Population-based analysis of ocular Chlamydia trachomatis in trachoma-endemic West African communities identifies genomic markers of disease severity.

BACKGROUND: Chlamydia trachomatis (Ct) is the most common infectious cause of blindness and bacterial sexually transmitted infection worldwide. Ct strain-specific differences in clinical trachoma suggest that genetic polymorphisms in Ct may contribute to the observed variability in severity of clinical disease. METHODS: Using Ct whole genome sequences obtained directly from conjunctival swabs, we studied Ct genomic diversity and associations between Ct genetic polymorphisms with ocular localization and disease severity in a treatment-naïve trachoma-endemic population in Guinea-Bissau, West Africa. RESULTS: All Ct sequences fall within the T2 ocular clade phylogenetically. This is consistent with the presence of the characteristic deletion in trpA resulting in a truncated non-functional protein and the ocular tyrosine repeat regions present in tarP associated with ocular tissue localization. We have identified 21 Ct non-synonymous single nucleotide polymorphisms (SNPs) associated with ocular localization, including SNPs within pmpD (odds ratio, OR = 4.07, p* = 0.001) and tarP (OR = 0.34, p* = 0.009). Eight synonymous SNPs associated with disease severity were found in yjfH (rlmB) (OR = 0.13, p* = 0.037), CTA0273 (OR = 0.12, p* = 0.027), trmD (OR = 0.12, p* = 0.032), CTA0744 (OR = 0.12, p* = 0.041), glgA (OR = 0.10, p* = 0.026), alaS (OR = 0.10, p* = 0.032), pmpE (OR = 0.08, p* = 0.001) and the intergenic region CTA0744-CTA0745 (OR = 0.13, p* = 0.043). CONCLUSIONS: This study demonstrates the extent of genomic diversity within a naturally circulating population of ocular Ct and is the first to describe novel genomic associations with disease severity. These findings direct investigation of host-pathogen interactions that may be important in ocular Ct pathogenesis and disease transmission.

Haematological indices and haematinic levels after mini gastric bypass: a matched comparison with Roux-en-Y gastric bypass.

Many surgeons believe mini gastric bypass (MGB) is more likely to cause micronutrient malabsorption compared to Roux-en-Y gastric bypass (RYGB). Till date, there is no published study evaluating haematological indices and haematinic levels in patients undergoing MGB and comparing these with a matched cohort of RYGB. Two hundred patients who underwent MGB between October 2012 and October 2015 were matched to 200 patients who underwent RYGB for age, sex, body mass index and time of surgery. We then compared haemoglobin, mean corpuscular volume, iron, ferritin, vitamin B12 and folic acid levels preoperatively and at 6 monthly intervals after surgery until 2 years. The percentage total weight loss was significantly higher in the MGB group compared to the RYGB group at all time points. At 2 years, MGB and RYGB both led to an increase in anaemia rates but the difference was only significant for MGB group. Compared to RYGB, MGB patients were more likely to be anaemic at 2 years, although the difference was not significant statistically (16.6% vs. 12.7%; P value = 0.55). There was a trend for lower iron and folate levels in MGB group compared to RYGB group but the difference was statistically significant at some of the time periods only (significantly lower folate at 6 and 12 months and lower iron at 6 months in the MGB group). MGB leads to a significant increase in anaemia rates in a supplemented cohort. There is a trend towards lower iron and folate levels and higher anaemia rates in MGB group in comparison with RYGB. Larger studies with longer follow-up should evaluate results of MGB with a shorter biliopancreatic limb.

Fitness consequences of dispersal: is leaving home the best of a bad lot?

Using 20 years of demographic and genetic data from four populations of banner-tailed kangaroo rats (Dipodomys spectabilis), we asked whether dispersing individuals gain benefits during adulthood that might compensate for the substantial survival costs they experience as juveniles. Compared to philopatric animals, within- and between-population dispersers gained no measureable advantages in adult survival, fecundity, or probability of recruiting offspring to adulthood. Restricting analyses to members of two central populations living more than 15 times the median dispersal distance from the study site edge, and using peripheral populations only to detect dispersal or offspring recruitment "offsite," did not change this result. Population density during year of birth had small negative effects on adult survival and fecundity, but there were no interactions with dispersal phenotype. We found no evidence that dispersers gained access to superior habitat or that their offspring suffered less inbreeding depression. Our results are consistent with fitness advantages being indirect; by leaving, dispersers release their kin from competition. Our results are also consistent with the possibility that dispersal is the "best of a bad lot." If dispersal is a conditional strategy, then the benefits may be obscured in observational data that compare dispersing individuals to philopatric individuals rather than to individuals whose dispersal phenotype is experimentally manipulated.

How much can parentage analyses tell us about precapture dispersal?

Estimating rates of movement among populations is never simple, and where young animals cannot all be captured at their birth sites, traditional field methods potentially underestimate dispersal rates. Genetic assignment tests appear to hold promise for detecting 'precapture' dispersal, and recent evidence suggests that even on the scale of dispersal between populations, genetic parentage analyses can also be informative. Herein, we examine the performance of both types of analysis with data from a 17-year study of dispersal in banner-tailed kangaroo rats Dipodomys spectabilis. We compare estimates of precapture dispersal from (i) the commonly used parentage analysis program cervus (ii) a pedigree-reconstruction program, MasterBayes, that combines genetic with spatial and other nongenetic information and (iii) genetic assignment procedures implemented by the program geneclass2, with (iv) rates of dispersal observed through recapture of a subset of animals initially marked shortly after weaning. geneclass2 estimates a larger proportion of precapture dispersers than MasterBayes, but both approaches as well as those based on field data alone, suggest that approximately 10% of adults in local populations are immigrants and that interpopulation dispersal is slightly female-biassed. All genetic procedures detect precapture dispersal between populations, but dispersers identified by MasterBayes are particularly compatible with what is independently known about body mass at dispersal, dispersal distance and distance between parents. Parentage analyses have considerable potential to infer the value of this otherwise elusive demographic parameter when most candidate parents can be genotyped and when nongenetic information, especially the distance separating candidate mothers and fathers, can be incorporated into the procedure.

General quantitative genetic methods for comparative biology: phylogenies, taxonomies and multi-trait models for continuous and categorical characters.

Although many of the statistical techniques used in comparative biology were originally developed in quantitative genetics, subsequent development of comparative techniques has progressed in relative isolation. Consequently, many of the new and planned developments in comparative analysis already have well-tested solutions in quantitative genetics. In this paper, we take three recent publications that develop phylogenetic meta-analysis, either implicitly or explicitly, and show how they can be considered as quantitative genetic models. We highlight some of the difficulties with the proposed solutions, and demonstrate that standard quantitative genetic theory and software offer solutions. We also show how results from Bayesian quantitative genetics can be used to create efficient Markov chain Monte Carlo algorithms for phylogenetic mixed models, thereby extending their generality to non-Gaussian data. Of particular utility is the development of multinomial models for analysing the evolution of discrete traits, and the development of multi-trait models in which traits can follow different distributions. Meta-analyses often include a nonrandom collection of species for which the full phylogenetic tree has only been partly resolved. Using missing data theory, we show how the presented models can be used to correct for nonrandom sampling and show how taxonomies and phylogenies can be combined to give a flexible framework with which to model dependence.

Bayesian paternity analysis and mating patterns in a parasitic nematode, Trichostrongylus tenuis.

Mating behaviour is a fundamental aspect of the evolutionary ecology of sexually reproducing species, but one that has been under-researched in parasitic nematodes. We analysed mating behaviour in the parasitic nematode Trichostrongylus tenuis by performing a paternity analysis in a population from a single red grouse host. Paternity of the 150 larval offspring of 25 mothers (sampled from one of the two host caeca) was assigned among 294 candidate fathers (sampled from both caeca). Each candidate father's probability of paternity of each offspring was estimated from 10-locus microsatellite genotypes. Seventy-six (51%) offspring were assigned a father with a probability of >0.8, and the estimated number of unsampled males was 136 (95% credible interval (CI) 77-219). The probability of a male from one caecum fathering an offspring in the other caecum was estimated as 0.024 (95% CI 0.003-0.077), indicating that the junction of the caeca is a strong barrier to dispersal. Levels of promiscuity (defined as the probability of two of an adult's offspring sharing only one parent) were high for both sexes. Variance in male reproductive success was moderately high, possibly because of a combination of random mating and high variance in post-copulatory reproductive success. These results provide the first data on individual mating behaviour among parasitic nematodes.

Analogues of Y27632 increase gap junction communication and suppress the formation of transformed NIH3T3 colonies.

BACKGROUND: Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells. METHODS: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras. In vitro kinase inhibitor profiling, co-culture of transformed cells with non-transformed cells and a novel Lucifer yellow/PKH67 dye transfer method were used to investigate their mode of action. RESULTS: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn. No toxicity was observed against either transformed or non-transformed cells and the effect was dependent on co-culture of these two cell types. In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4). Dye transfer analysis showed they increased gap junction intercellular communication (GJIC) between transformed and non-transformed cells. CONCLUSIONS: These data are the first to suggest that transient blockade of specific kinases can induce a persistent inhibition of non-contact inhibited transformed colony formation and can also remove pre-formed colonies. These effects could potentially be mediated by the observed increase in GJIC between transformed and non-transformed cells. Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

HIV in prisons: the London experience.

The commissioning of health services for all prisoners in publicly run prisons in England was transferred to local Primary Care Trusts in April 2006, pledging to provide an equivalent standard of health care as that in the community. We reviewed our experience of providing a specialist in-reach HIV service by performing a retrospective case notes review of all HIV-positive prisoners who accessed care from the prison genitourinary medicine service in three London prisons. A total of 112 HIV-positive prisoners were seen by the prison health-care service between April 2004 and 2006. This is the first study to look at how well HIV services are being provided during this transitional period of commissioning health services and provides insight into the challenges facing prison health-care providers. Good HIV outcomes are possible in prison but frequent transfers within the prison system and lack of effective HIV training among prison staff represent barriers to good care.

How to separate genetic and environmental causes of similarity between relatives.

Related individuals often have similar phenotypes, but this similarity may be due to the effects of shared environments as much as to the effects of shared genes. We consider here alternative approaches to separating the relative contributions of these two sources to phenotypic covariances, comparing experimental approaches such as cross-fostering, traditional statistical techniques and more complex statistical models, specifically the 'animal model'. Using both simulation studies and empirical data from wild populations, we demonstrate the ability of the animal model to reduce bias due to shared environment effects such as maternal or brood effects, especially where pedigrees contain multiple generations and immigration rates are low. However, where common environment effects are strong, a combination of both cross-fostering and an animal model provides the best way to avoid bias. We illustrate ways of partitioning phenotypic variance into components of additive genetic, maternal genetic, maternal environment, common environment, permanent environment and temporal effects, but also show how substantial confounding between these different effects may occur. Whilst the flexibility of the mixed model approach is extremely useful for incorporating the spatial, temporal and social heterogeneity typical of natural populations, the advantages will inevitably be restricted by the quality of pedigree information and care needs to be taken in specifying models that are appropriate to the data.

STS markers for the wheat yellow rust resistance gene Yr5 suggest a NBS-LRR-type resistance gene cluster.

Two sequence-tagged site (STS) markers for the wheat yellow rust resistance (R) gene Yr5 have been derived through the identification and characterization of linked amplified fragment length polymorphisms (AFLPs). The sequences of the 2 AFLP markers partially overlap with one another, but belong to discrete loci: S19M93-140 completely cosegregates with Yr5, whereas S23M41-310 maps at a distance of 0.7 cM. The DNA sequence of S23M41-310 shows significant homology with the 3' end of nucleotide-binding site (NBS) - leucine-rich repeat (LRR) - type R-genes, in particular with orthologues of the rice bacterial blight R-gene Xa-I. The distinct genetic location of the 2 AFLP loci suggests that Yr5 falls within an R-gene cluster. Because neither sequence forms part of a detectable transcription product, we propose that the Yr5 R-gene cluster includes R-gene analogues and pseudogenes. A Yr5 flanking simple sequence repeat (SSR) marker has also been identified, which allows Yr5 to be effectively incorporated, along with other R-genes for yellow rust, into elite wheat genetic backgrounds, through marker-assisted selection.

Testing the phenotypic gambit: phenotypic, genetic and environmental correlations of colour.

Evolutionary theory is primarily concerned with genetic processes, yet empirical testing of this theory often involves data collected on phenotypes. To make this tenable, the implicit assumption is often made that phenotypic patterns are good predictors of genetic patterns; an assumption that coined the phenotypic gambit. Although this assumption has been validated for traits with high heritability, such as morphology, its generality for traits with low heritabilities, such as life-history and behavioural traits, remains controversial. Using a large-scale cross-fostering experiment, we were able to measure genetic, common environmental and phenotypic correlations between four colour traits and two skeletal traits in a wild population of passerine birds, the blue tit (Parus caeruleus). Colour traits had little heritable variation but common environment effects were found to be important; skeletal traits showed the opposite pattern. Positive correlations because of a shared natal environment were found between all traits, obscuring negative genetic correlations between some colour and skeletal traits. Consequently, phenotypic patterns were poor surrogates for genetic patterns and we suggest that this may be common if trade-offs or substantial parental effects exist. For this group of traits, the phenotypic gambit cannot be made and we suggest caution when inferring genetic patterns from phenotypic data, especially for behavioural and life-history traits.

Towards unbiased parentage assignment: combining genetic, behavioural and spatial data in a Bayesian framework.

Inferring the parentage of a sample of individuals is often a prerequisite for many types of analysis in molecular ecology, evolutionary biology and quantitative genetics. In all but a few cases, the method of parentage assignment is divorced from the methods used to estimate the parameters of primary interest, such as mate choice or heritability. Here we present a Bayesian approach that simultaneously estimates the parentage of a sample of individuals and a wide range of population-level parameters in which we are interested. We show that joint estimation of parentage and population-level parameters increases the power of parentage assignment, reduces bias in parameter estimation, and accurately evaluates uncertainty in both. We illustrate the method by analysing a number of simulated test data sets, and through a re-analysis of parentage in the Seychelles warbler, Acrocephalus sechellensis. A combination of behavioural, spatial and genetic data are used in the analyses and, importantly, the method does not require strong prior information about the relationship between nongenetic data and parentage.

Additional targets of the Arabidopsis autonomous pathway members, FCA and FY.

A central player in the Arabidopsis floral transition is the floral repressor FLC, the MADS-box transcriptional regulator that inhibits the activity of genes required to switch the meristem from vegetative to floral development. One of the many pathways that regulate FLC expression is the autonomous promotion pathway composed of FCA, FY, FLD, FPA, FVE, LD, and FLK. Rather than a hierarchical set of activities the autonomous promotion pathway comprises sub-pathways of genes with different biochemical functions that all share FLC as a target. One sub-pathway involves FCA and FY, which interact to regulate RNA processing of FLC. Several of the identified components (FY, FVE, and FLD) are homologous to yeast and mammalian proteins with rather generic roles in gene regulation. So why do mutations in these genes specifically show a late-flowering phenotype in Arabidopsis? One reason, found during the analysis of fy alleles, is that the mutant alleles identified in flowering screens can be hypomorphic, they still have partial function. A broader role for the autonomous promotion pathway is supported by a microarray analysis which has identified genes mis-regulated in fca mutants, and whose expression is also altered in fy mutants.

Strong environmental determination of a carotenoid-based plumage trait is not mediated by carotenoid availability.

Carotenoid-based colours are recognized as having an important signalling function, yet the nature of the mechanisms that maintain their honesty is not well understood. By combining a carotenoid-feeding experiment with a quantitative genetic experiment in a wild population of blue tits (Parus caeruleus), we were able to test predictions that differentiate between proposed mechanisms. If variation in carotenoid ingestion underlies variation in carotenoid-based colour expression, then carotenoid-supplemented birds should have reduced variance in colour. In this study, carotenoid supplementation produced a small but significant change in plumage colouration, but no significant change in variation. These results suggest that variation in carotenoid acquisition is not an important source of variation for this colour trait, and that variation in post-ingestion processes are likely to be more important. The low heritability of this colour trait suggests environmental factors are likely to underlie the majority of variation in these processes.

Resonance contributions to anti-Stokes/Stokes ratios under surface enhanced Raman scattering conditions.

Anti-Stokes/Stokes asymmetries under surface enhanced Raman scattering (SERS) conditions are studied for a wide variety of SERS-active media and different analytes. Evidence is provided for the existence of underlying resonances that create these asymmetries. We show here that these resonances are associated with the electromagnetic coupling between the analyte (probe) and the metal. The work demonstrates the use of the anti-Stokes/Stokes ratio as a tool to understand the hierarchy of resonances in the SERS problem, which is essential for quantification purposes.

Evaluation of self-directed clinical education: validation of an instrument.

AIM: To explore the evaluation of self-directed, integrated clinical education. METHODS: We delivered a quantitative and qualitative, self-report questionnaire to students through their web-based learning management system. The questionnaire was distributed 4 times over 1 year, each time in 2 parts. A generic part evaluated boundary conditions for learning, teaching activities and "real patient learning". Factor analysis with varimax rotation was used to validate the constructs that made up the scale and to stimulate hypotheses about how they interrelated. A module-specific part evaluated real patient learning of the subject matter in the curriculum. RESULTS: A total of 101 students gave 380 of a possible 404 responses (94%). The generic data loaded onto 4 factors, corresponding to: firm quality; hospital-based teaching and learning; community and out-patient learning, and problem-based learning (PBL). A 5-item quality index had content, construct and criterion validity. Quality differed greatly between firms. Self-evaluation of module-specific, real patient learning was also valid. It was strongly influenced by the specialty interests of hospital firms. CONCLUSIONS: Quality is a multidimensional construct. Self-report evaluation of real patient learning is feasible, and could be capitalised on to promote reflective self-direction. The social and material context of learning is an important dimension of educational quality.

Novel syntheses of cis and trans isomers of combretastatin A-4.

A high-yielding, two-step stereoselective synthesis of the anticancer drug (Z)-combretastatin A-4 (1) has been devised. The method uses the Perkin condensation of 3,4,5-trimethoxyphenylacetic acid and 3-hydroxy-4-methoxybenzaldehyde followed by decarboxylation of the cinnamic acid intermediate using copper and quinoline. The iodine-catalyzed isomerization of the Z isomer 1 results in complete conversion to the E isomer. The Suzuki cross-coupling of an aryl boronic acid and vinyl bromide has also been successfully employed to produce both Z and E isomers of combretastatin A-4 stereoselectively. Both methods are far superior to the current five-step Wittig synthesis in which both isomers are produced nonstereoselectively.

The Medical Emergency Team: 12 month analysis of reasons for activation, immediate outcome and not-for-resuscitation orders.

OBJECTIVE: To describe the reasons for, and immediate outcome following Medical Emergency Team (MET) activation. METHODS: Retrospective analysis of MET calls in 1998. RESULTS: There were 713 MET calls to 559 in-patients. Of the 559 patients 252 (45%) were admitted to ICU and 49 (6.9%) died during the MET response. The three commonest criteria for calling the MET were a fall in GCS>2 (n=155); a systolic blood pressure<90 mmHg (n=142) and a respiratory rate>35 (n=109). Cardiac arrests accounted for 61 calls and had an immediate mortality of 59%. The most common MET criterion associated with admission to ICU was a respiratory rate >35. Of patients who received MET calls based only on the 'worried' criterion 16% were admitted to ICU. The MET felt that a not-for-resuscitation order would have been appropriate in 130 cases (23%). NFR orders were documented during 27 of the MET calls. CONCLUSIONS: The MET system provides objective and subjective criteria by which medical and nursing staff can identify patients who become acutely unwell. A high proportion of these patients will require admission to Intensive Care. The MET system also provides the opportunity to identify patients for whom an NFR order should be considered.

Linked parallel synthesis and MTT bioassay screening of substituted chalcones.

A 644-membered library of chalcones was prepared by parallel synthesis using the Claisen-Schmidt base-catalyzed aldol condensation of substituted acetophenones and benzaldehydes. The cytotoxicity of these chalcones was conveniently determined upon the crude products directly in 96-well microtiter test plates by the conventional MTT assay. This method revealed seven chalcones of IC(50) less than 1 microM of which 4'-hydroxy-2,4,6,3'-tetramethoxychalcone (5a) was the most active [IC(50) (K562), 30 nM]; it causes cell cycle arrest at the G(2)/M point and binds to tubulin at the colchicine binding site.

Large-scale mutagenesis directed at specific chromosomes in wheat.

A novel approach has been developed to allow for the efficient selection of loss-of-function wheat mutants in the M1 generation, following either physical or chemical mutagenesis. This has generated an order of magnitude increase in the efficiency of identification of mutants, and also greatly increases the likelihood that selected individuals reflect mutation events at the target locus, rather than at genes acting elsewhere in the disease resistance pathway. The approach relies only on prior knowledge of the chromosomal location of the target gene, and uses the polyploidy of wheat to construct populations for mutagenesis in which large numbers of individuals are hemizygous for the target gene. The idea is illustrated with the mass identification of mutants at three independent genes for race-specific resistance to yellow rust, and one gene for resistance to powdery mildew.