QTL sequencing strategy to map genomic regions associated with resistance to ascochyta blight in chickpea

Whole‐genome sequencing‐based bulked segregant analysis (BSA) for mapping quantitative trait loci (QTL) provides an efficient alternative approach to conventional QTL analysis as it significantly reduces the scale and cost of analysis with comparable power to QTL detection using full mapping population. We tested the application of next‐generation sequencing (NGS)‐based BSA approach for mapping QTLs for ascochyta blight resistance in chickpea using two recombinant inbred line populations CPR‐01 and CPR‐02. Eleven QTLs in CPR‐01 and six QTLs in CPR‐02 populations were mapped on chromosomes Ca1, Ca2, Ca4, Ca6 and Ca7. The QTLs identified in CPR‐01 using conventional biparental mapping approach were used to compare the efficiency of NGS‐based BSA in detecting QTLs for ascochyta blight resistance. The QTLs on chromosomes Ca1, Ca4, Ca6 and Ca7 overlapped with the QTLs previously detected in CPR‐01 using conventional QTL mapping method. The QTLs on chromosome Ca4 were detected in both populations and overlapped with the previously reported QTLs indicating conserved region for ascochyta blight resistance across different chickpea genotypes. Six candidate genes in the QTL regions identified using NGS‐based BSA on chromosomes Ca2 and Ca4 were validated for their association with ascochyta blight resistance in the CPR‐02 population. This study demonstrated the efficiency of NGS‐based BSA as a rapid and cost‐effective method to identify QTLs associated with ascochyta blight in chickpea.     

Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver

Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.     

Synthesis and evaluation of bidentate ligands designed to interact with PDZ domains

We designed bidentate ligands to target PDZ domains through two binding sites: site S0, delimited by the GLGF loop, and site S1, a zone situated around loop β(B)/β(C). A molecular docking study allowed us to design a generic S0 binder, to which was attached a variable size linker, itself linked to an amino acid aimed to interact with the S1 site of PDZ domains. A series of 15 novel bidentate ligands was prepared in 6-11 steps in good overall yield (24-43%). Some of these ligands showed an inhibitory activity against serotonin 5-HT2A receptor/PSD-95 interaction. This was assessed by pull-down assay using a synthetic decapeptide corresponding to the C-terminal residues of the receptor as a bait.     

Identification of PDZ ligands by docking-based virtual screening for the development of novel analgesic agents

Disrupting the interaction between the PDZ protein, PSD-95, and its target ligands (such as the glutamate NMDA receptor or the serotonin 5-HT_2A receptor) was found to reduce hyperalgesia in various models of neuropathic pain. Here, we set out to identify lead molecules which would interact with PSD-95, and hence, would potentially display analgesic activity. We describe the virtual screening of the Asinex and Cambridge databases which together contain almost one million molecules. Using three successive docking filters and visual inspection, we identified three structural classes of molecules and synthesized a potential lead compound from each class. The binding of the molecules with the PDZ domains of PSD-95 was assessed by ¹H–¹⁵N HSQC NMR experiments. The analgesic activity of the best ligand, quinoline 2, was evaluated in vivo in a model of neuropathic pain and showed promising results.     

Seroprevalence of Toxoplasma gondii in pigs, sheep, goats, and cattle from Grenada and Carriacou, West Indies

Prevalence of Toxoplasma gondii infection in pregnant women in Grenada is considered high. Little is known of the epidemiology of T. gondii infection in Caribbean Islands. Serum samples of 750 food animals in Grenada and Carriacou were tested for antibodies to T. gondii by the modified agglutination test (MAT). Antibodies to T. gondii (MAT, 1∶25 or higher) were found in 23.1% of 247 pigs, 44.1% of 204 sheep, 42.8% of 180 goats, and 8.4% of 119 cattle. Seroprevalence increased with age, indicating postnatal acquisition of T. gondii. Antibody titers of 1∶200 or higher were present in 65 of 90 seropositive sheep, 61 of 77 seropositive goats, and 23 of 57 seropositive pigs. However, none of the cattle had a MAT titer of 1∶200, suggesting that bovines are a poor host for T. gondii. Results indicate that pigs, sheep, and goats could be important sources of T. gondii infection if their meat is consumed undercooked.     

Confocal microscopic investigation of tubulin distribution and effect of paclitaxel on posttranslationally modified tubulins in sodium arsenite resistant Leishmania donovani

The affinity of arsenic towards the cytoskeleton leading to disturbance of tubulin polymerization is well known. Tubulin undergoes extensive posttranslational modifications which effect stability and dynamics of microtubules but little is known about the effect of antimicrotubule drugs on their distribution and function in kinetoplastid parasites such as Leishmania. The current study was undertaken to investigate the effect of continuous sodium arsenite exposure on the tubulin distribution profile in wild type and sodium arsenite resistant Leishmania donovani together with effect of paclitaxel, a tubulin-polymerizing agent, on that distribution using confocal microscopy. Immunofluorescence studies using specific monoclonal antibodies against alpha-tubulin and posttranslationally modified tubulins (acetylated and tyrosinated) have revealed distinct differences in the organization of microtubule arrays in wild type and sodium arsenite resistant L. donovani that is further affected by paclitaxel treatment. Microtubules are arranged in spiral arrays in wild type as compared to the longitudinal arrays in arsenite resistant L. donovani. The difference in microtubular structure organization may explain the parasite response to continuous drug pressure and illustrate the fundamental impact of arsenite on microtubules in arsenite resistant L. donovani.     

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.