factor in neurodegeneration is still unclear. Structural conversions and protein self-assembly resulting in the formation of amyloid oligomers and fibrils are important events in the pathophysiology of the disease. This comprehensive review sheds light on the evolving landscape of potential treatment modalities, highlighting the ongoing clinical trials and the potential socio-economic impact of novel therapeutic interventions in the realm of neurodegenerative diseases. Furthermore, many drugs are undergoing different levels of clinical trials that would certainly help in treating these disorders and will surely improve the socio-impact of human life.

Quercetin and resveratrol ameliorate nickel-mediated hypercontraction in isolated Wistar rat aorta.

PURPOSE: The ameliorative potential of quercetin and resveratrol on isolated endothelium-intact aortic rings incubated with nickel was examined. METHOD: The effect of varying concentrations of quercetin and resveratrol was investigated on isolated Wistar rat aortic rings using an organ bath system over vasoconstrictor phenylephrine (PE) at 1 µM. To delineate the mechanism of action, isolated aortic rings were pre-incubated with pharmacological modulators, such as verapamil 1 µM, apocynin 100 µM, indomethacin 100 µM or N-G-nitro-L-arginine methyl ester (L-NAME) 100 µM, separately, before incubation with 100 µM quercetin and 30 µM resveratrol. To assess the ameliorative and prophylactic potentials of quercetin and resveratrol, aortic rings were also incubated with quercetin or resveratrol for 40 min, followed by incubation with nickel for 40 min. RESULTS: At 100 µM, quercetin caused 29% inhibition of contraction, while resveratrol at 30 µM caused 55% inhibition of contraction in aortic rings compared with control. Aortic rings incubated with contractile modulators, such as verapamil, apocynin, indomethacin or N-G-nitro-L-arginine methyl ester (L-NAME), along with quercetin or resveratrol at their concentrations producing maximum relaxant effect, showed that both of these natural compounds exert their relaxant effect by inhibiting the generation of reactive oxygen species (ROS) from endothelial and smooth muscle cells, blocking voltage-gated calcium channels, and increasing the release of nitric oxide (NO). The mediation of hypercontraction by nickel is due to the increased ROS and the influx of calcium through voltage-dependent calcium channels. These natural compounds are shown to counter the nickel-induced effects, appearing as effective ameliorators. CONCLUSION: In this study, we found that quercetin and resveratrol act as ameliorators of nickel-mediated hypercontraction by decreasing ROS and enhancing NO release from endothelial cells.

Elucidation of Mechanisms in Cu (II) Caused Hypercontraction of Rat Tracheal Rings.

Airway smooth muscle contraction is one of the primary factors involved in the initiation and progression of asthma which in turn is regulated by increased cytosolic Ca2+ concentration from intracellular stores and through transmembrane ion channels. Calcium-independent factors such as reactive oxygen species (ROS) generation, nitric oxide (NO) depletion and cyclooxygenase (COX) pathways also contribute to tracheal smooth muscle contraction. Studies on copper toxicity suggest significance of this essential micronutrient overdose in acute respiratory disorders, allergic asthma and ciliary motion in tracheal explants. However, the mechanism of copper caused hypercontraction upon direct exposure to tracheal smooth muscle is largely unknown. In this study we investigate the effect of copper exposure on isolated tracheal rings and relative contributions of various factors in acetylcholine-induced contractions. Results obtained suggest that rise in intracellular calcium concentration via voltage-operated Ca2+ channel (VOCC), store-operated Ca2+ channel (SOCC), stretch-activated channels (SAC) and TRP channel (transient receptor potential channel) activation is the major factor in copper-mediated hypercontraction. ROS generation or COX-dependent pathways do not appear to significantly contribute to Cu2+ caused hypercontraction.

Immobilization of invertase in calcium alginate and calcium alginate-kappa-carrageenan beads and its application in bioethanol production.

Invertase from Saccharomyces cerevisiae was entrapped in Ca-alginate and Ca-alginate-kappa-carrageenan matrix. Optimum pH for the free and immobilized invertase was found to be 4.5 and 5.5, respectively. The optimum hydrolysis temperature was 55 °C for both the free and immobilized forms. Km values for free invertase and invertase entrapped in Ca-alginate and Ca-alginate-kappa-carrageenan beads were 15, 21, and 19 mM, respectively. Values of Vmax for free invertase and invertase entrapped in Ca-alginate and Ca-alginate-kappa-carrageenan beads were 238, 186, and 197 mM min-1, respectively. Invertase entrapped in Ca-alginate-kappa-carrageenan matrix had the highest pH and thermal stability, higher reusability with 71% retention in activity after nine batches of reuse and higher storage stability with 86% activity retention after 12 weeks at 4 °C, pH 4.5. Fermentation of cane molasses by yeast for bioethanol formation in the presence of free invertase at 30 °C, pH 5.0, led to an increase in ethanol production by 3%. However, the production increased by 9% when invertase entrapped in Ca-alginate-kappa-carrageenan was used as a catalyst.HighlightsInvertase from Saccharomyces cerevisiae was entrapped in Ca-alginate beads.For efficient encapsulation of invertase, kappa-carrageenan was used in combination with alginate as a matrix.Entrapment in Ca-alginate-kappa-carrageenan increased pH and thermal stability of invertase.Invertase entrapped in Ca-alginate-kappa-carrageenan was used for bioethanol production from cane molasses.

Application of Invertase Immobilized on Chitosan Using Glutaraldehyde or Tris(Hydroxymethyl)Phosphine as Cross-Linking Agent to Produce Bioethanol.

Invertase was immobilized on chitosan using glutaraldehyde or tris(hydroxymethyl)phosphine as cross-linker. The optimum pH for free and immobilized enzyme was found to be 4.5 and 5.5, respectively. The optimum hydrolysis temperature was 55 °C for both the free and immobilized forms. Km and Vmax values for free invertase, and invertase immobilized on glutaraldehyde- and THP-activated chitosan were 15, 19, and 20 mM, respectively, and 238, 204, and 212 mM min-1, respectively. The THP-immobilized enzyme had the highest pH and thermal stability, higher reusability with 70% retention in activity after 9 batches of reuse and higher storage stability with 90% retention in activity after 12 weeks at 4 °C, pH 4.5. Fermentation of cane molasses by yeast to form ethanol in the presence of free invertase at 30°C, pH 5.0 led to an increase in ethanol production by 3% and the production increased by 10.7% when immobilized invertase was used as catalyst. Graphical Abstract.

Cobalt-Induced Hypercontraction is Mediated by Generationof Reactive Oxygen Species and Influx of Calcium in Isolated RatAorta.

To investigate the mechanism of cobalt-mediated phenylephrine (PE)-induced contraction in endothelium-intact isolated Wistar rat aortic rings. Effect of dose-dependent concentrations of cobalt on PE-induced contraction was investigated in isolated Wistar rat aortic rings using an organ bath system. Aortic rings were pre-incubated with verapamil (1 µM and 20 µM), gadolinium, apocynin, indomethacin or N-G-nitro-L-arginine methyl ester (L-NAME) separately before incubation with cobalt. Endothelium-intact aortic rings were incubated with 800 nM, 1 µM, 10 µM, 50 µM cobalt; we observed 20%, 22%, 32% and 27% increased contractions respectively, while no effect was seen in tension recording on cobalt exposure. Incubation of endothelium-intact aortic rings with 100 µM apocynin and 100 µM L-NAME suggested the role of NADPH oxidase in generation of reactive oxygen species (ROS) and decrease in bioavailability of nitric oxide (NO) from eNOS on exposure to cobalt. Aortic rings pre-incubated with 1 µM and 20 µM verapamil suggested role of both L-type and T-type calcium channels in influx of extracellular calcium in smooth muscle cells. We observed no role of store-operated calcium channels (SOCC) in calcium influx due to cobalt exposure and cyclooxygenase in generation of prostanoids in isolated aortic rings. Cobalt caused rise of PE-induced contractions as a result of the endothelial generation of ROS, by decreasing bioavailability of NO. Generation of ROS may be responsible for causing the influx of extracellular calcium through L-type and T-type Ca2+ channels in smooth muscle cells.

Level of phospho-STAT3 (Tyr705) correlates with copy number and physical state of human papillomavirus 16 genome in cervical precancer and cancer lesions.

Our earlier studies indicated an important role of inducible transcription factor STAT3 in the establishment of persistent infection of human papillomavirus (HPV) type 16 and promotion of cervical carcinogenesis. Since HPV load and its physical state are two potential determinants of this virally-induced carcinogensis, though with some exceptions, we extended our study to examine the role of active STAT3 level in cervical precancer and cancer lesions and it's association with HPV viral load and physical state. An elevated level of active STAT3 was measured by assessing phospho-STAT3-Y705 (pSTAT3), in tumor tissues harboring higher viral load irrespective of the disease grade. Physical state analysis of HPV16 by assessing the degree of amplification of full length E2 and comparing it with E6 (E2:E6 ratio), which predominantly represent episomal form of HPV16, revealed low or undetectable pSTAT3. A strong pSTAT3 immunoreactivity was found in tissues those harbored either mixed or predominantly integrated form of viral genome. Cumulative analysis of pSTAT3 expression, viral load and physical state demonstrated a direct correlation between pSTAT3 expression, viral load and physical state of HPV. The study suggests that there exists a strong clinical correlation between level of active STAT3 expression and HPV genome copy number, and integrated state of the virus that may play a pivotal role in promotion/maintanence of tumorigenic phenotype.

Role of calcium channels and endothelial factors in nickel induced aortic hypercontraction in Wistar rats.

AIM: To investigate the mechanism of nickel augmented phenylephrine (PE)-induced contraction in isolated segments of Wistar rat aorta. MATERIALS AND METHODS: Effect of varying concentrations of nickel on PE-induced contraction were investigated in isolated segments of Wistar rat aorta using an organ bath system. Aortic rings were pre-incubated with verapamil (1 µM and 20 µM), gadolinium, apocynin, indomethacin or N-G-nitro-L-arginine methyl ester (L-NAME) separately before incubation with nickel. RESULTS: Endothelium intact aortic rings incubated with 100 nM, 1 µM or 100 µM of nickel exhibited 80%, 43% and 28% increase in PE-induced contraction, respectively, while no such enhancing responses were observed in endothelium denuded aorta. Incubation of aortic rings with 1 µM and 20 µM verapamil suggested an involvement of influx of calcium through T-type calcium channels in smooth muscle cells, while aortic rings pre-incubated with gadolinium showed no role of store operated calcium channels in the nickel effect on PE-induced contractions. The enhancing effect of nickel on PE-induced contractions was inhibited by apocynin, indomethacin or L-NAME. CONCLUSION: Nickel has caused augmentation of PE-induced contractions as a result of the endothelial generation of reactive oxygen species (ROS) and cyclooxygenase 2 (COX2) dependent endothelium contracting factors (EDCFs), which increases the influx of extracellular calcium through T-type Ca2+ channels in smooth muscle cells.

Detection of mutations in gyrB using denaturing high performance liquid chromatography (DHPLC) among Salmonella enterica serovar Typhi and Paratyphi A.

Background: Fluoroquinolone resistance is mediated by mutations in the quinolone-resistance determining region (QRDR) of the topoisomerase genes. Denaturing high performance liquid chromatography (DHPLC) was evaluated for detection of clinically important mutations in gyrB among Salmonella. Methods: Salmonella Typhi and S. Paratyphi A characterised for mutation in QRDR of gyrA, parC and parE were studied for mutation in gyrB by DHPLC and validated by sequencing. Results: The DHPLC analysis was able to resolve the test mutant from isolates with wild type gyrB and distinguished mutants from other mutant by peak profile and shift in retention time. Three sequence variants were detected at codon 464, and a novel mutation Ser→Thr was also detected. gyrB mutation was associated with non classical quinolone resistance (NALS-CIPDS) in 34 isolates of S. Typhi only and was distinct from classical quinolone resistance associated with gyrA mutations (NALR-CIPDS). Conclusions: DHPLC is effective for the detection of mutation and can reduce the need for sequencing to detect clinically significant gyrB mutations. GenBank accession nos: KF993966, KF993965 and KF993964.

Lifestyle and Sporadic Colorectal Cancer in India.

BACKGROUND: The study evaluated the patient, lifestyle and tumor profile in patients undergoing upfront surgery for sporadic colorectal cancer (CRC) in Indian population. MATERIALS AND METHODS: One hundred consecutive patients were included. Details related to their demographic profile, habits, signs and symptoms, tumor profile, further treatment and follow up were recorded. RESULTS: The majority of the patients had colonic cancer (68%), advanced tumor stage 3 and 4 (46%), moderately differentiated tumors (70%) with absence of lymphatic invasion (60%) and metastasis (90%). Correlations between tumor location and abdominal pain (p-value 0.002), bleeding per rectum (p-value <0.001), difficulty in micturition (p-value 0.012) and constipation (p-value 0.007) were found to be statistically significant. Abdominal pain was more frequently reported in patients with metastasis (p-value 0.031). Loss of weight statistically correlated with absence of lymphatic invasion (p-value 0.047). Associations between tumor stage and alcohol intake (p-value 0.050) and non vegetarian diet (p-value 0.006); lymphatic invasion and intake of spicy food (p-value 0.040) and non vegetarian diet (p-value 0.001) and metastasis and alcohol intake (p-value 0.041) were also observed. Age and tumor grade were also correlated (p-value 0.020). CONCLUSIONS: Minimizing the adverse lifestyle factors can help in reducing the overall incidence of CRC in the Indian population.

Diversity and antifungal resistance patterns of prevalent opportunistic pathogenic yeasts colonizing the oral cavities of asymptomatic human immunodeficiency virus-infected individuals, and their relation to CD4(+) counts.

BACKGROUND: Yeasts are important opportunistic pathogens, in individuals infected with human immunodeficiency virus (HIV). Yeast species inhabiting the oral mucosa of HIV-infected persons can act as source of oral lesions, especially as the individual progresses towards immunocompromised state. Present study was conducted to evaluate the diversity of yeasts in oral cavities of asymptomatic HIV-infected persons and their association with CD4(+) cell counts. MATERIALS AND METHODS: 100 HIV seropositive subjects and 100 healthy controls were screened for oral yeast carriage using standard procedures. RESULTS: Of the 100 HIV-seropositive persons screened, 48 were colonized by different yeasts, either alone or in association with another species. Candida albicans was the most common species (56.90%) while non C. albicans Candida (NCAC) accounted for 39.65%. Among NCAC, Candida tropicalis and Candida krusei were most common. One isolate each of rare opportunistic pathogenic yeasts, Geotrichum candidum and Saccharomyces cereviseae, was recovered. The control group had an oral candidal carriage rate of 23%; C. albicans was the predominant species, followed by Candida glabrata, C. tropicalis and Candida parapsilosis. Antifungal susceptibility testing revealed no resistance in C. albicans, to the commonly used antifungal agents, whereas resistance or dose dependent susceptibility to fluconazole was observed in some of the NCAC species. CONCLUSION: Oral carriage of opportunistic pathogenic yeasts was greater in HIV-seropositive persons heading towards immunocompromised state, as evidenced by their CD4(+) cell count. The predominant yeast isolated in this study (C. albicans), was found to be susceptible to commonly used antifungals.

Increased A3AR-dependent vasoconstriction in diabetic mice is promoted by myeloperoxidase.

Vascular dysfunction importantly contributes to mortality and morbidity in various cardiac and metabolic diseases. Among endogenous molecules regulating vascular tone is adenosine, with the adenosine A3 receptor (A3AR) exerting cardioprotective properties in ischemia and reperfusion. However, overexpression of A3AR is suggested to result in vascular dysfunction and inflammation. The leukocyte enzyme myeloperoxidase (MPO) is an important modulator of vascular function with nitric oxide-consuming and proinflammatory properties. Increased MPO plasma levels are observed in patients with cardiovascular disorders like heart failure, acute coronary syndromes, and arrhythmias. Given that vascular dysfunction and inflammation are also hallmarks of diabetes, the role of MPO in adenosine-dependent vasomotor function was investigated in a murine model of diabetes mellitus. Wild-type (WT) and MPO-deficient (Mpo) mice were treated with Streptozotocin (STZ), which induced an increase of MPO plasma levels in WT mice and led to enhanced aortic superoxide generation as assessed by dihydroethidium staining in STZ-treated WT mice as compared with controls. The vasoconstriction of aortic segments in response to the A3AR agonist Cl-IB-MECA (2-Chloro-N6-(3-iodobenzyl)-N-methyl-5-carbamoyladenosine) as determined by isometric force measurements was augmented in diabetic WT as compared with diabetic Mpo mice. Moreover, A3AR protein expression was enhanced in STZ-treated mice but was attenuated by MPO deficiency. The current data reveal an MPO-mediated increase of vascular A3AR expression under diabetic conditions, which leads to enhanced vasoconstriction in response to A3AR agonists and discloses an additional mechanism of MPO-mediated vascular dysfunction.

Modulation of Pb(II) caused aortal constriction by eugenol and carvacrol.

Exposure to lead is known to cause vasoconstriction, exact mechanism of which remains to be elucidated. In this study, we investigate contractile responses of rat aortal rings equilibrated with Pb(II) in organ bath system, explore pathways responsible for hypercontraction and examine two ameliorators of lead-induced hypercontraction. At 1 µmol L(-1) Pb(II), aortal rings showed an average increase of 50% in isometric contraction. Incubation of rings, unexposed to Pb(II), with 1 µmol L(-1) sodium nitroprusside (nitric oxide (NO) donor), 100 µmol L(-1) apocynin (reactive oxygen species (ROS) inhibitor), and 100 µmol L(-1) indomethacin (cyclooxygenase inhibitor) lead to decrease in phenylephrine-induced contraction by 31, 27, and 29%, respectively. This decrease of contraction for Pb(II)-exposed rings was 48, 53, and 38%, respectively, indicating that ROS- and NO-dependent components of contractions are significantly elevated in Pb(II)-induced hypercontraction. Cyclooxygenase-dependent contractile component did not show significant elevation. Eugenol and carvacrol are plant-derived phenols known to possess antioxidant activity and hence could act as possible ameliorators of hypercontraction. At saturating concentrations of 100 µmol L(-1), eugenol and carvacrol caused a decrease in contraction by 38 and 42% in unexposed rings and 46 and 50% in Pb(II)-exposed rings. Co-incubation of rings with eugenol/carvacrol and various inhibitors suggests that both these active principles exert their relaxant effect via quenching of ROS and stimulation of NO synthesis. To conclude, Pb(II) is shown to induce hypercontraction of aortal rings through elevation of ROS and depletion of NO. This hypercontraction is effectively mitigated by eugenol and carvacrol.

Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges.

OBJECTIVE: To establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance. METHODS: Eight hundred and ninety-one isolates of S. Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NALS (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from gyrA, gyrB, parC and parE and from the plasmid borne determinants: qnrA,B,S; aac(6')-Ib-cr and qepA. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci. RESULTS: Eighty isolates with a nalidixic acid MIC of <32 mg/L (NALS) and a ciprofloxacin MIC of >0.064 mg/L CIPI (ciprofloxacin reduced susceptibility) were found. In 36 NALS CIPI isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (n = 34), mutation in gyrB at codon 464, NAL MIC of 3-12 mg/L and CIP MIC of 0.064-0.5 mg/L.; and Group C, mutation in gyrA at codon 83 (n = 2) NAL MIC of 16 mg/L and CIP MIC of 0.25-0.38 mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA. CONCLUSION: The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India.

Inhibition of As(III) and Hg(II) caused aortic hypercontraction by eugenol, linalool and carvone.

Acute and chronic exposure to arsenic and mercury is known to produce vasoconstriction. There is, however, no clarity concerning the pathways leading to this increased contraction. In this study we elicit and compare maximum contractility of rat aortas under resting conditions in the presence of arsenic and mercury, and delineate pathways mediating this effect. Phenylephrine (PE) induced hypercontraction of 37% and 32% were obtained when isolated aortic segments were exposed to 25 ?M As(III) and 6 nM Hg(II), respectively. Isometric contraction measurements in presence of apocynin, verapamil and sodium nitroprusside indicates that the major causes of increased contraction are reactive oxygen species (ROS) and depletion of nitric oxide (NO). Calcium influx plays a minor role in arsenic and mercury caused hypercontraction. In unexposed aorta, eugenol causes relaxation by inhibiting ROS and elevating NO, linalool by blocking voltage dependent calcium channel (VDCC) and elevating NO, and carvone by blocking calcium influx through VDDC. Since the arsenic and mercury hypercontraction is mediated by increased ROS and depleted NO, we hypothesize that molecules which neutralize ROS or elevate NO will be better ameliorators. In line with this argument, we found eugenol to be the best ameliorator of arsenic and mercury hypercontraction followed by linalool and carvone.

Effect of lower doses of vanadate in combination with Azadirachta indica leaf extract on hepatic and renal antioxidant enzymes in streptozotocin-induced diabetic rats.

The present study was undertaken to investigate short-term (21 days) effects of oral administration of Azadirachta indica leaf extract and vanadate, separately and in combination, on the activities of antioxidant enzymes in streptozotocin-induced diabetic rats. Vanadate is a remarkable antidiabetic agent and shows insulin mimetic effect. However, severe toxicity is associated with vanadate when used in high concentration while at lower concentration the hypoglycemic property of vanadate is reduced. So, we used a low dose of vanadate in combination with A. indica leaf extract and evaluated their effect on the antioxidant defense system. Streptozotocin-diabetic rats were treated separately with insulin, vanadate (0.6 mg/ml), A. indica, and with combined dose of vanadate (0.2 mg/ml) and A. indica. At the end of the experiment, rats were sacrificed and serum glucose levels and activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were determined in cytosolic fraction of liver and kidney. Diabetic rats showed hyperglycemic condition and alteration in antioxidant enzyme activities. Treatment with antidiabetic compounds resulted in the reduction of glucose levels and restoration of enzyme activities to normal. Results showed that combined treatment of vanadate and A. indica leaf extract was the most effective in normalizing altered antioxidant enzyme system.

Kras gene mutation and RASSF1A, FHIT and MGMT gene promoter hypermethylation: indicators of tumor staging and metastasis in adenocarcinomatous sporadic colorectal cancer in Indian population.

OBJECTIVE: Colorectal cancer (CRC) development involves underlying modifications at genetic/epigenetic level. This study evaluated the role of Kras gene mutation and RASSF1A, FHIT and MGMT gene promoter hypermethylation together/independently in sporadic CRC in Indian population and correlation with clinicopathological variables of the disease. METHODS: One hundred and twenty four consecutive surgically resected tissues (62 tumor and equal number of normal adjacent controls) of primary sporadic CRC were included and patient details including demographic characteristics, lifestyle/food or drinking habits, clinical and histopathological profiles were recorded. Polymerase chain reaction - Restriction fragment length polymorphism and direct sequencing for Kras gene mutation and Methylation Specific-PCR for RASSF1A, FHIT and MGMT genes was performed. RESULTS: Kras gene mutation at codon 12 & 13 and methylated RASSF1A, FHIT and MGMT gene was observed in 47%, 19%, 47%, 37% and 47% cases, respectively. Alcohol intake and smoking were significantly associated with presence of Kras mutation (codon 12) and MGMT methylation (p-value <0.049). Tumor stage and metastasis correlated with presence of mutant Kras codon 12 (p-values 0.018, 0.044) and methylated RASSF1A (p-values 0.034, 0.044), FHIT (p-values 0.001, 0.047) and MGMT (p-values 0.018, 0.044) genes. Combinatorial effect of gene mutation/methylation was also observed (p-value <0.025). Overall, tumor stage 3, moderately differentiated tumors, presence of lymphatic invasion and absence of metastasis was more frequently observed in tumors with mutated Kras and/or methylated RASSF1A, FHIT and MGMT genes. CONCLUSION: Synergistic interrelationship between these genes in sporadic CRC may be used as diagnostic/prognostic markers in assessing the overall pathological status of CRC.

Strengthening of antioxidant defense by Azadirachta indica in alloxan-diabetic rat tissues.

BACKGROUND: Azadirachta indica has been reported to correct altered glycaemia in diabetes. OBJECTIVE: The aqueous extract of A. indica leaf and bark has been evaluated for its effect on antioxidant status of alloxan diabetic rats and compared with insulin treatment. MATERIALS AND METHODS: The oral effective dose of A. indica leaf (500 mg/kg body weight) and A. indica bark (100 mg/kg body weight) were given once daily for 21 days to separate groups of diabetic rats. At the end of the experimental period blood glucose level and activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PD), and membrane lipid peroxidation were determined in different fractions of liver and kidney tissues. RESULTS: Diabetic rats showed high blood glucose (P<0.01), increased level of malondialdehyde (P<0.05) and a significant decrease in the activity of antioxidant enzymes. Treatment with insulin, A. indica leaf extract (AILE), and A. indica bark extract (AIBE) restored the above altered parameters close to the control ones. CONCLUSIONS: Both AILE and AIBE were found significantly effective in reducing hyperglycemia-induced oxidative stress. The findings suggest further investigations for the possible use of A. indica as alternative medicine to prevent long-term complications of diabetes.

Association of adiponectin gene functional polymorphisms (-11377C/G and +45T/G) with nonalcoholic fatty liver disease.

Adiponectin levels are reduced in NAFLD patients and genetic variants of adiponectin have been frequently associated with type 2 diabetes and insulin resistance. To determine the genotypic frequencies of adiponectin functional polymorphisms (-11377C/G and +45T/G) and their subsequent effect on disease progression and plasma adiponectin levels in the patients with NAFLD. A total of 137 NAFLD patients and 250 matched controls were enrolled in the study. DNA sequencing and genotyping were performed to identify the genetic variants. The plasma adiponectin levels were assessed by ELISA. Homozygous mutant genotype of adiponectin SNPs, -11377C/G and +45T/G, were significantly more prevalent in NAFLD patients than controls (Bonferroni corrected p=0.014 and 0.018, respectively). Plasma adiponectin levels were significantly lower in the NAFLD patients as compared to controls. Moreover, presence of 'G' allele at position -11377C/G and +45T/G was found to be associated with necroinflammatory grade and reduced adiponectin levels, (p values 0.02 and 0.01) respectively. -11377G and +45G alleles are associated with severity of liver disease and hypoadiponectemia, in the patients with NAFLD, respectively.