Nagihan Gülsoy

Genome Sequence of the Fish Pathogen Flavobacterium columnare ATCC 49512

Flavobacterium columnare is a Gram-negative, rod-shaped, motile, and highly prevalent fish pathogen causing columnaris disease in freshwater fish worldwide. Here, we present the complete genome sequence of F. columnare strain ATCC 49512.

High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes

ABSTRACT A high-throughput bioluminescence screening procedure for identification of virulence genes in bacteria was developed and applied to the fish pathogen Edwardsiella ictaluri. A random transposon mutant library expressing bioluminescence was constructed and robotically arrayed on 384-well plates. Mutants were cultivated and mixed with catfish serum and neutrophils in 96-well plates, and bioluminescence was used to detect mutants that are more susceptible to killing by these host factors. The virulence and vaccine efficacy of selected mutants were determined in channel catfish. Transposon insertion sites in 13 mutants attenuated in the natural host were mapped to the E. ictaluri genome. Ten unique genes were mutated, including genes encoding a negative regulator of sigmaE activity, a glycine cleavage system protein, tricarboxylic acid cycle enzymes, an O polysaccharide biosynthesis enzyme, proteins encoded on the native plasmid pEI1, and a fimbrial chaperon protein. Three of these mutants were found to have potential as live attenuated vaccines. This study demonstrates a novel application of bioluminescence to identify bacterial genes required for host resistance; as a result, efficacious and genetically defined live attenuated vaccine candidates were developed.

Proteomic analysis of the fish pathogen Flavobacterium columnare

BackgroundFlavobacterium columnare causes columnaris disease in cultured and wild fish populations worldwide. Columnaris is the second most prevalent bacterial disease of commercial channel catfish industry in the United States. Despite its economic importance, little is known about the expressed proteins and virulence mechanisms of F. columnare. Here, we report the first high throughput proteomic analysis of F. columnare using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS.ResultsProteins identified in this study and predicted from the draft F. columnare genome were clustered into functional groups using clusters of orthologous groups (COGs), and their subcellular locations were predicted. Possible functional relations among the identified proteins were determined using pathway analysis. The total number of unique F. columnare proteins identified using both 2-D LC and 2-DE approaches was 621, of which 10.95% (68) were identified by both methods, while 77.29% (480) and 11.76% (73) were unique in 2-D LC and 2-DE, respectively. COG groupings and subcellular localizations were similar between our data set and proteins predicted from the whole genome. Twenty eight pathways were significantly represented in our dataset (P < 0.05).ConclusionResults from this study provide experimental evidence for many proteins that were predicted from the F. columnare genome annotation, and they should accelerate functional and comparative studies aimed at understanding virulence mechanisms of this important pathogen.

Particle morphology influence on mechanical and biocompatibility properties of injection molded Ti alloy powder

Titanium and Titanium alloys exhibits properties that are excellent for various bio-applications. Metal injection molding is a processing route that offers reduction in costs, with the added advantage of near net-shape components. Different physical properties of Titanium alloy powders, shaped and processed via injection molding can achieve high complexity of part geometry with mechanical and bioactivity properties, similar or superior to wrought material. This study describes that the effect of particle morphology on the microstructural, mechanical and biocompatibility properties of injection molded Ti-6Al-4V (Ti64) alloy powder for biomaterials applications. Ti64 powders irregular and spherical in shape were injection molded with wax based binder. Binder debinding was performed in solvent and thermal method. After debinding the samples were sintered under high vacuum. Metallographic studies were determined to densification and the corresponding microstructural changes. Sintered samples were immersed in a simulated body fluid (SBF) with elemental concentrations that were comparable to those of human blood plasma for a total period of 15 days. Both materials were implanted in fibroblast culture for biocompatibility evaluations were carried out. The results show that spherical and irregular powder could be sintered to a maximum theoretical density. Maximum tensile strength was obtained for spherical shape powder sintered. The tensile strength of the irregular shape powder sintered at the same temperature was lower due to higher porosity. Finally, mechanical tests show that the irregular shape powder has lower mechanical properties than spherical shape powder. The sintered irregular Ti64 powder exhibited better biocompatibility than sintered spherical Ti64 powder. Results of study showed that sintered spherical and irregular Ti64 powders exhibited high mechanical properties and good biocompatibility properties.

Genotoxic effects of boric acid and borax in zebrafish, Danio rerio using alkaline comet assay.

The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish.

Effect of Zr, Nb and Ti addition on injection molded 316L stainless steel for bio-applications: Mechanical, electrochemical and biocompatibility properties.

The research investigated the effect of Zr, Nb and Ti additions on mechanical, electrochemical properties and biocompatibility of injection molded 316L stainless steel. Addition of elemental powder is promoted to get high performance of sintered 316L stainless steels. The amount of additive powder plays a role in determining the sintered microstructure and all properties. In this study, 316L stainless steel powders used with the elemental Zr, Nb and Ti powders. A feedstock containing 62.5 wt% powders loading was molded at different injection molded temperature. The binders were completely removed from molded components by solvent and thermal debinding at different temperatures. The debinded samples were sintered at 1350°C for 60 min. Mechanical, electrochemical property and biocompatibility of the sintered samples were performed mechanical, electrochemical, SBF immersion tests and cell culture experiments. Results of study showed that sintered 316L and 316L with additives samples exhibited high corrosion properties and biocompatibility in a physiological environment.

Comparative Analysis of the Flavobacterium columnare Genomovar I and II Genomes

Columnaris disease caused by Gram-negative rod Flavobacterium columnare is one of the most common diseases of catfish. F. columnare is also a common problem in other cultured fish species worldwide. F. columnare has three major genomovars; we have sequenced a representative strain from genomovar I (ATCC 49512, which is avirulent in catfish) and genomovar II (94-081, which is highly pathogenic in catfish). Here, we present a comparative analysis of the two genomes. Interestingly, F. columnare ATCC 49512 and 94-081 meet criteria to be considered different species based on the Average Nucleotide Identity (90.71% similar) and DNA–DNA Hybridization (42.6% similar). Genome alignment indicated the two genomes have a large number of rearrangements. However, function-based comparative genomics analysis indicated that the two strains have similar functional capabilities with 2,263 conserved orthologous clusters; strain ATCC 49512 has 290 unique orthologous clusters while strain 94-081 has 391. Both strains carry type I secretion system, type VI secretion system, and type IX secretion system. The two genomes also have similar CRISPR capacities. The F. columnare strain ATCC 49512 genome contains a higher number of insertion sequence families and phage regions, while the F. columnare strain 94-081 genome has more genomic islands and more regulatory gene capacity. Transposon mutagenesis using Tn4351 in pathogenic strain 94-081 yielded six mutants, and experimental infections of fish showed hemolysin and glycine cleavage protein mutants had 15 and 10% mortalities, respectively, while the wild-type strain caused 100% mortalities. Our comparative and mutational analysis yielded important information on classification of genomovars I and II F. columnare as well as potential virulence genes in F. columnare strain 94-081.

Effect of boron addition on injection molded 316L stainless steel: mechanical, corrosion properties and in vitro bioactivity.

The research was investigated the effect of boron additions on sintering characteristics, mechanical, corrosion properties and biocompatibility of injection molded austenitic grade 316L stainless steel. Addition of boron is promoted to get high density of sintered 316L stainless steels. The amount of boron plays a role in determining the sintered microstructure and all properties. In this study, 316L stainless steel powders have been used with the elemental NiB powders. A feedstock containing 62.5 wt% powders loading was molded at different injection molded temperature. The binders were completely removed from molded components by solvent and thermal debinding at different temperature. The debinded samples were sintered at different temperature for 60 min. Mechanical property, microstructural characterization and electrochemical property of the sintered samples were performed using tensile testing, hardness, optical, scanning electron microscopy and electrochemical corrosion experiments. Sintered samples were immersed in a simulated body fluid (SBF) with elemental concentrations that were comparable to those of human blood plasma for a total period of 15 days. Both materials were implanted in fibroblast culture for biocompatibility evaluations were carried out. Results of study showed that sintered 316L and 316L with NiB addition samples exhibited high mechanical and corrosion properties in a physiological environment. Especially, 316L with NiB addition can be used in some bioapplications.

Experimentation and Analysis of Powder Injection Molded Ti10Nb10Zr Alloy: A Promising Candidate for Electrochemical and Biomedical Application

This paper describes the microstructural, mechanical and corrosion properties of injection molded Ti10Nb10Zr alloys. Ti10Nb10Zr powder was injection molded with wax-based binder. The critical powder loading for injection molding was 55 vol% for feedstock. Binder debinding was performed in solvent and thermal method. After debinding the samples were sintered at different temperatures and times in vacuum atmosphere (10−5 mbar) to obtain fully dense parts. Metallographic studies were conducted to determine the extent of densification and the corresponding microstructural changes. The electrochemical property and biocompatibility of the sintered samples were performed electrochemically, by selfbody-fluid immersion tests and cell culture experiments. The results show that Ti10Nb10Zr alloys could be sintered to a maximum 99% of theoretical density. Maximum ultimate tensile strength, elongation and hardness obtained were 748 MPa, 14.3 and 114 HRB respectively at 1500 C for 3 h. Additionally, the sintered Ti10Nb10Zr alloys exhibited high mechanical and corrosion properties in a physiological environment.

Ultrastructural aspects of previtellogenic oocyte growth in hermaphrodite sharpsnout seabream, Diplodus puntazzo (Teleostei, Sparidae)

This paper describes various aspects of previtellogenic oocyte growth in sharpsnout seabream, Diplodus puntazzo , is an important marine culture fish species in the Mediterranean. The ultrastructural characteristics of nuclear morphology, nuclear-cytoplasmic ratio and the starting of the follicle envelope formation were described in detail. These cells do not significantly differ from those of the other teleost species. The ultrastructural aspects provide new information on the reproductive biology of Sparidae.