LED-Fluorescence Microscopy for Diagnosis of Pulmonary Tuberculosis under Programmatic Conditions in India

Background

Light-emitting diode fluorescence microscopy (LED-FM) has been shown to be more sensitive than conventional bright field microscopy using Ziehl-Neelsen (ZN) stain in detecting sputum smear positive tuberculosis in controlled laboratory conditions. In 2012, Auramine O staining based LED-FM replaced conventional ZN microscopy in 200 designated microscopy centres (DMC) of medical colleges operating in collaboration with India’s Revised National Tuberculosis Control Programme. We aimed to assess the impact of introduction of LED-FM services on sputum smear positive case detection under program conditions.

Methods

This was a before and after comparison study. In 15 randomly selected medical college DMCs, all presumptive TB patients who underwent sputum smear examination in the years 2011 (before LED-FM) and 2012 (after LED-FM) were compared. An additional 15 comparable DMCs that implemented conventional ZN sputum smear microscopy were also selected for comparison between 2011 and 2012.

Results

The proportion of presumptive TB patients (PTP)found sputum smear positive increased by 30%- from 13.6% (3432/25159) in 2011 to 17.8% (4706/26426) in 2012 (P value <0.01) in the sites that implemented LED-FM microscopy, whereas in DMCs where the ZN staining procedure is followed the proportion of sputum smear positive had remained unchanged (13.0%versus 12.6%;P value0.31).

Conclusion

Use of LED-FM significantly increased the proportion of smear positive cases among presumptive TB patients under routine program conditions in high workload laboratories. The study provides operational evidence needed to scale-up the use of LED-FM in similar settings in India and beyond.     

Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation

Human sexual determination is initiated by a cascade of genes that lead to the development of the fetal gonad. Whereas development of the female external genitalia does not require fetal ovarian hormones, male genital development requires the action of testicular testosterone and its more potent derivative dihydrotestosterone (DHT). The "classic" biosynthetic pathway from cholesterol to testosterone in the testis and the subsequent conversion of testosterone to DHT in genital skin is well established. Recently, an alternative pathway leading to DHT has been described in marsupials, but its potential importance to human development is unclear. AKR1C2 is an enzyme that participates in the alternative but not the classic pathway. Using a candidate gene approach, we identified AKR1C2 mutations with sex-limited recessive inheritance in four 46,XY individuals with disordered sexual development (DSD). Analysis of the inheritance of microsatellite markers excluded other candidate loci. Affected individuals had moderate to severe undervirilization at birth; when recreated by site-directed mutagenesis and expressed in bacteria, the mutant AKR1C2 had diminished but not absent catalytic activities. The 46,XY DSD individuals also carry a mutation causing aberrant splicing in AKR1C4, which encodes an enzyme with similar activity. This suggests a mode of inheritance where the severity of the developmental defect depends on the number of mutations in the two genes. An unrelated 46,XY DSD patient carried AKR1C2 mutations on both alleles, confirming the essential role of AKR1C2 and corroborating the hypothesis that both the classic and alternative pathways of testicular androgen biosynthesis are needed for normal human male sexual differentiation.     

Decolorization and partial degradation of monoazo dyes in sequential fixed-film anaerobic batch reactor (SFABR)

Decolorization of two monoazo dyes, acid orange 6 (AO6) and acid orange 7 (AO7), were studied in sequential fixed-film anaerobic batch reactor (SFABR) with varying dye concentrations and 500 mg/L glucose as the co-substrate. More than 90% dye decolorization could be achieved, even at 300 mg/L, with both AO6 and AO7 and dye decolorization rates were 168 mg/L/d and 176 mg/L/d, respectively. COD removals with these two monoazo dyes were significantly different, as 75% and 35% decrease were observed with AO6 and AO7, respectively. UV-visible spectral as well as HPLC analysis of SFABR treated effluent showed the accumulation of 4-aminobenzenesulfonate (4-ABS) from AO6 and AO7. Aminoresorcinol (AR) formed from AO6 decolorization could not be detected at the end of SFABR cycle. This along with high COD removal indicated its further degradation. Formation of pink coloration on exposure to air indicated the presence of 1-amino-2-naphthol (AN) in AO7 fed reactor effluent. Thus both 4-ABS and AN were resistant to further degradation under anaerobic conditions. Presence of nitrate did not decrease the observed decolorization at the end of 24h SFABR cycle, although initial rate was decreased. This indicates the suitability of SFABR configuration for the treatment of azo-dye containing wastewaters in the presence of nitrate.     

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance,Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Background

Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress.

Methodology

The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.

Results

The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.

Conclusions

Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.     

Microbial secondary metabolites ameliorate growth, <Emphasis Type="Italic">in planta</Emphasis> contents and lignification in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

In the present investigation, metabolites of Streptomyces sp. MTN14 and Trichoderma harzianum ThU significantly enhanced biomass yield (3.58 and 3.48 fold respectively) in comparison to the control plants. The secondary metabolites treatments also showed significant augmentation (0.75–2.25 fold) in withanolide A, a plant secondary metabolite. Lignin deposition, total phenolic and flavonoid content in W. somnifera were maximally induced in treatment having T. harzianum metabolites. Also, Trichoderma and Streptomyces metabolites were found much better in invoking in planta contents and antioxidants compared with their live culture treatments. Therefore, identification of new molecular effectors from metabolites of efficient microbes may be used as biopesticide and biofertilizer for commercial production of W. somnifera globally.     

Reduction of nitric oxide level leads to spontaneous resumption of meiosis in diplotene-arrested rat oocytes cultured in?vitro

The present study was aimed to investigate whether a decrease of nitric oxide (NO) level is beneficial for sponateous resumptiom of meiosis in diplotene-arrested oocytes cultured in vitro. For this purpose, diplotene-arrested oocytes were collected from ovary of immature female rats after a single subcutaneous injection of 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48 h. In vitro effects of S-nitroso-l-acetyl penicillamine (SNAP; an NO donor) and aminoguanidine (AG; an inducible NOS [iNOS] inhibitor), intracellular NO, cyclic guanosine monophosphate (cGMP), Cdc25B, Thr-14/Tyr-15 and Thr-161 phosphorylated cyclin-dependent kinase-1 (CDK1), and cyclin B1 levels were analyzed. The SNAP inhibited spontaneous meiotic resumption form diplotene arrest in a concentration-dependent manner, while AG-induced meiotic resumption form diplotene in 0.1 mmol/L 3-isobutyl-1-methylxanthine (IBMX)-treated oocytes in a concentration-dependent manner. The intracellular NO as well as cGMP levels were decreased significantly during spontaneous meiotic resumption from diplotene arrest. The reduction of Cdc25B expression level was associated with the accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. However, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels were reduced significantly during meiotic resumption from diplotene arrest. Taken together, these data suggest that the inhibition of iNOS expression leads to a decrease of NO and cGMP levels thereby decreasing Cdc25B level. The reduced CDC25 B level leads to accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. As a result, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels are decreased leading to maturation-promoting factor (MPF) inactivation. The inactive MPF finally induced meiotic resumption from diplotene stage in rat oocytes cultured in vitro.     

Discovery of genomic regions and candidate genes controlling shelling percentage using QTL‐seq approach in cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) is an important grain legume providing high‐quality cooking oil, rich proteins and other nutrients. Shelling percentage (SP) is the 2nd most important agronomic trait after pod yield and this trait significantly affects the economic value of peanut in the market. Deployment of diagnostic markers through genomics‐assisted breeding (GAB) can accelerate the process of developing improved varieties with enhanced SP. In this context, we deployed the QTL‐seq approach to identify genomic regions and candidate genes controlling SP in a recombinant inbred line population (Yuanza 9102 × Xuzhou 68‐4). Four libraries (two parents and two extreme bulks) were constructed and sequenced, generating 456.89–790.32 million reads and achieving 91.85%–93.18% genome coverage and 14.04–21.37 mean read depth. Comprehensive analysis of two sets of data (Yuanza 9102/two bulks and Xuzhou 68‐4/two bulks) using the QTL‐seq pipeline resulted in discovery of two overlapped genomic regions (2.75 Mb on A09 and 1.1 Mb on B02). Nine candidate genes affected by 10 SNPs with non‐synonymous effects or in UTRs were identified in these regions for SP. Cost‐effective KASP (Kompetitive Allele‐Specific PCR) markers were developed for one SNP from A09 and three SNPs from B02 chromosome. Genotyping of the mapping population with these newly developed KASP markers confirmed the major control and stable expressions of these genomic regions across five environments. The identified candidate genomic regions and genes for SP further provide opportunity for gene cloning and deployment of diagnostic markers in molecular breeding for achieving high SP in improved varieties.     

Evolution of incompatibility systems in plants: Complementarity and the mating locus in flowering plants and fungi

Summary The restriction of sexual pairing by a specificity gene is considered to be an ancient development in the plant kingdom. The diversity and general parallelism of incompatibility systems seen amongst the phyla at the present time can be rationalized in terms of the association of various derived forms of the ancestral specificity unit with differing spectra of accessory factors controlling sexual physiology in the different phyla. Sexual morphogenesis has become divided into distinct phases under the control of complementary genes. These phases are initiated by a regulatory system of Co-ordinator genes which control the order in which groups of morphogenetic genes are expressed during development. The entire sexual cycle will be completed only if all the complementary groups are activated in the appropriate sequence. The present article discusses essential features of the evolution of the breeding locus in different phyla. These features are consistent in themselves with the present data and are not dependent on the proposed ancient origin of the specificity gene.The above hypothesis throws light on the (1) evolution of the complex mating loci in flowering plants and fungi; (2) evolution of complementary incompatibility and heteromorphic incompatibility in flowering plants; (3) anomalous cross-compatibility behaviour of mutants in the fungus Schizophyllum commune; (4) nature of homothallism in higher fungi; (5) mode of origin of new functional self-incompatibility alleles; and (6) homogenic and heterogenic incompatibility.