Classification of leprosy into multibacillary and paucibacillary groups: an analysis

Classification of leprosy patients into multibacillary and paucibacillary determines the duration of their treatment. Misclassification leads to increased risk of relapse due to insufficient treatment if a multibacillary patient is classified as paucibacillary. This also prolongs the time the patient is infective. Over the years, the criteria used for classification (for treatment purpose) of leprosy patients have changed significantly from bacterial index measuring approach through number of skin lesions. The reliability of both of these criteria has been questioned. Several studies have shown that the presence of antibodies to the Mycobacterium leprae -specific antigens correlates with the bacterial load of a leprosy patient. Further, there are reports where results of serology and bacteriological approaches have been found to agree substantially. Thus, serology seems to be a worthwhile convenient alternative tool for classification of leprosy into multibacillary or paucibacillary. Nevertheless, in view of the limitations of various classification criteria, follow-up studies are called for to understand the efficiency of various approaches in preventing relapse after treatment. The method ensuring the lowest rate of relapse could be adopted for future use in classifying these patients.     

5′-Phosphorylation of Oligonucleotides with Phosphorous Acid in Automated DNA Synthesis

Abstract

Coupling of phosphorous acid in automated DNA synthesis using H-phosphonate methodology leads to 5′-5′ linked dimers and 5′-H-phosphonates. The yield is dependent on the phosphorous acid concentration, chain length of the oligomer, and pore size of the support. 5′-Phosphate oligomers are obtained from the H-phosphonate oligomers by silylation and oxidation.     

High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries

We have previously established a minimalist approach to antibody engineering by using a phage-displayed framework to support complementarity determining region (CDR) diversity restricted to a binary code of tyrosine and serine. Here, we systematically augmented the original binary library with additional levels of diversity and examined the effects. The diversity of the simplest library, in which only heavy chain CDR positions were randomized by the binary code, was expanded in a stepwise manner by adding diversity to the light chain, by diversifying non-paratope residues that may influence CDR conformations, and by adding additional chemical diversity to CDR-H3. The additional diversity incrementally improved the affinities of antibodies raised against human vascular endoethelial growth factor and the structure of an antibody-antigen complex showed that tyrosine side-chains are sufficient to mediate most of the interactions with antigen, but a glycine residue in CDR-H3 was critical for providing a conformation suitable for high-affinity binding. Using new high-throughput procedures and the most complex library, we produced multiple high-affinity antibodies with dissociation constants in the single-digit nanomolar range against a wide variety of protein antigens. Thus, this fully synthetic, minimalist library has essentially recapitulated the capacity of the natural immune system to generate high-affinity antibodies. Libraries of this type should be highly useful for proteomic applications, as they minimize inherent complexities of natural antibodies that have hindered the establishment of high-throughput procedures. Furthermore, analysis of a large number of antibodies derived from these well-defined and simplistic libraries allowed us to uncover statistically significant trends in CDR sequences, which provide valuable insights into antibody library design and into factors governing protein-protein interactions.     

Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and gamma-glutamylcysteine synthetase expression

We have developed a genetics-based phytoremediation strategy for arsenic in which the oxyanion arsenate is transported aboveground, reduced to arsenite, and sequestered in thiol-peptide complexes. The Escherichia coli arsC gene encodes arsenate reductase (ArsC), which catalyzes the glutathione (GSH)-coupled electrochemical reduction of arsenate to the more toxic arsenite. Arabidopsis thaliana plants transformed with the arsC gene expressed from a light-induced soybean rubisco promoter (SRS1p) strongly express ArsC protein in leaves, but not roots, and were consequently hypersensitive to arsenate. Arabidopsis plants expressing the E. coli gene encoding gamma-glutamylcysteine synthetase (gamma-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared with wild type. However, plants expressing SRS1p/ArsC and ACT2p/gamma-ECS together showed substantially greater arsenic tolerance than gamma-ECS or wild-type plants. When grown on arsenic, these plants accumulated 4- to 17-fold greater fresh shoot weight and accumulated 2- to 3-fold more arsenic per gram of tissue than wild type or plants expressing gamma-ECS or ArsC alone. This arsenic remediation strategy should be applicable to a wide variety of plant species.     

Single‐nucleotide polymorphism discovery in Leptographium longiclavatum,a mountain pine beetle‐associated symbiotic fungus,using whole‐genome resequencing

Single‐nucleotide polymorphisms (SNPs) are rapidly becoming the standard markers in population genomics studies; however, their use in nonmodel organisms is limited due to the lack of cost‐effective approaches to uncover genome‐wide variation, and the large number of individuals needed in the screening process to reduce ascertainment bias. To discover SNPs for population genomics studies in the fungal symbionts of the mountain pine beetle (MPB), we developed a road map to discover SNPs and to produce a genotyping platform. We undertook a whole‐genome sequencing approach of Leptographium longiclavatum in combination with available genomics resources of another MPB symbiont, Grosmannia clavigera. We sequenced 71 individuals pooled into four groups using the Illumina sequencing technology. We generated between 27 and 30 million reads of 75 bp that resulted in a total of 1, 181 contigs longer than 2 kb and an assembled genome size of 28.9 Mb (N₅₀ = 48 kb, average depth = 125x). A total of 9052 proteins were annotated, and between 9531 and 17 266 SNPs were identified in the four pools. A subset of 206 genes (containing 574 SNPs, 11% false positives) was used to develop a genotyping platform for this species. Using this roadmap, we developed a genotyping assay with a total of 147 SNPs located in 121 genes using the Illumina^® Sequenom iPLEX Gold. Our preliminary genotyping (success rate = 85%) of 304 individuals from 36 populations supports the utility of this approach for population genomics studies in other MPB fungal symbionts and other fungal nonmodel species.     

Starvation Resistance in a Stenothermal Species from the Indian Subcontinent: Mechanistic Basis of Clinal Variation

Parallel clines for starvation resistance and lipid content are well documented among drosophilids on the Indian subcontinent. However, the mechanistic basis of these clines has not been investigated so far. Here, we investigate the utilization of lipids during starvation as a function of duration of stress in D. ananassae. We found higher lipid content responsible for high starvation resistance at lower latitudes. Lipids were utilized during starvation only; not during any other climatic stresses like desiccation or thermal stresses. We also found a cline for consumption of total body lipids; as more content (out of total amount of lipids) was utilized by flies at lower latitudes and lesser at higher latitudes. But, there was no latitudinal cline for threshold lipid amount in the case of females while for males there was a positive cline. Lastly, parallel clines have evolved under contrasting climatic conditions i.e. drier and colder northern localities have flies with lower lipid and reduced starvation resistance while hot and humid localities favor flies with higher lipid levels and greater starvation tolerance. Thus, the evolution of clines associated with starvation and lipid content might have resulted due to specific ecological conditions i.e. humidity gradient on the Indian subcontinent.     

Adaptations to environmental stress in altitudinal populations of two Drosophila species

Abstract.  Opposite clinal variation for desiccation and starvation tolerance are observed in four altitudinal populations (219–2202 m), each of two sympatric and cold adapted species: Drosophila takahashii and Drosophila nepalensis from northern India. The high-altitude populations are more tolerant to desiccation than those from lower altitudes, whereas the reverse trend occurs for starvation tolerance. The magnitude of tolerances are significantly high in D. nepalensis, which is better adapted to cold conditions. During winter months (November to February), there are significant decreases in T _max, T _min and relative humidity along the altitudinal transect. Higher desiccation resistance can develop under cold conditions over short-range, altitudinally varying, geographical areas (250 km) compared with our previously reported long-range (>2000 km), latitudinal variations under tropical climatic conditions. However, significant starvation tolerances are favoured by small body size, higher dispersal rate and higher ambient temperature of the site of origin of populations. Significant correlations of two climatic factors (the mean monthly coefficients of variation of temperature and relative humidity) with these two physiological traits can best explain the observed altitudinal clinal variations under natural conditions.     

The landscape of transposable elements in the finished genome of the fungal wheat pathogen Mycosphaerella graminicola

Background

In addition to gene identification and annotation, repetitive sequence analysis has become an integral part of genome sequencing projects. Identification of repeats is important not only because it improves gene prediction, but also because of the role that repetitive sequences play in determining the structure and evolution of genes and genomes. Several methods using different repeat-finding strategies are available for whole-genome repeat sequence analysis. Four independent approaches were used to identify and characterize the repetitive fraction of the Mycosphaerella graminicola (synonym Zymoseptoria tritici) genome. This ascomycete fungus is a wheat pathogen and its finished genome comprises 21 chromosomes, eight of which can be lost with no obvious effects on fitness so are dispensable.

Results

Using a combination of four repeat-finding methods, at least 17% of the M. graminicola genome was estimated to be repetitive. Class I transposable elements, that amplify via an RNA intermediate, account for about 70% of the total repetitive content in the M. graminicola genome. The dispensable chromosomes had a higher percentage of repetitive elements as compared to the core chromosomes. Distribution of repeats across the chromosomes also varied, with at least six chromosomes showing a non-random distribution of repetitive elements. Repeat families showed transition mutations and a CpA → TpA dinucleotide bias, indicating the presence of a repeat-induced point mutation (RIP)-like mechanism in M. graminicola. One gene family and two repeat families specific to subtelomeres also were identified in the M. graminicola genome. A total of 78 putative clusters of nested elements was found in the M. graminicola genome. Several genes with putative roles in pathogenicity were found associated with these nested repeat clusters. This analysis of the transposable element content in the finished M. graminicola genome resulted in a thorough and highly curated database of repetitive sequences.

Conclusions

This comprehensive analysis will serve as a scaffold to address additional biological questions regarding the origin and fate of transposable elements in fungi. Future analyses of the distribution of repetitive sequences in M. graminicola also will be able to provide insights into the association of repeats with genes and their potential role in gene and genome evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1132) contains supplementary material, which is available to authorized users.     

Divergence for tolerance to thermal-stress related traits in two Drosophila species of immigrans group

We investigated associations of the species specific divergence of thermal adaptations of Drosophila nasuta and Drosophila immigrans with seasonal as well as geographical changes in the relative abundance. In D. immigrans, cold resistance and egg-to-adult viability after a cold-shock have shown a positive cline with latitude, whereas no such clinal patterns were evident for heat resistance as well as egg-to-adult viability after heat stress. In contrast, basal heat tolerance and egg-to-adult viability after a heat-shock increased towards the equator i.e. from north to south in D. nasuta. Further, we also found species-specific divergence in seasonal climatic selection responses for heat as well as cold resistance i.e. warmer and higher humidity conditions of the rainy season have evidenced ∼2-fold higher heat tolerance of D. nasuta, but lower temperature as well as lower humidity conditions of the autumn significantly increased (∼3-fold) cold tolerance of D. immigrans. Geographical variations were significant for the absolute and relative hardening capacity for cold tolerance in D. immigrans, but for heat resistance of D. nasuta. Results of multiple regression analysis of changes in thermal tolerance level in latitudinal populations as a simultaneous function of climatic variables (T_ave and RH) have shown that average yearly relative humidity levels significantly affect heat tolerance of D. nasuta, but both T_ave and RH contribute to changes in the cold tolerance of D. immigrans. Thus, our results suggest that climatic selection responses for basal and hardened thermo-tolerance levels affect relative abundance of D. nasuta and D. immigrans across a latitudinal transect as well as seasons.