Seq4SNPs: new software for retrieval of multiple, accurately annotated DNA sequences, ready formatted for SNP assay design

Background  

In moderate-throughput SNP genotyping there was a gap in the workflow, between choosing a set of SNPs and submitting their sequences to proprietary assay design software, which was not met by existing software. Retrieval and formatting of sequences flanking each SNP, prior to assay design, becomes rate-limiting for more than about ten SNPs, especially if annotated for repetitive regions and adjacent variations. We routinely process up to 50 SNPs at once.     

Liver asialoglycoprotein receptor levels correlate with severity of alcoholic liver damage in rats.

It has been demonstrated that the oral administration of ethanol (Lieber-DeCarli liquid diet) to rats results in a decreased expression and content of the asialoglycoprotein receptor (ASGP-R) in the resultant fatty liver. In the present study, we wanted to determine whether the extent of impaired receptor content was correlated with the severity of liver pathology by using the intragastric feeding model. When ASGP-R protein and mRNA levels were measured in animals infused with ethanol or dextrose in the presence of fish oil (FO) or medium-chain triglyceride as the source of fat, more significant impairments to the ASGP-R were observed in the FO-ethanol group compared with the medium-chain triglyceride-ethanol group. Furthermore, only the FO-ethanol group showed pathological liver changes. These results demonstrate that a correlation exists between the progression of alcohol-associated liver injury, as defined by the severity of liver pathology, and an ethanol-induced decline in ASGP-R content.     

A case of spontaneous coronary artery disease regression

Coronary angiographic trials have demonstrated that lowering cholesterol can slow the progression of atherosclerosis, limit the formation of new lesions and enhance atherosclerotic regression together with reducing the incidence of clinical events (Waters D, 1996). Spontaneous regression of coronary atherosclerotic lesions is rare. We report the case of a patient with a severe within-stent restenotic lesion whose coronary disease spontaneously regressed 12 months after initial diagnosis, allowing for medical treatment of symptoms rather than repeated intervention. (Int J Cardiovasc Interventions 1999; 2: 121-123)     

Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.      

Maintenance of the virulence plasmid in Shigella flexneri is influenced by Lon and two functional partitioning systems

Members of the genus Shigella carry a large plasmid, pINV, which is essential for virulence. In Shigella flexneri, pINV harbours three toxin‐antitoxin (TA) systems, CcdAB, GmvAT and VapBC that promote vertical transmission of the plasmid. Type II TA systems, such as those on pINV, consist of a toxic protein and protein antitoxin. Selective degradation of the antitoxin by proteases leads to the unopposed action of the toxin once genes encoding a TA system have been lost, such as following failure to inherit a plasmid harbouring a TA system. Here, we investigate the role of proteases in the function of the pINV TA systems and demonstrate that Lon, but not ClpP, is required for their activity during plasmid stability. This provides the first evidence that acetyltransferase family TA systems, such as GmvAT, can be regulated by Lon. Interestingly, S. flexneri pINV also harbours two putative partitioning systems, ParAB and StbAB. We show that both systems are functional for plasmid maintenance although their activity is masked by other systems on pINV. Using a model vector based on the pINV replicon, we observe temperature‐dependent differences between the two partitioning systems that contribute to our understanding of the maintenance of virulence in Shigella species.     

Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention

To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.     

Penaeid and carid community changes in the St Lucia estuarine lake system,South Africa,under low water level,extended closed periods and marine reconnection conditions

Penaeid and carid communities were assessed in St Lucia over a wide range of saline conditions, water level conditions and mouth states, including a six-month marine connection and various connections to the Mfolozi Estuary. Samples were collected biannually in spring and autumn from November 2004 to May 2012 using seine nets. Seven penaeid and seven carid species were recorded, of which Palaemon pacificus, P. peringueyi, Penaeus indicus and Metapenaeus monoceros were the most abundant. The decline from 2004 to 2007 in both species recorded and densities of freshwater, estuarine and marine species during the initial closed period was linked to increased salinity, lack of recruitment and reduced estuarine surface area. Increased wave action and high seas following Cyclone Gamede opened the mouth in March 2007 after a 57-month closed period, resulting in recruitment of marine species, predominantly P. indicus, followed by a decline in densities after reclosure in August 2007. Increases in marine and freshwater species were evident after Mfolozi flood connections during 2008, 2009 and 2010. Highest densities were recorded after breaching in 2007 and after flood connections, highlighting the importance of a marine link to maintain the recruitment of penaeid species.     

Controls of Nucleosome Positioning in the Human Genome

Nucleosomes are important for gene regulation because their arrangement on the genome can control which proteins bind to DNA. Currently, few human nucleosomes are thought to be consistently positioned across cells; however, this has been difficult to assess due to the limited resolution of existing data. We performed paired-end sequencing of micrococcal nuclease-digested chromatin (MNase–seq) from seven lymphoblastoid cell lines and mapped over 3.6 billion MNase–seq fragments to the human genome to create the highest-resolution map of nucleosome occupancy to date in a human cell type. In contrast to previous results, we find that most nucleosomes have more consistent positioning than expected by chance and a substantial fraction (8.7%) of nucleosomes have moderate to strong positioning. In aggregate, nucleosome sequences have 10 bp periodic patterns in dinucleotide frequency and DNase I sensitivity; and, across cells, nucleosomes frequently have translational offsets that are multiples of 10 bp. We estimate that almost half of the genome contains regularly spaced arrays of nucleosomes, which are enriched in active chromatin domains. Single nucleotide polymorphisms that reduce DNase I sensitivity can disrupt the phasing of nucleosome arrays, which indicates that they often result from positioning against a barrier formed by other proteins. However, nucleosome arrays can also be created by DNA sequence alone. The most striking example is an array of over 400 nucleosomes on chromosome 12 that is created by tandem repetition of sequences with strong positioning properties. In summary, a large fraction of nucleosomes are consistently positioned—in some regions because they adopt favored sequence positions, and in other regions because they are forced into specific arrangements by chromatin remodeling or DNA binding proteins.