Development of sesbania mosaic virus nanoparticles for imaging.

The capsids of viruses have a high degree of symmetry. Therefore, virus nanoparticles (VNPs) can be programmed to display many imaging agents precisely. Plant VNPs are biocompatible, biodegradable and non-infectious to mammals. We have carried out bioconjugation of sesbania mosaic virus (SeMV), a well characterized plant virus, with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries. Monitoring of cellular internalization of labelled SeMV nanoparticles (NPs) by confocal microscopy and flow cytometry showed that the particles have a natural preference for entry into MDA-MB-231 (breast cancer) cells, although they could also enter various other cell lines. The fluorescence of SeMV NPs labelled via the cysteines with Cy5.5 dye was found to be more stable and was detectable with greater sensitivity than that of particles labelled via the lysines with Alexa Fluor. Live-cell imaging using SeMV internally labelled with Cy5.5 showed that it could bind to MDA-MB-231 cells in less than 5 minutes and enter the cells within 15 minutes. The particles undergo endolysosomal degradation by 6 h as evidenced by their co-localization with LAMP-1. Far-western blot analysis with a HeLa cell membrane protein fraction showed that SeMV interacts with 54-, 35- and 33-kDa proteins, which were identified by mass spectrometry as vimentin, voltage-dependent anion-selective channel protein (VDAC1), and annexin A2 isoform 2 (ANXA2), respectively, suggesting that the particles may bind and enter the cell through these proteins. The results presented here demonstrate that the SeMV NPs provide a new platform technology that could be used to develop in vivo imaging and targeted drug delivery agents for cancer diagnosis and therapy.

Comparative structural and enzymatic studies on Salmonella typhimurium diaminopropionate ammonia lyase reveal its unique features.

Cellular metabolism of amino acids is controlled by a large number of pyridoxal 5'-phosphate (PLP) dependent enzymes. Diaminopropionate ammonia lyase (DAPAL), a fold type II PLP-dependent enzyme, degrades both the D and L forms of diaminopropionic acid (DAP) to pyruvate and ammonia. Earlier studies on the Escherichia coli DAPAL (EcDAPAL) had suggested that a disulfide bond located close to the active site may be crucial for maintaining the geometry of the substrate entry channel and the active site. In order to obtain further insights into the catalytic properties of DAPAL, structural and functional studies on Salmonella typhimurium DAPAL (StDAPAL) were initiated. The three-dimensional X-ray crystal structure of StDAPAL was determined at 2.5 Šresolution. As expected, the polypeptide fold and dimeric organization of StDAPAL is similar to those of EcDAPAL. A phosphate group was located in the active site of StDAPAL and expulsion of this phosphate is probably essential to bring Asp125 to a conformation suitable for proton abstraction from the substrate (D-DAP). The unique disulfide bond of EcDAPAL was absent in StDAPAL, although the enzyme displayed comparable catalytic activity. Site directed mutagenesis of the cysteine residues involved in disulfide bond formation in EcDAPAL followed by functional and biophysical studies further confirmed that the disulfide bond is not necessary either for substrate binding or for catalysis. The activity of StDAPAL but not EcDAPAL was enhanced by monovalent cations suggesting subtle differences in the active site geometries of these two closely related enzymes.

Structural and biophysical characterization of Rv3716c, a hypothetical protein from Mycobacterium tuberculosis.

Latent tuberculosis (TB) is the main hurdle in reaching the goal of "Stop TB 2050". Tuberculin skin and Interferon-gamma release assay tests used currently for the diagnosis of TB infection cannot distinguish between active disease and latent tuberculosis infection (LTBI) and hence new and sensitive protein markers need to be identified for the diagnosis. A protein Rv3716c from Mycobacterium tuberculosis (MtbRv3716c) has been identified as a potential surrogate marker for the diagnosis of LTBI. Here, we present characterization of MtbRv3716c (∼13 kDa) using both biophysical and X-Ray crystallographic methods. EMSA study showed that MtbRv3716c binds to double stranded DNA. X-ray diffraction data collected on a crystal of MtbRv3716c at 1.9 Å resolution was used for structure determination using the molecular replacement method. Significant electron density was not observed for the N-terminal 21 and C-terminal 41 residues in the final electron density map. The C- terminal disordered region is proline rich and displays characteristics of intrinsically disordered proteins. Although the crystal asymmetric unit contained a protomer, a tight dimer could be generated by the application of the crystal two-fold symmetry parallel to the b axis. Packing of dimers in the crystal is mediated by a cadmium ion (Cd2+) occurring at the interface of two dimers. Molecular packing analysis reveals large cavities that are probably occupied by the disordered segments of the N- and C-termini. Structural comparison with other homologous hypothetical DNA binding proteins (PDB codes: 1PUG, 1YBX) highlights structural features that might be significant for DNA binding.

Structural studies on the decameric S. typhimurium arginine decarboxylase (ADC): Pyridoxal 5'-phosphate binding induces conformational changes.

Enteric pathogens such as Salmonella typhimurium colonize the human gut in spite of the lethal acidic pH environment (pH < 2.5) due to the activation of inducible acid tolerance response (ATR) systems. The pyridoxal 5'-phosphate (PLP)-dependent enzyme, biodegradative arginine decarboxylase (ADC, encoded by AdiA), is a component of an ATR system. The enzyme consumes a cytoplasmic proton in the process of arginine degradation to agmatine. Arginine-agmatine antiporter (AdiC) exchanges the product agmatine for arginine. In this manuscript, we describe the structure of Salmonella typhimurium ADC (StADC). The decameric structure assembled from five dimers related by a non crystallographic 5-fold symmetry represents the first apo-form of the enzyme. The structure suggests that PLP-binding is not a prerequisite for oligomerization. Comparison with E. coli ADC reveals that PLP-binding is accompanied by the movement and ordering of two loops (residues 150-159 and 191-197) and a few active site residues such as His256 and Lys257. A number of residues important for substrate binding are disordered in the apo-StADC structure indicating that PLP binding is important for substrate binding. Unlike the interactions between 5-fold related protomers, interactions that stabilize the dimeric structure are not pH dependent.

Structural studies on tobacco streak virus coat protein: Insights into the pleomorphic nature of ilarviruses.

Tobacco streak virus (TSV), the type member of Ilarvirus genus, is a major plant pathogen. TSV purified from infected plants consists of a ss-RNA genome encapsidated in spheroidal particles with diameters of 27, 30 and 33nm constructed from multiple copies of a single species of coat protein (CP) subunits. Apart from protecting the viral genome, CPs of ilarviruses play several key roles in the life cycle of these viruses. Unlike the related bromo and cucumoviruses, ilarvirus particles are labile and pleomorphic, which has posed difficulties in their crystallization and structure determination. In the current study, a truncated TSV-CP was crystallized in two distinct forms and their structures were determined at resolutions of 2.4Å and 2.1Å, respectively. The core of TSV CP was found to possess the canonical ß-barrel jelly roll tertiary structure observed in several other viruses. Dimers of CP with swapped C-terminal arms (C-arm) were observed in both the crystal forms. The C-arm was found to be flexible and is likely to be responsible for the polymorphic and pleomorphic nature of TSV capsids. Consistent with this observation, mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses. This communication represents the first report on the structure of the CP from an ilarvirus.

Biodistribution and toxicity evaluation of sesbania mosaic virus nanoparticles in mice.

Sesbania mosaic virus (SeMV), a 30-nm spherical plant sobemovirus, is suitable for developing functionalized nanoparticles for biomedical applications. However, the in vivo behavior of SeMV and the clinical impact following its delivery via the oral or intravenous route are not known. To address this question, we examined the biodistribution, toxicity and histopathological changes in SeMV treated mice. No toxic effects were observed in mice administered high doses (100 mg and 200 mg per kg body weight orally or 40 mg and 80 mg per kg body weight intravenously) of SeMV, and they were found to be normal. Analysis of fecal sample showed that SeMV was cleared in 16 h when 20 mg of the virus per kg body weight was administered orally. RT-PCR analysis of blood samples showed that SeMV was present up to 72 h in mice inoculated either intravenously (8 mg/kg body weight) or orally (20 mg/kg body weight). Further, SeMV was found to be localized up to 72 h in spleen and liver tissues of intravenously inoculated mice only. Biochemical and hematological parameters were found to be normal at 6 and 72 h after administration of SeMV. Furthermore, no noticeable changes were observed in histological sections of brain, liver, spleen, lungs and kidney tissue samples collected at 6 and 72 h from SeMV administered mice when compared to control mice. Thus, SeMV appears to be a safe and non-toxic platform that can be tailored as a nanocarrier for in vivo biomedical applications.

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.

Insights into stabilizing interactions in the distorted domain-swapped dimer of Salmonella typhimurium survival protein.

The survival protein SurE from Salmonella typhimurium (StSurE) is a dimeric protein that functions as a phosphatase. SurE dimers are formed by the swapping of a loop with a pair of ß-strands and a C-terminal helix between two protomers. In a previous study, the Asp230 and His234 residues were mutated to Ala to abolish a hydrogen bond that was thought to be crucial for C-terminal helix swapping. These mutations led to functionally inactive and distorted dimers in which the two protomers were related by a rotation of 167°. New salt bridges involving Glu112 were observed in the dimeric interface of the H234A and D230A/H234A mutants. To explore the role of these salt bridges in the stability of the distorted structure, E112A, E112A/D230A, E112A/H234A, E112A/D230A/H234A, R179L/H180A/H234A and E112A/R179L/H180A/H234A mutants were constructed. X-ray crystal structures of the E112A, E112A/H234A and E112A/D230A mutants could be determined. The dimeric structures of the E112A and E112A/H234A mutants were similar to that of native SurE, while the E112A/D230A mutant had a residual rotation of 11° between the B chains upon superposition of the A chains of the mutant and native dimers. The native dimeric structure was nearly restored in the E112A/H234A mutant, suggesting that the new salt bridge observed in the H234A and D230A/H234A mutants was indeed responsible for the stability of their distorted structures. Catalytic activity was also restored in these mutants, implying that appropriate dimeric organization is necessary for the activity of SurE.

Structural characterization of a mitochondrial 3-ketoacyl-CoA (T1)-like thiolase from Mycobacterium smegmatis.

Thiolases catalyze the degradation and synthesis of 3-ketoacyl-CoA molecules. Here, the crystal structures of a T1-like thiolase (MSM-13 thiolase) from Mycobacterium smegmatis in apo and liganded forms are described. Systematic comparisons of six crystallographically independent unliganded MSM-13 thiolase tetramers (dimers of tight dimers) from three different crystal forms revealed that the two tight dimers are connected to a rigid tetramerization domain via flexible hinge regions, generating an asymmetric tetramer. In the liganded structure, CoA is bound to those subunits that are rotated towards the tip of the tetramerization loop of the opposing dimer, suggesting that this loop is important for substrate binding. The hinge regions responsible for this rotation occur near Val123 and Arg149. The Lα1-covering loop-Lα2 region, together with the Nß2-Nα2 loop of the adjacent subunit, defines a specificity pocket that is larger and more polar than those of other tetrameric thiolases, suggesting that MSM-13 thiolase has a distinct substrate specificity. Consistent with this finding, only residual activity was detected with acetoacetyl-CoA as the substrate in the degradative direction. No activity was observed with acetyl-CoA in the synthetic direction. Structural comparisons with other well characterized thiolases suggest that MSM-13 thiolase is probably a degradative thiolase that is specific for 3-ketoacyl-CoA molecules with polar, bulky acyl chains.

Crystal structures of SCP2-thiolases of Trypanosomatidae, human pathogens causing widespread tropical diseases: the importance for catalysis of the cysteine of the unique HDCF loop.

Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites.

Stakeholders' perspective on the daily regimen of tuberculosis treatment- A qualitative approach.

INTRODUCTION: In the context of changing over from an intermittent treatment regimen to a daily regimen, it becomes crucial to understand the impact of a daily regimen on the treatment process and outcome. It enables health professionals to strengthen strategies, to enhance the quality of treatment as well as the quality of life of TB patients. The perspective of each stakeholder involved in the process is important in assessing the impact of the daily regimen. OBJECTIVES: To understand patients' and providers' perspectives on the daily regimen of Tuberculosis treatment. METHODOLOGY: A qualitative study was conducted between March 2020 to June 2020, including in-depth interviews with TB patients on treatment and DOT providers, and Key Informant Interview(KII) with TB Health Visitors(TBHV) and family members of TB patients. A thematic-network analysis approach was utilized to get the results. RESULTS: Two sub-themes emerged: (i) Acceptance of the daily regimen of treatment; (ii) operational difficulties of the daily regimen. No injections in the regimen, fewer side effects of drugs as dose depends on weight band, family members can be treatment supporter, awareness about disease and treatment, the drugs are as same as private drugs available, adherence has improved, monthly DBT were found to some of the enablers in the study. The Barriers found in the study were traveling daily to get drugs, loss of daily wages, accompanying patients daily, tracing private patients, pyridoxine is not given free in this regimen, increased workload for treatment providers, etc. CONCLUSION: The study points out that acceptance of the patient to the daily regimen is better as they have lesser side effects. The operational difficulties in the implementation of the daily regimen can be addressed by providing family members as treatment supporters.

Prevalence and Determinants of Avian Allergic Manifestation in a Selected Rural Population in Mysuru: A Cross-Sectional Survey.

Background: Avian diseases should be known to people who raise birds as pets or for commercial purposes. Exposure to high levels of airborne dust and endotoxins is known to cause allergies. This study, therefore, intends to assess avian allergy and its associated factors among poultry workers and bird fanciers in urban Mysuru. Methods: A cross-sectional study was done over three months among 60 poultry workers and 60 bird fanciers in a selected rural population of the Mysuru district. The participants were selected by simple random sampling method and their data were obtained by interview method using a pre-tested semi-structured questionnaire. Chi-square test/Fisher's exact test was used to check the associations between the demographic variables' avian allergy. Results: The survey revealed that 31.67% of poultry workers and 53.33% of bird fanciers had symptoms. The majority of the study participants were illiterate and were not using personal protective equipment (PPE) regularly. The results showed a significant association between education (P = 0.013) and the use of PPE (P = 0.020) with avian allergy. Observing the health hazards, 42.10% had respiratory problems, 42.10% had skin problems, and 26.31% had eye problems. Further, a significant association was also found between education and knowledge about diseases (P = 0.001). Conclusion: The avian allergy among the bird fanciers was higher when compared to poultry workers. It is necessary to organize routine counseling, awareness, and training programs about diseases and prevention strategies related to their work and living environments and keep them updated.

Effects of mobile Health (mHealth) application on cervical cancer prevention knowledge and screening among women social support groups with low-socioeconomic status in Mysuru city, Southern India.

BACKGROUND: Cervical cancer is an important area of action because of the mortality and morbidity of the disease, and the potential for effective prevention by screening. Involving the social support groups by health education improves cervical cancer screening and early detection of the disease in the community. In the ongoing efforts to strengthen cervical cancer prevention, control, and management, digital health and technology will have a significant role to play. OBJECTIVE: To assess the effectiveness of the mHealth-based intervention on cervical cancer preparedness among women social support groups. MATERIALS & METHODS: A pre-post interventional study was conducted among women social support groups from lower socio-economic status, identified from the field practice area. Purposive sampling technique was employed. A Cervical Cancer Awareness Measure (CAM) instrument was used to assess the cancer preparedness among the social support group women After taking inputs from the stakeholders' mobile health application was developed. The mHealth educational intervention was given to 102 women. Both pre-and post-test questionnaires were administered through mHealth application to assess the change in knowledge after a gap of 1 month to 2 months. The data obtained was coded and entered into Microsoft Excel worksheet 2016 and was later imported and analyzed using SPSS version 22 (licensed to the Institute). The difference in median scores of knowledge and practice were interpreted as statistically significant at p value of < 0.05. RESULTS & CONCLUSION: Before the intervention only 13 (12.7%) of them had heard about cervical cancer. There was a significant increase in the knowledge about warning signs & symptoms, risk factors of cervical cancer, and HPV vaccination. Around 5% increase in Pap smear test uptake.

Relationship between Frailty, Glycemic Control, and Nutritional Status among the Elderly with Diabetes Mellitus Residing in an Urban Community of Mysuru.

Background: In India, the elderly (aged 60 and above) constitute 8.2% of the total population and are expected to increase to 10% by the year 2020. Globally, around 450 million people are suffering from diabetes mellitus. Frailty is regarded as a predisability state and, therefore, if identified early, may avert many adverse health outcomes in the elderly. Diabetes and frailty are found to be close associates. Materials and Methods: This community-based cross-sectional study was conducted among 104 elderlies with diabetes mellitus residing in an urban slum situated in Mysuru for a period of 6 months. Pretested structured questionnaire was used to collect the information on sociodemographic characteristics and details of diabetes. The Tilburg's Frailty Scale was used to assess frailty, and the Mini Nutritional Assessment Scale was used to assess the nutritional status. Results: The prevalence of frailty among the study population was 53.8%. 51% of the subjects were found to have their glycemic status under control, 16.3% were malnourished, and 70.2% were at risk of malnutrition (RMN). The majority of the subjects with malnourishment were frail (76.5%) followed by those at RMN, 36 (49.3%). Gender, marital status, engaging in occupation, socio economic status, poor glycemic control were found to be significantly associated with frailty. Conclusion: The prevalence of frailty is significantly higher among elderly diabetics. The poorer glycemic control is a significant factor associated with frailty, and malnourished elderlies are more at risk of developing frailty.

Teaching Hands-Only CPR (HOCPR) skills to 8th-grade students in urban Bengaluru: Development of a comprehensive Hands-Only CPR programme for high school students.

Background and Aims: Out-of-hospital cardiac arrest is one of the leading causes of death in India. Only 1.3% of these arrests receive bystander cardiopulmonary resuscitation (CPR). Bystander CPR increases a victim's chances of survival; training school children in Hands-Only CPR (HOCPR) is a proven method of increasing bystander CPR rates. Heart Rescue India is an international project working to improve care for cardiovascular diseases, and as a part of it, a ten module Cardiovascular disease (CVD) educational programme, including HOCPR training, was conducted in ten schools in 2017-18. The objective of our study was to assess the effectiveness of HOCPR training for 8th-grade high school students. Methods: Four hundred fourteen of the 530 enroled students from ten schools of Bengaluru participated in the study. The participants attended a one-hour didactic session about the recognition of cardiac arrest and HOCPR in three simple steps. Subsequently, students received hands-on training for HOCPR. The sessions included pre- and post-assessment of knowledge and skills. The results were statistically analysed using paired t-test and the McNemar test. Results: The mean overall pre-assessment score for knowledge was 62.07 ± 28.38%, and the post-assessment score was 72.42 ± 26.58% (P < 0.001). In addition, there was a statistically significant improvement in the post-training scores for HOCPR in all three parameters, namely compressions per minute, depth and chest recoil. Conclusion: The study demonstrated a simple yet effective HOCPR programme for high school children.

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity.

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4Å resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes.

Revisiting the mechanism of the triosephosphate isomerase reaction: the role of the fully conserved glutamic acid 97 residue.

An analysis of 503 available triosephosphate isomerase sequences revealed nine fully conserved residues. Of these, four residues-K12, H95, E97 and E165-are capable of proton transfer and are all arrayed around the dihydroxyacetone phosphate substrate in the three-dimensional structure. Specific roles have been assigned to the residues K12, H95 and E165, but the nature of the involvement of E97 has not been established. Kinetic and structural characterization is reported for the E97Q and E97D mutants of Plasmodium falciparum triosephosphate isomerase (Pf TIM). A 4000-fold reduction in k(cat) is observed for E97Q, whereas the E97D mutant shows a 100-fold reduction. The control mutant, E165A, which lacks the key catalytic base, shows an approximately 9000-fold drop in activity. The integrity of the overall fold and stability of the dimeric structure have been demonstrated by biophysical studies. Crystal structures of E97Q and E97D mutants have been determined at 2.0 Å resolution. In the case of the isosteric replacement of glutamic acid by glutamine in the E97Q mutant a large conformational change for the critical K12 side chain is observed, corresponding to a trans-to-gauche transition about the Cγ-Cδ (χ(3)) bond. In the E97D mutant, the K12 side chain maintains the wild-type orientation, but the hydrogen bond between K12 and D97 is lost. The results are interpreted as a direct role for E97 in the catalytic proton transfer cycle. The proposed mechanism eliminates the need to invoke the formation of the energetically unfavourable imidazolate anion at H95, a key feature of the classical mechanism.

Preliminary X-ray crystallographic studies on acetate kinase (AckA) from Salmonella typhimurium in two crystal forms.

Acetate kinase (AckA) catalyzes the reversible transfer of a phosphate group from acetyl phosphate to ADP, generating acetate and ATP, and plays a central role in carbon metabolism. In the present work, the gene corresponding to AckA from Salmonella typhimurium (StAckA) was cloned in the IPTG-inducible pRSET C vector, resulting in the attachment of a hexahistidine tag to the N-terminus of the expressed enzyme. The recombinant protein was overexpressed, purified and crystallized in two different crystal forms using the microbatch-under-oil method. Form I crystals diffracted to 2.70 Å resolution when examined using X-rays from a rotating-anode X-ray generator and belonged to the monoclinic space group C2, with unit-cell parameters a = 283.16, b = 62.17, c = 91.69 Å, ß = 93.57°. Form II crystals, which diffracted to a higher resolution of 2.35 Å on the rotating-anode X-ray generator and to 1.90 Å on beamline BM14 of the ESRF, Grenoble, also belonged to space group C2 but with smaller unit-cell parameters (a = 151.01, b = 78.50, c = 97.48 Å, ß = 116.37°). Calculation of Matthews coefficients for the two crystal forms suggested the presence of four and two protomers of StAckA in the asymmetric units of forms I and II, respectively. Initial phases for the form I diffraction data were obtained by molecular replacement using the coordinates of Thermotoga maritima AckA (TmAckA) as the search model. The form II structure was phased using a monomer of form I as the phasing model. Inspection of the initial electron-density maps suggests dramatic conformational differences between residues 230 and 300 of the two crystal forms and warrants further investigation.

Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase.

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to 2.5 Šresolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme.

Learning with lockdown: Utility of whatsApp status-based multiple-choice question discussion on COVID-19 among medical students and teaching faculty.

BACKGROUND: Following the COVID-19 pandemic, the Government of India announced lockdown from March 25, 2020, which included measures such as social distancing, canceling mass gatherings, and closure of schools and colleges. Since the present generation of students spends a lot of time on social media, especially WhatsApp, the utility of the same to discuss the various public health domains of COVID-19 through multiple-choice questions (MCQs) was explored. MATERIALS AND METHODS: This longitudinal study was conducted among 154 participants (undergraduates, interns, postgraduates, and teaching faculty) for 3 months. MCQs on COVID-19 were disseminated through WhatsApp status of the investigator, and feedback regarding the same was taken at the end of 3 months. The activity log was maintained, and the information was downloaded as a comma-separated value (CSV) file and exported to MS Excel. Descriptive statistics were applied. The location of the participants was entered into the Microsoft Excel sheet and converted into CSV file. The Geographical data were analyzed in the Quantum Geographical information System (QGIS). RESULTS: Among the 154 study participants, 48% were undergraduates, 10.4% were interns, 30.6% were postgraduates, and 11% were teaching faculty. Eighty-nine percent of the participants felt that WhatsApp can be used as a platform for discussing MCQs. 89%, 93%, and 89.6% of the participants felt that the exercise improved their analytical skills, helped in self-directed learning, and improved collaborative learning, respectively. CONCLUSIONS: Majority of the participants responded that WhatsApp can be used as a platform to discuss MCQs and that the exercise helped in collaborative and self-directed learning.