Glutamate-gated chloride channel subunit cDNA sequencing of Cochliomyia hominivorax (Diptera: Calliphoridae): cDNA variants and polymorphisms

The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel) is one of the major myiasis-causing flies that injures livestock and leads to losses of ~US$ 2.7 billions/year in the Neotropics. Ivermectin (IVM), a macrocyclic lactone (ML), is the most used preventive insecticide for this parasite and targets the glutamate-gated chloride (GLUCLα) channels. Several authors have associated altered GluClα homologues to MLs resistance in invertebrates, although studies about resistance in NWS are limited to other genes. Here, we aimed to characterise the NWS GluClα (ChGluClα) cDNA and to search for alterations associated with IVM resistance in NWS larvae from a bioassay. The open reading frame of the ChGluClα comprised 1,359 bp and encoded a sequence of 452 amino acids. The ChGluClα cDNAs of the bioassay larvae showed different sequences that could be splice variants, which agree with the occurrence of alternative splicing in GluClα homologues. In addition, we found cDNAs with premature stop codons and the K242R SNP, which occurred more frequently in the surviving larvae and was located close to mutation (L256F) involved in ML resistance. Although these alterations were in low frequency, the ChGluClα sequencing will allow further studies to find alterations in the gene of resistant natural populations.     

Terahertz radiation influence on stressed drosophila life span

Virgin fruit fly females and males were stressed by placement into a confined space without food for 3 h. Part of stressed flies were subjected to terahertz irradiation (0.1–2.2 THz) during 30 min. Life span of individual flies was evaluated. Terahertz radiation had some positive influence on male survival during the stage of monotonic decrease in flies number and negative effect during the stage of relatively stable number of flies. The survival of irradiated females on the stage of sharp decline in the number of flies was higher than in stressed and control females. Authors propose that terahertz radiation has an indirect effect on gene expression and signaling pathways which control the survival and life span of Drosophila.     

Molecular evidence to reconcile taxonomic instability in mahseer species (Pisces: Cyprinidae) of India

The mahseers are an important group of fishes endemic to Asia with most species considered threatened. Conservation plans to save declining wild populations are hindered by unstable taxonomy, and detailed systematic review could form a solid platform for future management and conservation. D-loop and cytochrome c oxidase I (COI) mtDNA sequences were examined in nine mahseer species of Tor, Neolissochilus, and Naziritor. Pseudogenes amplified in a portion of the species limited the utility of the D-loop region. ABGD analysis, NJ, ML, and MP methods and genetic distance (TrN?+?I?+?G) using COI data revealed concordant species delimiting patterns. The three genera were monophyletic, separated as distinct clades (TrN?+?I?+?G 0.064 to 0.106), and Naziritor was flagged as a separate genus, distinct from Puntius (TrN?+?I?+?G 0.196). Out of seven nominal species known for Tor cogeners from India, only five were recovered with mtDNA data (TrN?+?I?+?G 0.000 to 0.037) and two species could not be distinguished with the molecular data set employed. Tor mosal, synonymized as Tor putitora, was rediscovered as a distinct species (TrN?+?I?+?G 0.031) based on its type locality. Tor mussulah was confirmed as a separate species (TrN?+?I?+?G 0.019 to 0.026). Two valid species, Tor macrolepis and T. mosal mahanadicus, were not distinct from T. putitora (TrN?+?I?+?G 0.00). The high divergence with mtDNA data failed to validate T. mosal mahanadicus as a subspecies of T. mosal (TrN?+?I?+?G 0.031). Morphological outliers discovered within the distribution range of Tor tor (TrN?+?I?+?G 0.022 to 0.025) shared the same lineage with T. putitora (TrN?+?I?+?G 0.002 to 0.005), indicating a new extended distribution of the Himalayan mahseer T. putitora in the rivers of the Indian central plateau. The findings indicate the need for integrating molecular and morphological tools for taxonomic revision of the Tor and Naziritor genera, so that taxa are precisely defined for accurate in situ and ex situ conservation decisions.     

Effects of metabolic pathway precursors and polydimethylsiloxane (PDMS) on poly-(gamma)-glutamic acid production by Bacillus subtilis BL53

The aims of this study were to evaluate the effects of the addition of metabolic precursors and polydimethylsiloxane (PDMS) as an oxygen carrier to cultures of Bacillus subtilis BL53 during the production of γ-PGA. Kinetics analyses of cultivations of different media showed that B. subtilis BL53 is an exogenous glutamic acid-dependent strain. When the metabolic pathway precursors of γ-PGA synthesis, l-glutamine and a-ketoglutaric acid, were added to the culture medium, production of the biopolymer was increased by 20 % considering the medium without these precursors. The addition of 10 % of the oxygen carrier PDMS to cultures caused a two-fold increase in the volumetric oxygen mass transfer coefficient (k_La), improving γ-PGA production and productivity. Finally, bioreactor cultures of B. subtilis BL53 adopting the combination of optimized medium E, added of glutamine, α-ketoglutaric acid, and PDMS, showed a productivity of 1 g L^?1 h^?1 of g-PGA after only 24 h of cultivation. Results of this study suggest that the use of metabolic pathway precursors glutamine and a-ketolgutaric acid, combined with the addition of PDMS as an oxygen carrier in bioreactors, can improve γ-PGA production and productivity by Bacillus strains .     

The influence of repetitive thermal stresses on the dominance of reef-building Acropora spp. (Scleractinia) on coral reefs of the Maldive Islands

The distribution and size-age structure of Acropora corals were studied in two Maldivian atolls that differ in their geographic position and sea surface temperature regimes. The frequency and strength of thermal anomalies for the last 2 decades had a significant influence on the abundance, mortality rates, and age structures of acroporid communities. The long-term temperature amplitude was higher and the maxima were more pronounced in the northernmost Ihavandippolu Atoll than those in the equatorial South Huvadhoo Atoll. These differences resulted in a 10.4% mean cover of Acropora at Ihavandippolu Atoll, whereas the Acropora cover in the South Huvadhoo Atoll reached 59.5%. In the northern atoll, the coral mortality rate after the 2010 thermal anomaly was 3 times higher than that in the southern atoll. Younger acroporid colonies (up to 2 years old) dominated the northern atoll reefs, while the southern atoll showed a high proportion of older mature colonies. In both atolls, healthy table colonies of Acropora cytherea with a disk diameter greater than 2 m were observed that apparently survived three thermal anomalies since 1998. The mechanisms of acclimatization of Acropora and the prospects for its dominance in the Maldives under changing environmental conditions are discussed.     

Detection and characterization of phosphatidylcholine in various strains of the genus Chlamydomonas (Volvocales,Chlorophyceae)

The laboratory strains of Chlamydomonas reinhardtii have been reported to contain no phosphatidylcholine (PC), which is considered to be replaced by another zwitterionic lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). According to the recently published classification, the strains belonged to the subgroup Reinhardtinia. Screening for PC in 13 selected strains of Chlamydomonas in the NIES Algal Collection, which are different in habitats and belong to different phylogenetic subgroups in the genus, revealed the presence of PC in four strains: a strain in the subgroup Polytominia, and three strains in Reinhardtinia. PC was not detected in three other strains of Reinhardtinia analyzed. The presence/absence of PC was not related to the phylogenetic relationship based on 18S rRNA. DGTS was detected in all the strains analyzed. The rare isomer of linolenic acid, 18:3(5,9,12), which has been found in the DGTS of C. reinhardtii, was found in the PC of the two strains and in the DGTS of the five strains. The occurrence of this fatty acid seems limited to a branch of Reinhardtinia. Acquisition and loss of PC in various strains of Chlamydomonas are discussed from the viewpoint of evolution of PC biosynthetic pathway.     

Relationship of Cob characters with Grain Morphology in Maize (Zea mays,Poaceae)

Twenty varieties of maize (Zea mays, Poaceae) were studied through 11 attributes in three to seven randomly selected plants of each variety with a view to understanding the effect of cob characters on technologically desirable grain qualities. Canonical discriminant analysis showed thatproductivity (determined by total grain weight/cob, cob diameter and average grain weight) was the most discriminating among varieties followed by round grains fraction (represented by whole top and middle flat grains, number of rows and grain count/surface area), middle flat grains (composed of middle flat grains and grain count/surface area) and shape of the cob (determined by shape index, total grain weight/cob and cob diameter), which accounted for 35.1, 18.3, 12.2, and 9.8% of the total variance, respectively. In the light of these results, tentative norms have been suggested to evolve maize varieties of superior technological properties and yet retain high productivity. A cylindrical cob of large diameter with highest number of grains/area and smallest possible number of rows together constituted an ideal combination to achieve the objectives. Such possibilities in the light of available information are discussed.     

Estimates of heavy metal tolerance and chromium(VI) reducing ability of Pseudomonas aeruginosa CCTCC AB93066: chromium(VI) toxicity and environmental parameters optimization

The potential role of parameters in the reduction of hexavalent chromium [Cr(VI)] by Pseudomonas aeruginosa is not well documented. In this study, laboratory batch studies were conducted to assess the effect of a variety of factors, e.g., carbon sources, salinity, initial Cr(VI) concentrations, co-existing ions and a metabolic inhibitor, on microbial Cr(VI) reduction to Cr(III) by P. aeruginosa AB93066. Strain AB93066 tolerated up to 400 mg/L of Cr(VI) in nutrient broth medium compared to only 150 mg/L of Cr(VI) in nutrient agar. This bacteria exhibited different levels of resistance against Pb(II) (200 mg/L), Cd(II) (100 mg/L), Ni(II) (100 mg/L), Cu(II) (100 mg/L), Co(II) (50 mg/L) and Hg(II) (5 mg/L). Cr(VI) reduction was significantly promoted by the addition of glucose and glycerine but was strongly inhibited by the presence of methanol and phenol. The rate of Cr(VI) reduction increased with increasing concentrations of Cr(VI) and then decreased at higher concentrations. The presence of Ni(II) stimulated Cr(VI) reduction, while Pb(II), Co(II) and Cd(II) had adverse impact on reduction ability of this strain. Cr(VI) reduction was also inhibited by high levels of NaCl, various concentrations of sodium azide and 20 mM of SO₄ ^2?, MoO₄ ^2?, NO₃ ^?, PO₄ ^3?. No significant relationship was observed between Cr(VI) reduction and redox potential of the culture medium. Scanning electron microscopy showed visible morphological changes in the cells due to chromate stress. Fourier transform infrared spectroscopy analysis revealed chromium species was likely to form complexes with certain functional groups such as carboxyl and amino groups on the surface of P. aeruginosa AB93066. Overall, above results are beneficial to the bioremediation of chromate-polluted industrial wastewaters.     

Association of interleukin 1 receptor antagonist (IL1RN) gene polymorphism with recurrent pregnancy loss risk in the North Indian Population and a meta-analysis

An appropriate ratio of interleukin 1 beta to interleukin 1 receptor antagonist (IL1Ra) is required for successful pregnancy. Our objective was to study the genetic association between IL1RN variable numbers of tandem repeat (VNTR) polymorphism and recurrent pregnancy loss (RPL). To analyze the association between IL1RN VNTR allele and RPL, we investigated the IL1RN VNTR polymorphism in 136 RPL patients and in 200 healthy control women. Meta-analysis on this polymorphism was conducted to support our findings. PCR based approach was used to analyze IL1RN VNTR polymorphism and it was further confirmed by sequencing. Systematic review and meta-analysis was done using electronic database (Pub-Med, Google Scholar and Ovid) up to February 27, 2013. This meta-analysis was assessed by comprehensive meta-analysis software version 2. For meta-analysis 549 cases and 1,450 controls were included. The frequency of IL1RN genotype 2/2 was significantly higher in RPL compared to control group (AORs 3.10, 95 % CI 1.58–6.11, p = 0.001). The presence of rare allele also increased the risk of RPL significantly (ORs 1.63, 95 % CI 1.16–2.29, p = 0.004). The meta-analysis stratified by ethnicity showed that individuals with allele 2 had increased risk of RPL (OR 1.29, 95 % CI 1.04–1.61, p = 0.01), in Asians population by using fixed model. However the data of the present study clearly suggests that IL1RN VNTR polymorphism is a genetic risk factor for pregnancy loss in the study population.     

Phenotypic and genotypic characteristics of Arcanobacterium haemolyticum isolated from clinical samples in a Danish hospital

Six Arcanobacterium haemolyticum strains isolated from six patients of two hospitals in Denmark were identified phenotypically, also including matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and by genotypic methods. The latter were performed by sequencing 16S rDNA and glyceraldehyde 3-phosphate dehydrogenase encoding gene gap and by amplification of an A. haemolyticum specific region of 16S–23S rDNA intergenic spacer region and 23S rDNA. The six A. haemolyticum strains were further investigated for the presence of seven potential virulence genes encoding arcanolysin, phospholipase D, hemolysin A, CAMP factor family protein, collagen binding protein, neuraminidase A and neuraminidase H which appeared to be present in two (seven virulence genes), two (six virulence genes) and two strains (four virulence genes), respectively. The phenotypic and genotypic properties described in the present study might help to reliably identify and further characterize A. haemolyticum isolated from human patients, a species which seems to be of increasing importance.     

Bone Metabolism Status and Associated Risk Factors in Elderly Patients with Chronic Obstructive Pulmonary Disease (COPD)

The prevalence of osteoporosis in older patients with chronic obstructive pulmonary disease (COPD) is higher than in the age-matched elderly patients, but the exact cause in relation to COPD is not clear. We hypothesized that the underlying causes for this difference are related to bone metabolism with the possible risk factors that include the duration of COPD, GOLD grade, cor pulmonale, the frequencies of acute exacerbations within the past year, smoking and inhaled corticosteroid therapy. We conducted a matched-pair study of 100 patients aged older than 65 years at the Southwest Hospital from May to November 2012. The enrolled patients with COPD were matched to controls for age and gender. Clinical characteristics of cohorts were recorded. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry and osteoporosis was diagnosed according to the definition of WHO. All cohorts accepted bone metabolism marker measurement, including Procollagen type 1 aminoterminal propeptide (P1NP), β-C-telopeptides of type I collagen (βCTX), and N-terminal midmolecule fragment osteocalcin (N-MID OC). Statistical analysis was calculated using the student’s t test, ANOVA and multiple regression analysis at a significance level set at a p < 0.05. Circulating biochemical markers of bone formation (P1NP), resorption (βCTX) and turnover (N-MID OC) were significantly lower in the COPD group than control group, while mean 25-OH Vitamin D was similar in two groups. The P1NP, βCTX, and N-MID OC were still lower in men with COPD, but only P1NP was lower in women with COPD compared to that of controls. Multiple regression analysis in COPD group suggests that age, the frequency of acute exacerbation, and BMD are independent risk factors for P1NP. The frequency of acute exacerbation within the past one year and 25-OH D level are independent risk factors for βCTX; the frequency of acute exacerbation is the only independent risk factor for N-MID OC. These were significant differences in bone metabolism in patients with or without COPD. These results should help us to further understand the cause of osteoporosis and fractures and conduce to prevent osteoporosis in patients with COPD.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells

Cystic fibrosis transmembrane conductance regulator (CFTR) acts as a cAMP-dependent chloride channel, has been studied in various types of cells. CFTR is abundantly expressed in vascular smooth muscle cells and closely linked to vascular tone regulation. However, the functional significance of CFTR in basilar vascular smooth muscle cells (BASMCs) remains elusive. Accumulating evidence has shown the direct role of CFTR in cell apoptosis that contributes to several main pathological events in CF, such as inflammation, lung injury and pancreatic insufficiency. We therefore investigated the role of CFTR in BASMC apoptotic process induced by hydrogen peroxide (H₂O₂). We found that H₂O₂-induced cell apoptosis was parallel to a significant decrease in endogenous CFTR protein expression. Silencing CFTR with adenovirus-mediated CFTR specific siRNA further enhanced H₂O₂-induced BASMC injury, mitochondrial cytochrome c release into cytoplasm, cleaved caspase-3 and -9 protein expression and oxidized glutathione levels; while decreased cell viability, the Bcl-2/Bax ratio, mitochondrial membrane potential, total glutathione levels, activities of superoxide dismutase and catalase. The pharmacological activation of CFTR with forskolin produced the opposite effects. These results strongly suggest that CFTR may modulate oxidative stress-related BASMC apoptosis through the cAMP- and mitochondria-dependent pathway and regulating endogenous antioxidant defense system.     

Influence of bovine lactoferrin on the growth of selected probiotic bacteria under aerobic conditions

Bovine lactoferrin (bLf) is a natural glycoprotein, and it shows broad-spectrum antimicrobial activity. However, reports on the influences of bLf on probiotic bacteria have been mixed. We examined the effects of apo-bLf (between 0.25 and 128 mg/mL) on both aerobic and anaerobic cultures of probiotics. We found that bLf had similar effects on the growth of probiotics under aerobic or anaerobic conditions, and that it actively and significantly (at concentrations of >0.25 mg/mL) retarded the growth rate of Bifidobacterium bifidum (ATCC 29521), B. longum (ATCC 15707), B. lactis (BCRC 17394), B. infantis (ATCC 15697), Lactobacillus reuteri (ATCC 23272), L. rhamnosus (ATCC 53103), and L. coryniformis (ATCC 25602) in a dose-dependent manner. Otherwise, minimal inhibitory concentrations (MICs) were 128 or >128 mg/mL against B. bifidum, B. longum, B. lactis, L. reuteri, and L. rhamnosus (ATCC 53103). With regard to MICs, bLf showed at least four-fold lower inhibitory effect on probiotics than on pathogens. Intriguingly, bLf (>0.25 mg/mL) significantly enhanced the growth of Rhamnosus (ATCC 7469) and L. acidophilus (BCRC 14065) by approximately 40–200 %, during their late periods of growth. Supernatants produced from aerobic but not anaerobic cultures of L. acidophilus reduced the growth of Escherichia coli by about 20 %. Thus, bLf displayed a dose-dependent inhibitory effect on the growth of most probiotic strains under either aerobic or anaerobic conditions. An antibacterial supernatant prepared from the aerobic cultures may have significant practical use.     

Dietary l-glutamine supplementation modulates microbial community and activates innate immunity in the mouse intestine

This study was conducted to determine effects of dietary supplementation with 1 % l-glutamine for 14 days on the abundance of intestinal bacteria and the activation of intestinal innate immunity in mice. The measured variables included (1) the abundance of Bacteroidetes, Firmicutes, Lactobacillus, Streptococcus and Bifidobacterium in the lumen of the small intestine; (2) the expression of toll-like receptors (TLRs), pro-inflammatory cytokines, and antibacterial substances secreted by Paneth cells and goblet cells in the jejunum, ileum and colon; and (3) the activation of TLR4-nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), and phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt) signaling pathways in the jejunum and ileum. In the jejunum, glutamine supplementation decreased the abundance of Firmicutes, while increased mRNA levels for antibacterial substances in association with the activation of NF-κB and PI3K-Akt pathways. In the ileum, glutamine supplementation induced a shift in the Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes, and enhanced mRNA levels for Tlr4, pro-inflammatory cytokines, and antibacterial substances participating in NF-κB and JNK signaling pathways. These results indicate that the effects of glutamine on the intestine vary with its segments and compartments. Collectively, dietary glutamine supplementation of mice beneficially alters intestinal bacterial community and activates the innate immunity in the small intestine through NF-κB, MAPK and PI3K-Akt signaling pathways.     

Centric diatoms from Lake Frolikha (Transbaikal area) and peculiarities of distribution of some taxa in Asia

The species composition of centric diatoms from Lake Frolikha (Transbaikal area) has been studied. The lake is located on the eastern shore of Lake Baikal and connected with it by a river. Twenty-three species of Centrophyceae from 7 genera (Aulacoseira, Cyclostephanos, Cyclotella, Discostella, Handmania, Pliocaenicus, and Stephanodiscus) have been found. The most represented genus is Aulacoseira (11 species). Fifteen species and 4 genera are new for the lake record. All revealed species are known in other lakes of the Baikal region; however Baikal endemics are absent in Lake Frolikha. The flora of centric diatoms in Lake Frolikha can be divided into two groups. The first group includes taxa common in Lake Baikal, and the second group includes taxa not typical for Lake Baikal. The level of differences between flora in Lake Baikal and Lake Frolikha is high (43%) despite the close location and connection to the river. An analysis of distribution of interesting species in Asia is presented.     

Study of effect of substitution of the penultimate amino acid residue on expression,structure, and functional properties of Yersinia pseudotuberculosis OmpY porin

The purpose of the study was to compare the expression of two Yersinia pseudotuberculosis proteins, wild-type porin OmpY and the mutant porin OmpY designated as OmpY-Q having the uncharged amino acid residue Gln instead of positively charged Arg at the penultimate position in the same heterologous host. According to the literature, a similar substitution (Lys to Gln) of the penultimate amino acid residue in Neisseria meningitidis porin PorA drastically improved the assembly of the protein in the E. coli outer membrane in vivo. Site-directed mutagenesis was used to replace Arg by Gln (R338Q) in OmpY, and the conditions for optimal expression and maturation of OmpY-Q were selected. It was found that the growth rates of E. coli strains producing OmpY and OmpY-Q and the expression levels of the porins were approximately equal. Comparative analysis of recombinant OmpY and OmpY-Q did not show significant differences in structure, antigenic, and functional properties of the porins, or any noticeable effect of the R338Q substitution in OmpY on its assembly in the E. coli outer membrane in vivo. The probable causes of discrepancies between our results and the previous data on porin PorA are discussed considering the known mechanisms of biogenesis of porins at the periplasmic stage.