Diversity analysis of dairy and non-dairy strains of Lactococcus lactis ssp. lactis by multilocus sequence analysis (MLSA)

The genetic diversity of 31 identified strains of Lactococcus lactis ssp. lactis isolated from different dairy and non-dairy sources were investigated at gene level using multilocus sequence analysis (MLSA) and PCR-RFLP based on the differences in four selected partial protein coding gene sequences: araT, encoding aromatic amino acid-specific aminotransferase; dtpT, encoding di/tri peptide transporter; yueF, encoding non-proteolytic protein, peptidase, M16 family; and pdhA, encoding pyruvate dehydrogenase E1 component α-subunit. A set of seven test strains from different isolation sources and one reference strain, L. lactis ssp. lactis NCDC 094, were analyzed by MLSA. The strains showed distinct diversity among themselves and exhibited a greater percent similarity with reference strains L. lactis ssp. lactis CV56 (CP002365.1), IL1403 (AE005176.1), and KF147 (CP001834.1) in comparison with L. lactis ssp. cremoris NZ9000 (CP002094.1), MG1363 (AM406671.1), and SK11 (CP00425.1). The MLSA revealed one distinct genomic lineage within strains exclusively of L. lactis ssp. lactis. This analysis also revealed no source-wise genetic relationship in the test strains analyzed. Further, PCR-RFLP of araT, dtpT, yueF and pdhA also characterized the single genomic lineage exclusively of L. lactis ssp. lactis within a total of 24 test strains.     

Evidence of salicylic acid pathway with EDS1 and PAD4 proteins by molecular dynamics simulation for grape improvement

Biotic stress is a major cause of heavy loss in grape productivity. In order to develop biotic stress-resistant grape varieties, the key defense genes along with its pathway have to be deciphered. In angiosperm plants, lipase-like protein phytoalexin deficient 4 (PAD4) is well known to be essential for systemic resistance against biotic stress. PAD4 functions together with its interacting partner protein enhanced disease susceptibility 1 (EDS1) to promote salicylic acid (SA)-dependent and SA-independent defense pathway. Existence and structure of key protein of systemic resistance EDS1 and PAD4 are not known in grapes. Before SA pathway studies are taken in grape, molecular evidence of EDS1: PAD4 complex is to be established. To establish this, EDS1 protein sequence was retrieved from NCBI and homologous PAD4 protein was generated using Arabidopsis thaliana as template and conserved domains were confirmed. In this study, computational methods were used to model EDS1 and PAD4 and simulated the interactions of EDS1 and PAD4. Since no structural details of the proteins were available, homology modeling was employed to construct three-dimensional structures. Further, molecular dynamic simulations were performed to study the dynamic behavior of the EDS1 and PAD4. The modeled proteins were validated and subjected to molecular docking analysis. Molecular evidence of stable complex of EDS1:PAD4 in grape supporting SA defense pathway in response to biotic stress is reported in this study. If SA defense pathway genes are explored, then markers of genes involved can play pivotal role in grape variety development especially against biotic stress leading to higher productivity.     

Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b

The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, A. (2002) J. Biol. Chem. 277, 6822-6829). Asp(1080) had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp(1080) to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp(1080) mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp(1077) had no effect on basal activity or calmodulin activation. We propose that the conserved Asp(1080), even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.     

Comparison of three rat models of cerebral vasospasm

A substantial number of rat models have been used to research subarachnoid hemorrhage-induced cerebral vasospasm; however, controversy exists regarding which method of selection is appropriate for this species. This study was designed to provide extensive information about the three most popular subarachnoid hemorrhage rat models: the endovascular puncture model, the single-hemorrhage model, and the double-hemorrhage model. In this study, the basilar artery and posterior communicating artery were chosen for histopathological examination and morphometric analysis. Both the endovascular puncture model and single-hemorrhage model developed significant degrees of vasospasm, which were less severe when compared with the double-hemorrhage model. The endovascular puncture model and double-hemorrhage model both developed more vasospasms in the posterior communicating artery than in the basilar artery. The endovascular puncture model has a markedly high mortality rate and high variability in bleeding volume. Overall, the present study showed that the double-hemorrhage model in rats is a more suitable tool with which to investigate mechanism and therapeutic approaches because it accurately correlates with the time courses for vasospasm in humans.     

Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions,local adaptation and geographic structure

Recent advances in sequencing allow population‐genomic data to be generated for virtually any species. However, approaches to analyse such data lag behind the ability to generate it, particularly in nonmodel species. Linkage disequilibrium (LD, the nonrandom association of alleles from different loci) is a highly sensitive indicator of many evolutionary phenomena including chromosomal inversions, local adaptation and geographical structure. Here, we present linkage disequilibrium network analysis (LDna), which accesses information on LD shared between multiple loci genomewide. In LD networks, vertices represent loci, and connections between vertices represent the LD between them. We analysed such networks in two test cases: a new restriction‐site‐associated DNA sequence (RAD‐seq) data set for Anopheles baimaii, a Southeast Asian malaria vector; and a well‐characterized single nucleotide polymorphism (SNP) data set from 21 three‐spined stickleback individuals. In each case, we readily identified five distinct LD network clusters (single‐outlier clusters, SOCs), each comprising many loci connected by high LD. In A. baimaii, further population‐genetic analyses supported the inference that each SOC corresponds to a large inversion, consistent with previous cytological studies. For sticklebacks, we inferred that each SOC was associated with a distinct evolutionary phenomenon: two chromosomal inversions, local adaptation, population‐demographic history and geographic structure. LDna is thus a useful exploratory tool, able to give a global overview of LD associated with diverse evolutionary phenomena and identify loci potentially involved. LDna does not require a linkage map or reference genome, so it is applicable to any population‐genomic data set, making it especially valuable for nonmodel species.     

Indole-3-piperazinyl derivatives: novel chemical class of 5-HT(6) receptor antagonists

N₁-Arylsulfonyl-3-piperazinyl indole derivatives were designed and identified as a novel class of 5-HT₆ receptors ligands. All the compounds have high affinity and antagonist activity towards 5-HT₆ receptor. The compound 7a (K_i = 3.4 nM, functional assay IC₅₀ = 310 nM) shows enhanced cognitive effect when tested in NORT and Morris water maze models. Synthesis, SAR and PK profile of these novel compounds constitute the subject matter of this Letter.     

MAP Kinase analyser: A tool for plant kinase and substrate analysis

MAPK (Mitogen Activated Protein Kinase) is a Ser/Thr kinase, which plays a crucial role in plant growth and development, transferring the extra cellular stimuli into intracellular response etc. Manual identification of these MAPK in the plant genome is tedious and time taking process. There are number of online servers which predict the P-site (phosphorylation site), find the motifs and domain but there is no specific tool which can identify all them together. In order to identify the P-Site, phosphorylation site consensus sequences and domain of the MAPK in plant genome, we developed a tool, MAP Kinase analyzer. MAP kinase analyzer take protein sequence as input in the fasta format and the output of tool includes: 1) The prediction of the phosphorylation site viz., Serine (S), Threonine (T), and Tyrosine (Y), Contex, Position, Score and phosphorylating kinase as well as the graphical output; 2) Phosphorylation site consensus sequence pattern for different kinases and 3) Domain information about the MAPK's. The MAP kinase analyser tool and supplementary files can be downloaded from http://www.bioinfogbpuat/mapk_OWN_1/.     

Influence of Nutrient Availability and Quorum Sensing on the Formation of Metabolically Inactive Microcolonies Within Structurally Heterogeneous Bacterial Biofilms: An Individual-Based 3D Cellular Automata Model

The resistance of bacterial biofilms to antibiotic treatment has been attributed to the emergence of structurally heterogeneous microenvironments containing metabolically inactive cell populations. In this study, we use a three-dimensional individual-based cellular automata model to investigate the influence of nutrient availability and quorum sensing on microbial heterogeneity in growing biofilms. Mature biofilms exhibited at least three structurally distinct strata: a high-volume, homogeneous region sandwiched between two compact sections of high heterogeneity. Cell death occurred preferentially in layers in close proximity to the substratum, resulting in increased heterogeneity in this section of the biofilm; the thickness and heterogeneity of this lowermost layer increased with time, ultimately leading to sloughing. The model predicted the formation of metabolically dormant cellular microniches embedded within faster-growing cell clusters. Biofilms utilizing quorum sensing were more heterogeneous compared to their non-quorum sensing counterparts, and resisted sloughing, featuring a cell-devoid layer of EPS atop the substratum upon which the remainder of the biofilm developed. Overall, our study provides a computational framework to analyze metabolic diversity and heterogeneity of biofilm-associated microorganisms and may pave the way toward gaining further insights into the biophysical mechanisms of antibiotic resistance.     

GD3 recruits reactive oxygen species to induce cell proliferation and apoptosis in human aortic smooth muscle cells

Sialic acid containing glycosphingolipids (gangliosides) are expressed on the surface of all mammalian cells and have been implicated in regulating various biological phenomena; however, the detailed signaling mechanisms involved in this process are not known. We report here a novel aspect of disialoganglioside, GD3-mediated regulation of cell proliferation and cell death via the recruitment of reactive oxygen species (ROS). A low concentration (2.5-10 microm) of GD3, incubated with human aortic smooth muscle cells for a short period of time (10-30 min), stimulates superoxide generation via the activation of both NADPH oxidase and NADH oxidase activity. This leads to downstream signaling leading to cell proliferation and apoptosis. However, [(3)H]GD3 incubated with the cells under such conditions was found in a trypsin-sensitive fraction that was separable from endogenous GD3. The exact mechanism causing ROS generation and downstream signaling remains to be elucidated. The uptake of GD3 was accompanied by a 2.5-fold stimulation in the activity of mitogen-activated protein (MAP) kinase and 5-fold stimulation in cell proliferation. Preincubation of cells with membrane-permeable antioxidants, pyrrolidine dithiocarbamate, and N-acetylcysteine abrogated the superoxide generation and cell proliferation. In contrast, at higher concentrations (50-200 microm) GD3 inhibited the generation of superoxides but markedly stimulated the generation of nitric oxide (NO) (10-fold compared with control). This in turn stimulated mitochondrial cytochrome c release and intrachromosomal DNA fragmentation, which lead to apoptosis. In sum, at a low concentration, GD3 recruits superoxides to activate p44 MAPK and stimulates cell proliferation. In contrast, at high concentrations GD3 recruits nitric oxide to scavenge superoxide radicals that triggered signaling events that led to apoptosis. These observations might have relevance in regard to the potential role of GD3 in aortic smooth muscle cell proliferation and apoptosis that may contribute to plaque rupture in atherosclerosis.