Involvement of the smeAB multidrug efflux pump in resistance to plant antimicrobials and contribution to nodulation competitiveness in Sinorhizobium meliloti

The contributions of multicomponent-type multidrug efflux pumps to antimicrobial resistance and nodulation ability in Sinorhizobium meliloti were comprehensively analyzed. Computational searches identified genes in the S. meliloti strain 1021 genome encoding 1 pump from the ATP-binding cassette family, 3 pumps from the major facilitator superfamily, and 10 pumps from the resistance-nodulation-cell division family, and subsequently, these genes were deleted either individually or simultaneously. Antimicrobial susceptibility tests demonstrated that deletion of the smeAB pump genes resulted in increased susceptibility to a range of antibiotics, dyes, detergents, and plant-derived compounds and, further, that specific deletion of the smeCD or smeEF genes in a ΔsmeAB background caused a further increase in susceptibility to certain antibiotics. Competitive nodulation experiments revealed that the smeAB mutant was defective in competing with the wild-type strain for nodulation. The introduction of a plasmid carrying smeAB into the smeAB mutant restored antimicrobial resistance and nodulation competitiveness. These findings suggest that the SmeAB pump, which is a major multidrug efflux system of S. meliloti, plays an important role in nodulation competitiveness by mediating resistance toward antimicrobial compounds produced by the host plant.     

Prevalence of known mutations in the MEFV gene in a population screening with high rate of carriers

The Familial Mediterranean Fever (FMF) shows an autosomal recessive pattern of inheritance and affects certain ethnic groups. Disease is caused by mutations in MEFV gene and more than 180 mutations have been defined in affected individuals. Current study aimed to determine the frequency-type of the mutations for MEFV gene in Sivas??middle Anatolian city. The cohort was composed of 3340 patients. MEFV gene mutations were studied by multiplex PCR based reverse hybridization stripAssay method. Patients?? clinical features were; family history: 68%, erysipelas-like erythema: 17.6%, fever: 89.9%, abdominal pain: 84.2%, peritonitis: 90.2%, arthritis: 33%, pleuritis: 14.2%, parental consanguinity: 21.2%. Current results revealed that M694V is the most frequent mutation (43.12%), followed by E148Q (20.18), M680I(G/C) (15.00%) and V726A (11.32%). The study population has a high rate of carriers and the E148Q mutation frequency was found to be highest when compared to the other regions of Turkey and other Mediterranean groups.     

Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease

A multiplex PCR (mPCR) method was designed for the simultaneous detection of 4 major fish pathogens, Flavobacterium psychrophilum, Lactococcus garvieae, Pseudomonas aeruginosa, and P. putida. Each of the 4 pairs of oligonucleotide primers exclusively amplified the 16S rDNA gene of their targeted microorganism. The average detection limits for each organism amplified by mPCR were 2 colony-forming units (CFU) of F. psychrophilum, 3 CFU of L. garvieae, 3 CFU of P. aeruginosa, and 5 CFU of P. putida in mixed cultures. Multiplex PCR did not produce any nonspecific amplification products when tested against 28 related species of bacteria. High amounts of DNA from 1 bacterial species had a significant effect on the amplification sensitivity of the other bacterial species when these were present in lower concentrations in the multiplex reaction. The mPCR assay proved useful for the detection of the bacteria in naturally infected fish. The assay is a sensitive, specific, and reproducible diagnostic tool for the simultaneous detection of 4 pathogenic bacteria that cause disease in fish and offers a potentially useful alternative to the conventional culture-based method.     

Plexin-D1 is a novel regulator of germinal centers and humoral immune responses

Long-lived humoral immune responses depend upon the generation of memory B cells and long-lived plasma cells during the germinal center (GC) reaction. These memory compartments, characterized by class-switched IgG and high-affinity Abs, are the basis for successful vaccination. We report that a new member of the plexin family of molecules, plexin-D1, controls the GC reaction and is required for secondary humoral immune responses. Plexin-D1 was not required for B cell maturation, marginal zone precursor development, dark and light zone formation, Igλ(+) and Igκ(+) B cell skewing, B1/B2 development, and the initial extrafollicular response. Plexin-D1 expression was increased following B cell activation, and PlxnD1(-/-) mice exhibited defective GC reactions during T-dependent immune activation. PlxnD1(-/-) B cells showed a defect in migration toward the GC chemokines, CXCL12, CXCL13, and CCL19. Accordingly, PlxnD1(-/-) mice exhibited defective production of IgG1 and IgG2b, but not IgG3 serum Ab, accompanied by reductions in long-lived bone marrow plasmacytes and recall humoral memory responses. These data show a new role for immune plexins in the GC reaction and generation of immunologic memory.     

Design and synthesis of the tumor-activated prodrug of dihydropyrimidine dehydrogenase (DPD) inhibitor,RO0094889 for combination therapy with capecitabine

A series of tumor-activated prodrugs of the inhibitors of dihydropyrimidine dehydrogenase (DPD), an enzyme catabolizing 5-fluorouracil (5-FU: 4g), has been designed and synthesized. RO0094889 (11c) is a prodrug of 5-vinyluracil (4c), a known DPD inhibitor, and was designed to generate 4c selectively in tumor tissues by sequential conversion of 11c by three enzymes: esterase, cytidine deaminase and thymidine phosphorylase, the latter two of which are known to be highly expressed in various tumor tissues. When capecitabine (1), a tumor-activated prodrug of 5-FU, was co-administered orally with 11c, 5-FU in tumor tissues was significantly increased with only a slight increase of 5-FU in plasma as compared with oral capecitabine alone.     

Gene Silencing Mediated by Endogenous MicroRNAs under Heat Stress Conditions in Mammalian Cells

Heat shock, sudden change in temperature, triggers various responses in cells for protecting the cells from such a severe circumstance. Here we investigated gene silencing mediated by endogenous microRNAs (miRNAs) in mammalian cells exposed to a mild hyperthermia, by means of miRNA activity assay using a luciferase reporter gene as well as miRNA expression analysis using a DNA microarray. Our findings indicated that the gene silencing activities involving miRNAs were enhanced without increasing in their expression levels under heat-stress conditions. Additionally, the gene silencing activity appeared to be independent of the cytoprotective action involving heat shock proteins that are immediately activated in heat-shocked cells and that function as molecular chaperons for restoring heat-denatured proteins to normal proteins. Our current findings suggested the possibility that gene silencing involving endogenous miRNAs might play a subsidiary role in heat-shocked cells for an aggressive inhibition of the expression of heat-denatured proteins.     

Investigation of the VDR gene polymorphisms association with susceptibility to colorectal cancer

The effects of 1,25-dihydroxyvitamin D3 are mediated by binding to a specific intracellular vitamin D receptor (VDR), which has been identified in a variety of tissues. Certain polymorphisms in the VDR gene have been associated with various neoplasms. For this purpose, we studied whether VDR TaqI or FokI genotype are associated with serum 25-hydroxyvitamin D3 in 52 controls and 26 patients with colorectal cancer. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), and agarose gel electrophoresis tecniques were used to detect these polymorphisms. We measured 25-hydroxyvitamin D3 serum levels by ELISA. The frequencies of the FF, Ff and ff genotypes were 73.1%, 11.5%, 15.4% in colorectal cancer patients and 38.5%, 59.6%, 1.9% in healthy controls, respectively. We observed the T allele in 50% and 58.7%, and the t allele in 50% and 41.3% of colorectal cancer patients and the control group, respectively. In patients with colorectal cancer who have TT genotype, serum 25-hydroxyvitamin D3 level was lower than those with Tt/tt genotype (p:0.016). The frequency of subjects with TTFf or TtFf genotype in colorectal cancer patients was very low compared with all other genotypes (OR = 0.112; 95%CI 0.030-0.419). These data suggest that VDR TtFf or TTFf genotypes may protect against colorectal carcinogenesis. However, further studies are necessary to confirm these findings.     

Contrasted levels of genetic diversity in a benthic Mediterranean octocoral: Consequences of different demographic histories?

Understanding the factors explaining the observed patterns of genetic diversity is an important question in evolutionary biology. We provide the first data on the genetic structure of a Mediterranean octocoral, the yellow gorgonian Eunicella cavolini, along with insights into the demographic history of this species. We sampled populations in four areas of the Mediterranean Sea: continental France, Algeria, Turkey, and the Balearic and Corsica islands. Along French coasts, three sites were sampled at two depths (20 and 40 m). We demonstrated a high genetic structure in this species (overall F_ST = 0.13), and most pairwise differentiation tests were significant. We did not detect any difference between depths at the same site. Clustering analyses revealed four differentiated groups corresponding to the main geographical areas. The levels of allelic richness and heterozygosity were significantly different between regions, with highest diversity in Algeria and lowest levels in Turkey. The highest levels of private allelic richness were observed in Algeria followed by Turkey. Such contrasted patterns of genetic diversity were not observed in other Mediterranean octocorals and could be the result of different evolutionary histories. We also provide new empirical evidence of contrasting results between tests and model‐based studies of demographic history. Our results have important consequences for the management of this species.     

Ethanol production from rice hull using Pichia stipitis and optimization of acid pretreatment and detoxification processes

The goal of this study was to produce ethanol from rice hull hydrolysates (RHHs) using Pichia stipitis strains and to optimize dilute acid hydrolysis and detoxification processes by response surface methodology (RSM). The optimized conditions were found as 127.14°C, solid:liquid ratio of 1:10.44 (w/v), acid ratio of 2.52% (w/v), and hydrolysis time of 22.01 min. At these conditions, the fermentable sugar concentration was 21.87 g/L. Additionally, the nondetoxified RHH at optimized conditions contained 865.2 mg/L phenolics, 24.06 g/L fermentable sugar, no hydroxymethylfurfural (HMF), 1.62 g/L acetate, 0.36 g/L lactate, 1.89 g/L glucose, and 13.49 g/L fructose + xylose. Furthermore, RHH was detoxified with various methods and the best procedures were found to be neutralization with CaO or charcoal treatment in terms of the reduction of inhibitory compounds as compared to nondetoxified RHH. After detoxification procedures, the content of hydrolysates consisted of 557.2 and 203.1 mg/L phenolics, 19.7 and 21.60 g/L fermentable sugar, no HMF, 0.98 and 1.39 g/L acetate, 0 and 0.04 g/L lactate, 1.13 and 1.03 g/L glucose, and 8.46 and 12.09 g/L fructose + xylose, respectively. Moreover, the base‐line mediums (control), and nondetoxified and detoxified hydrolysates were used to produce ethanol by using P. stipitis strains. The highest yields except that of base‐line mediums were achieved using neutralization (35.69 and 38.33% by P. stipitis ATCC 58784 and ATCC 58785, respectively) and charcoal (37.55% by P. stipitis ATCC 58785) detoxification methods. Results showed that the rice hull can be utilized as a good feedstock for ethanol production using P. stipitis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:872–882, 2016