Distinct Distribution Pattern of Hepatitis B Virus Genotype C and D in Liver Tissue and Serum of Dual Genotype Infected Liver Cirrhosis and Hepatocellular Carcinoma Patients

Aims

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.

Methods

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.

Results

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.

Conclusions

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.     

Insights into the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain

BackgroundPhosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P₂ through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P₂/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways.MethodsSurface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used.ResultsIn this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P₂/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC₅₀ ranges from 53 to 159 nM for PI(4,5)P₂/PLCδ1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂], PI(4,5)P₂ specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C δ1 (PLCδ1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLCδ1-PH domain from plasma membrane to cytosol in A549 cells.ConclusionsOverall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment.General significanceThese results suggest specific but differential binding of these compounds to the PLCδ1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.     

Correlation of hydrogen-bonding propensity and anticancer profile of tetrazole-tethered combretastatin analogues

A series of 1,5-disubstituted tetrazole-tethered combretastatin analogues with extended hydrogen-bond donors at the ortho-positions of the aryl A and B rings were developed and evaluated for their antitubulin and antiproliferative activity. We wanted to test whether intramolecular hydrogen-bonding used as a conformational locking element in these analogues would improve their activity. The correlation of crystal structures with the antitubulin and antiproliferative profiles of the modified analogues suggested that hydrogen-bond-mediated conformational control of the A ring is deleterious to the bioactivity. In contrast, although there was no clear evidence that intramolecular hydrogen bonding to the B ring enhanced activity, we found that increased substitution on the B ring had a positive effect on antitubulin and antiproliferative activity. Among the various analogues synthesized, compounds 5d and 5e, having hydrogen-bonding donor groups at the ortho and meta-positions on the 4-methoxy phenyl B ring, are strong inhibitors of tubulin polymerization and antiproliferative agents having IC₅₀ value in micromolar concentrations.     

An insight into the binding of 6-hydroxyflavone with hen egg white lysozyme: a combined approach of multi-spectroscopic and computational studies

Abstract

The interaction of 6-hydroxyflavone (6HF) with hen egg white lysozyme (HEWL) has been executed using multi-spectroscopic and computational methods. Steady state fluorescence studies indicated that static quenching mechanism is involved in the binding of 6HF with HEWL, which was further supported by excited state lifetime and UV–vis absorption studies. The binding constant (K_b) of the HEWL–6HF complex was observed to be 6.44?±?0.09?×?10⁴ M^?1 at 293?K, which decreases with the increase in temperature. The calculation of the thermodynamic quantities showed that the binding is exothermic in nature with a negative enthalpy change (ΔH = ?11.91?±?1.02?kJ mol^?1) along with a positive entropy change (ΔS = +51.36?±?2.43 J K^?1 mol^?1), and the major forces responsible for the binding are hydrogen bonding and hydrophobic interactions. The possibility of energy transfer from tryptophan (Trp) residue to the 6HF ligand was observed from Fo¨rster’s theory. The inclusion of 6HF within the binding site of HEWL induces some micro-environmental changes around the Trp residues as indicated by synchronous and three-dimensional (3D) fluorescence studies. The changes in secondary structural components of HEWL are observed on binding with 6HF along with a reduction in % α-helical content. Computational studies correlate well with the experimental finding, and the ligand 6HF is found to bind near to Trp 62 and Trp 63 residues of HEWL. Altogether, the present study provides an insight into the interaction dynamics and energetics of the binding of 6HF to HEWL.

Communicated by Ramaswamy H. Sarma     

Mode of action of iron on arylsulphatases under acute lead acetate treated conditions in rats

Arylsulphatases A, B and C were found to be inhibited in liver and kidney tissues under lead acetate-treated conditions (both in vivo and in vitro) in rats. When lead acetate-treated animals (in vivo) were supplemented with ferric ammonium citrate (in vivo), a remarkable recovery was found in the activities of all arylsulphatases A, B and C whereas ferric ammonium citrate itself had no effect on the activities of arylsulphatases. When both the in vivo and in vitro lead acetate-treated arylsulphatases were supplemented with the purified ferritins (in vitro) it was observed that lead-induced inhibition of the activities of arylsulphatases was successfully reversed. It was also found that ferritins were able to bind a large quantity of lead. These results indicated that ferritins were directly involved for reactivation of arylsulphatases which were inhibited by lead. It was well established that a response to iron administration in rats was an immediate de novo stimulation of ferritin biosynthesis. Iron might therefore protect the enzymatic activities of arylsulphatases by enhancing the level of ferritin in liver and kidney tissues which is known to bind a large quantity of lead thereby ameliorating their toxic effects in the living system.     

Socioeconomic differentials in nutritional status of children in the states of West Bengal and Assam, India

Malnutrition among children is prevalent in almost all the states in India. This study assesses the extent and causes of malnutrition in two eastern Indian states with similar climates, namely West Bengal and Assam, using data from the National Family Health Survey 1998-99 (NFHS-2). The three indices of malnutrition taken for analysis are weight-for-height (WHZ), height-for-age (HAZ) and weight-for-age (WAZ). These are assumed to depend on birth order, preceding birth interval, parent's educational status, working status of the mother, mother's age at delivery of the children, source of drinking water, toilet facilities and standard of living of the household. Logistic regression was carried out separately for each of the three indices on the explanatory variables for both the states. It was found that not all variables are equally important in determining whether a baby is underweight, or suffering from acute or chronic malnutrition. Also, the importance of variables is not the same in the two states. It was observed that the coefficients associated with the variables in determining weight-for-height are not significant compared with those for weight-for-age and height-for-age.     

Effects of pH and ionic strength on the assembly and bundling of FtsZ protofilaments: a possible role of electrostatic interactions in the bundling of protofilaments

Assembly, bundling and stability of FtsZ protofilaments are important for the formation and functioning of the cytokinetic Z-ring during bacterial division. We found that the bundling of FtsZ protofilaments decreased strongly with increasing pH from 6.0 to 7.9, while the assembly of FtsZ monomers did not decrease considerably. In addition, the disassembly of FtsZ protofilaments was strongly suppressed at pH 6.0 as compared to the elevated pHs. The far-UV circular dichroism spectra of the native FtsZ and the tryptophan emission spectra of mutated FtsZ (Y371W) did not change by increasing pH from 6 to 7.9 indicating that the structure of FtsZ was not altered significantly. Further, the inhibition of bundling of FtsZ protofilaments predominantly, and the inhibition of assembly to a lesser extent by salt indicated that electrostatic interactions are important for the assembly and bundling of FtsZ protofilaments. These observations are supported by the results of computational docking of Escherichia coli dimer structure. The results suggest that the basic intracellular pH (7.4-7.8) of E. coli may play a role in regulating the assembly dynamics of FtsZ in the Z-ring by reducing protofilament stability and bundling in bacterial cytoplasm.