Susceptibility of rat-derived cells to replication by human immunodeficiency virus type 1

Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.     

A novel delta(3),delta(2)-enoyl-CoA isomerase involved in the biosynthesis of the cyclohexanecarboxylic acid-derived moiety of the polyketide ansatrienin A

The side chain of the antifungal polyketide ansatrienin A produced by Streptomyces collinus contains a cyclohexanecarboxylic acid (CHC) derived moiety. This CHC in the coenzyme A activated form (CHC-CoA) is derived from shikimic acid via a pathway in which the penultimate step is the isomerization of 2-cyclohexenylcarbonyl-CoA to 1-cyclohexenylcarbonyl-CoA. We have purified a 28 kDa 2-cyclohexenylcarbonyl-CoA isomerase (ChcB) from S. collinus and cloned and sequenced the corresponding chcB gene. The predicted amino acid sequence of ChcB showed moderate sequence identity to members of the hydratase/isomerase superfamily of enzymes. The recombinant ChcB was overexpressed in Escherichia coli and purified to homogeneity using metal chelate chromatography. Kinetic analysis demonstrated that recombinant ChcB had wide substrate specificity and could catalyze a double bond isomerization using 2-cyclohexenylcarbonyl-CoA (K(m) 116 +/- 68 microM, k(cat)( )()3.7 +/- 1.0 min(-)(1)), trans-3-hexenyl-CoA (K(m) 39 +/- 10 microM, k(cat)( )()12.8 +/- 1 min(-)(1)), and vinylacetyl-CoA (K(m) 156 +/- 34 microM, k(cat)( )()29 +/- 3 min(-)(1)) as substrates. ChcB activity in cell extracts of S. collinus SP1, an insertionally disrupted chcB mutant, was shown to decrease by more than 99% (as compared to the wild-type strain) using all three of these substrates. The S. collinus SP1 strain, unlike the wild-type strain, could not produce omega-cyclohexyl fatty acids but was still able to grow efficiently on methyl oleate as a sole carbon source. These observations demonstrate that the S. collinus ChcB is required for catalyzing the isomerization of 2-cyclohexenylcarbonyl-CoA to 1-cyclohexenylcarbonyl-CoA during CHC-CoA biosynthesis but not for degradation of unsaturated fatty acids. The chcB gene does not appear to be associated with the ansatrienin biosynthetic gene cluster, which has previously been shown to contain at least one gene known to be essential for CHC-CoA biosynthesis. This finding represents a notable exception to the general rule regarding the clustering of polyketide biosynthetic pathway genes.     

Effects of an educational intervention for general practitioners in adolescent health care principles: a randomized controlled study

Objective To evaluate the effectiveness of an educational intervention in adolescent health designed for general practitioners, in accordance with evidence-based practice in continuing medical education. Design Randomized, controlled trial with baseline testing and 7- and 13-month follow-ups. Setting The intervention was delivered in local community settings to general practitioners in metropolitan Melbourne, Australia. Participants A total of 108 self-selected general practitioners. Intervention A multifaceted educational program (2.5 hours per week for 6 weeks) in the principles of adolescent health care, followed 6 weeks later by a 2-hour session of case discussion and debriefing. Outcome measures Objective ratings of videotaped consultations with standardized adolescent patients and self-completion questionnaires were used to measure general practitioners' knowledge, skill, and self-perceived competency; satisfaction with the program; and self-reported change in practice. Results 103 of 108 physicians (95%) completed all phases of the intervention and evaluation protocol. The intervention group showed significantly greater improvements than the control group in all outcomes at the 7-month follow-up (all subjects P<0.03), except for the standardized patients' rating of rapport and satisfaction (P=0.12). 104 participants (96%) found the program appropriate and relevant. At the 13-month follow-up, most improvements were sustained, the standardized patients' rating of confidentiality fell slightly, and the objective assessment of competence further improved. 106 physicians (98%) reported a change in practice attributable to the intervention. Conclusions General practitioners were willing to complete continuing medical education in adolescent health and its evaluation. The design of the intervention, using evidence-based educational strategies, proved effective and expeditious in achieving sustainable and large improvements in knowledge, skill, and self-perceived competency.     

Linkage of recessive Robinow syndrome to a 4 cM interval on chromosome 9q22

Autosomal recessive Robinow syndrome is a form of mesomelic dwarfism with multiple rib and vertebral anomalies. Using autozygosity mapping we have identified a genetic locus (RBNW1) for this syndrome at chromosome 9q22 in seven consanguineous families from Oman. Our results indicate that the gene lies within a 4 cM region between markers D9S1836 and D9S1803 (maximum multipoint LOD score 12.3). In addition, we have analysed two non-Omani families with autosomal recessive Robinow and found no genetic heterogeneity.     

Making blind robots see: the synergy between fluorescent dyes and imaging devices in automated proteomics

Proteomics investigations endeavor to provide a global understanding of gene product synthesis rate, degradation rate, functional competence, posttranslational modification, subcellular distribution and physical interactions with other cell components. Protein expression encompasses an enormous dynamic range. Since rare proteins cannot be amplified by any type of PCR method, sensitive detection is critical to proteome projects. Fluorescence methods deliver streamlined detection protocols, superior detection sensitivity, broad linear dynamic range and excellent compatibility with modern microchemical identification methods such as mass spectrometry. Two general approaches to fluorescence detection of proteins are currently practiced: the covalent derivatization of proteins with fluorophores or noncovalent interaction of fluorophores either via the SDS micelle or through direct electrostatic interaction with proteins. One approach for quantifying fluorescence is to use a photomultiplier tube detector combined with a laser light scanner. In addition, fluorescence imaging is performed using a charge-coupled device camera combined with a UV light or xenon arc source. Fluorescent dyes with bimodal excitation spectra may be broadly implemented on a wide range of analytical imaging devices, permitting their widespread application to proteomics studies and incorporation into semiautomated analysis environments.     

Differential responses of Abies fabri and Rhododendron calophytum at two sites with contrasting pollution deposition and available calcium in southwestern China

The correlation between forest decline and calcium (Ca) depletion under long-term acid deposition remains elusive in China due to the high level of Ca deposition. We compared two species (Abies fabri and Rhododendron calophytum) for their growth pattern and base elements concentration in both polluted (Mt. Emei) and unpolluted (Mt. Gongga) sites in Sichuan, southwestern China. A. fabri grown at Mt. Emei had poorer crown condition, slower radial growth rate, and lower seedling density under long-term acid deposition, which correlated closely with the reduced Ca concentration in foliage and tree-ring, in comparison with those at Mt. Gongga. In contrast, R. calophytum showed a stable Ca level and thus displayed normal growth between the two sites. The differential capability of these two species to acclimate to poor Ca environment is one of the keys to understanding the long-term ecological effect of changing atmospheric acid and Ca deposition in the subalpine forest in southwestern China.     

Cryptic genetic diversity in Rattus of the San Francisco Bay region, California

Invasive species can have complex invasion histories, harbor cryptic levels of diversity, and pose taxonomic problems for pest management authorities. Roof rats, Rattus rattus sensu lato, are common invasive pests of the San Francisco Bay Area in California, USA. They are a significant health risk and pest management efforts impose a large financial investment from public institutions and private individuals. Recent molecular genetic and taxonomic studies of black rats in their native range in Asia have shown that the species is a complex of two karyotypic forms and four mitochondrial genetic lineages that may represent four distinct species. We used mtDNA sequences and nuclear microsatellite variation to identify which mitochondrial lineages of the R. rattus group are present in the San Francisco Bay Area and to test for gene flow among them. We recovered specimens with mtDNA sequences representing two of the major mtDNA lineages of the R. rattus group. Microsatellite variation, however, was not structured in concordance with mtDNA lineages, suggesting a more complex history involving hybridization and introgression between these lineages. Although Aplin et al. (2011) and Lack et al. (2012) reported R. rattus Lineage II in North America, this is the first detailed examination of possible gene flow amongst lineages in this region.     

Evaluation of species distribution models by resampling of sites surveyed a century ago by Joseph Grinnell

Species distribution models (SDMs) are commonly applied to predict species’ responses to anticipated global change, but lack of data from future time periods precludes assessment of their reliability. Instead, performance against test data in the same era is assumed to correlate with accuracy in the future. Moreover, high‐confidence absence data is required for testing model accuracy but is often unavailable since a species may be present when undetected. Here we evaluate the performance of eight SDMs trained with historic (1900–1939) or modern (1970–2009) climate data and occurrence records for 18 mammalian species. Models were projected to the same or the opposing time period and evaluated with data obtained from surveys conducted by Joseph Grinnell and his colleagues in the Sierra Nevada of California from 1900 to 1939 and modern resurveys from 2003 to 2011. Occupancy modeling was used to confidently assign absences at test sites where species were undetected. SDMs were evaluated using species’ presences combined with this high‐confidence absence (HCA) set, a low‐confidence set in which non‐detections were assumed to indicate absence (LCA), and randomly located ‘pseudoabsences’ (PSA). Model performance increased significantly with the quality of absences (mean AUC ± SE: 0.76 ± 0.01 for PSA, 0.79 ± 0.01 for LCA, and 0.81 ± 0.01 for HCA), and apparent differences between SDMs declined as the quality of test absences increased. Models projecting across time performed as well as when projecting within the same time period when assessed with threshold‐independent metrics. However, accuracy of presence and absence predictions sometimes declined in cross‐era projections. Although most variation in performance occurred among species, autecological traits were only weakly correlated with model accuracy. Our study indicates that a) the quality of evaluation data affects assessments of model performance; b) within‐era performance correlates positively but unreliably with cross‐era performance; and c) SDMs can be reliably but cautiously projected across time.     

Impact of crimson clover dying mulch on two eggplant insect herbivores

Eggplant Solanum melongena L., is often colonized by two early season insect defoliators. The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), and flea beetles Epitrix spp., emerge from their overwintering sites in early spring and seek out emerging host plants such as eggplant. During the 2009 and 2010 growing season, field studies were conducted to investigate the impact of inter‐planting eggplant into a crimson clover (CC), Trifolium incarnatum L., winter cover crop on populations of flea beetles, CPB and their associated arthropod predators. The experiment consisted also of two levels of insecticide usage such as an application of azadirachtins plus pyrethrins followed by several applications of spinosad or no insecticide sprays as subplot treatments. During both study years, significantly fewer (adults, larvae and egg masses) were found on eggplant inter‐planted into CC than in bare‐ground (BG) eggplant plots. Although flea beetle abundance was greater in BG eggplant during 2010, they appeared to be less influenced by the presence of CC than were CPB. Additionally, there was no apparent impact of insecticide treatment on CPB populations on eggplant inter‐planted into CC. However, there was a decline in CPB following treatments with insecticides in BG eggplant plots. This suggests that a winter cover crop such as CC can be used to help manage CPB in eggplant, however, using this tactic in tandem with insecticide sprays may not result in greater CPB management.