Development of a vaccine for Chlamydia trachomatis: Challenges and current progress

Chlamydia trachomatis remains an enigmatic bacterial pathogen with no vaccine yet available to treat human ocular and genital tract infections caused by tissue-tropic serovars of the organism. Globally, it is the leading cause of preventable blindness as well as the leading cause of bacterial sexually transmitted infections. The pathogen has a range of virulence factors that enable it to successfully evade both the innate and adaptive immune system of the host. The host immune system, although protective, paradoxically is also associated closely with the pathologies of trachoma and pelvic inflammatory disease – disease sequelae of some chlamydial infections and reinfections in some genetically susceptible hosts. In this review, we focus on what is known currently about the pathogenesis of ocular and genital infections caused by this mucosal pathogen. We also discuss novel insights into the pathogenesis of infections caused by the genital and ocular serovars of C. trachomatis, including a discussion of both pathogen and host factors, such as the human microbiota at these mucosal sites as well as the current immunological challenges facing vaccine development. Finally, we discuss the current progress toward development of a vaccine against C. trachomatis. A wide range of recombinant protein antigens are being identified and, hence, are available for vaccine trials. A plasmid-free live strain has recently been produced and evaluated in the mouse (Chlamydia muridarum) and monkey (C. trachomatis) models. The data for ocular infections in the monkey model was particularly encouraging, although the path to regulatory approval of a live vaccine is still uncertain. While still a major challenge, vaccines for ocular and genital C. trachomatis infections are looking more promising.

Verticillium dahliae (defoliating strain) - cotton, vegetables

DAFF scholarship to train in the recognition and identification of defoliating strains of Verticillium dahliae in cotton and vegetable crops.

Inheritance and relative dominance, expressed as toxicity response and delayed development, of phosphine resistance in immature stages of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

Fumigation of stored grain with phosphine (PH 3) is used widely to control the lesser grain borer Rhyzopertha dominica. However, development of high level resistance to phosphine in this species threatens control. Effective resistance management relies on knowledge of the expression of resistance in relation to dosage at all life stages. Therefore, we determined the mode of inheritance of phosphine resistance and strength of the resistance phenotype at each developmental stage. We achieved this by comparing mortality and developmental delay between a strongly resistant strain (R-strain), a susceptible strain (S-strain) and their F 1 progenies. Resistance was a maternally inherited, semi-dominant trait in the egg stage but was inherited as an autosomal, incompletely recessive trait in larvae and pupae. The rank order of developmental tolerance in both the sensitive and resistant strains was eggs > pupae > larvae. Comparison of published values for the response of adult R. dominica relative to our results from immature stages reveals that the adult stage of the S-strain is more sensitive to phosphine than are larvae. This situation is reversed in the R-strain as the adult stage is much more resistant to phosphine than even the most tolerant immature stage. Phosphine resistance factors at LC 50 were eggs 400×, larvae 87× and pupae 181× with respect to reference susceptible strain (S-strain) adults indicating that tolerance conferred by a particular immature stage neither strongly nor reliably interacts with the genetic resistance element. Developmental delay relative to unfumigated control insects was observed in 93% of resistant pupae, 86% of resistant larvae and 41% of resistant eggs. Increased delay in development and the toxicity response to phosphine exposure were both incompletely recessive. We show that resistance to phosphine has pleiotropic effects and that the expression of these effects varies with genotype and throughout the life history of the insect. © 2012.

Characterization of genomic diversity in extraintestinal pathogenic Escherichia coli (ExPEC) and development of a diagnostic DNA microarray for the differentiation of ExPEC isolates causing urinary tract infections

Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Thin Film Strain Gauges--an Overview

This review gives a brief description of the historical development followed by the origin and the principle of operation of strain gauges. The features of an ideal strain gauge for measurement purposes and the general classes of strain gauges are given. The remaning part is devoted to an important development in strain gauge technology, namely thin film strain gauges. After highlighting the advantages of thin film strain gauges, a review of current data is given. Detailed description of metallic thin film strain gauges is provided and avaliable information on alloy semiconductor and cermet films for their possible use as strain gauge elements has also been included. The importance of ion implantation in tailoring the properties of strain gauges is highlighted. 33 ref.--AA

Characterization of a mercury-reducing Bacillus cereus strain isolated from the Pulicat Lake sediments, south east coast of India

Pulicat Lake sediments are often severely polluted with the toxic heavy metal mercury. Several mercury-resistant strains of Bacillus species were isolated from the sediments and all the isolates exhibited broad spectrum resistance (resistance to both organic and inorganic mercuric compounds). Plasmid curing assay showed that all the isolated Bacillus strains carry chromosomally borne mercury resistance. Polymerase chain reaction and southern hybridization analyses using merA and merB3 gene primers/probes showed that five of the isolated Bacillus strains carry sequences similar to known merA and merB3 genes. Results of multiple sequence alignment revealed 99% similarity with merA and merB3 of TnMERI1 (class II transposons). Other mercury resistant Bacillus species lacking homology to these genes were not able to volatilize mercuric chloride, indicating the presence of other modes of resistance to mercuric compounds.

Characterization of a mercury-reducing Bacillus cereus strain isolated from the Pulicat Lake sediments, south east coast of India

Pulicat Lake sediments are often severely polluted with the toxic heavy metal mercury. Several mercury-resistant strains of Bacillus species were isolated from the sediments and all the isolates exhibited broad spectrum resistance (resistance to both organic and inorganic mercuric compounds). Plasmid curing assay showed that all the isolated Bacillus strains carry chromosomally borne mercury resistance. Polymerase chain reaction and southern hybridization analyses using merA and merB3 gene primers/probes showed that five of the isolated Bacillus strains carry sequences similar to known merA and merB3 genes. Results of multiple sequence alignment revealed 99% similarity with merA and merB3 of TnMERI1 (class II transposons). Other mercury resistant Bacillus species lacking homology to these genes were not able to volatilize mercuric chloride, indicating the presence of other modes of resistance to mercuric compounds.

Hot deformation characteristics of INCONEL alloy MA 754 and development of a processing map

The characteristics of hot deformation of INCONEL alloy MA 754 have been studied processing maps obtained on the basis of flow stress data generated in compression in the temperature range 700-degrees-C to 1150-degrees-C and strain rate range 0.001 to 100 s-1. The map exhibited three domains. (1) A domain of dynamic recovery occurs in the temperature range 800-degrees-C to 1075-degrees-C and strain rate range 0.02 to 2 s-1, with a peak efficiency of 18 pct occurring at 950-degrees-C and 0.1 s-1. Transmission electron microscope (TEM) micrographs revealed stable subgrain structure in this domain with the subgrain size increasing exponentially with an increase in temperature. (2) A domain exhibiting grain boundary cracking occurs at temperatures lower than 800-degrees-C and strain rates lower than 0.01 s-1. (3) A domain exhibiting intense grain boundary cavitation occurs at temperatures higher than 1075-degrees-C. The material did not exhibit a dynamic recrystallization (DRX) domain, unlike other superalloys. At strain rates higher than about 1 s-1, the material exhibits flow instabilities manifesting as kinking of the elongated grains and adiabatic shear bands. The material may be safely worked in the domain of dynamic recovery but can only be statically recrystallized.

Microstructure Development, Nanomechanical, and Dynamic Compression Properties of Spark Plasma Sintered TiB2-Ti-Based Homogeneous and Bi-layered Composites

The growing threats due to increased use of small-caliber armor piercing projectiles demand the development of new light-weight body armor materials. In this context, TiB2 appears to be a promising ceramic material. However, poor sinterability and low fracture toughness remain two major issues for TiB2. In order to address these issues together, Ti as a sinter-aid is used to develop TiB2-(x wt pct Ti), (x = 10, 20) homogeneous composites and a bi-layered composite (BLC) with each layer having Ti content of 10 and 20 wt pct. The present study uniquely demonstrates the efficacy of two-stage spark plasma sintering route to develop dense TiB2-Ti composites with an excellent combination of nanoscale hardness (similar to 36 GPa) and indentation fracture toughness (similar to 12 MPa m(1/2)). In case of BLC, these properties are not compromised w.r.t. homogeneous composites, suggesting the retention of baseline material properties even in the bi-layer design due to optimal relief of residual stresses. The better indentation toughness of TiB2-(10 wt pct Ti) and TiB2-(20 wt pct Ti) composites can be attributed to the observed crack deflection/arrest, indicating better damage tolerance. Transmission electron microscope investigation reveals the presence of dense dislocation networks and deformation twins in alpha-Ti at the grain boundaries and triple pockets, surrounded by TiB2 grains. The dynamic strength of around 4 GPa has been measured using Split Hopkinson Pressure Bar tests in a reproducible manner at strain rates of the order of 600 s(-1). The damage progression under high strain rate has been investigated by acquiring real time images for the entire test duration using ultra-high speed imaging. An attempt has been made to establish microstructure-property correlation and a simple analysis based on Mohr-Coulomb theory is used to rationalize the measured strength properties.

New insights into the development of microstructure and deformation texture in nickel-60 wt.% cobalt alloy

The present study investigates the critical role of deformation twinning and Bs-type shear bands in the evolution of deformation texture in a low stacking fault energy Ni-60Co alloy up to very large rolling strain (epsilon(t) approximate to 4). The alloy develops a strong brass-type rolling texture, and its formation is initiated at the early stages of deformation. Extensive twinning is observed at the intermediate stages of deformation, which causes significant texture reorientation towards alpha-fiber. A pseudo-in-situ electron back-scattered diffraction technique adopted to capture orientation changes within individual grains during the early stages suggests that twinning should be subsequently aided by crystallographic slip to attain alpha-fiber (< 1 1 0 >parallel to ND) orientations. Beyond 40% reduction, deformation is dominated by Bs-type shear bands, and the banding coincides with the evolution of < 1 1 1 >parallel to ND components. The volume fraction of shear bands is significant at higher strains, and crystallites within the bands preferentially show < 1 1 0 >parallel to ND components. The absence of the Cu {1 1 2}< 1 1 1 > component in the initial texture, and subsequently during rolling, indicates that, for the evolution of a brass-type texture, the presence of the Cu component is not a necessary condition. The final rolling texture is a synergistic effect of deformation twinning and shear banding. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Roles of the troponin isoforms during indirect flight muscle development in Drosophila

Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, a majority of the myofibrillar proteins in the indirect flight muscles (IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform.

Development of alloys with high strength at elevated temperatures by tuning the bimodal microstructure in the Al-Cu-Ni eutectic system

The development of high-strength aluminum alloys that can operate at 250 degrees C and beyond remains a challenge to the materials community. In this paper we report preliminary development of nanostructural Al-Cu-Ni ternary alloys containing alpha-Al, binary Al2Cu and ternary Al2Cu4Ni intermetallics. The alloys exhibits fracture strength of similar to 1 GPa with similar to 9% fracture strain at room temperature. At 300 degrees C, the alloy retains the high strength. The reasons for such significant mechanical properties are rationalized by unraveling the roles and response of various microstructural features. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Influence of external weld flash on the in-plane plane-strain formability of friction stir welded sheets

The main aim of the present work is to analyze the influence of external weld flash on the formability of friction stir welding sheets through in-plane plane-strain formability tests. The load-extension behavior and forming limit strains are measured to quantify the formability. The influence of friction stir welding parameters on the height of weld flash was also studied. The base materials used for welding trials are AA6061T6 and AA5052H32 alloy sheets of 2.1-mm thickness. It is observed that the influence of external weld flash on the maximum load and total extension for all the friction stir welding conditions is negligible. The effect of weld flash on the limiting major strain is also insignificant. But the presence of weld flash has changed the limiting minor strain, more toward plane-strain condition, indicating the change in strain-path toward plane-strain. This is due to the strain taken by weld flash, along with the major strain, minor strain, and thickness strain in the friction stir welding sheet plane because of constancy of volume. The formation of weld flash and its height are affected synergistically by the axial force and temperature development during friction stir welding. The higher the axial force and temperature, the higher the flash height.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development

A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'- region, named sopB4 and sopB5, were each fused to the grIpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ApmrG-H111-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-y, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Thl response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.