Dissecting the Role of Critical Residues and Substrate Preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis

Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys487 resulted in ∼95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys172 and Ala302 to be involved in the binding of ATP and Ser404 in the binding of CoenzymeA. The influence of mutations of the residues Val209 and Trp377 emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C26/C24 fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys172, Ala302 and Lys487 and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs

Acute post-disaster medical needs of patients with diabetes: emergency department use in New York City by diabetic adults after Hurricane Sandy.

OBJECTIVE: To evaluate the acute impact of disasters on diabetic patients, we performed a geospatial analysis of emergency department (ED) use by New York City diabetic adults in the week after Hurricane Sandy. RESEARCH DESIGN AND METHODS: Using an all-payer claims database, we retrospectively analyzed the demographics, insurance status, and medical comorbidities of post-disaster ED patients with diabetes who lived in the most geographically vulnerable areas. We compared the patterns of ED use among diabetic adults in the first week after Hurricane Sandy\u27s landfall to utilization before the disaster in 2012. RESULTS: In the highest level evacuation zone in New York City, postdisaster increases in ED visits for a primary or secondary diagnosis of diabetes were attributable to a significantly higher proportion of Medicare patients. Emergency visits for a primary diagnosis of diabetes had an increased frequency of certain comorbidities, including hypertension, recent procedure, and chronic skin ulcers. Patients with a history of diabetes visited EDs in increased numbers after Hurricane Sandy for a primary diagnosis of myocardial infarction, prescription refills, drug dependence, dialysis, among other conditions. CONCLUSIONS: We found that diabetic adults aged 65 years and older are especially at risk for requiring postdisaster emergency care compared to other vulnerable populations. Our findings also suggest that there is a need to support diabetic adults particularly in the week after a disaster by ensuring access to medications, aftercare for patients who had a recent procedure, and optimize their cardiovascular health to reduce the risk of heart attacks

Comparative mapping of Brassica juncea and Arabidopsis thaliana using Intron Polymorphism (IP) markers: homoeologous relationships, diversification and evolution of the A, B and C Brassica genomes

Background: Extensive mapping efforts are currently underway for the establishment of comparative genomics between the model plant, Arabidopsis thaliana and various Brassica species. Most of these studies have deployed RFLP markers, the use of which is a laborious and time-consuming process. We therefore tested the efficacy of PCR-based Intron Polymorphism (IP) markers to analyze genome-wide synteny between the oilseed crop, Brassica juncea (AABB genome) and A. thaliana and analyzed the arrangement of 24 (previously described) genomic block segments in the A, B and C Brassica genomes to study the evolutionary events contributing to karyotype variations in the three diploid Brassica genomes. Results: IP markers were highly efficient and generated easily discernable polymorphisms on agarose gels. Comparative analysis of the segmental organization of the A and B genomes of B. juncea (present study) with the A and B genomes of B. napus and B. nigra respectively (described earlier), revealed a high degree of colinearity suggesting minimal macro-level changes after polyploidization. The ancestral block arrangements that remained unaltered during evolution and the karyotype rearrangements that originated in the Oleracea lineage after its divergence from Rapa lineage were identified. Genomic rearrangements leading to the gain or loss of one chromosome each between the A-B and A-C lineages were deciphered. Complete homoeology in terms of block organization was found between three linkage groups (LG) each for the A-B and A-C genomes. Based on the homoeology shared between the A, B and C genomes, a new nomenclature for the B genome LGs was assigned to establish uniformity in the international Brassica LG nomenclature code. Conclusion: IP markers were highly effective in generating comparative relationships between Arabidopsis and various Brassica species. Comparative genomics between the three Brassica lineages established the major rearrangements, translocations and fusions pivotal to karyotype diversification between the A, B and C genomes of Brassica species. The inter-relationships established between the Brassica lineages vis-à-vis Arabidopsis would facilitate the identification and isolation of candidate genes contributing to traits of agronomic value in crop Brassicas and the development of unified tools for Brassica genomics

QTL Landscape for Oil Content in Brassica juncea: Analysis in Multiple Bi-Parental Populations in High and “0” Erucic Background

Increasing oil content in oilseed mustard (Brassica juncea) is a major breeding objective—more so, in the lines that have “0” erucic acid content (< 2% of the seed oil) as earlier studies have shown negative pleiotropic effect of erucic acid loci on the oil content, both in oilseed mustard and rapeseed. We report here QTL analysis of oil content in eight different mapping populations involving seven different parents—including a high oil content line J8 (~49%). The parental lines of the mapping populations contained wide variation in oil content and erucic acid content. The eight mapping populations were categorized into two sets—five populations with individuals segregating for erucic acid (SE populations) and the remaining three with zero erucic acid segregants (ZE populations). Meta-analysis of QTL mapped in individual SE populations identified nine significant C-QTL, with two of these merging most of the major oil QTL that colocalized with the erucic acid loci on the linkage groups A08 and B07. QTL analysis of oil content in ZE populations revealed a change in the landscape of the oil QTL compared to the SE populations, in terms of altered allelic effects and phenotypic variance explained by ZE QTL at the “common” QTL and observation of “novel” QTL in the ZE background. The important loci contributing to oil content variation, identified in the present study could be used in the breeding programmes for increasing the oil content in high erucic and “0” erucic backgrounds

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of ∼3.5 Å in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action

Prevalence, Distribution and Functional Significance of the −237C to T Polymorphism in the IL-12Rβ2 Promoter in Indian Tuberculosis Patients

Cytokine/cytokine receptor gene polymorphisms related to structure/expression could impact immune response. Hence, the −237 polymorphic site in the 5′ promoter region of the IL-12Rβ2 (SNP ID: rs11810249) gene associated with the AP-4 transcription motif GAGCTG, was examined. Amplicons encompassing the polymorphism were generated from 46 pulmonary tuberculosis patients, 35 family contacts and 28 miscellaneous volunteers and sequenced. The C allele predominated among patients, (93.4%, 43/46), and in all volunteers and contacts screened, but the T allele was exclusively limited to patients, (6.5%, 3/46). The functional impact of this polymorphism on transcriptional activity was assessed by Luciferase-reporter and electrophoretic mobility shift assays (EMSA). Luciferase-reporter assays showed a significant reduction in transcriptional efficiency with T compared to C allele. The reduction in transcriptional efficiency with the T allele construct (pGIL-12Rb2-T), in U-87MG, THP-1 and Jurkat cell lines, were 53, 37.6, and 49.8% respectively, compared to the C allele construct (pGIL-12Rb2-C). Similarly, densitometric analysis of the EMSA assay showed reduced binding of the AP-4 transcription factor, to T compared to the C nucleotide probe. Reduced mRNA expression in all patients (3/3) harboring the T allele was seen, whereas individuals with the C allele exhibited high mRNA expression (17/25; 68%, p = 0.05). These observations were in agreement with the in vitro assessment of the promoter activity by Luciferase-reporter and EMSA assays. The reduced expression of IL-12Rβ2 transcripts in 8 patients despite having the C allele was attributed to the predominant over expression of the suppressors (IL-4 and GATA-3) and reduced expression of enhancers (IFN-α) of IL-12Rβ2 transcripts. The 17 high IL-12Rβ2 mRNA expressers had significantly elevated IFN-α mRNA levels compared to low expressers and volunteers. Notwithstanding the presence of high levels of IL-12Rβ2 mRNA in these patients elevated IFN-α expression could modulate their immune responses to Mycobacterium tuberculosis

Dielectric constants and refractive indices of 1-propanol or 2-propanol + cyclohexane, benzene, toluene, <i>o-, m- </i>and <i>p</i>-xylene at 308.15 K

974-978Dielectric constants and refractive indices data at 308.15 K for 1-propanol or 2-propanol with cyclohexane, benzene, toluene, o-, m- and p-xylene have been reported. The analysis of data shows the presence of strong specific interactions between propanol and aromatic hydrocarbon. Frohlich equation is used to calculate the apparent dipole moment of these binary mixtures

Molar excess volumes of 1-propanol or 2-propanol + cyclohexane or aromatic hydrocarbons at 303.15 K

508-512Molar excess volume data for 1-propanol or 2-propanol + cyclohexane or benzene or toluene or o- or m-or p-xylen at 303.15 K have been reported. VE data for these binary systems are also interpreted in terms of Mecke-Kempter type association model with a Flory contribution term. The predicted VEvalues, to some extent, are in good agreement with the experimental data

Molar excess free energy of mixing of 1-propanol or 2-propanol + aromatic hydrocarbons at 308.15 K in terms of an association model with a Flory contribution term

219-229<span style="font-size:12.0pt;font-family: " times="" new="" roman";mso-fareast-font-family:"times="" roman";mso-ansi-language:="" en-in;mso-fareast-language:en-in;mso-bidi-language:ar-sa"="" lang="EN-IN">Excess molar Gibbs free energy of mixing for 1-propanol or 2-propanol + benzene, + toluene, + o-, m-and pxylenes at 308.15 K are calculated by Barker's method from vapour pressure data measured by a static method. The free energy of mixing for these binary systems are also predicted in terms of Mecke-Kempter type of association model with Flory contribution term using two interaction parameters. The predicted value agrees reasonably well with the experimental values.</span