Identification of selenosugars and other low-molecular weight selenium metabolites in high-selenium cereal crops

Several novel selenium containing compounds were characterized in staple crops (wheat, rice and maize) grown on soils naturally rich in selenium. A dedicated method based on the coupling of liquid chromatography with multiplexed detection (ICP-MS, ESI-Orbitrap MS(/MS)) was developed for the speciation of low-molecular weight (<5 kDa) selenium metabolites. Nine species present in different proportions as a function of the crop type were identified by cation-exchange HPLC-ESI-Orbitrap MS on the basis of the accurate molecular mass and MS/MS spectra. The natural origin of these species was then validated by varying extraction conditions and by using hydrophilic interaction LC (HILIC)-ESI-Orbitrap MS(/MS). Among the identified compounds, Se-containing monosaccharides (hexose moiety, m/z 317 and m/z 358) or Se-containing disaccharides (hexose-pentose moiety, m/z 407 and m/z 408) were the first selenosugars reported in edible plants. It is also the first report of the presence of 2,3-dihydroxypropionyl-selenolanthionine (m/z 345) in rice. Because these crops can be an important source of selenium in animal and human nutrition, the understanding of the origin and the fate of these species during metabolic processes will be of great interest.     

Characterization,development and mapping of Unigene-derived microsatellite markers in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

Molecular variation within known genes controlling specific functions provide candidate gene-based markers which are tightly linked with the trait of interest. Unigene-derived microsatellite markers, with their unique identity and positions, offer the advantage of unraveling variation in the expressed component of the genome. We characterized ≥12-bp-long microsatellite loci from 13,899 unique sequences of sorghum [Sorghum bicolor (L.) Moench] available in the NCBI unigene database for their abundance and possible use in sorghum breeding. Analysis of 12,464 unigenes (≥200-bp) using MISA software identified 14,082 simple sequence repeats (SSRs) in 7,370 unigenes, from which 1,519 unigene SSR markers were developed. The average frequency of SSR was 1 per1.6 kb and 1.0 per 1.1 unigene; hexamers followed by trimers were found in abundance, of which 33.3% AT-rich and CCG repeats were the most abundant. Of the 302 unigene SSRs tested, 60 (19.8%) were polymorphic between the two parents, M35-1 and B35 of a recombinant inbred line (RIL) mapping population. A mapping population consisting of 500 RILs was developed using the above two parents, and a subset of random 245 RILs was used for genotyping with polymorphic SSRs. We developed a linkage map containing 231 markers, of which 228 (174 genomic and 54 genic) were microsatellites and three were morphological markers. Markers were distributed over 21 linkage groups, and spanned a genetic distance of 1235.5 cM. This map includes 81 new SSRs, of which 35 (21 unigene and 14 genomic) were developed in the present study and 46 from other studies. The order of the SSR markers mapped in the present study was confirmed physically by BLAST search against the whole-genome shotgun sequence of sorghum. Many unigene sequences used for marker development in this study include genes coding for important regulatory proteins and functional proteins that are involved in stress-related metabolism. The unigene SSR markers used together with other SSR markers to construct the sorghum genetic map will have applications in studies on comparative mapping, functional diversity analysis and association mapping, and for quantitative trait loci detection for drought and other agronomically important traits in sorghum.     

Feasibility and Effectiveness of Provider Initiated HIV Testing and Counseling of TB Suspects in Vizianagaram District, South India

Background

Though internationally recommended, provider initiated HIV testing and counseling (PITC) of persons suspected of tuberculosis (TB) is not a policy in India; HIV seroprevalence among TB suspects has never been reported. The current policy of PITC for diagnosed TB cases may limit opportunities of early HIV diagnosis and treatment. We determined HIV seroprevalence among persons suspected of TB and assessed feasibility and effectiveness of PITC implementation at this earlier stage in the TB diagnostic pathway.

Methods

All adults examined for diagnostic sputum microscopy (TB suspects) in Vizianagaram district (population 2.5 million), in November-December 2010, were offered voluntary HIV counseling and testing (VCT) and assessed for TB diagnosis.

Results

Of 2918 eligible TB suspects, 2465(85%) consented to VCT. Among these, 246(10%) were HIV-positive. Of the 246, 84(34%) were newly diagnosed as HIV (HIV status not known previously). To detect a new case of HIV infection, the number needed to screen (NNS) was 26 among ‘TB suspects’, comparable to that among ‘TB patients’. Among suspects aged 25–54 years, not diagnosed as TB, the NNS was 17.

Conclusion

The seroprevalence of HIV among ‘TB suspects’ was as high as that among ‘TB patients’. Implementation of PITC among TB suspects was feasible and effective, detecting a large number of new HIV cases with minimal additional workload on staff of HIV testing centre. HIV testing of TB suspects aged 25–54 years demonstrated higher yield for a given effort, and should be considered by policy makers at least in settings with high HIV prevalence.     

Factors associated with delays in treatment initiation after tuberculosis diagnosis in two districts of India

Background

Excessive time between diagnosis and initiation of tuberculosis (TB) treatment contributes to ongoing TB transmission and should be minimized. In India, Revised National TB Control Programme (RNTCP) focuses on indicator start of treatment within 7 days of diagnosis for patients with sputum smear-positive PTB for monitoring DOTS implementation.

Objectives

To determine length of time between diagnosis and initiation of treatment and factors associated with delays of more than 7 days in smear-positive pulmonary TB.

Methods

Using existing programme records such as the TB Register, treatment cards, and the laboratory register, we conducted a retrospective cohort study of all patients with smear-positive pulmonary TB registered from July-September 2010 in two districts in India. A random sample of patients with pulmonary TB who experienced treatment delay of more than 7 days was interviewed using structured questionnaire.

Results

2027 of 3411 patients registered with pulmonary TB were smear-positive. 711(35%) patients had >7 days between diagnosis and treatment and 262(13%) had delays >15 days. Mean duration between TB diagnosis and treatment initiation was 8 days (range = 0–128 days). Odds of treatment delay >7 days was 1.8 times more likely among those who had been previously treated (95% confidence interval [CI] 1.5–2.3) and 1.6 (95% CI 1.3–1.8) times more likely among those diagnosed in health facilities without microscopy centers. The main factors associated with a delay >7 days were: patient reluctance to start a re-treatment regimen, patients seeking second opinions, delay in transportation of drugs to the DOT centers and delay in initial home visits. To conclude, treatment delay >7 days was associated with a number of factors that included history of previous treatment and absence of TB diagnostic services in the local health facility. Decentralized diagnostic facilities and improved referral procedures may reduce such treatment delays.     

Inhibition of type IA topoisomerase by a monoclonal antibody through perturbation of DNA cleavage-religation equilibrium

Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e. the introduction of the DNA supercoils). A number of diverse molecules target DNA gyrase. However, inhibitors that arrest the activity of bacterial topoisomerase I at low concentrations remain to be identified. Towards this end, as a proof of principle, monoclonal antibodies that inhibit Mycobacterium smegmatis topoisomerase I have been characterized and the specific inhibition of Mycobacterium smegmatis topoisomerase I by a monoclonal antibody, 2F3G4, at a nanomolar concentration is described. The enzyme-bound monoclonal antibody stimulated the first transesterification reaction leading to enhanced DNA cleavage, without significantly altering the religation activity of the enzyme. The stimulated DNA cleavage resulted in perturbation of the cleavage-religation equilibrium, increasing single-strand nicks and protein-DNA covalent adducts. Monoclonal antibodies with such a mechanism of inhibition can serve as invaluable tools for probing the structure and mechanism of the enzyme, as well as in the design of novel inhibitors that arrest enzyme activity.     

Optimization of a new heteropolysaccharide production by a native isolate of Leuconostoc sp. CFR-2181

Aim:  This work is aimed at optimizing the production of a new heteropolysaccharide (HePS) of Leuconostoc sp. CFR-2181 by standardizing the fermentation conditions in a low cost semi-synthetic medium.
Methods and Results:  Both nutritional and cultural parameters, such as carbon source and its concentration, initial pH of the exopolysaccharide (EPS) medium, fermentation temperature and fermentation period were optimized. Fermentation of the EPS medium (pH 6·7), containing sucrose at 5% (w/v) and 5% (v/v) inoculum, at 25 ° C resulted in maximum production of HePS (18·38 g l⁻¹) by the isolate in 4 h of fermentation.
Conclusions:  The isolate was found to produce good amount of HePS in just 4 h in a low cost semi-synthetic EPS medium.
Significance and Impact of the Study:  This is the first report on rapid production of HePS by any lactic culture, which can significantly reduce the cost of the EPS.     

Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function

Thyroid-stimulating hormone (TSH) controls thyroid growth and hormone secretion through binding to its G protein-coupled receptor (TSHR) and production of cyclic AMP (cAMP). Serum TSH is a sensitive indicator of thyroid function, and overt abnormalities in thyroid function lead to common endocrine disorders affecting approximately 10% of individuals over a life span. By genotyping 362,129 SNPs in 4,300 Sardinians, we identified a strong association (p = 1.3 x 10(-11)) between alleles of rs4704397 and circulating TSH levels; each additional copy of the minor A allele was associated with an increase of 0.13 muIU/ml in TSH. The single-nucleotide polymorphism (SNP) is located in intron 1 of PDE8B, encoding a high-affinity cAMP-specific phosphodiesterase. The association was replicated in 4,158 individuals, including additional Sardinians and two genetically distant cohorts from Tuscany and the Old Order Amish (overall p value = 1.9 x 10(-20)). In addition to association of TSH levels with SNPs in PDE8B, our genome scan provided evidence for association with PDE10A and several biologically interesting candidates in a focused analysis of 24 genes. In particular, we found evidence for association of TSH levels with SNPs in the THRB (rs1505287, p = 7.3 x 10(-5)), GNAQ (rs10512065, p = 2.0 x 10(-4)), TG (rs2252696, p = 2.2 x 10(-3)), POU1F1 (rs1976324, p = 3.9 x 10(-3)), PDE4D (rs27178, p = 8.3 x 10(-3)), and TSHR (rs4903957, p = 8.6 x 10(-3)) loci. Overall, the results suggest a primary effect of PDE8B variants on cAMP levels in the thyroid. This would affect production of T4 and T3 and feedback to alter TSH release by the pituitary. PDE8B may thus provide a candidate target for the treatment of thyroid dysfunction.     

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.