The Genome of Thermosipho africanus TCF52B: Lateral Genetic Connections to the Firmicutes and Archaea

Lateral gene transfers (LGT) (also called horizontal gene transfers) have been a major force shaping the Thermosipho africanus TCF52B genome, whose sequence we describe here. Firmicutes emerge as the principal LGT partner. Twenty-six percent of phylogenetic trees suggest LGT with this group, while 13% of the open reading frames indicate LGT with Archaea.Thermosipho africanus TCF52B was isolated from produced fluids of a high-temperature oil reservoir in the North Sea using fish waste as the only substrate (4). Phylogenetic analyses based on the 16S rRNA gene sequence and DNA-DNA hybridization placed it as a strain of Thermosipho africanus, which was first isolated from a shallow marine hydrothermal system in Djibouti, Africa (8, 21).The complete genome sequence of this strain was determined by the conventional whole-genome shotgun strategy. Genomic libraries containing 1- to 4-kb and 40-kb fragments were constructed, and sequence chromatograms were produced using a MegaBACE 1000 capillary DNA sequencer (GE Healthcare). Nucleotide skews were computed as described previously (11). Automated open reading frame (ORF) identification and annotation were performed using the annotation software Manatee made available by TIGR (23). Pseudogenes were identified by doing BLAST searches of neighboring ORFs with the same or similar annotations and by using the program Psi-phi (9, 10), and clustered regularly interspaced short palindromic repeat loci (CRISPRs) were identified using the web site http://crispr.u-psud.fr/crispr/CRISPRHomePage.php with the default parameters (6). Maximum-likelihood (ML) trees (WAG [Γ+Ι model, four categories]) were constructed from protein-coding ORFs using PHYML and the PhyloGenie package (5). Recently, several Thermotogales genomes have become available in GenBank. As these genomes had not been published yet, we did not include them in any “genome-scale” analyses (i.e., the phylogenetic analyses). We did, however, include them in the BLAST analyses of mobile Thermosipho africanus genes.The genome of Thermosipho africanus strain TCF52B is a single circular chromosome consisting of 2,016,657 bp with an average G+C content of 30.8%. Strand asymmetries, such as GC skew and tetramer skews, are pronounced and show two clear singularity points, located at roughly 8 kb and 1033 kb from the +1 site (see Fig. S1 in the supplemental material). Since these two points are diametrically opposed on the circular chromosome, dividing it into two halves with opposite compositional skews, they make good candidates for the putative origin and termination of replication. The 1,033-kb region is likely to harbor the origin, since GC skew becomes positive past this location, as in most bacterial genomes with a known origin.The genome contains 2,000 potential coding sequences, of which 1913 are putative protein-coding ORFs, 30 are putatively assigned as pseudogenes, and 57 encode RNA. A comparison to the genome of Thermotoga maritima is given in Table ​Table1.1. The Thermosipho africanus genome is about 156 kb larger than the Thermotoga maritima genome and carries 36 more ORFs. The genome contains duplicated regions comprising paralogous gene copies, CRISPRs, and mobile genetic elements, which collectively provide considerable indirect evidence for genomic instability and acquisition of exogenous genetic information.

TABLE 1.

General features of the Thermosipho africanus genome, with a comparison to Thermotoga maritima

Analysis of a diverse global Pisum sp. collection and comparison to a Chinese local P. sativum collection with microsatellite markers

Twenty-one informative microsatellite loci were used to assess and compare the genetic diversity among Pisum genotypes sourced from within and outside China. The Chinese germplasm comprised 1243 P. sativum genotypes from 28 provinces and this was compared to 774 P. sativum genotypes that represented a globally diverse germplasm collection, as well as 103 genotypes from related Pisum species. The Chinese P. sativum germplasm was found to contain genotypes genetically distinct from the global gene pool sourced outside China. The Chinese spring type genotypes were separate from the global gene pool and from the other main Chinese gene pool of winter types. The distinct Chinese spring gene pool comprised genotypes from Inner Mongolia and Sha'anxi provinces, with those from Sha'anxi showing the greatest diversity. The other main gene pool within China included both spring types from other northern provinces and winter types from central and southern China, plus some accessions from Inner Mongolia and Sha'anxi. A core collection of Chinese landraces chosen to represent molecular diversity was compared both to the wider Chinese collection and to a geographically diverse core collection of Chinese landraces. The average gene diversity and allelic richness per locus of both the micro-satellite based core and the wider collection were similar, and greater than the geographically diverse core. The genetic diversity of P. sativum within China appears to be quite different to that detected in the global gene pool, including the presence of several rare alleles, and may be a useful source of allelic variation for both major gene and quantitative traits.     

Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berries

Background  

Fresh fruits are well accepted as a good source of the dietary antioxidant ascorbic acid (Asc, Vitamin C). However, fruits such as grapes do not accumulate exceptionally high quantities of Asc. Grapes, unlike most other cultivated fruits do however use Asc as a precursor for the synthesis of both oxalic (OA) and tartaric acids (TA). TA is a commercially important product in the wine industry and due to its acidifying effect on crushed juice it can influence the organoleptic properties of the wine. Despite the interest in Asc accumulation in fruits, little is known about the mechanisms whereby Asc concentration is regulated. The purpose of this study was to gain insights into Asc metabolism in wine grapes (Vitis vinifera c.v. Shiraz.) and thus ascertain whether the developmental demand for TA and OA synthesis influences Asc accumulation in the berry.     

Trends in loss to follow-up among migrant workers on antiretroviral therapy in a community cohort in Lesotho

Background

The provision of antiretroviral therapy (ART) to migrant populations raises particular challenges with respect to ensuring adequate treatment support, adherence, and retention in care. We assessed rates of loss to follow-up for migrant workers compared with non-migrant workers in a routine treatment programme in Morjia, Lesotho.

Design

All adult patients (≥18 years) initiating ART between January 1, 2008, and December 31, 2008, and followed up until the end of 2009, were included in the study. We described rates of loss to follow-up according to migrant status by Kaplan-Meier estimates, and used Poisson regression to model associations between migrant status and loss to follow-up controlling for potential confounders identified a priori.

Results

Our cohort comprised 1185 people, among whom 12% (148) were migrant workers. Among the migrant workers, median age was 36.1 (29.6–45.9) and the majority (55%) were male. We found no statistically significant differences between baseline characteristics and migrant status. Rates of lost to follow up were similar between migrants and non-migrants in the first 3 months but differences increased thereafter. Between 3 and 6 months after initiating antiretroviral therapy, migrants had a 2.78-fold increased rate of defaulting (95%CI 1.15–6.73); between 6 and 12 months the rate was 2.36 times greater (95%CI 1.18–4.73), whereas after 1 year the rate was 6.69 times greater (95%CI 3.18–14.09).

Conclusions

Our study highlights the need for programme implementers to take into account the specific challenges that may influence continuity of antiretroviral treatment and care for migrant populations.     

Physiologic profile of professional cricketers

This study aims to provide a physiologic profile of professional cricketers and note positional differences at the start of the 2007/08 competitive season. Fifteen participants (9 bowlers, 6 batsmen) aged 25.0 ± 5.0 years (mean ± SD) took part in this study. Participants (bowlers and batsmen) completed a series of field-based fitness assessments: body composition (sum of 7 skinfolds, 72.5 ± 16.5 and 65.5 ± 19.3 mm, respectively), flexibility (sit and reach 8.1 ± 10.3 and 6.0 ± 6.2 cm, respectively), predicted maximal oxygen uptake (multistage shuttle run, 54.1 ± 2.8 and 56.1 ± 4.5 ml-1·kg-1·min-1, respectively), upper- (medicine ball throw, 7.7 ± 0.6 and 7.0 ± 0.1 m, respectively) and lower-body strength (countermovement jump, 45.7 ± 5.8 and 43.9 ± 4.1 cm, respectively), speed (sprint 17.7 m, 2.76 ± 0.6 and 2.77 ± 0.1 s, respectively), and explosive power (repeated jump, 31.0 ± 2.0 and 34.1 ± 4.8 cm, respectively). The data provided the physical fitness profile for each player, which, compared with normative data, identified that this cohort of professional cricketers had some superior fitness parameters compared with the general population, and where applicable, were comparable with other professional athletes. In addition, after effect size calculations, the results showed that some physical fitness differences existed between playing positions. Cricket professionals possess a superior level of physical fitness and strength, and conditioning coaches should seek to progress these physical parameters and further identify position-specific physical requirements to progress the modern game.     

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.     

Laboratory Studies on the use of Two New Arenas to Evaluate the Impact of the Predatory Mites Blattisocius tarsalis and Cheyletus eruditus on Residual Populations of the Stored Product Mite Acarus siro

Residual populations of storage mites sheltering in crevices and cracks escape conventional control treatments and are implicated in the infestation of newly harvested grain. In a series of 24 h laboratory tests, the performance of solitary adults of two predatory mite species, Cheyletus eruditus (Schrank) and Blattisocius tarsalis (Berlese), were assessed for controlling small numbers of the flour mite Acarus siro (L.). Tests were carried out in the presence or absence of prey refuges or grain debris to afford shelter to the flour mites. While C. eruditus had a significant effect on the motile stages of A. siro, in contrast B. tarsalis had a significant effect on the eggs. The maximum percentage of motile stages of A. siro eaten by C. eruditus was 82%, whereas the minimum percentage of A. siro eggs eaten by B. tarsalis was 99%. While the performance of C. eruditus in predating on motile stages of the flour mite was hindered by the presence of the prey refuge (38% eaten) and grain debris (25% eaten), the performance of B. tarsalis in predating on flour mite eggs was unaffected (100% eaten in presence of prey refuge or grain debris). In prolonged exposures (36 days) the performance of 2, 4 or 8 adult predators, either a single species or a combination of both, was assessed for their ability to control a population of the flour mite developing up to F₂ from an initial inoculum of 80 females and 20 males, allowed to oviposit for 72 h in the absence of predatory mites. The maximum reduction in prey population of 80% was achieved with eight B. tarsalis. Combining the two predatory species did not enhance the reduction of A. siro population.     

In vivo murine CD23 destabilization enhances CD23 shedding and IgE synthesis

To investigate the effects of in vivo CD23 destabilization on CD23 shedding and IgE production, an anti-CD23 stalk monoclonal (19G5), previously shown to enhance proteolysis of CD23 in vitro, was utilized. Compared to isotype control-treated mice, BALB/cJ mice injected with 19G5 displayed significantly enhanced serum soluble CD23 and IgE. Soluble CD23 and IgE levels were also increased in 19G5-treated C57BL/6J mice (intermediate IgE responders); however, the kinetics of the responses differed between the high (BALB/cJ) and intermediate responder mice, suggesting a potential role for CD23 in regulating IgE responder status. The 19G5-induced IgE response was dependent on IL-4 and independent of CD21 as demonstrated through use of IL-4Ralpha and CD21/35-deficient mice, respectively. Overall, the data provide a direct demonstration for CD23's role in regulating IgE production in vivo and suggest that therapies aimed at stabilizing cell surface CD23 would be beneficial in controlling allergic disease.     

Myeloperoxidase-derived 2-chlorohexadecanal forms Schiff bases with primary amines of ethanolamine glycerophospholipids and lysine

Numerous studies have suggested relationships between myeloperoxidase, inflammation, and atherosclerosis. MPO-derived reactive chlorinating species (RCS) attack membrane plasmalogens releasing alpha-chloro-fatty aldehydes (alpha-Cl-FALDs) including 2-chlorohexadecanal (2-ClHDA). The molecular targets of alpha-Cl-FALDs are not known. The current study demonstrates 2-ClHDA adducts with ethanolamine glycerophospholipids and Fmoc-lysine. Utilizing electrospray ionization mass spectrometry, chlorinated adducts were observed that are apparent Schiff base adducts. Reduction of these Schiff base adducts with sodium cyanoborohydride resulted in a novel, stable adduct produced by the elimination of HCl. NMR further confirmed this structure. 2-ClHDA adducts with ethanolamine glycerophospholipids were also substrates for phospholipase D (PLD). The hydrolysis products were derivatized to pentafluorobenzoyl esters, and further structurally confirmed by GC-MS. Multiple molecular species of 2-ClHDA-N-modified ethanolamine glycerophospholipids were observed in endothelial cells treated with 2-ClHDA. These results show novel Schiff base adducts of alpha-Cl-FALDs with primary amines, which may represent an important fate of alpha-Cl-FALDs.     

Species divergence and relationships in Stephanomeria (Compositae): PgiC phylogeny compared to prior biosystematic studies

We present a maximum likelihood tree of 41 PgiC sequences for the monophyletic Stephanomeria, with 10 perennial and six annual species, widely distributed in western North America and exemplary of different speciation processes. The phylogenetic analysis represents the first use of PgiC sequences for Compositae. The annual species were originally delimited by biosystematic studies that provided evidence of their reproductive compatibility and chromosome structural homology. The perennial species are highly distinctive in morphology and have not been examined similarly. The PgiC tree provides more resolution than our previous ITS/ETS tree and reflects both past and ongoing hybridization and/or incomplete lineage sorting. Two major PgiC clades were resolved in Stephanomeria. One clade contains the genes from the annual species plus the perennial, insular endemic S. guadalupensis, which appears closely related to a monophyletic S. virgata. Stephanomeria exigua is not monophyletic. The second clade includes the genes from all other sampled perennial species and a monophyletic subclade of four genes from two annual species. The results are compared to previous studies, also using PgiC, of Clarkia (Onagraceae). Both molecular systematic and biosystematic approaches are essential to discern the very different courses of evolution in these two, well-studied genera of western North America.