The first draft of the pigeonpea genome sequence

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety ??Asha??. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550?bp and >10-fold genome coverage, resulting in 510,809,477?bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable ??Arhar?? simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.     

An approach to delineate primers for a group of poorly conserved sequences incorporating the common motif region

Glutathione synthetase (gshB) has previously been reported to confer tolerance to acidic soil condition in Rhizobium species. Cloning the gene coding for this enzyme necessitates the designing of proper primer sets which in turn depends on the identification of high quality sequence similarity in multiple global alignments. In this experiment, a group of homologous gene sequences related to gshB gene (accession no: gi-86355669:327589-328536) of Rhizobium etli CFN 42, were extracted from NCBI nucleotide sequence databases using BLASTN and were analyzed for designing degenerate primers. However, the T-coffee multiple global alignment results did not show any block of conserved region for the above sequence set to design the primers. Therefore, we attempted to identify the location of common motif region based on multiple local alignments employing the MEME algorithm supported with MAST and Primer3. The results revealed some common motif regions that enabled us to design the primer sets for related gshB gene sequences. The result will be validated in wet lab.     

Potentiation of antinociceptive effect of NSAIDs by a specific lipooxygenase inhibitor, acetyl 11-keto-beta boswellic acid

The present study was aimed to assess the combined effects of cyclooxygenase and 5-lipoxygenase (COX/5-LOX) inhibitors in different animal models of nociception. Naproxen, nimesulide and rofecoxib are well-established antinociceptive agents acting via COX inhibition. AKBA (acetyl-keto-beta-boswellic acid) is a 5-LOX inhibitor. AKBA (50-200 mg/kg) produced a dose dependent and significant antinociceptive effect in different animal models of nociception. Based on the earlier reports from our laboratory, sub effective doses of all the three COX Inhibitors were selected and they were administered (naproxen, 5 mg/kg; nimesulide, 1 mg/kg; and rofecoxib, 1 mg/kg) with AKBA (100 mg/kg). This produced a more significant antinociceptive effect as compared to per se effect observed in all the three models of nociception. However, the effect of combination of nimesulide with AKBA was more pronounced as compared to naproxen and rofecoxib and their combination with AKBA. The present finding provided an evidence for the potentiation of antinociceptive effect of NSAIDs with AKBA. Such a combination may help to reduce the therapeutic doses of conventional NSAIDs and also reduce side effects (gastric, cardiac and renal) that are popularly associated with the NSAIDs.     

Osteogenic activity of constituents from Butea monosperma

Phytochemical investigation from the stem bark of Butea monosperma, led to the isolation and identification of three new compounds named buteaspermin A (1), buteaspermin B (2) and buteaspermanol (3), along with 19 known compounds. The structure of compounds 1–22 were established on the basis of their spectroscopic data. The isolated compounds 2–17 were evaluated using neonatal (1–3 day old) rat calvaria derived primary osteoblast cultures. Five of these compounds 7, 10–13 showed promising osteogenic activity, attributed to increased osteoblast proliferation, differentiation and mineralization as evidenced by marked increase in expression of alkaline phosphatase, an early phase differentiation marker, and alizarin Red S staining of osteoblasts cultured for 48 h and von Kossa silver staining of nodules formed 15 days after culture with these compounds. Quantification of mineralization by optical density measurement of Alizarin Red S extracted from stained osteoblasts cultured for 7 days in presence of these compounds showed significant (P < 0.05, vs corresponding vehicle control group) increase in mineralization. On the basis of biological results, structure–activity relationships are discussed.     

Silver nanoparticles as a new generation of antimicrobials

Silver has been in use since time immemorial in the form of metallic silver, silver nitrate, silver sulfadiazine for the treatment of burns, wounds and several bacterial infections. But due to the emergence of several antibiotics the use of these silver compounds has been declined remarkably. Nanotechnology is gaining tremendous impetus in the present century due to its capability of modulating metals into their nanosize, which drastically changes the chemical, physical and optical properties of metals. Metallic silver in the form of silver nanoparticles has made a remarkable comeback as a potential antimicrobial agent. The use of silver nanoparticles is also important, as several pathogenic bacteria have developed resistance against various antibiotics. Hence, silver nanoparticles have emerged up with diverse medical applications ranging from silver based dressings, silver coated medicinal devices, such as nanogels, nanolotions, etc.     

The Metalloregulatory Zinc Site in Streptococcus pneumoniae AdcR, a Zinc-activated MarR Family Repressor

Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain-heart infusion) under microaerobic conditions revealed upregulation of 13 genes, including adcR and adcCBA, encoding a high-affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and an approximately twofold increase in cell-associated Zn(II). Apo- and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K_DNA-Zn = 2.4 × 10⁸ M^- 1 (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with stepwise Zn(II) affinities K_Zn1 and K_Zn2 of ≥ 10⁹ M^- 1 at pH 6.0 and ≥ 10¹² M^- 1 at pH 8.0, and one to three lower affinity Zn(II) depending on the pH. X-ray absorption spectroscopy of the high-affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA-binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high-affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild type apo-AdcR, but fail to conformationally switch upon Zn(II) binding. These studies implicate His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.     

Mycogenic metal nanoparticles: progress and applications

Nanotechnology is relevant to diverse fields of science and technology. Due to the many advantages over non-biological systems, several research groups have exploited the use of biological systems for the synthesis of nanoparticles. Among the different microbes used for the synthesis of nanoparticles, fungi are efficient candidates for fabrication of metal nanoparticles both intra- and extracellulary. The nanoparticles synthesized using fungi present good polydispersity, dimensions and stability. The potential applications of nanotechnology and nanoparticles in different fields have revolutionized the health care, textile and agricultural industries and they are reviewed here.     

Metabolic pathway reconstruction of eugenol to vanillin bioconversion in Aspergillus niger

Identification of missing genes or proteins participating in the metabolic pathways as enzymes are of great interest. One such class of pathway is involved in the eugenol to vanillin bioconversion. Our goal is to develop an integral approach for identifying the topology of a reference or known pathway in other organism. We successfully identify the missing enzymes and then reconstruct the vanillin biosynthetic pathway in Aspergillus niger. The procedure combines enzyme sequence similarity searched through BLAST homology search and orthologs detection through COG & KEGG databases. Conservation of protein domains and motifs was searched through CDD, PFAM & PROSITE databases. Predictions regarding how proteins act in pathway were validated experimentally and also compared with reported data. The bioconversion of vanillin was screened on UV-TLC plates and later confirmed through GC and GC-MS techniques. We applied a procedure for identifying missing enzymes on the basis of conserved functional motifs and later reconstruct the metabolic pathway in target organism. Using the vanillin biosynthetic pathway of Pseudomonas fluorescens as a case study, we indicate how this approach can be used to reconstruct the reference pathway in A. niger and later results were experimentally validated through chromatography and spectroscopy techniques.     

Origins of delays in monolayer kinetics: phospholipase A2 paradigm

The interfacial kinetic paradigm is adopted to model the kinetic behavior of pig pancreatic phospholipase A(2) (PLA2) at the monolayer interface. A short delay of about a minute to the onset of the steady state is observed under all monolayer reaction progress conditions, including the PLA2-catalyzed hydrolysis of didecanoylphosphatidyl-choline (PC10) and -glycerol (PG10) monolayers as analyzed in this paper. This delay is independent of enzyme concentration and surface pressure and is attributed to the equilibration time by stationary diffusion of the enzyme added to the stirred subphase to the monolayer through the intervening unstirred aqueous layer. The longer delays of up to several hours, seen with the PC10 monolayers at >15 mN/m, are influenced by surface pressure as well as enzyme concentration. Virtually all features of the monolayer reaction progress are consistent with the assumption that the product accumulates in the substrate monolayer, although the products alone do not spread as a compressible monolayer. These results rule out models that invoke slow "activation" of PLA2 on the monolayer. The observed steady-state rate on monolayers after the delays is <1% of the rate observed with micellar or vesicles substrates of comparable substrate. Together these results suggest that the monolayer steady-state rate includes contributions from steps other than those of the interfacial turnover cycle. Additional considerations that provide understanding of the pre-steady-state behaviors and other nonideal effects at the surface are also discussed.