Genetic Characterization of Hepatitis B Virus in Peripheral Blood Leukocytes: Evidence for Selection and Compartmentalization of Viral Variants with the Immune Escape G145R Mutation

The compartmentalization of viral variants in distinct host tissues is a frequent event in many viral infections. Although hepatitis B virus (HBV) classically is considered hepatotropic, it has strong lymphotropic properties as well. However, unlike other viruses, molecular evolutionary studies to characterize HBV variants in compartments other than hepatocytes or sera have not been performed. The present work attempted to characterize HBV sequences from the peripheral blood leukocytes (PBL) of a large set of subjects, using advanced molecular biology and computational methods. The results of this study revealed the exclusive compartmentalization of HBV subgenotype Ae/A2-specific sequences with a potent immune escape G145R mutation in the PBL of the majority of the subjects. Interestingly, entirely different HBV genotypes/subgenotypes (C, D, or Aa/A1) were found to predominate in the sera of the same study populations. These results suggest that subgenotype Ae/A2 is selectively archived in the PBL, and the high prevalence of G145R indicates high immune pressure and high evolutionary rates of HBV DNA in the PBL. The results are analogous to available literature on the compartmentalization of other viruses. The present work thus provides evidence in favor of the compartment-specific abundance, evolution, and emergence of the potent immune escape mutant. These findings have important implications in the field of HBV molecular epidemiology, transmission, transfusion medicine, organ transplantation, and vaccination strategies.Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family and classically has been described to be hepatotropic, causing a wide range of clinical and subclinical manifestations of liver disease (57). Nevertheless, studies of HBV-infected human subjects and woodchucks infected with Woodchuck hepatitis virus (WHV; an animal model of hepadnaviral infection) have reported different molecular forms of replicative intermediates in the lymphatic cells and have established that hepadnaviruses are strongly lymphotropic in nature (29). Moreover, the results of studies of human subjects as well as with animal models have revealed that the life-long occult persistence of replication- and transmission-competent viruses in lymphatic cells is a strict consequence of hepadnaviral infections (29).More interestingly, in animal models, lymphatic system-restricted occult hepadnaviral infection has been found to be transmissible vertically as an asymptomatic, serologically occult infection exclusively confined to the lymphatic system (29). Earlier we provided evidence that occult HBV persisting in the lymphatic cells are transmissible, specifically to the PBL through horizontal intrafamilial modes (9). These observations clearly indicate important immunological, pathogenic, and epidemiological implications of lymphatic system-restricted hepadnaviral infections. Although the involvement of specific viral variants has been suggested to explain this lymphatic system-restricted hepadnaviral infection and transmission (29), the classical belief that hepatocytes are the primary target and only reservoir of HBV has precluded the genetic characterization of hepadnaviruses from extrahepatic sites.Fascinatingly, despite being classically considered a hepatotropic virus, hepatitis C virus (HCV), belonging to the family Flaviviridae, also shows occult persistence and lymphotropism very similar to that of hepadnaviruses (37). Similarly to WHV, HBV, and HCV, other viruses, including HIV (human immunodeficiency virus), small ruminant lentivirus, and Epstein-Barr virus, also have been shown to infect and persist in different anatomical compartments of the body in addition to their classical target cells (38, 40, 43, 45, 50). Furthermore, recent molecular evolutionary analyses based on envelope sequences of these viruses (e.g., HIV, HCV, small ruminant lentivirus, Epstein-Barr virus, etc.) have established clearly that these viruses undergo selection and independent evolution in diverse tissues, leading to the tissue-specific compartmentalization of viral populations (38, 40, 43, 45, 50). In contrast to other viruses, to the best of our knowledge, methodical molecular evolutionary studies to characterize HBV sequences isolated from extrahepatic sites of HBV-infected subjects have not been reported in the literature.We hypothesized that similar to other viruses, HBV also undergo independent evolution in different compartments of the body under the influence of differential immune pressure. To examine our hypothesis, we used the most easily available lymphatic cells, the peripheral blood leukocytes (PBL), determined the HBV envelope sequences from HBV DNA isolated from these cells, and performed advanced genetic, phylogenetic, and mutational analysis. The results of this work demonstrate a highly compartment-specific preponderance of HBV genetic variants in serum and PBL of the same study population, providing evidence in favor of the compartmentalization of HBV genetic variants. The results and important implications of these findings are discussed in this work.     

Synthesis and characterizations of novel quinoline derivatives having mixed ligand activities at the κ and μ receptors: Potential therapeutic efficacy against morphine dependence

Based on an established 3D pharmacophore, a series of quinoline derivatives were synthesized. The opioidergic properties of these compounds were determined by a competitive binding assay using ¹²⁵I-Dynorphine, ³H-DAMGO and ¹²⁵I-DADLE for κ, μ, and δ receptors, respectively. Results showed varying degree of activities of the compounds to κ and μ opioid receptors with negligible interactions at the δ receptor. The compound, S4 was the most successful in inhibiting the two most prominent quantitative features of naloxone precipitated withdrawal symptoms - stereotyped jumping and body weight loss. Determination of IC₅₀ of S4 revealed a greater affinity towards μ compared to κ receptor. In conclusion, quinoline derivatives of S4 like structure offer potential tool for treatment of narcotic addictions.     

Analysis of oligomeric proteins during unfolding by pH and temperature

During thermal transition and variation of pH, structural properties of 35 proteins and their complexes (bound with substrate and co-factor) were analyzed in detail. During pH alteration, these proteins were shown to have substantial differences in conformations. pH conformers were analyzed in detail. Free energy and other energy parameters were also estimated for these proteins at various pH and temperatures. Detailed structural analysis and binding interfaces of various substrates, inhibitors and cofactor of these proteins were also investigated using docking and molecular dynamic simulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Crystal structure and microstructure of cholesteryl oleyl carbonate

The crystal structure as well as the microstructure, i.e., size and strain, of crystallites of cholesteryl oleyl carbonate was determined from X-ray powder diffraction data. The X-ray line broadening was analyzed through the refinement of TCH-pseudo-Voigt function parameters (isotropic effects) and the refinement of multipolar functions, i.e., symmetrized cubic harmonics (anisotropic effects). The crystal structure turns out to be primitive monoclinic, space group Pc, type I monolayer having two molecules per unit cell with parameters: a = 18.921 ± 0.006 Å, b = 12.952 ± 0.003 Å, c = 9.276 ± 0.002 Å and β = 91.32 ± 0.03°. The average size of a well ground specimen of crystallites was 60 nm. The average micro-strain, e.g., 45 × 10⁻⁴ has been tentatively attributed to fatty chain conformational disorder. The unit cell parameters, including the lamellar thickness, of COC crystal is very closely similar to those of another, structurally similar cholesterol ester, e.g., cholesteryl oleate (CO) crystal, space group P2₁, type II monolayer. Type I monolayer structure has been established for COC on the basis of the intensity calculations of the XRD profiles of both CO and COC. The dipolar and structural disorder in a 4:1 molar, binary mixture of CO and COC can be accommodated in an induced smectic phase with a lamellar thickness, which is nearly equal to that of pure CO or pure COC.     

A novel role for Bruton's tyrosine kinase in hepatocyte growth factor-mediated immunoregulation of dendritic cells

The limited success of dendritic cell (DC)-based immunotherapy in multiple myeloma is partly due to hepatocyte growth factor (HGF)-induced DC dysfunction. From a therapeutic standpoint, it is important to understand the molecular events involved in inhibition of DC activation/maturation by HGF. Because Bruton's tyrosine kinase (Btk) negatively regulates maturation and immunostimulatory function of DCs, a role for Btk in HGF-induced inhibition of both murine and human DCs was investigated. We demonstrate that Btk is a novel proximal component of HGF-induced c-MET (HGF receptor) signaling. Following HGF treatment, Btk binds to c-MET and becomes activated. Btk activation in turn blocks the NF-κB pathway and subsequent DC activation via the c-Src-PI3K-AKT-mammalian target of rapamycin (mTOR) pathway. Notably, Btk activation is necessary for HGF-induced association of c-Src and PI3K with c-MET. Furthermore, we provide the first evidence that HGF inhibits DC activation by inducing autocrine interleukin (IL)-10 secretion, which requires activation of Btk. Blocking activation of Btk and its downstream the c-Src-PI3K-AKT-mTOR pathway prevents HGF-induced IL-10 secretion by DCs. In addition, neutralization of IL-10 secretion from DCs impaired the inhibitory effect of HGF on DCs. Thus, our study identifies a novel role for Btk in HGF-induced DC inhibition.     

Effect of probiotic fermented milk and chlorophyllin on gene expressions and genotoxicity during AFB₁-induced hepatocellular carcinoma

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B? (AFB?) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B? at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB? control group. The expression of c-myc bax, bcl-2, cyclin D1, p53 and rasp-21 genes was also studied. A significant (P<0.05) reduction in DNA damage was observed in probiotic fermented milk with chlorophyllin group as compared to aflatoxin B? control group. The c-myc, bcl-2, cyclin D1 and rasp-21 level was found to be highest in AFB? control group as compared to the treatment group. The results advocate the enhanced protective potential of probiotic fermented milk and chlorophyllin against AFB?-induced molecular alterations in hepatic cells during carcinogenesis.     

Cestodes (Caryophyllidea) of the stinging catfish Heteropneustes fossilis (Siluriformes: Heteropneustidae) from Asia

The stinging catfish Heteropneustes fossilis (Bloch) (Siluriformes: Heteropneustidae) has been reported to harbor as many as 19 species of caryophyllidean tapeworms (Cestoda) of 11 genera in tropical Asia (Indomalayan zoogeographical region). However, a critical review of the species composition has shown that only 1 species, Lucknowia fossilisi Gupta, 1961 (Lytocestidae), is a specific parasite of H. fossilis. Three other species, Djombangia penetrans Bovien, 1926 (syn., Djombangia caballeroi Sahay and Sahay, 1977 ), Pseudocaryophyllaeus ritai Gupta and Singh, 1983 (syn. Pseudocaryophyllaeus lucknowensis Gupta and Sinha, 1984 ), and Pseudocaryophyllaeus tenuicollis (Bovien, 1926) Ash, Scholz, Oros and Kar, 2011 (syn. P. mackiewiczi Gupta and Parmar, 1982 ), were found only once. Lucknowia fossilisi is redescribed on the basis of new material collected in West Bengal and voucher specimens from Maharashtra, India. A total of 9 species of Capingentoides, Lucknowia, Lytocestus, Pseudoadenoscolex, Pseudocaryophyllaeus, Pseudoheteroinverta, and Sukhapatae are newly synonymized with L. fossilisi and previous synonymies of 9 other species, proposed by Hafeezulah (1993), are confirmed. Generic diagnosis of Lucknowia Gupta, 1961 is amended. In addition, 1 species of Pseudobatrachus and 2 species of the monotypic genera Pseudoneckinverta and Sudhaena are invalidated as nomina nuda.     

Anthelmintic efficacy of Flemingia vestita: genistein-induced effect on the activity of nitric oxide synthase and nitric oxide in the trematode parasite,Fasciolopsis buski

The root-tuber peel of Flemingia vestita, an indigenous leguminous plant of Meghalaya (Northeast India), has usage in local traditional medicine as curative against worm infections. The peel and its active component, genistein, have been shown to cause flaccid paralysis, deformity of tegumental architecture and alterations in the activity of several enzymes in platyhelminth parasites. To investigate further the mode of action and anthelmintic efficacy of the plant-derived components, the crude peel extract of F. vestita and genistein were tested, hitherto for the first time, in respect of the unique neuronal messenger nitric oxide (NO) and the enzyme nitric oxide synthase (NOS) in Fasciolopsis buski, the large intestinal fluke of swine and human host. NADPH-diaphorase histochemical staining (a selective marker for NOS in neuronal tissues), which was demonstrable in the neuronal cell bodies in the cerebral ganglia, the brain commissure, the main nerve cords and in the innervation of the pharynx, ventral sucker, terminal genitalia and genital parenchyma of the parasite, showed a stronger activity in the treated worms. In biochemical analysis also, the NOS activity showed a significant increase in the parasites treated with the test materials and reference drug, compared to the untreated controls. The increase in NOS activity in the treated parasites can be attributed to an inducing effect of the plant-derived components.     

Post-Discharge Mortality in Children with Severe Malnutrition and Pneumonia in Bangladesh

Background

Post-discharge mortality among children with severe illness in resource-limited settings is under-recognized and there are limited data. We evaluated post-discharge mortality in a recently reported cohort of children with severe malnutrition and pneumonia, and identified characteristics associated with an increased risk of death.

Methods

Young children (<5 years of age) with severe malnutrition (WHO criteria) and radiographic pneumonia on admission to Dhaka Hospital of icddr,b over a 15-month period were managed according to standard protocols. Those discharged were followed-up and survival status at 12 weeks post-discharge was determined. Verbal autopsy was requested from families of those that died.

Results

Of 405 children hospitalized with severe malnutrition and pneumonia, 369 (median age, 10 months) were discharged alive with a follow-up plan. Of these, 32 (8.7%) died in the community within 3 months of discharge: median 22 (IQR 9–35) days from discharge to death. Most deaths were reportedly associated with acute onset of new respiratory or gastrointestinal symptoms. Those that died following discharge were significantly younger (median 6 [IQR 3,12] months) and more severely malnourished, on admission and on discharge, than those that survived. Bivariate analysis found that severe wasting on admission (OR 3.64, 95% CI 1.66–7.97) and age <12 months (OR 2.54, 95% CI 1.1–8.8) were significantly associated with post-discharge death. Of those that died in the community, none had attended a scheduled follow-up and care-seeking from a traditional healer was more common (p<0.001) compared to those who survived.

Conclusion and Significance

Post-discharge mortality was common in Bangladeshi children following inpatient care for severe malnutrition and pneumonia. The underlying contributing factors require a better understanding to inform the potential of interventions that could improve survival.     

Phylogeny,phenotypic and nutritional characteristics of estuarine soil actinomycetes having broad-spectrum antimicrobial activity derived from an ecologically guided bioprospecting programme

No investigation has been done to identify ecological factors selecting for broad-spectrum antimicrobial activity of actinomycetes in the estuaries. Previously, we established that, in the Sundarbans (the world’s largest tidal mangrove forest), high antagonistic potential (AP) sampling sites were influenced by tides, while the low AP sites were not. We now report molecular phylogenetic analysis, morphological, physiological and biochemical characteristics of actinomycetes of high AP sites. The effects of soil organic carbon, nitrogen and ionic content (which strongly influenced AP) on the strength and spectrum of activity of the isolates were also studied in shake flasks. Molecular phylogenetic analysis showed sequences of our strains to be 96–99% similar to the 16S rDNA sequences of Streptomyces. Results showed variation among sporophore sizes, ornamentation and number of spores. Jaccard’s similarity coefficients of the isolates varied from 0.512 to 0.884 indicating disparity in the biochemical and physiological characteristics, possibly due to spatial separation of the sampling sites. Top soil in the intertidal zone of estuaries is generated from settling and consolidation of fluid mud and Streptomyces would be expected to produce broad-spectrum antimicrobials in such virgin soil. Isolates should be collected from the narrow band between the mean high and low tide marks to maximize chances of finding broad-spectrum activity. Considering the study on nutritional requirements vis-à-vis the field studies, it was concluded that results of this investigation corroborated the field observations where soil nitrogen, rather than organic carbon or ionic concentration, played a major role in determining the antimicrobial spectrum.