SNP genotyping on pooled DNAs: comparison of genotyping technologies and a semi automated method for data storage and analysis

We have compared the accuracy, efficiency and robustness of three methods of genotyping single nucleotide polymorphisms on pooled DNAs. We conclude that (i) the frequencies of the two alleles in pools should be corrected with a factor for unequal allelic amplification, which should be estimated from the mean ratio of a set of heterozygotes (k); (ii) the repeatability of an assay is more important than pinpoint accuracy when estimating allele frequencies, and assays should therefore be optimised to increase the repeatability; and (iii) the size of a pool has a relatively small effect on the accuracy of allele frequency estimation. We therefore recommend that large pools are genotyped and replicated a minimum of four times. In addition, we describe statistical approaches to allow rigorous comparison of DNA pool results. Finally, we describe an extension to our ACeDB database that facilitates management and analysis of the data generated by association studies.     

Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli

Applications of genetic engineering or metabolic engineering have increased in both academic and industrial institutions. Most current metabolic engineering studies have focused on enzyme levels and on the effect of the amplification, addition, or deletion of a particular pathway. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. It is conceivable that in cofactor-dependent production systems, cofactor availability and the proportion of cofactor in the active form may play an important role in dictating the overall process yield. Hence, the manipulation of these cofactor levels may be crucial in order to further increase production. We have demonstrated that manipulation of cofactors can be achieved by external and genetic means and these manipulations have the potential to be used as an additional tool to achieve desired metabolic goals. We have shown experimentally that the NADH/NAD(+) ratio can be altered by using carbon sources with different oxidation states. We have shown further that the metabolite distribution can be influenced by a change in the NADH/NAD(+) ratio as mediated by the oxidation state of the carbon source used. We have also demonstrated that the total NAD(H/(+)) levels can be increased by the overexpression of the pncB gene. The increase in the total NAD(H/(+)) levels can be achieved even in a complex medium, which is commonly used by most industrial processes. Finally, we have shown that manipulation of the CoA pool/flux can be used to increase the productivity of a model product, isoamyl acetate.     

A Toolkit for ARB to Integrate Custom Databases and Externally Built Phylogenies

Researchers are perpetually amassing biological sequence data. The computational approaches employed by ecologists for organizing this data (e.g. alignment, phylogeny, etc.) typically scale nonlinearly in execution time with the size of the dataset. This often serves as a bottleneck for processing experimental data since many molecular studies are characterized by massive datasets. To keep up with experimental data demands, ecologists are forced to choose between continually upgrading expensive in-house computer hardware or outsourcing the most demanding computations to the cloud. Outsourcing is attractive since it is the least expensive option, but does not necessarily allow direct user interaction with the data for exploratory analysis. Desktop analytical tools such as ARB are indispensable for this purpose, but they do not necessarily offer a convenient solution for the coordination and integration of datasets between local and outsourced destinations. Therefore, researchers are currently left with an undesirable tradeoff between computational throughput and analytical capability. To mitigate this tradeoff we introduce a software package to leverage the utility of the interactive exploratory tools offered by ARB with the computational throughput of cloud-based resources. Our pipeline serves as middleware between the desktop and the cloud allowing researchers to form local custom databases containing sequences and metadata from multiple resources and a method for linking data outsourced for computation back to the local database. A tutorial implementation of the toolkit is provided in the supporting information, S1 Tutorial. Availability: http://www.ece.drexel.edu/gailr/EESI/tutorial.php.     

Halobacteriovorax,an underestimated predator on bacteria: potential impact relative to viruses on bacterial mortality

Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients.     

Vibrio parahaemolyticus and Vibrio vulnificus in South America: water,seafood and human infections

The bacterial species, Vibrio parahaemolyticus and Vibrio vulnificus, are ubiquitous in estuaries and coastal waters throughout the world, but they also happen to be important human pathogens. They are concentrated by filter‐feeding shellfish which are often consumed raw or undercooked, providing an important potential route of entry for an infective dose of these bacteria. Vibrio parahaemolyticus can cause abdominal cramping, nausea, diarrhoea, vomiting, chills and fever. Vibrio vulnificus can cause similar gastrointestinal‐related symptoms, but can also spread to the bloodstream, resulting in primary septicaemia, and it can also cause disease via wound infections. The objective of this article is to summarize, for the first time, the incidence and importance of V. parahaemolyticus and V. vulnificus in South America, in environmental waters and seafood, especifically molluscan shellfish, as well as human infection cases and outbreaks. It appears that infections from V. parahaemolyticus have been more strongly related to shellfish ingestion and have been more frequently reported on the Pacific coast of South America. Conversely, V. vulnificus has been more frequently acquired by water contact with open wounds and its presence has been more heavily reported along the Atlantic coast of South America, and while documented to cause serious mortality, have been relatively few in number. The impacts of El Nino Southern Oscillation (ENSO) have been observed to cause an increase in V. parahaemolyticus outbreaks on the Pacific coast of South America. The implementation of a regulated monitoring approach, along with the use of faster, more accurate and virulence‐specific detection approaches, such as PCR confirmation, should be considered to detect the presence of pathogenic Vibrio strains in environmental and seafood samples for protection of public health. Furthermore, improved clinical surveillance with suspected cases should be implemented. This review highlights the need for more research and monitoring of vibrios in South America, in water, shellfish and clinical samples.     

Resolvin E1 regulates inflammation at the cellular and tissue level and restores tissue homeostasis in vivo

Resolvin E1 (RvE1) is a potent proresolving mediator of inflammation derived from omega-3 eicosapentaenoic acid that acts locally to stop leukocyte recruitment and promote resolution. RvE1 displays potent counter-regulatory and tissue-protective actions in vitro and in vivo. Periodontal disease is a local inflammatory disease initiated by bacteria characterized by neutrophil-mediated tissue injury followed by development of a chronic immune lesion. In this study, we report the treatment of established periodontitis using RvE1 as a monotherapy in rabbits compared with structurally related lipids PGE(2) and leukotriene B(4). PGE(2) and leukotriene B(4) each enhanced development of periodontitis and worsened the severity of disease. Promotion of resolution of inflammation as a therapeutic target with RvE1 resulted in complete restoration of the local lesion, and reduction in the systemic inflammatory markers C-reactive protein and IL-1beta. This report is the first to show that resolution of inflammation by a naturally occurring endogenous lipid mediator results in complete regeneration of pathologically lost tissues, including bone.     

Soil bacterial changes upon snowmelt: laboratory studies of the effects of early and late meltwater fractions

The effects of polluted and leached snowmelt waters on an upland soil bacterial community were determined in laboratory soil cores using simulated solutions. 24 isolate characters were determined before, and after, 32 days of solution application.The soil bacterial community exposed to polluted meltwaters (pH 2.3) showed a significant increase in its ability to metabolise the carbohydrates tested, and a significant decrease in the number of Gran-positive coccal forms. The percentage of Micrococcus isolates declined from 27% of the community to 2%, whilst Pseudomonas and Enterobacteriaceae isolates increased in number.The soil bacterial community exposed to leached snowmelt waters (pH 5.4) showed a significant increase in biochemical potential, but the ability to metabolise carbohydrates showed no change.     

Modification of BRCA1-associated breast cancer risk by the polymorphic androgen-receptor CAG repeat

Compared with the general population, women who have inherited a germline mutation in the BRCA1 gene have a greatly increased risk of developing breast cancer. However, there is also substantial interindividual variability in the occurrence of breast cancer among BRCA1 mutation carriers. We hypothesize that other genes, particularly those involved in endocrine signaling, may modify the BRCA1-associated age-specific breast cancer risk. We studied the effect of the CAG repeat-length polymorphism found in exon 1 of the androgen-receptor (AR) gene (AR-CAG). AR alleles containing longer CAG repeat lengths are associated with a decreased ability to activate androgen-responsive genes. Using a sample of women who inherited germline BRCA1 mutations, we compared AR-CAG repeat length in 165 women with and 139 women without breast cancer. We found that women were at significantly increased risk of breast cancer if they carried at least one AR allele with >/=28 CAG repeats. Women who carried an AR-CAG allele of >/=28, >/=29, or >/=30 repeats were given a diagnosis 0.8, 1.8, or 6.3 years earlier than women who did not carry at least one such allele. All 11 women in our sample who carried at least one AR-CAG allele with >/=29 repeats had breast cancer. Our results support the hypothesis that age at breast cancer diagnosis is earlier among BRCA1 mutation carriers who carry very long AR-CAG repeats. These results suggest that pathways involving androgen signaling may affect the risk of BRCA1-associated breast cancer.