Structure and Genetic Diversity of Natural Populations of Morus alba in the Trans-Himalayan Ladakh Region

Sequence-related amplified polymorphism markers were used to assess the genetic structure in three natural populations of Morus alba from trans-Himalaya. Multilocation sampling was conducted across 14 collection sites. The overall genetic diversity estimates were high: percentage polymorphic loci 89.66%, Nei’s gene diversity 0.2286, and Shannon’s information index 0.2175. At a regional level, partitioning of variability assessed using analysis of molecular variance (AMOVA), revealed 80% variation within and 20% among collection sites. Pattern appeared in STRUCTURE, BARRIER, and AMOVA, clearly demonstrating gene flow between the Indus and Suru populations and a geographic barrier between the Indus-Suru and Nubra populations, which effectively hinders gene flow. The results showed significant genetic differentiation, population structure, high to restricted gene flow, and high genetic diversity. The assumption that samples collected from the three valleys represent three different populations does not hold true. The fragmentation present in trans-Himalaya was more natural and less anthropogenic.     

Anthocyanin production in a callus line of <Emphasis Type="Italic">Panax sikkimensis</Emphasis> Ban

A root-derived callus line of Panax sikkimensis that stably accumulates anthocyanins was established by small cell aggregate selection method. The selected line showed a growth index of 221.36 and an anthocyanin content of 2.76 mg/g fw (7.076% dw) in 50–60 d of growth on a modified MS medium containing 4.5 μM 2,4-dichlorophenoxy acetic acid and 1.2 μM kinetin under 16-h light and 8-h dark photoperiodic conditions. Incubation under continuous light increased the growth index to 435.57 but led to a marginal dilution of anthocyanin content to 2.192 mg/g fw (6.928% dw). The purple-red pigment had absorption maximum at 528 nm. The selected callus line has shown sustained growth and productivity for more than 6 yr now. Interestingly, pigment accumulation in the selected line did not hinder the ginsenoside production in the callus tissue (0.9–1.2% fw).     

Biodegradation of pyridine raffinate by two bacterial co-cultures of Bacillus cereus (DQ435020) and Alcaligenes faecalis (DQ435021)

This study deals with the optimization of bacterial degradation of pyridine raffinate by previously isolated two aerobic bacteria ITRCEM1 (Bacillus cereus) and ITRCEM2 (Alcaligens faecalis) with accession number DQ4335020 and DQ435021, respectively. The degradation of pyridine raffinate was studied by axenic and mixed bacterial consortium at different nutritional and environmental conditions after the removal of formaldehyde from pyridine raffinate (FPPR). Results revealed that the optimum degradation of pyridine raffinate was observed by mixed bacterial culture in presence of glucose (1% w/v) and peptone (0.2% w/v) at 20% FPPR, pH 7.0, temperature 30°C and 120 rpm at 168 h incubation period . The HPLC analysis of degraded pyridine raffinate samples has indicated the complete removal of α, β and γ picoline. Further, the GC–MS analysis of FPPR pyridine raffinate has shown the presence of pyrazine acetonitrile (6.74), 1,3-dioxepin (8.68), 2-pyridine carboxaldehyde (11.26), propiolactone (12.06), 2-butanol (13.10), benzenesulfonic acid (16.22) and 1,4-dimethyl pyperadine while phenol (17.64) and 3,4-dimethyl benzaldehyde as metabolic products of FPPR.     

Pathogen specific, IRF3-dependent signaling and innate resistance to human kidney infection

The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3(-/-) mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNβ-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3(-/-) mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

Chromatographic separation and sensitive determination of teriflunomide,an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive, selective and high throughput liquid chromatography tandem mass spectrometry (LC–ESI-MS/MS) method has been developed for the determination of teriflunomide, an active metabolite of leflunomide in human plasma. Plasma samples were prepared by liquid–liquid extraction of teriflunomide and valsartan as internal standard (IS) in ethyl acetate from 200 μL human plasma. The chromatographic separation was achieved on an Inertsil ODS-3 C18 (50 mm × 4.6 mm, 3 μm) analytical column using isocratic mobile phase, consisting of 20 mM ammonium acetate–methanol (25:75, v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for teriflunomide (m/z 269.0 → 82.0) and IS (m/z 434.1 → 350.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over a wide dynamic concentration range of 10.1–4001 ng/mL. Matrix effect was assessed by post-column infusion experiment and the mean process efficiency were 91.7% and 88.2% for teriflunomide and IS respectively. The method was rugged and rapid with a total run time of 2.0 min and is applied to a bioequivalence study of 20 mg leflunomide (test and reference) tablet formulation in 12 healthy Indian male subjects under fasting condition.     

Genetic diversity of <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> isolates from certain agro-climatic regions of India by using RAPD markers

Genetic diversity analysis of Macrophomina phaseolina isolates obtained from different host range and diverse geographical locations in India was carried out using RAPD fingerprinting. Of the thirteen 10-mer random primers used, primer OPB-08 gave the maximum polymorphism and the UPGMA clustering could separate 50 isolates in to ten groups at more than 65% similarity level. The ten clusters correlated well with the geographical locations with exceptions for isolates obtained from Eastern and Western Ghats. There was a segregation of isolates from these two geographical locations in to two clusters thus, distributing 10 genotypes in to eight geographical locations. All the isolates M. phaseolina irrespective of their host and geographical origin, exhibited two representative monomorphic bands at 250 bp and 1 kb, presence of these bands suggests that isolates might have evolved from a common ancestor but due to geographical isolation fallowed by natural selection and genetic drift might have segregated in to subpopulations. Genetic similarity in the pathogenic population reflects the dispersal of single lineage in all locations in India.