Isolation and identification of an endophytic fungus Pezicula sp. in Forsythia viridissima and its secondary metabolites

In a survey of endophytic fungal biodiversity, an antimicrobial endophytic isolate zjwcf069 was obtained from twigs of Forsythia viridissima, Zhejiang Province, Southeast China. Zjwcf069 was then identified as Pezicula sp. through combination of morphological and phylogenetic analysis based on ITS-rDNA. Zjwcf069 here represented the first endophytic fungus in Pezicula isolated from host F. viridissima. From the fermentation broth, four compounds were obtained through silica gel column chromatography and Sephadex LH-20 under the guide of bioassay. Their structures were elucidated by spectroscopic analysis as mellein (1), ramulosin (2), butanedioic acid (3), and 4-methoxy-1(3H)-isobenzofuranone (4). Compound 4 here stood for the very first time as natural product from microbes. In vitro antifungal assay showed that compound 1 displayed growth inhibition against 9 plant pathogenic fungi, especially Botrytis cinerea and Fulvia fulva with EC₅₀ values below 50 μg/mL. Endophytic fungi in medicinal plants were good resources for bioactive secondary metabolites.     

Production of salidroside and polysaccharides in Rhodiola sachalinensis using airlift bioreactor systems

Rhodiola sachalinensis is widely used in traditional Chinese medicine, and salidroside and polysaccharides are its important bioactive compounds. This study used airlift bioreactor systems to produce mass bioactive compounds through callus culture. Several factors affecting callus biomass and bioactive compound accumulation were investigated. Callus growth was vigorous in a bioreactor system, and the growth ratio was 2.8-fold higher in bioreactor culture than in agitated-flask culture. Callus biomass and polysaccharide content were favorable at 0.1 air volume per culture volume per min (vvm), whereas favorable salidroside content was observed at a high air volume (0.2 vvm). The maximum yields of salidroside (7.90 mg l^?1) and polysaccharide (2.87 g l^?1) were obtained at 0.1 vvm. Inoculum density greatly affected callus biomass and bioactive compound accumulation, and the highest biomass and contents or yields of salidroside and polysaccharide were determined at a high inoculum density of 12.5 g l^?1. The level of hydrogen ion concentration (pH) at 5.8 improved callus biomass accumulation. Acidic medium (pH 4.8) stimulated salidroside synthesis but higher pH level (7.8) promoted polysaccharide accumulation. The highest yields of both bioactive compounds were obtained at pH 5.8. Methyl jasmonate (MeJA) participated in synthesis promotion of bioactive compounds, and the contents and yields of salidroside [4.75 mg g^?1 dry weight (DW), 58.43 mg l^?1] and polysaccharides (392.41 mg g^?1 DW, 4.79 g l^?1) were at maximum at 125 and 150 μmol of MeJA. Therefore, bioreactor systems can be used to produce R. sachalinensis bioactive compounds, and callus culture in a bioreactor can be as an alternative method for supplying materials for commercial drug production.     

Comparative chemical analysis and antifungal activity of Ochtodes secundiramea (Rhodophyta) extracts obtained using different biomass processing methods

The activities of bioactive compounds can be influenced by countless factors, including post-collection sample treatment. In this study, the activities of Ochtodes secundiramea extracts obtained using different processes are compared; the biomass was macerated immediately, lyophilized in liquid nitrogen, or dried in an oven at 50 °C after collection. The chemical profiles of the extracts were evaluated using gas chromatography-mass spectrometry (GC-MS), and their biological activities were assessed using qualitative and quantitative bioautography thin-layer chromatography (TLC) assays against phytopathogenic fungi. Preparative high-performance liquid chromatography (HPLC) was employed to isolate the major compound observed in semi-quantitative (CG-MS). The biomass processing methods were shown to influence the chemical profiles of the extracts that promote the oxidation of halogenated monoterpenes; however, C₁₀H₁₅Br₂Cl was the major compound identified in all of the extracts regardless of the preparation method used. Similarly, changes in qualitative antifungal activity were observed depending on the preparation method, even though the minimum amount of material required for the inhibition of fungal growth (the activity detection limit) was 5 μg in all cases. The activity detection limits for the HPLC-purified majority compound were 5 and 10 μg against Cladosporium sphaerospermum and Colletotrichum lagenarium, respectively; these values are similar to those obtained for the crude extracts. These results point towards the possibility of using these crude extracts for the control of anthracnose in cucumbers post-harvest.     

Litoribrevibacter albus gen. nov. sp. nov., isolated from coastal seawater,Fujian province,China

A Gram-stain negative, short rod-shaped aerobic bacterium with flagella, designated strain Y32^T, was isolated from coastal seawater in Xiamen, Fujian Province of China. 16S rRNA gene sequence comparisons showed that strain Y32^T is a member of the family Oceanospirillaceae, forming a distinct lineage with species of the genus Litoribacillus. The 16S rRNA gene sequence similarities between strain Y32^T and other strains were all less than 94.0 %. Strain Y32^T was found to grow optimally at 28 °C, at pH 7.0–8.0 and in the presence of 4–5 % (w/v) NaCl. The major fatty acids were identified as Summed Feature 3 (comprising C_16:1 ω7c and/or C_16:1 ω6c, 49.4 %), C_16:0 (17.7 %), C_14:0 (6.9 %) and C_18:1 ω9c (5.4 %). The major respiratory quinone was identified as ubiquinone-8 (Q-8). The major polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of strain Y32^T was determined to be 55.6 mol%. According to its morphology, physiology, fatty acid composition, polar lipids composition and 16S rRNA gene sequence data, strain Y32^T represents a novel species of a new genus in the family Oceanospirillaceae, for which the name Litoribrevibacter albus gen. nov. sp. nov. is proposed. The type strain of Litoribrevibacter albus is Y32^T (=MCCC 1F01211^T=NBRC 110071^T).     

Mechanisms of Na+ uptake,ammonia excretion,and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi,Hemigrammus rhodostomus,and Moenkhausia diktyota)

Mechanisms of Na⁺ uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J _in), efflux (J _out), and net flux rates (J _net) of Na⁺ and Cl^?, and the net excretion rate of ammonia (J _Amm). The fish were collected directly from the Rio Negro, and studied in their native “blackwater” which is acidic (pH 4.5), ion-poor (Na⁺, Cl^? ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l^?1). J _in ^Na , J _in ^Cl , and J _Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J _in ^Na was unaffected by amiloride (10^?4 M, NHE and Na⁺ channel blocker), but both J _in ^Na and J _in ^Cl were virtually eliminated (85–99 % blockade) by AgNO₃ (10^?7 M). A time course study on cardinal tetras demonstrated that J _in ^Na blockade by AgNO₃ was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10^?4 M) caused a 50 % reduction in J _in ^Na _.. Additionally, J _in ^Na was unaffected by phenamil (10^?5 M, Na⁺ channel blocker), bumetanide (10^?4 M, NKCC blocker), hydrochlorothiazide (5 × 10^?3 M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J _in ^Na (by acetazolamide and AgNO₃ respectively), had any inhibitory effect on J _Amm. Therefore, Na⁺ uptake in Rio Negro tetras depends on an internal supply of H⁺ from CA, but does not fit any of the currently accepted H⁺-dependent models (NHE, Na⁺ channel/V-type H⁺-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current “Na⁺/NH₄ ⁺ exchange metabolon” paradigm. Na⁺, K⁺-ATPase and V-type H⁺-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH₄Cl, 10^?3 M) significantly increased J _in ^Na , and NH₄ ⁺ was equally or more effective than K⁺ in activating branchial Na⁺,(K⁺) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na⁺ uptake, but that Na⁺ uptake (by an as yet unknown H⁺-dependent apical mechanism) depends on ammonia excretion, driven by active NH₄ ⁺ entry via basolateral Na⁺,(K⁺)-ATPase.     

Screening survey of co-production of fusaric acid,fusarin C,and fumonisins B1, B2 and B3 by Fusarium strains grown in maize grains

Fusarium species isolated from Belgian maize were screened for their ability to produce fusarin C, fusaric acid, fumonisins B₁ (FB₁), FB₂ and FB₃ in maize grains. First, cultivation of Fusarium species in Myro liquid medium allowed overcoming the shortage of the standard of fusarin C on the market. All Fusarium verticillioides produced much higher contents of mycotoxins in Myro compared to Fusarium graminearum or Fusarium venenatum. The optimization of the LC-MS/MS method resulted in low limits of detection and quantification for fusarin C, fusaric acid, FB₁, FB₂ and FB₃ determination in maize grains. Its application for screening the potential toxin production ability evidenced that the concentrations of the analytes were significantly increased at various levels when F. verticillioides strains were cultivated in maize grains and reached 441 mg kg^?1 for fusaric acid, 74 mg kg^?1 for fusarin C, 1,301 mg kg^?1 for FB₁, 367 mg kg^?1 for FB₂ and 753 mg kg^?1 for FB₃.     

Association of vitamin D receptor FokI and ApaI polymorphisms with lung cancer risk in Tunisian population

Many studies reported that Vitamin D Receptor (VDR) gene polymorphisms might influence the cancer risk due to their antiproliferative, antiangiogenic, and apoptotic effects. The aim of this study was to explore the genetic association of VDR polymorphisms with lung cancer risk in Tunisian population. The genotype and haplotype frequencies of four VDR polymorphisms, FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) were studied using polymerase chain reaction and restriction fragment length polymorphism analysis in 240 patients with lung cancer and 280 healthy controls. The distribution of genotype frequencies differed significantly between lung cancer subjects and controls (FokI P _adj  = 0.002; ApaI P _adj  = 0.013). Haplotype analyses revealed a significant association between G-A-C and A-C-T haplotypes and lung cancer risk (P _corr  = 0.0128, P _corr  = 0.008). When patients were stratified according to gender, age, and smoking, significant associations were detected with FokI and TaqI polymorphisms. We found a lack of association between BsmI, TaqI polymorphisms and lung cancer risk (P > 0.05). Only, the attributable proportion due to interaction and the synergic index for interaction between ApaI polymorphism and smoking were statistically significant (P _adj  = 0.74, 95 % CI = 0.38–1.20) and (P _adj  = 0.63, 95 % CI = 0.05–1.21), respectively. Both the additive interaction measures suggested the existence of a biological interaction between SNP ApaI, but not FokI, and smoking. The multiplicative interaction measure was not statistically significant (P > 0.05). This is the first study in Tunisia, which suggested that VDR FokI and ApaI polymorphisms might be risk factors for lung cancer development.     

Hydraulic adjustments in aspen (Populus tremuloides) seedlings following defoliation involve root and leaf aquaporins

Main conclusion

Changes in root and leaf hydraulic properties and stimulation of transpiration rates that were initially triggered by defoliation were accompanied by corresponding changes in leaf and root aquaporin expression. Aspen (Populus tremuloides) seedlings were subjected to defoliation treatments by removing 50, 75 % or all of the leaves. Root hydraulic conductivity (L_pr) was sharply reduced in plants defoliated for 1 day and 1 week. The decrease in L _pr could not be prevented by stem girdling and it was accompanied in one-day-defoliated plants by a large decrease in the root expression of PIP1,2 aquaporin and an over twofold decrease in hydraulic conductivity of root cortical cells (L _pc). Contrary to L _pr and L _pc, 50 and 75 % defoliation treatments profoundly increased leaf lamina conductance (K _lam) after 1 day and this increase was similar in magnitude for both defoliation treatments. Transpiration rates (E) rapidly declined after the removal of 75 % of leaves. However, E increased by over twofold in defoliated plants after 1 day and the increases in E and K _lam were accompanied by five- and tenfold increases in the leaf expression of PIP2;4 in 50 and 75 % defoliation treatments, respectively. Defoliation treatments also stimulated net photosynthesis after 1 day and 3 weeks, although the increase was not as high as E. Leaf water potentials remained relatively stable following defoliation with the exception of a small decrease 1 day after defoliation which suggests that root water transport did not initially keep pace with the increased transpirational water loss. The results demonstrate the importance of root and leaf hydraulic properties in plant responses to defoliation and point to the involvement of PIP aquaporins in the early events following the loss of leaves.     

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning,expression, and biochemical characterization

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with K _m and k _cat values of 0.197 mM and 2,804.32 s^?1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn²⁺ and inhibited by Cu²⁺, Zn²⁺, Ni²⁺, and Fe²⁺. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.     

Identification of methylation quantitative trait loci (mQTLs) influencing promoter DNA methylation of alcohol dependence risk genes

Interaction of DNA methylation and sequence variants that are methylation quantitative trait loci (mQTLs) may influence susceptibility to diseases such as alcohol dependence (AD). We used genome-wide genotype data from 268 African Americans (AAs: 129 AD cases and 139 controls) and 143 European Americans (EAs: 129 AD cases and 14 controls) to identify mQTLs that were associated with promoter CpGs in 82 AD risk genes. 282 significant mQTL–CpG pairs (9.9 × 10^?100 ≤ P _nominal ≤ 7.7 × 10^?8) in AAs and 313 significant mQTL–CpG pairs (2.7 × 10^?53 ≤ P _nominal ≤ 9.9 × 10^?8) in EAs were identified [i.e., mQTL–CpG associations survived multiple-testing correction, q values (false discovery rate) ≤ 0.05]. The most significant mQTL was rs1800759, which was strongly associated with CpG cg12011299 in both AAs (P _nominal = 9.9 × 10^?100; q = 6.7 × 10^?91) and EAs (P _nominal = 2.7 × 10^?53; q = 1.4 × 10^?44). Rs1800759 (previously known to be associated to AD) and CpG cg12011299 (distance: 37 bp) are both located in alcohol dehydrogenase (ADH) 4 gene (ADH4) promoter region. In general, the strength of association between mQTLs and CpGs was inversely correlated with the distance between them. Association was also influenced by race and AD. Additionally, 48.3 % of the mQTLs identified in AAs and 65.6 % of the mQTLs identified in EAs were predicted to be expression QTLs. Three mQTLs (rs2173201, rs4147542, and rs4147541 in ADH1B-AHD1C gene cluster region) found in AAs were previously identified by our genome-wide association studies as being significantly associated with AD in AAs. Thus, DNA methylation, which can be influenced by sequence variants and is implicated in gene expression regulation, appears to at least partially underlie the association of genetic variation with AD.     

Extracellular endoglucanase activity from Paenibacillus polymyxa BEb-40: production,optimization and enzymatic characterization

Endoglucanase activity produced by Paenibacillus polymyxa BEb-40 was studied. In submerged culture with minimal medium supplemented with carboxymethylcellulose (CMC), this microorganism produced up to 0.37 U/mL endoglucanase activity with high specific activity (14.3 U/mg_{total protein}). Detection of endoglucanase activity through zymography revealed at least 14 isoenzymes with molecular weights between 38 and 220 kDa. This high variety of secreted endoglucanases has not been described previously in Paenibacillus genus. The optimum conditions, determined by response surface methodology, were 48 °C and pH 3.4, which allowed an increase of 33.7 % in the relative endoglucanase activity obtained with respect to the standard conditions. Nevertheless, high levels of hydrolysis of at least 70 % of the maximum activity could be obtained at wide ranges of pH (2–9) and temperature (40–60 °C). Under optimal conditions, high levels of CMC hydrolysis were reached, of about 40 %, after only 12 h of reaction with substrate/total protein ratios between 19 and 76. Kinetic analysis revealed that endoglucanase activity followed a mixed inhibition model (K _m = 8.4 mM, K _ic = 0.03 mM, K _iu = 0.35 mM, V _max = 33.3 U/mg_{total protein}). These results allow to consider P. polymyxa BEb-40 as a promising microorganism for the production of endoglucanases, with possibilities of application in the breakdown of lignocellulosic biomass. The high specific activity at wide ranges of pH and temperature can allow its use in a wide variety of processes, under both acidic and alkaline conditions, as well as in mesophilic and thermophilic temperatures, further reducing the amount of enzymes used.     

Pelagibaca abyssi sp. nov., of the family Rhodobacteraceae,isolated from deep-sea water

A Gram-stain negative, oval-shaped, aerobic, catalase and oxidase-positive bacterium, designated JLT2014^T, was isolated from a deep-seawater sample (obtained at a 2,000 m depth) of the Southeastern Pacific Ocean. The dominant fatty acids were identified as C_18:1ω7c/C_18:1ω6c, C_16:0 and C_10:0 3-OH, which altogether represented 60.1 % of the total. The predominant respiratory quinone was identified as Q-10. The G+C content of genomic DNA was determined to be 66.4 mol %. The major polar lipids were identified as phosphatidylethanolamine and diphosphatidylglycerol. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the novel isolate can be affiliated with the Roseobacter clade within the family Rhodobacteraceae. Strain JLT2014^T exhibited highest 16S rRNA gene sequence similarity value to Pelagibaca bermudensis HTCC2601^T (sequence similarity value: 97.6 %). The DNA–DNA relatedness value between strain JLT2014^T and P. bermudensis HTCC2601^T was 46.9 ± 2 %. Based on phenotypic properties and phylogenetic analysis, the name Pelagibaca abyssi sp. nov. is proposed, with JLT2014^T(=LMG 27363^T=CGMCC 1.12376^T) as the type strain.     

NOD2 and CCDC122-LACC1 genes are associated with leprosy susceptibility in Brazilians

Leprosy is a complex disease with phenotypes strongly influenced by genetic variation. A Chinese genome-wide association study (GWAS) depicted novel genes and pathways associated with leprosy susceptibility, only partially replicated by independent studies in different ethnicities. Here, we describe the results of a validation and replication study of the Chinese GWAS in Brazilians, using a stepwise strategy that involved two family-based and three independent case–control samples, resulting in 3,614 individuals enrolled. First, we genotyped a family-based sample for 36 tag single-nucleotide polymorphisms (SNPs) of five genes located in four different candidate loci: CCDC122-LACC1, NOD2, TNFSF15 and RIPK2. Association between leprosy and tag SNPs at NOD2 (rs8057431) and CCDC122-LACC1 (rs4942254) was then replicated in three additional, independent samples (combined OR_AA = 0.49, P = 1.39e?06; OR_CC = 0.72, P = 0.003, respectively). These results clearly implicate the NOD2 pathway in the regulation of leprosy susceptibility across diverse populations.     

Association between the MMP-9−1562 C>T polymorphism and the risk of stroke: a meta-analysis

Matrix metalloproteinase (MMP)-9 so far is identified as extremely large and complicated MMP family member. Recently, dozens of studies have explored the association between a promoter polymorphism (?1562 C>T) in MMP-9 and stroke susceptibility. However, the conclusions of these studies still remain equivocal. Therefore, our current meta-analysis was conducted to investigate whether or not the MMP-9 promoter polymorphism is related to the risk of stroke. Electronic databases (PubMed, EMBASE, Web of Science, Cochrane Library and the Chinese Biomedical Literature Database) were searched to obtain all the available studies investigating this polymorphism and stroke from inception to October 2013. Overall and subgroup analyses were rigorously conducted after data extraction. Pooled odds ratio (OR) corresponding to 95 % confidence interval (CI) were estimated. The statistical analysis was performed using Review Manager 5.2. Totally, seven studies involving 1,624 cases and 1,525 controls were identified. The overall results suggested that there was no association of the C?1562T variant on stroke risk under the T allele versus C allele [OR _{T vs. C} 0.98, 95 % CI (0.84, 1.15), P = 0.84], the dominant model [OR _{TT+TC vs. CC} 0.95, 95 % CI (0.81, 1.13), P = 0.59], the recessive model [OR _{TT vs. TC+CC} 1.55, 95 % CI (0.86, 2.81), P = 0.15], the homozygote comparison [OR _{TT vs. CC} 1.48, 95 % CI (0.82, 2.68), P = 0.20] and the heterozygote comparison [OR _{TC vs. CC} 0.93, 95 % CI (0.78, 1.10), P = 0.38]. In the subgroup analyses by ethnicity, age, stroke type and source of controls, no significant relations were observed in any genetic models. Our results indicated that MMP-9?1562 C>T polymorphism was not a risk factor for stroke. Further studies should focus on gene–gene and gene–environment interactions, and provide a more convincing explanation for this association.     

The energetics of a Malagasy rodent,Macrotarsomys ingens (Nesomyinae): a test of island and zoogeographical effects on metabolism

This study provides first insights into the energetics of the Nesomyinae, a subfamily of rodents endemic to Madagascar. The ancestral nesomyine colonized Madagascar from Africa ca. 30–15 mya at the onset of Oligocene global cooling. We tested the hypothesis that, contrary to what might be expected from Island Biogeography theory, post-colonization character displacement of thermoregulatory traits was constrained by phylogenetic inertia through climate adaptation. The study was conducted in the Parc National d’Ankarafantsika, Madagascar. We measured the basal metabolic rate (BMR) and body temperature (T _b) patterns of naturally warm-acclimated, freshly captured adult long-tailed big-footed mice Macrotarsomys ingens (67.4 g). The mean ± SD BMR of M. ingens was 0.298 ± 0.032 Watts (n = 12), 31.7 % lower than that predicted by a phylogenetically independent allometric equation. Body mass was correlated with BMR. The lower critical limit of thermoneutrality (T _lc) was 30.7 °C. The mean ± SD T _b = 36.1 ± 0.8 °C (n = 12) compared well with the mean T _b values for myomorph rodents from the Afrotropical zone, but was lower than those of the Neotropical and Palearctic zones. M. ingens became pathologically hypothermic when exposed to ambient temperatures lower than 18 °C. The soil temperature at depths of 250 mm and deeper did not decrease below 22 °C throughout the austral winter. The thermoregulatory data for M. ingens did not differ from those that characterize mainland Afrotropical rodents. However, BMR and T _b were lower than those of Holarctic rodents. Thus, contrary to expectations of Island Biogeography theory that rapid character displacement often occurs in morphological and behavioural traits when mammals colonize islands, M. ingens displayed climate-related physiological traits indicative of phylogenetic inertia. Presumably the tropical conditions that prevailed on Madagascar at the time of colonisation differed very little from those of the African mainland, and hence there was no strong driving force for change. Unlike small tenrecs and lemurs that radiated on Madagascar prior to the Oligocene, traits associated with an insular existence, such as daily torpor and hibernation, were not evident in M. ingens.     

Vibrio madracius sp. nov. Isolated from Madracis decactis (Scleractinia) in St Peter & St Paul Archipelago,Mid-Atlantic Ridge,Brazil

Three novel isolates (A-354^T, A-328, and A-384) were retrieved from apparently healthy scleractinian Madracis decactis in the remote St Peter & St Paul Archipelago, Mid-Atlantic Ridge, Brazil. The novel isolates formed a distinct lineage based on the phylogenetic reconstruction using the 16S rRNA and pyrH gene sequences. They fell into the Mediterranei clade and their closest phylogenetic neighbour was V. mediterranei species, sharing upto 98.1 % 16S rRNA gene sequence similarity. Genomic analysis including in silico DDH, MLSA, AAI and genomic signature distinguished A-354^T from V. mediterranei LMG 19703 (=AK1) with values of 33.3, 94.2, 92 %, and 11.3, respectively. Phenotypically, the novel isolates can be differentiated from V. mediterranei based on the four following features. They do not grow at 8 % NaCl; use d-gluconic acid but not l-galactonic acid lactone as carbon source; and do not have the fatty acid C_18:0. Differentiation from both the other Mediterranei clade species (V. maritimus and V. variabilis) is supported by fifteen features. The novel species show lysine decarboxylase and tryptophan deaminase, but not gelatinase and arginine dihydrolase activity; produce acetoin; use α-d-lactose, N-acetyl-d-galactosamine, myo-Inositol, d-gluconic acid, and β-hydroxy-d,l-butyric acid; and present the fatty acids C_14:0 iso, C_15:0 anteiso, C_16:0 iso, C_17:0 anteiso, and C_17:1x8c . Whole-cell protein profiles, based on MALDI-TOF, showed that the isolates are not clonal and also distinguished them from the closes phylogenetic neighbors. The name Vibrio madracius sp. nov. is proposed to encompass these novel isolates. The G+C content of the type strain A-354^T (=LMG 28124^T=CBAS 482^T) is 44.5 mol%.     

Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite,Plasmodium vivax,and characterization of its properties

Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni²⁺-NTA resin giving a yield of 25–30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 × 10⁸ min^?1 M^?1, 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors.