The structural basis of mode of activation and functional diversity: a case study with HtrA family of serine proteases

HtrA (High temperature requirement protease A) proteins that are primarily involved in protein quality control belong to a family of serine proteases conserved from bacteria to humans. HtrAs are oligomeric proteins that share a common trimeric pyramidal architecture where each monomer comprises a serine protease domain and one or two PDZ domains. Although the overall structural integrity is well maintained and they exhibit similar mechanism of activation, subtle conformational changes and structural plasticity especially in the flexible loop regions and domain interfaces lead to differences in their active site conformation and hence in their specificity and functions.     

Response of Barley Genotypes to Fusarium Head Blight under Natural Infection and Artificial Inoculation Conditions

Forty-eight spring barley genotypes were evaluated for deoxynivalenol (DON) concentration under natural infection across 5 years at Harrington, Prince Edward Island. These genotypes were also evaluated for Fusarium head blight (FHB) severity and DON concentration under field nurseries with artificial inoculation of Fusarium graminearum by the grain spawn method across 2 years at Ottawa, Ontario, and one year at Hangzhou, China. Additionally, these genotypes were also evaluated for FHB severity under greenhouse conditions with artificial inoculation of F. graminearum by conidial suspension spray method across 3 years at Ottawa, Ontario. The objective of the study was to investigate if reactions of barley genotypes to artificial FHB inoculation correlate with reactions to natural FHB infection. DON concentration under natural infection was positively correlated with DON concentration (r = 0.47, P < 0.01) and FHB incidence (r = 0.56, P < 0.01) in the artificially inoculated nursery with grain spawn method. Therefore, the grain spawn method can be used to effectively screen for low DON. FHB severity, generated from greenhouse spray, however, was not correlated with DON concentration (r = 0.12, P > 0.05) under natural infection and it was not correlated with DON concentration (r = −0.23, P > 0.05) and FHB incidence (r = 0.19, P > 0.05) in the artificially inoculated nursery with grain spawn method. FHB severity, DON concentration, and yield were affected by year, genotype, and the genotype × year interaction. The effectiveness of greenhouse spray inoculation for indirect selection for low DON concentration requires further studies. Nine of the 48 genotypes were found to contain low DON under natural infection. Island barley had low DON and also had high yield.     

Distinct Roles for Rho Versus Rac/Cdc42 GTPases Downstream of Vav2 in Regulating Mammary Epithelial Acinar Architecture

Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.     

A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity

Low reaction yields and the high cost of obtaining a single type of pure CD make γ-CD costly. Using rational design and with the aid of 3D modeling structures, recombinant CGTase from Bacillus sp. G1 was molecularly engineered with the aim of producing a higher percentage of γ-CD. A single mutation at subsite −3, denoted H43T, was found to increase γ-CD production from 10% to approximately 39% using tapioca starch. This novel increment was probably the result of reduced steric hindrance to the formation of γ-CD because of the shortened side chain together with the shortened loop at positions 86–89, at substrate-binding subsite −3. A mutation (Tyr188 → Trp) and a deletion at loop 139–144 showed little effect on product specificity; however, mutagenesis at these sites affected cyclization, coupling and hydrolysis activities as well as the kinetic properties of the mutant CGTase. Based on rational design, three further mutations of the mutant H43T (denoted H43T/Δ(139–144)/S134T/A137V/L138D/V139I, H43T/S85G and H43T/Y87F) were constructed and produced γ-CD with yields of 20%, 20% and 39%, respectively. The mutant H43T/Δ(139–144)/S134T/A137V/L138D/V139I had very low cyclization and coupling activities, however their hydrolysis activity was retained. Double mutation (H43T/S85G) caused the enzyme to exhibit higher starch hydrolysis activity, approximately 26 times higher than the native CGTase G1. Although the mutants H43T and H43T/Y87F could produce the same percentage (39%) of γ-CD, the latter was more efficient as the total amount of CD produced was higher based on the V_max and k_cat values.     

Phylogeny and infrageneric classification of Correa Andrews (Rutaceae) on the basis of nuclear and chloroplast DNA

This paper presents phylogenies of the small but ecologically and horticulturally important Australian genus Correa (Rutaceae). Consensus phylogenies generated using parsimony were congruent with their counterparts generated by Bayesian analysis, although usually less well resolved. The phylogeny generated from the second internal transcribed spacer region of the nuclear ribosomal DNA supported the monophyly of Correa and identified two well supported clades (one comprising C. lawrenceana and C. baeuerlenii and the other containing all other species of the genus). Phylogenetic reconstructions based on the combined trnL-trnF spacer and the trnK intron (including the matK gene) regions of chloroplast DNA also supported the monophyly of Correa and of the C. lawrenceana/C. baeuerlenii clade, but the topology among the other species differed markedly from that in the ITS-based phylogeny. The major clades identified in the chloroplast phylogenies seemed to follow geographic patterns rather than species boundaries, with different samples of C. glabra bearing chloroplast genotypes from different clades. These patterns are likely to be because of independent evolution of the chloroplast and nuclear genomes, and are typical of cases of introgressive hybridisation among species or incomplete lineage sorting of chloroplast genomes leading to incongruence between chloroplast and nuclear phylogenies. Thus, the phylogenies based on nuclear DNA should reflect species relations better than the chloroplast phylogeny in Correa, and we propose a new subgeneric classification of the genus on the basis of the ITS-based phylogeny and morphology. Correa subgenus Persistens Othman, Duretto and G.J. Jord., containing C. lawrenceana and C. baeuerlenii, is formally described.     

Screening of marine actinobacteria for amylase enzymes inhibitors

Amylase inhibitor producing actinobacteria were isolated and characterized from terrestrial environment and there is no much report found from marine environment, hence in the present study, 17 strains isolated from the rhizosphere sediments of mangroves were tested for their amylase inhibition ability. Seawater requirement test for the growth of actinobacteria found that the strains SSR-3, SSR-12 and SSR-16 requires at least 50% and SSR-6 requires at least 25% seawater for their growth. The inhibition activity of both prokaryotic and eukaryotic amylase was tested by using Bacillus subtilis and Aspergillus niger. The maximum amylase activity (40mm) produced by the A. niger was taken as positive control, when the test actinobacteria strains grown in the medium they inhibited amylase activity and was evidenced by the reduction in inhibition zone (14–37 mm) similarly the amylase produced by the Bacillus subtilis was also recorded maximum (35 mm) amylase activity and was taken as positive control, and the test atinobacterial strains reduced enzyme action(12–33 mm) it varied levals. This indicates that the actinobacteria strains were controlled amylase enzyme activity in both the cases. The strain SSR-10 was highly effective and SSR-8 was less effective in inhibiting eukaryotic amylase produced by A. niger. The strain SSR-2 was effective and SSR-6 showed very less effect in inhibiting the prokaryotic amylase produced by the B subtilis.     

Identifying Elements of Patient-Centered Care in Underserved Populations: A Qualitative Study of Patient Perspectives

Patient-centered care is an important goal in the delivery of healthcare. However, many patients do not engage in preventive medical care. In this pilot study, we conducted twenty in depth, semi-structured qualitative interviews at the University of Illinois at Chicago Health Sciences campus in a four month time frame. Many patients were underserved and underinsured, and we wanted to understand their experiences in the healthcare system. Using content analysis, several themes emerged from the interview data. Participants discussed the need for empathy and rapport with their providers. They identified provider behaviors that fostered a positive clinical relationship, including step-by step explanations of procedures, attention to body language and clinic atmosphere, and appropriate time management. Participants identified cost as the most common barrier to engaging in preventive care and discussed children and social support as motivating factors. A long-term relationship with a provider was an important motivator for preventive care, suggesting that the therapeutic alliance was essential to many patients. Conversely, many participants discussed a sense of dehumanization in the healthcare system, reporting that their life circumstances were overlooked, or that they were judged based on insurance status or ethnicity. We discuss implications for provider training and healthcare delivery, including the importance of patient-centered medical homes.     

Studies on the efficiency of different extraction procedures on the anti microbial activity of selected medicinal plants

Many doubts still persist even today when it comes to selection of the solvents for extracting the active constituents from various Indian medicinal plants. This study was aimed at assessing and establishing the best solvent for extracting the active constituents from 10 plant extracts. Thin layer chromatography (TLC) was used to separate and establish the active constituents present in each of the medicinal plants. Active constituents from each plant were extracted by using three different solvent systems namely diethyl ether, chloroform and hexane and were tested against three species of gram negative and three species of gram positive bacteria (Escherichia coli, Pseudomonas aureginosa, Streptococcus pneumoniae, Aeromonas hydrophila, Staphylococcus aereus, Bacillus cereus) by means of agar well diffusion assay. Studies on the antioxidant activity studies were also carried out for these plant extracts by using Diphenylpicryl-hydrazyl (DPPH) method. For the antimicrobial activity, the study revealed that among the selected plants, Azadiracta indica, Pongamia pinnata, Aloe barbadensis had the maximum antibacterial activity. Among the extraction procedures diethyl ether was found to be the best solvent that could be used for the extraction procedure. On the antioxidant activity part, Coleus amboinicus and Calotropis procera were found to have high antioxidant activity of 91.64% and 88.72% respectively and the further results are reported and discussed.     

Freshwater ascomycetes: Jahnula apiospora (Jahnulales, Dothideomycetes), a new species from Prince Edward Island, Canada

A new ascomycete species, Jahnula apiospora (Jahnulales, Dothideomycetes), collected from submerged wood in a freshwater creek on Prince Edward Island, Canada, is described and illustrated. The characteristic features of the new species are globose to subglobose, black, ostiolate, membranous ascomata with broad, brown, subtending hyphae; a peridial wall composed of an outer layer of thick-walled cells occluded by black, amorphous material along the upper two-thirds of the ascoma; trabeculate pseudoparaphyses; cylindrical to narrowly fusoid, fissitunicate asci; and brown, one-septate, apiosporous ascospores without a gelatinous sheath or appendages.