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.

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.

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.

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.

Association analysis of p16 (CDKN2A) and RB1 polymorphisms with susceptibility to cervical cancer in Indian population.

The potent tumor suppressors P16 and RB1 are the key regulators of cell cycle machinery in eukaryotes. Polymorphisms in these genes play an important role in the outcome of various diseases including cancer. In the present study, we evaluated the association of p16 and RB1 polymorphisms with cervical cancer susceptibility in Indian population. We screened 150 histologically confirmed cervical cancer cases along with equal number of healthy controls with normal cervical cytology. PCR-RFLP method was employed for genotyping of SNPs in p16 C540G (rs11515), C580T (rs3088440) in the 3'-UTR of exon 3 and RB1 A153104G (rs4151580) located in the intron 18 and confirmed by direct sequencing. Both patients and controls were screened for HPV infection. In this case-control study 84.67% (127/150) of cases were found to be positive for HPV DNA sequence. Women carrying p16 C540G carrier genotypes 540 (CG/GG) may have protective effect for the development of cervical cancer (P=0.0001, OR=0.31, 95% CI=0.17-0.56). And SNP at C580T of p16 gene was found to be negatively associated with the risk of cervical cancer (P=0.0004, OR=0.04, 95% CI=0.002-0.63). p16 (540C/580T) has emerged as a major risk haplotype (P=0.033, OR=1.47, 95% CI=1.05-2.07) whereas p16 (540G/580T) as a chief protective haplotype (P=0.014, OR=0.39, 95% CI=0.18-0.83) for the development of cervical cancer among Indian women. Contrary to this, SNP at A153104G of RB1 gene showed statistically significant association (P=0.035, OR=1.69, 95% CI=1.06-2.68) with increased susceptibility for the development of cervical cancer. Our results suggest that single nucleotide polymorphisms in p16, RB1 genes may affect the susceptibility to cervical cancer collectively.

Identification of fibrinogen-binding proteins of Aspergillus fumigatus using proteomic approach.

Aspergillus fumigatus, the main etiological agent for various forms of human aspergillosis, gets access to the respiratory system of human host by inhalation of airborne conidia. These conidia possibly adhere to extracellular matrix (ECM) proteins. Among the ECM proteins involved in adherence, fibrinogen is thought to be crucial. Here, we studied whether A. fumigatus three-week culture filtrate (3wcf) proteins promote binding of A. fumigatus to ECM proteins and promote fungal growth. We observed that incubation of ECM with 3wcf proteins led to dose- and time-dependent increase in adherence of conidia to the ECM. In order to identify the catalogue of fibrinogen-binding A. fumigatus proteins, we carried out fibrinogen affinity blotting using two-dimensional gel electrophoresed 3wcf proteins. A total of 15 fibrinogen-binding protein spots corresponding to 7 unique proteins were identified in 3wcf using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF). Among these, 4 proteins, namely, beta-glucosidase, alpha-mannosidase, pectate lyase A and oryzin precursor were predicted to have cell wall or extracellular localization, whereas amidase family protein and two hypothetical proteins did not display the signal sequence. This study reports seven novel fibrinogen-binding proteins of A. fumigatus, some of which could be further explored for targeting the adhesion phenomenon as antifungal strategy.

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.

Rapid immobilization of enzymes onto solid supports by ultrasound waves.

Herein, we describe a non-conventional method for immobilization of enzymes onto different solid surfaces using ultrasound as a source of energy. When horseradish peroxidase (HRP) was taken on the surface of an activated support and allowed to float on a sonicator bath operating at a frequency of 40 KHz, it readily started binding itself to the surface. Maximum binding was observed in 10 min whereas a control experiment carried out similarly without ultrasound waves showed insignificant immobilization. Ultrasound wave-mediated immobilization is rapid and reproducible and is better suitable for versatile applications in different fields, including fabrication of enzyme-based biosensors or bioreactors.

Transcriptomic and proteomic profile of Aspergillus fumigatus on exposure to artemisinin.

Artemisinin, an antimalarial drug, and its derivatives are reported to have antifungal activity against some fungi. We report its antifungal activity against Aspergillus fumigatus (A. fumigatus), a pathogenic filamentous fungus responsible for allergic and invasive aspergillosis in humans, and its synergistic effect in combination with itraconazole (ITC), an available antifungal drug. In order to identify its molecular targets, we further analyzed transcript and proteomic profiles of the fungus on exposure to the artemisinin. In transcriptomic analysis, a total of 745 genes were observed to be modulated on exposure to artemisinin, and some of them were confirmed by real-time polymerase chain reaction analysis. Proteomic profiles of A. fumigatus treated with artemisinin showed modulation of 175 proteins (66 upregulated and 109 downregulated) as compared to the control. Peptide mass fingerprinting led to the identification of 85 proteins-29 upregulated and 56 downregulated, 65 of which were unique proteins. Consistent with earlier reports of molecular mechanisms of artemisinin and that of other antifungal drugs, we believe that oxidative phosphorylation pathway (64 kDa mitochondrial NADH dehydrogenase), cell wall-associated proteins and enzymes (conidial hydrophobin B protein, cell wall phiA protein, extracellular thaumatin domain protein, 1,3-beta-glucanosyltransferase Gel2) and genes involved in ergosterol biosynthesis (ERG6 and coproporphyrinogen III oxidase, HEM13) are potential targets of artemisinin for further investigations.

Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis.

Pathogenic strains of mycobacteria produce copious amounts of glutamine synthetase (GS) in the culture medium. The enzyme activity is linked to synthesis of poly-α-l-glutamine (PLG) in the cell walls. This study describes a glnA-1 mutant of Mycobacterium bovis that produces reduced levels of GS. The mutant was able to grow in enriched 7H9 medium without glutamine supplementation. The glnA-1 strain contained no detectable PLG in the cell walls and showed marked sensitivity to different chemical and physical stresses such as lysozyme, SDS and sonication. The sensitivity of the mutant to two antitubercular drugs, rifampicin and d-cycloserine, was also increased. The glnA-1 strain infected THP-1 cells with reduced efficiency and was also attenuated for growth in macrophages. A Mycobacterium smegmatis strain containing the M. bovis glnA-1 gene survived longer in THP-1 cells than the wild-type strain and also produced cell wall-associated PLG. The M. bovis mutant was not able to replicate in the organs of BALB/c mice and was cleared within 4-6 weeks of infection. Disruption of the glnA-1 gene adversely affected biofilm formation on polystyrene surfaces. The results of this study demonstrate that the absence of glnA-1 not only attenuates the pathogen but also affects cell surface properties by altering the cell wall chemistry of the organism via the synthesis of PLG; this may be a target for drug development.

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.

Transcription factor AP-1 in esophageal squamous cell carcinoma: alterations in activity and expression during human Papillomavirus infection.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related deaths in Jammu and Kashmir (J&K) region of India. A substantial proportion of esophageal carcinoma is associated with infection of high-risk HPV type 16 and HPV18, the oncogenic expression of which is controlled by host cell transcription factor Activator Protein-1 (AP-1). We, therefore, have investigated the role of DNA binding and expression pattern of AP-1 in esophageal cancer with or without HPV infection. METHODS: Seventy five histopathologically-confirmed esophageal cancer and an equal number of corresponding adjacent normal tissue biopsies from Kashmir were analyzed for HPV infection, DNA binding activity and expression of AP-1 family of proteins by PCR, gel shift assay and immunoblotting respectively. RESULTS: A high DNA binding activity and elevated expression of AP-1 proteins were observed in esophageal cancer, which differed between HPV positive (19%) and HPV negative (81%) carcinomas. While JunB, c-Fos and Fra-1 were the major contributors to AP-1 binding activity in HPV negative cases, Fra-1 was completely absent in HPV16 positive cancers. Comparison of AP-1 family proteins demonstrated high expression of JunD and c-Fos in HPV positive tumors, but interestingly, Fra-1 expression was extremely low or nil in these tumor tissues. CONCLUSION: Differential AP-1 binding activity and expression of its specific proteins between HPV--positive and HPV--negative cases indicate that AP-1 may play an important role during HPV-induced esophageal carcinogenesis.

Identification and characterization of a laminin-binding protein of Aspergillus fumigatus: extracellular thaumatin domain protein (AfCalAp).

Aspergillus fumigatus, an opportunistic fungal pathogen, infects the human host via inhalation of airborne conidia. Adhesion of fungal conidia, to host cells and extracellular matrix (ECM) components associated with host tissue surfaces, is thought to be the primary step in the pathogenesis and dissemination of infection. To identify novel adhesion proteins (adhesins) of A. fumigatus, we screened its proteome in silico using SPAAN (software program for prediction of adhesins and adhesin-like proteins using neural networks). One of the predicted adhesin-encoding genes with a P(ad) (probability of being adhesin) value >0.9, the gene encoding extracellular thaumatin domain protein (AfCalA), was cloned and expressed in Escherichia coli. Recombinant AfCalAp showed significant binding with laminin and murine lung cells. Anti-AfCalAp antibodies inhibited the binding of AfCalAp to laminin in a dose-dependent manner. Significant binding of anti-AfCalAp antibodies to 2 h swollen conidia suggests the presence of AfCalAp on the conidial surface. AfCalA transcript was not detectable in resting conidia but was detected in conidia incubated with RPMI 1640 medium in the presence and absence of lung epithelial cell line (A539)-derived ECM. Elevated levels of IgE antibodies specific to AfCalAp were observed in the sera of two out of seven patients with allergic bronchopulmonary aspergillosis. The study confirms the relevance of the bioinformatic approach for predicting fungal adhesins and establishes AfCalAp as a novel laminin-binding protein of A. fumigatus.

Aberrant promoter methylation and reduced expression of p16 gene in esophageal squamous cell carcinoma from Kashmir valley: a high-risk area.

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancer in Jammu and Kashmir region of India and has multi-factorial etiology involving dietary habits, genetic factors, and gene environmental interactions. Inactivation of the p16 gene expression by aberrant promoter methylation plays an important role in the progression of esophageal carcinoma. In the present investigation, we have studied the role of p16 promoter methylation in 69 histopathologically confirmed ESCC tissues and compared it with corresponding normal adjacent tissues for DNA methylation in the CpG island in the p16 promoter region by methylation-specific polymerase chain reaction (MSP) and p16 protein expression by immunoblotting. The results showed loss of p16 expression in 67% (46/69) of tumor tissues compared to only 3% in control tissues (2/69). Promoter methylation was observed in 52% (36/69) of tumor tissues and it gradually increased with the increasing severity of histological grades of the cancer (P = 0.0001). Loss of p16 expression with promoter methylation was observed in 26 of 36 cases (72%). Analysis of patients dietary habits revealed a strong association between promoter methylation and high consumption of hot salted tea (P < 0.05) which is a most favourite drink commonly consumed by Kashmiri people.

Genetic variant of CCND1: association with HPV-mediated cervical cancer in Indian population.

The potential association of single nucleotide polymorphisms (SNPs) (G870A and G1722C) of CCND1 with susceptibility to cervical cancer was investigated. The study included 200 cervical cancer cases along with an equal number of healthy controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct sequencing were employed for genotyping. We found that women carrying the 870AA genotype have a 2.49-fold increased risk for the development of cervical cancer (odds ratio (OR) 2.49; 95% confidence interval (CI) 1.51-4.09; p = 0.0004) compared with GG+GA genotypes. For the 1722 locus, the frequency of the polymorphic 'C' allele was strongly associated with a reduced risk of cervical cancer (p = 0.019; OR 0.71; 95% CI 0.54-0.94). Our data suggest that CCND1 G870A polymorphism could act as a risk factor for the development of cervical cancer. And G1722C polymorphism may play a protective role against the development of human papillomavirus-associated cervical cancer among Indian women.

Hepatitis B virus genotypes and hepatitis B surface antigen mutations in family contacts of hepatitis B virus infected patients with occult hepatitis B virus infection.

BACKGROUND: The association and profile of surface gene mutations with viral genotypes have been studied in patients with chronic hepatitis B virus (HBV) but not in subjects with occult HBV infection. AIM: This study aimed to investigate the association of surface gene mutations with viral genotypes in occult HBV infection. MATERIALS & METHODS: Of 293 family contacts of 90 chronic HBV index patients, 110 consented for the study. Of 110 subjects, 97 were hepatitis B surface antigen (HBsAg) negative. HBV genotyping was done using direct DNA sequencing. The S-gene was also sequenced in 13 chronic hepatitis B patients to serve as controls. RESULTS: Twenty-eight (28.8%) of the 97 subjects had occult HBV infection. Bidirectional sequencing of partial S-gene was successful in 13 of them. Seven (53.8%) of the viral sequences are genotype A1, two (15.3%) each having genotypes D5&D2 and one each (7.6%) having D1&G genotypes. Seven (53.8%) of the 13 HBsAg positive patients, had genotype D&6 (46.1%) genotype A. A128V & T143M mutations were observed in 5 of 13 (38.4%) subjects and A128V & P127S in 2 of 13 (15.3%) patients (P = 0.385). A128V mutation was seen in two (15.3%) subjects with D2 genotype, while T143M mutation was seen in three (23.07%) subjects with A1genotype. At aa125, three (23.07%) subjects with D5 genotype had methionine instead of threonine. There were wild type sequences in five (38.4%) subjects, one each of D1, G genotypes (20%) and four A1 (80%) genotypes. None of the subjects had G145R mutation. CONCLUSIONS: Occult HBV infection may be common in household contacts of chronic HBV infected patients. Equal prevalence of A&D sub-genotypes was present in occult HBV subjects and in chronic HBV patients. Mutations of the S-gene are genotype specific in both occult as well as chronic HBV infection.

Expressed sequence tags of Aspergillus fumigatus: Extension of catalogue and their evaluation as putative drug targets and/or diagnostic markers.

Aspergillus fumigatus a fungal pathogen is implicated in a spectrum of allergic and invasive disorders in humans. Validation of transcriptome of pathogen is essential for understanding its virulence mechanism and to identify new therapeutic targets/diagnostic markers. In order to rapidly identify genes of Aspergillus fumigatus we adopted sequencing of cDNA clones. Our earlier effort has lead to identification of 68 expressed sequence tags of Aspergillus fumigatus. Present study describes 52 more expressed sequence tags generated by sequencing 200 phage clones of a non-normalized cDNA library. One of the cDNA clones comprised of the complete coding region for tetratricopeptide repeat domain protein gene. Various homology search algorithms were employed to assign functions to expressed sequence tags coding for hypothetical proteins, and relevance of these expressed sequence tags or their protein products as drug targets/diagnostic markers was examined by searching for homologues in fungi and human.

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.