Structural and Kinetic Characterization of the SpeG Spermidine/Spermine N-acetyltransferase from Methicillin-Resistant Staphylococcus aureus USA300.

Polyamines are simple yet critical molecules with diverse roles in numerous pathogenic and non-pathogenic organisms. Regulating polyamine concentrations affects the transcription and translation of genes and proteins important for cell growth, stress, and toxicity. One way polyamine concentrations are maintained within the cell is via spermidine/spermine N-acetyltransferases (SSATs) that acetylate intracellular polyamines so they can be exported. The bacterial SpeG enzyme is an SSAT that exhibits a unique dodecameric structure and allosteric site compared to other SSATs that have been previously characterized. While its overall 3D structure is conserved, its presence and role in different bacterial pathogens are inconsistent. For example, not all bacteria have speG encoded in their genomes; in some bacteria, the speG gene is present but has become silenced, and in other bacteria, it has been acquired on mobile genetic elements. The latter is the case for methicillin-resistant Staphylococcus aureus (MRSA) USA300, where it appears to aid pathogenesis. To gain a greater understanding of the structure/function relationship of SpeG from the MRSA USA300 strain (SaSpeG), we determined its X-ray crystal structure in the presence and absence of spermine. Additionally, we showed the oligomeric state of SaSpeG is dynamic, and its homogeneity is affected by polyamines and AcCoA. Enzyme kinetic assays showed that pre-incubation with polyamines significantly affected the positive cooperativity toward spermine and spermidine and the catalytic efficiency of the enzyme. Furthermore, we showed bacterial SpeG enzymes do not have equivalent capabilities to acetylate aminopropyl versus aminbutyl ends of spermidine. Overall, this study provides new insight that will assist in understanding the SpeG enzyme and its role in pathogenic and non-pathogenic bacteria at a molecular level.

Recurrent Oral Ulcers and Its Association With Stress Among Dental Students in the Northeast Indian Population: A Cross-Sectional Questionnaire-Based Survey.

Aim The term "aphthous" is derived from the Greek word "aphtha," which means ulceration. The cause of aphthous ulcers is multifactorial, but emotional stress is one of the most important risk factors for its occurrence in young individuals. The aim of this study was to evaluate the association of recurrent oral ulcers (ROUs) with stress among dental students in the Northeast Indian Population. Materials and methods A total of 1,134 students were recruited for the study. Two sets of questionnaires were designed. The first set consisted of 11 questions related to demographic data and information about recurrent oral ulcers. The second set of questionnaire had 14 questions related to stress symptoms. Google Forms (Google, Inc., Mountain View, CA, USA) was used for recording the responses of the study participants. Questionnaires were sent to the participants through e-mail, and responses were recorded. Statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) version 21 (IBM SPSS Statistics, Armonk, NY, USA) and Microsoft Office Excel (Microsoft Corp., Redmond, WA, USA). The chi-square test was used to compare categorical variables. Results Among the 1,134 participants, 32.7% (371 students) reported a previous history of recurrent oral ulcers. Out of 371 participants who had given a history of oral ulcers, only 27.2% exhibited direct stress to ulcer association. On further assessment using the Perceived Stress Scale (PSS), it was found that a far greater percentage of students (i.e., more than 27.2%) were under some form of stress or the other. Conclusions The results of this study will help improve the quality of life among the study population, either by tackling their stress levels or by identifying adequate interventions.

Prevalence of tobacco-associated oral mucosal lesion in Hazaribagh population: A cross-sectional study.

Background: Smoking and chewing tobacco carry significant risks for the development of oral cancer and premalignant lesions. The present study was conducted to find the prevalence of tobacco-related habits in Hazaribagh population and its association with oral mucosal lesion. Methodology: The present study was carried out on patients who visited the Department of Oral Medicine And Radiology, Hazaribagh College of Dental Sciences And Hospital. A total of 5,000 subjects were screened for tobacco-related habits and associated mucosal changes. Detailed clinical history about tobacco-related habits was obtained. Oral mucosal lesions were screened using the WHO format for diagnosis of oral lesions. The findings were tabulated and analyzed statistically. Results: Of the 5,000 subjects enrolled for the study, 1,085 (21.7%) used tobacco in some forms. Habit of smoking tobacco was present among 273 (25.2%) and using smokeless tobacco among 811 (74.7%) individuals. Tobacco pouch keratosis (46.1%) was found to be most common lesion, followed by oral submucous fibrosis (OSMF) (16.1%), lichenoid reaction (14.1%), smokers palate/melanosis (12.2%), leukoplakia (7.2%), erythroplakia (2.3%), and oral cancer in (2%). Conclusion: The results provide insight into prevalent tobacco habits and associated oral mucosal lesions in Hazaribagh population. These may act as baseline data for the formulation of preventive programs and help future studies explore the prevalence of tobacco-associated lesions in vulnerable populations. Current knowledge, including findings from the present study, about the prevalence of tobacco use and various oral lesions associated with it may help primary health care physicians to promote among patients visiting them the awareness about the adverse effects of tobacco consumption and treatment options available for tobacco-related lesions at the early stage.

Intron exon boundary junctions in human genome have in-built unique structural and energetic signals.

Precise identification of correct exon-intron boundaries is a prerequisite to analyze the location and structure of genes. The existing framework for genomic signals, delineating exon and introns in a genomic segment, seems insufficient, predominantly due to poor sequence consensus as well as limitations of training on available experimental data sets. We present here a novel concept for characterizing exon-intron boundaries in genomic segments on the basis of structural and energetic properties. We analyzed boundary junctions on both sides of all the exons (3 28 368) of protein coding genes from human genome (GENCODE database) using 28 structural and three energy parameters. Study of sequence conservation at these sites shows very poor consensus. It is observed that DNA adopts a unique structural and energy state at the boundary junctions. Also, signals are somewhat different for housekeeping and tissue specific genes. Clustering of 31 parameters into four derived vectors gives some additional insights into the physical mechanisms involved in this biological process. Sites of structural and energy signals correlate well to the positions playing important roles in pre-mRNA splicing.

Quantification and distribution of mast cells in oral periapical inflammatory lesions.

Background: Periapical granuloma and periapical cysts develop as a sequela of inflammation of dental pulp, usually a consequence of an infected root canal. Mast cells (MCs) have most frequently been implicated in the pathophysiology of periapical lesions such as periapical granulomas and cysts. Aims: The present study was undertaken to emphasize the presence and quantification of MCs in periapical lesions (granulomas and cysts) using toluidine blue stain, with an aim to elucidate their role in the pathogenesis of these lesions. Materials and Methods: The study comprised a total of 55 previously diagnosed cases (30 cases of radicular cyst and 25 cases of periapical granuloma) selected from the departmental archives. The MCs were quantified in all the sections in the three zones, namely the epithelial, subepithelial, and deeper connective tissue zones. The results were sent for statistical analysis. Statistical Analysis: Wilcoxon paired t-test and Mann-Whitney U test were applied for intra- and intergroup comparison MCs in different zones of periapical granulomas and radicular cyst, respectively. Results: A statistically significant difference was observed between the two lesions in all the three zones, i.e., epithelial, subepithelial, and in deeper connective tissue zone with P = 0.001. Conclusion: Although MCs were present in all the three zones of both periapical cysts and granuloma, they were predominantly observed in radicular cysts than in periapical granuloma. This suggests a significant role of MC in initiation, development, and progression of these periapical lesions.

Structural basis for disulphide-CoA inhibition of a butyryl-CoA hexameric thioesterase.

Acyl-coenzyme A thioesterases (ACTs) catalyse the hydrolysis of thioester bonds between fatty-acyl chains and coenzyme A (CoA), producing a free fatty-acyl chain and CoA. These enzymes are expressed ubiquitously across prokaryotes and eukaryotes, and play important roles in lipid metabolism. There are 25 thioesterase families, subdivided based on their active site configuration, protein oligomerization, and substrate specificity. Understanding the mechanism of regulation within these families is important due to their roles in controlling the cell concentration of a range of fatty acids and CoA-bound compounds. Here we report a structural basis for a novel mode of inhibition of an ACT from Staphylococcus aureus. The enzyme displays a hotdog fold composed of five ß-strands wrapping around a central α-helix, and an additional 30 residue α-helix located at its C-terminus. We show that the enzyme is a hexamer and has specificity towards butyryl-CoA. Structural analysis revealed putative catalytic residues, and we show through site directed mutagenesis that Asn28, Asp43, and Thr60 are critical for activity. Additionally, we show that the Asn28Ala destabilises the enzyme oligomeric state into two distinct populations. Co-crystallization of the enzyme with the substrate butyryl-CoA produced a crystal with three CoA ligands bound in the enzyme active sites: CoA, butyryl-CoA, and disulphide-CoA, the latter of which inhibits enzyme activity. Our study provides new insights into the structure and specificity of hexameric thioesterases, inhibitory feedback mechanisms, and possible biotechnological applications in short-chain fatty acid production such as biofuels, pharmaceuticals, and industrial compounds.

Seasonal Transition in PM10 Exposure and Associated All-Cause Mortality Risks in India.

Lack of a consistent PM10 (particulate matter smaller than 10 µm) database at high spatial resolution hinders in assessing the environmental impact of PM10 in India. Here we propose an alternate approach to estimate the PM10 database. Aerosol extinction coefficients at the surface are calculated from midvisible aerosol optical depth from MERRA-2 reanalysis data using characteristics vertical profiles from CALIOP and then are converted to PM10 mass using aerosol property information and microphysical data. The retrieved PM10 are bias-corrected and evaluated ( R2 = 0.85) against coincident ground-based data maintained under the Central Pollution Control Board network. PM10 exposure exceeds Indian annual air quality standard in 72.3% districts. Transition in PM10 exposure from the monsoon (Jun-Sep) to postmonsoon season (Oct-Nov) translates to 1-2% higher all-cause mortality risk over the polluted Indo-Gangetic Basin (IGB). Mortality risk increases in the central to eastern IGB and central India and reduces in Delhi national capital region in the winter (Dec-Feb) relative to the postmonsoon season. Mortality risk decreases by 0.5-1.8% in most parts of India in the premonsoon season (Mar-May). Our results quantify the vulnerability in terms of seasonal transition in all-cause mortality risks due to PM10 exposure at district level for the first time in India.

Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase.

Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn24 and Asp39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase.

Importance of aerosol non-sphericity in estimating aerosol radiative forcing in Indo-Gangetic Basin.

Aerosols are usually presumed spherical in shape while estimating the direct radiative forcing (DRF) using observations or in the models. In the Indo-Gangetic Basin (IGB), a regional aerosol hotspot where dust is a major aerosol species and has been observed to be non-spherical in shape, it is important to test the validity of this assumption. We address this issue using measured chemical composition at megacity Delhi, a representative site of the western IGB. Based on the observation, we choose three non-spherical shapes - spheroid, cylinder and chebyshev, and compute their optical properties. Non-spherical dust enhances aerosol extinction coefficient (ßext) and single scattering albedo (SSA) at visible wavelengths by >0.05km-1 and >0.04 respectively, while it decreases asymmetry parameter (g) by ~0.1. Accounting non-sphericity leads top-of-the-atmosphere (TOA) dust DRF to more cooling due to enhanced backscattering and increases surface dimming due to enhanced ßext. Outgoing shortwave flux at TOA increases by up to 3.3% for composite aerosols with non-spherical dust externally mixed with other spherical species. Our results show that while non-sphericity needs to be accounted for, choice of shape may not be important in estimating aerosol DRF in the IGB.

Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-chain Fatty Acyl-CoAs and a Novel CoA-induced Fit Mechanism.

PaaI thioesterases are members of the TE13 thioesterase family that catalyze the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8-Å resolution, to reveal two double hotdog domains arranged in a back to back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle x-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn(37), Asp(52), and Thr(68) are important for catalysis, and size exclusion chromatography analysis and x-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 Å in the N terminus, and a loop region involving Tyr(38)-Tyr(39). This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family.

Structural characterization of a Gcn5-related N-acetyltransferase from Staphylococcus aureus.

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphylococcus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic ß-bulge splayed at the ß-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.

Purification, crystallization and preliminary X-ray diffraction analysis of the N-acetyltransferase SAV0826 from Staphylococcus aureus.

Staphylococcus aureus is a prevalent microorganism that is capable of causing a wide range of infections and diseases. Several strains of this bacterial species have developed antibiotic resistance to methicillin and vancomycin, and higher death rates are still being reported each year owing to multidrug-resistant strains. Certain GCN5-related N-acetyltransferases (GNATs) exhibit a broad substrate range, including aminoglycosides, histones, other proteins and serotonin, and have been implicated in antibiotic drug resistance. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of a GNAT from S. aureus (SaNAT) are reported. SaNAT was recombinantly expressed and crystallized by the hanging-drop vapour-diffusion method at 296 K, and the crystals diffracted to 1.7 Å resolution on the MX2 beamline at the Australian Synchrotron. The crystals belonged to space group P43212, with unit-cell parameters a = b = 84.86, c = 49.06 Å, α = ß = γ = 90°. A single molecule is likely to be present in the asymmetric unit. A full structural and functional analysis is currently being undertaken to provide novel insights into the protein function, which in turn may provide a basis for drug design.

Expression, purification, crystallization and preliminary X-ray analysis of the PaaI-like thioesterase SAV0944 from Staphylococcus aureus.

Staphylococcus aureus is the causative agent of many diseases, including meningitis, bacteraemia, pneumonia, food poisoning and toxic shock syndrome. Structural characterization of the PaaI-like thioesterase SAV0944 (SaPaaI) from S. aureus subsp. aureus Mu50 will aid in understanding its potential as a new therapeutic target by knowledge of its molecular details and cellular functions. Here, the recombinant expression, purification and crystallization of SaPaaI thioesterase from S. aureus are reported. This protein initially crystallized with the ligand coenzyme A using the hanging-drop vapour-diffusion technique with condition No. 40 of Crystal Screen from Hampton Research at 296 K. Optimal final conditions consisting of 24% PEG 4000, 100 mM sodium citrate pH 6.5, 12% 2-propanol gave single diffraction-quality crystals. These crystals diffracted to beyond 2 Å resolution at the Australian Synchrotron and belonged to space group P12(1)1, with unit-cell parameters a = 44.05, b = 89.05, c = 60.74 Å, ß = 100.5°. Initial structure determination and refinement gave an R factor and R(free) of 17.3 and 22.0%, respectively, confirming a positive solution in obtaining phases using molecular replacement.

Alteration in uptake and translocation of essential nutrients in cabbage by excess lead.

To observe the tolerance limit of lead phytotoxicity in cabbage (Brassica oleracea L.) var. Golden Aker plants were grown in refined sand with complete nutrient solution for 41 days. On 42nd day, pots with plants were separated into six lots. One lot was allowed to grow as such and was treated as control, in rest of the five lots, lead was applied at 0.1, 0.2, 0.4, 0.5 and 1.0 mM as lead nitrate. At d 75 (34 days after metal exposure), the lead toxicity symptoms as restricted growth was observed on plants at 1.0 mM lead supply. Excess lead (0.5 and 1.0 mM) developed interveinal chlorosis along the margins of young leaves. The affected leaves were reduced in size giving plant a rosette like appearance. Head size was markedly reduced at these (0.5 and 1.0 mM) levels of lead. At 0.5 mM the intensity of symptoms was markedly low. With an increase in lead supply, the concentration of lead and zinc was increased whereas that of P, S, Fe, Mn and Cu were decreased in various parts of cabbage. At 1.0 mM Pb, the concentration of lead was highest in roots and lowest in head. In leaves of cabbage the threshold of toxicity and toxicity values were 150 and 320 microg g-1 dry matter, respectively.