Identification of virulence factors in Vibrio cholerae isolated from Iraq during the 2007-2009 outbreak

Thousands of people were infected with Vibrio cholerae during the outbreak in Iraq in 2007-2009. Vibrio cholerae was shown to be variable in its content of virulence determinants and in its antibiotic sensitivity. This study was designed to isolate and characterize clinical and environmental V.?cholerae isolates and to determine antibiotic sensitivity, enzyme and toxin production, and the presence of virulence genes. Eighty clinical and five environmental bacterial isolates were collected and diagnosed by subjecting them to microscopic, biochemical, serological, and molecular analysis. The results revealed that 55% of clinical isolates belonged to the Inaba serotype, 32.5% to the Ogawa serotypes, and 12.5% to the Non-O1 serotype. All environmental V.?cholerae isolates belonged to the Non-O1 serotype. All environmental isolates were sensitive to all examined antimicrobial agents, while all clinical isolates showed a high sensitivity (100%) to ampicillin, gentamicin, cephalothin, tetracycline, erythromycin, and ciprofloxacin, and a high resistance (97.5%) to co-trimoxazole, nalidixic acid, and chloramphenicol. It was found that all V.?cholerae (O1) isolates were resistant to the Vibrio static O129 and all Non-O1 V.?cholerae isolates were sensitive to the Vibrio static O129. All clinical and environmental isolates produced hemolysin (100%) and lecithinase (100%), while they showed various production rates of protease (90% of clinical and 60% of environmental) and lipase (50% of clinical and 20% of environmental). The ompW gene was amplified in all the clinical and environmental V.?cholerae isolates, but not in other related and nonrelated bacteria. Multiplex PCR analysis showed that the toxR gene was amplified in all clinical and environmental isolates, while ctxA, ctxB, tcpA genes were amplified only in clinical (O1) isolates. This study indicates the differences in the production of some enzymes and toxins and in the content of virulence genes between clinical and environmental isolates in Iraq during the outbreak (2007-2009).     

A bacterial pathogen uses dimethylsulfoniopropionate as a cue to target heat-stressed corals

Diseases are an emerging threat to ocean ecosystems. Coral reefs, in particular, are experiencing a worldwide decline because of disease and bleaching, which have been exacerbated by rising seawater temperatures. Yet, the ecological mechanisms behind most coral diseases remain unidentified. Here, we demonstrate that a coral pathogen, Vibrio coralliilyticus, uses chemotaxis and chemokinesis to target the mucus of its coral host, Pocillopora damicornis. A primary driver of this response is the host metabolite dimethylsulfoniopropionate (DMSP), a key element in the global sulfur cycle and a potent foraging cue throughout the marine food web. Coral mucus is rich in DMSP, and we found that DMSP alone elicits chemotactic responses of comparable intensity to whole mucus. Furthermore, in heat-stressed coral fragments, DMSP concentrations increased fivefold and the pathogen''s chemotactic response was correspondingly enhanced. Intriguingly, despite being a rich source of carbon and sulfur, DMSP is not metabolized by the pathogen, suggesting that it is used purely as an infochemical for host location. These results reveal a new role for DMSP in coral disease, demonstrate the importance of chemical signaling and swimming behavior in the recruitment of pathogens to corals and highlight the impact of increased seawater temperatures on disease pathways.     

Strategies for enhancing the accumulation and retention of extracellular matrix in tissue-engineered cartilage cultured in bioreactors

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min(-1)) and gradually increasing (0.075-0.2 mL min(-1)) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0-4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8-5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min(-1). GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention.     

Variability in Microbial Community Composition and Function Between Different Niches Within a Coral Reef

To explore how microbial community composition and function varies within a coral reef ecosystem, we performed metagenomic sequencing of seawater from four niches across Heron Island Reef, within the Great Barrier Reef. Metagenomes were sequenced from seawater samples associated with (1) the surface of the coral species Acropora palifera, (2) the surface of the coral species Acropora aspera, (3) the sandy substrate within the reef lagoon and (4) open water, outside of the reef crest. Microbial composition and metabolic function differed substantially between the four niches. The taxonomic profile showed a clear shift from an oligotroph-dominated community (e.g. SAR11, Prochlorococcus, Synechococcus) in the open water and sandy substrate niches, to a community characterised by an increased frequency of copiotrophic bacteria (e.g. Vibrio, Pseudoalteromonas, Alteromonas) in the coral seawater niches. The metabolic potential of the four microbial assemblages also displayed significant differences, with the open water and sandy substrate niches dominated by genes associated with core house-keeping processes such as amino acid, carbohydrate and protein metabolism as well as DNA and RNA synthesis and metabolism. In contrast, the coral surface seawater metagenomes had an enhanced frequency of genes associated with dynamic processes including motility and chemotaxis, regulation and cell signalling. These findings demonstrate that the composition and function of microbial communities are highly variable between niches within coral reef ecosystems and that coral reefs host heterogeneous microbial communities that are likely shaped by habitat structure, presence of animal hosts and local biogeochemical conditions.     

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints

The effect of dynamic mechanical shear and compression on the synthesis of human tissue‐engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA–alginate scaffolds were precultured in shaking T‐flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak‐to‐peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T‐flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3‐ and 10‐fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4‐fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue‐engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Biotechnol. Bioeng. 2012; 109:1060–1073. © 2011 Wiley Periodicals, Inc.     

Improved seeding of chondrocytes into polyglycolic acid scaffolds using semi-static and alginate loading methods

Cell seeding and attachment in three-dimensional scaffolds is a key step in tissue engineering with implications for cell differentiation and tissue development. In this work, two new seeding methods were investigated using human chondrocytes and polyglycolic acid (PGA) fibrous mesh scaffolds. A simple semi-static seeding method using culture plates and tissue flasks was developed as an easy-to-perform modification of static seeding. An alginate-loading method was also studied, using alginate hydrogel as an adjuvant for entrapping cells within PGA scaffolds. Both the semi-static and PGA-alginate methods produced more homogeneous cell distributions than conventional static and dynamic seeding. Using 20 × 10(6) cells, whereas the seeding efficiency for static seeding was only 52%, all other techniques produced seeding efficiencies of ≥ 90%. With 40 × 10(6) cells, the efficiency of semi-static seeding declined to 74% while the dynamic and PGA-alginate methods retained their ability to accommodate high cell numbers. The seeded scaffolds were cultured in recirculation bioreactors to determine the effect of seeding method on cartilage production. Statically seeded scaffolds did not survive the 5-week cultivation period. Deposition of extracellular matrix in scaffolds seeded using the semi-static and PGA-alginate methods was more uniform compared with scaffolds seeded using the dynamic method. The new semi-static and PGA-alginate seeding methods developed in this work are recommended for tissue engineering because they provide substantial benefits compared with static seeding in terms of seeding efficiency, cell distribution, and cartilage deposition while remaining simple and easy to execute.     

Targeting Wnt/tenascin C-mediated cross talk between pancreatic cancer cells and stellate cells via activation of the metastasis suppressor NDRG1

A major barrier to successful pancreatic cancer (PC) treatment is the surrounding stroma, which secretes growth factors/cytokines that promote PC progression. Wnt and tenascin C (TnC) are key ligands secreted by stromal pancreatic stellate cells (PSCs) that then act on PC cells in a paracrine manner to activate the oncogenic β-catenin and YAP/TAZ signaling pathways. Therefore, therapies targeting oncogenic Wnt/TnC cross talk between PC cells and PSCs constitute a promising new therapeutic approach for PC treatment. The metastasis suppressor N-myc downstream-regulated gene-1 (NDRG1) inhibits tumor progression and metastasis in numerous cancers, including PC. We demonstrate herein that targeting NDRG1 using the clinically trialed anticancer agent di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) inhibited Wnt/TnC-mediated interactions between PC cells and the surrounding PSCs. Mechanistically, NDRG1 and DpC markedly inhibit secretion of Wnt3a and TnC by PSCs, while also attenuating Wnt/β-catenin and YAP/TAZ activation and downstream signaling in PC cells. This antioncogenic activity was mediated by direct inhibition of β-catenin and YAP/TAZ nuclear localization and by increasing the Wnt inhibitor, DKK1. Expression of NDRG1 also inhibited transforming growth factor (TGF)-β secretion by PC cells, a key mechanism by which PC cells activate PSCs. Using an in vivo orthotopic PC mouse model, we show DpC downregulated β-catenin, TnC, and YAP/TAZ, while potently increasing NDRG1 expression in PC tumors. We conclude that NDRG1 and DpC inhibit Wnt/TnC-mediated interactions between PC cells and PSCs. These results further illuminate the antioncogenic mechanism of NDRG1 and the potential of targeting this metastasis suppressor to overcome the oncogenic effects of the PC-PSC interaction.     

The discomfort index,mortality and the London summers of 1976 and 1978

The Discomfort Index (DI), and its associated heat load categories as worked out for conditions in Israel, was used in a study of the summer months of 1976 and 1978 in London. The cool summer of 1978 presented no heat load problems but the exceptionally warm summer of 1976, especially the period between 22 June and 9 July, produced several days of moderate heat load conditions. During this hot spell mortality from ischaemic heart disease, cerebrovascular accidents and respiratory disease all increased substantially. It is suggested that the heat load categories, although rarely attained, would be useful in predicting danger periods during heatwave conditions in the United Kingdom.     

Partially local three-way alignments and the sequence signatures of mitochondrial genome rearrangements

Background

Genomic DNA frequently undergoes rearrangement of the gene order that can be localized by comparing the two DNA sequences. In mitochondrial genomes different mechanisms are likely at work, at least some of which involve the duplication of sequence around the location of the apparent breakpoints. We hypothesize that these different mechanisms of genome rearrangement leave distinctive sequence footprints. In order to study such effects it is important to locate the breakpoint positions with precision.

Results

We define a partially local sequence alignment problem that assumes that following a rearrangement of a sequence F, two fragments L, and R are produced that may exactly fit together to match F, leave a gap of deleted DNA between L and R, or overlap with each other. We show that this alignment problem can be solved by dynamic programming in cubic space and time. We apply the new method to evaluate rearrangements of animal mitogenomes and find that a surprisingly large fraction of these events involved local sequence duplications.

Conclusions

The partially local sequence alignment method is an effective way to investigate the mechanism of genomic rearrangement events. While applied here only to mitogenomes there is no reason why the method could not be used to also consider rearrangements in nuclear genomes.

     

Towards a comprehensive picture of alloacceptor tRNA remolding in metazoan mitochondrial genomes

Remolding of tRNAs is a well-documented process in mitochondrial genomes that changes the identity of a tRNA. It involves a duplication of a tRNA gene, a mutation that changes the anticodon and the loss of the ancestral tRNA gene. The net effect is a functional tRNA that is more closely related to tRNAs of a different alloacceptor family than to tRNAs with the same anticodon in related species. Beyond being of interest for understanding mitochondrial tRNA function and evolution, tRNA remolding events can lead to artifacts in the annotation of mitogenomes and thus in studies of mitogenomic evolution. Therefore, it is important to identify and catalog these events. Here we describe novel methods to detect tRNA remolding in large-scale data sets and apply them to survey tRNA remolding throughout animal evolution. We identify several novel remolding events in addition to the ones previously mentioned in the literature. A detailed analysis of these remoldings showed that many of them are derived from ancestral events.