Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance.

In many organisms "Universal Stress Proteins" (USPs) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8Å and 2.4Å resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing.

Expression of microRNA-155 correlates positively with the expression of Toll-like receptor 7 and modulates hepatitis B virus via C/EBP-ß in hepatocytes.

Effective recognition of viral infection and successive activation of antiviral innate immune responses are vital for host antiviral defence, which largely depends on multiple regulators, including Toll-like receptors (TLRs) and microRNAs. Several early reports suggest that specific TLR-mediated immune responses can control hepatitis B virus (HBV) replication and express differentially with disease outcome. Considering the versatile function of miR-155 in the TLR-mediated innate immune response, we aimed to study the association between miR-155 and TLRs and their subsequent impact on HBV replication using both a HBV-replicating stable cell line (HepG2.2.15) and HBV-infected liver biopsy and serum samples. Our results showed that miR-155 was suppressed during HBV infection and a subsequent positive correlation of miR-155 with TLR7 activation was noted. Further, ectopic expression of miR-155 in vitro reduced HBV load as evidenced from reduced viral DNA, mRNA and subsequently reduced level of secreted viral antigens (HBsAg and HBeAg). Our results further suggested that CCAAT/enhancer-binding protein-ß (C/EBP-ß), a positive regulator of HBV transcription, was inhibited by miR-155. Taken together, our study established a correlation between miR-155 and TLR7 during HBV infection and also demonstrated in vitro that increased miR-155 level could help to reduce HBV viral load by targeting C/EBP-ß.

Occult HBV infection among anti-HBc positive HIV-infected patients in apex referral centre, Eastern India.

BACKGROUND: The prevalence of occult HBV, defined by the presence of HBV DNA in individuals with antibodies to HBV core antigen and with absence of HBV surface antigen, but its clinical significance and virological features in HIV-infected patients is still unclear. AIM: To investigate the prevalence, clinical significance and molecular characterization of occult hepatitis B virus infection in ART-Naive HIV-positive individuals. MATERIAL AND METHODS: Among the 1077 HIV-infected patients with different risk factors for HIV infection, 297 were HBsAg-ve ART-naive, of them 112 was randomly selected for the study. HBV DNA was tested by in-house PCR and quantified by qPCR. Molecular characterization was performed by sequencing the envelope and overlapping polymerase genes. RESULTS: We found the prevalence of occult HBV to be 10.7% among a randomly selected group of HBsAg-ve/antiHBc+ve HIV-infected patients. Overall 33.9% (38 of 112) of the patients were antiHBc positive indicating exposure to HBV infection. HBV DNA was detected in 12/38 (31.5%) antiHBc positive samples and 50% of them had CD4 T cell count < 200 cells/mm(3). HCV coinfection was low (2.7%). No surrogate marker for OBI could be identified. Presence of antiHBs antibodies did not rule out OBI. Liver biopsy in six cases showed varying stages of chronic hepatitis. Several mutations were detected but not the common immune escape mutant G145R. CONCLUSION: In conclusion the prevalence of OBI was significantly high among HIV coinfected patients, which highlights the importance of HBV DNA testing in these patients and indicates need for further prospective studies in larger cohorts to assess its clinical significance.

Subgenotypes of hepatitis B virus genotype D (D1, D2, D3 and D5) in India: differential pattern of mutations, liver injury and occult HBV infection.

Hepatitis B genotype D (HBV/D) is the most widespread genotype and exists as at least five subgenotypes (HBV/D1-D5). However, little is known about the association of virological characteristics with clinical differences among HBV/D subgenotypes. To investigate the virological characteristics of these subgenotypes and their clinical implications, we selected a cohort of 109 genotype D infected individuals from the state of West Bengal, India, including 68 HBsAg positive patients and 41 with occult HBV infection. Among the HBsAg positive subjects 28 had chronic hepatitis B virus infection, 40 were asymptomatic carriers based on clinical examination, liver function test and ultrasonograph results. Overall, HBV/D1 was found in 17%, HBV/D2 in 29%, HBV/D3 in 34% and HBV/D5 in 20% of the cases. HBV/D1 was significantly associated with chronic liver disease (P = 0.01), and in this subgenotype A1896 (PreC mutations) were most common. Although BCP mutations (A/C1753 and T1762/A1764) were found to be frequently associated with HBV/D2 (33% and 33%) and D5 (47% and 59%), no apparent clinical correlation was observed. On the other hand, occult HBV infection was significantly associated with HBV/D3 infection, along with low level of BCP and PreC mutations and several non-synonymous substitutions in the catalytic reverse transcriptase (RT) domain of polymerase gene. Similar nucleotide substitutions in the surface (S) gene region were observed from both northern and eastern Indian HBV/D3 isolates. In conclusion, HBV/D subgenotypes differ in their mutational patterns in the S, polymerase and the BCP/PreC regions that may influence their clinical outcomes.

Sinonasal Mass-a Recent Study of Its Clinicopathological Profile.

A variety of non-neoplastic and neoplastic conditions involving the nasal cavity, paranasal sinuses (PNS), and nasopharynx are commonly encountered in clinical practice. The objectives of the study were to identify various pathological conditions that present with sinus or nasal mass, to understand their varied clinical behavior, to know the distribution of various lesions among the different age and sex groups, and to study the management of sinonasal masses and its outcome on follow-up. A prospective study was carried out from September 2013 to August 2015, in the Otorhinolaryngology Department of Hi-Tech Medical College & Hospital, Bhubaneswar, Odisha. The study included patients of any age and sex presenting with nasal symptoms (suspected of a sinonasal mass). This study included all cases seen during the above duration. Complete history was taken and full clinical examination was carried out. Majority of the patients with sinonasal masses were in the age group 41-50 years. Male:female ratio was about 1.2:1. Nasal obstruction was the most common presentation. Most common non-neoplastic lesion was rhinosporidosis and most common benign lesion was hemangioma. Most common malignant lesion was maxillary carcinoma. The presenting features of all sinonasal lesions may be indistinguishable and pose diagnostic dilemma. Correlation of clinical, radiologic, and most importantly pathologic modalities is of utmost importance for accurate diagnosis.

Production of Recombinant Rhomboid Proteases.

Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.

Compact panicle architecture is detrimental for growth as well as sucrose synthase activity of developing rice kernels.

The increase of spikelet number in the panicles of modern super rice has made the architecture compact, as the extra spikelets are accommodated mostly on secondary branches than on primary branches. However, the grain yield did not improve because of poor grain filling, which was more visible in the basal spikelets than apical spikelets. The objective of this study was to examine the effect of the compactness and positional difference of spikelets in the panicle on grain filling by comparing the activity and genetic expression of starch synthesising enzymes in the developing kernels of lax-(Upahar and CR3856-45-11-2-7-2-5 (CR-45)) and compact-(Mahalaxmi and CR3856-29-14-2-1-1-1 (CR-29)) panicle cultivars. Upahar and Mahalaxmi are genetically related, whereas CR-45 and CR-29 are recombinant inbred lines. The grain carbohydrate concentration and activity of sucrose synthase (SUS) enzyme were estimated during the active period of grain filling. Further, expression of isoforms of SUS, ADP glucose pyrophosphorylase (APL and APS for large and small units respectively) and starch synthase (SS and GBSS for soluble and granule bound starch synthases respectively) were also assayed through PCR studies. The genotype approach used revealed grain SUS activity and starch concentration high and sugar concentration low in the lax- compared with compact-panicle cultivars and in the apical spikelets compared with basal ones. The margin of variation between apical and basal spikelets was higher in the compact- than the lax-panicle cultivars. Genetic expression of most of the isoforms of the enzymes was higher in the lax- than the compact-panicle cultivars as seen in RT-PCR studies. A quantitative appraisal of transcript levels of isoforms in the qRT-PCR identified greater expression of SUS3 in the basal spikelets of Upahar than that in Mahalaxmi and in CR-45 over CR-29, most prominently during the active period of grain filling. We conclude that proximal location as well as increased density of spikelets on panicles affected SUS3 expression in the basal spikelets. The metabolic dominance of a spikelet in rice panicle is dependent on the expression of the genes for different isoforms of starch synthesising enzymes, but the expression of SUS3 could be more specific than the others. SUS3 expression is most active during grain filling of the lax-panicle cultivars, but its dominance is reduced significantly in the kernels of the compact-panicle cultivars.

Potassium deficiency affects water status and photosynthetic rate of the vegetative sink in green house tomato prior to its effects on source activity.

The potassium requirement of green house tomatoes is very high for vegetative growth and fruit production. Potassium deficiency in plants takes long time for expression of visible symptoms. The objective of this study is to detect the deficiency early during the vegetative growth and define the roles of aquaporin and K-channel transporters in the process of regulation of water status and source-sink relationship. The tomato plants were grown hydroponically inside green house of Hiroshima University, Japan and subjected to different levels of K in the rooting medium. Potassium deficiency stress decreased photosynthesis, expansion and transport of ¹4C assimilates of the source leaf, but the effects became evident only after diameter expansion of the growing stem (sink) was down-regulated. The depression of stem diameter expansion is assumed to be associated with the suppression of water supply more than photosynthate supply to the organ. The stem diameter expansion is parameterized by root water uptake and leaf transpiration rates. The application of aquaporin inhibitor (AgNO3) decreased leaf water potential, stem expansion and root hydraulic conductance within minutes of application. Similar results were obtained for application of the K-channel inhibitors. These observations suggested a close relationship between stem diameter expansion and activities of aquaporins and K-channel transporters in roots. The deficiency of potassium might have reduced aquaporin activity, consequently suppressing root hydraulic conductance and water supply to the growing stem for diameter expansion and leaf for transpiration. We conclude that close coupling between aquaporins and K-channel transporters in water uptake of roots is responsible for regulation of stem diameter dynamics of green house tomato plants.

NSs encoded by groundnut bud necrosis virus is a bifunctional enzyme.

Groundnut bud necrosis virus (GBNV), a member of genus Tospovirus in the family Bunyaviridae, infects a large number of leguminosae and solanaceae plants in India. With a view to elucidate the function of nonstructural protein, NSs encoded by the small RNA genome (S RNA), the NSs protein of GBNV- tomato (Karnataka) was over-expressed in E. coli and purified by Ni-NTA chromatography. The purified rNSs protein exhibited an RNA stimulated NTPase activity. Further, this activity was metal ion dependent and was inhibited by adenosine 5' (beta, gamma imido) triphosphate, an ATP analog. The rNSs could also hydrolyze dATP. Interestingly, in addition to the NTPase and dATPase activities, the rNSs exhibited ATP independent 5' RNA/DNA phosphatase activity that was completely inhibited by AMP. The 5' alpha phosphate could be removed from ssDNA, ssRNA, dsDNA and dsRNA thus confirming that rNSs has a novel 5' alpha phosphatase activity. K189A mutation in the Walker motif A (GxxxxGKT) resulted in complete loss of ATPase activity, but the 5' phosphatase activity was unaffected. On the other hand, D159A mutation in the Walker motif B (DExx) resulted in partial loss of both the activities. These results demonstrate for the first time that NSs is a bifunctional enzyme, which could participate in viral movement, replication or in suppression of the host defense mechanism.

Unusual foreign body in throat.

Live fish in throat is a rare foreign body. Here we present two such cases. The foreign body were removed under general anaesthesia and nutrition was maintained by Nasogastric tube for few days. Patient returned to normal duty within a week.