Using RAD‐seq to develop sex‐linked markers in a haplodiplontic alga

For many taxa, including isomorphic haplodiplontic macroalgae, determining sex and ploidy is challenging, thereby limiting the scope of some population demographic and genetic studies. Here, we used double‐digest restriction site‐associated DNA sequencing (ddRAD‐seq) to identify sex‐linked molecular markers in the widespread red alga Agarophyton vermiculophyllum. In the ddRAD‐seq library, we included 10 female gametophytes, 10 male gametophytes, and 16 tetrasporophytes from one native and one non‐native site (N = 40 gametophytes and N = 32 tetrasporophytes total). We identified seven putatively female‐linked and 19 putatively male‐linked sequences. Four female‐ and eight male‐linked markers amplified in all three life cycle stages. Using one female‐ and one male‐linked marker that were sex‐specific, we developed a duplex PCR and tested the efficacy of this assay on a subset of thalli sampled at two sites in the non‐native range. We confirmed ploidy based on the visual observation of reproductive structures and previous microsatellite genotyping at 10 polymorphic loci. For 32 vegetative thalli, we were able to assign sex and confirm ploidy in these previously genotyped thalli. These markers will be integral to ongoing studies of A. vermiculophyllum invasion. We discuss the utility of RAD‐seq over other approaches previously used, such as RAPDs (random amplified polymorphic DNA), for future work designing sex‐linked markers in other haplodiplontic macroalgae for which genomes are lacking.     

Willingness of men who have sex with men (MSM) in the United States to be circumcised as adults to reduce the risk of HIV infection

Background

Circumcision reduces HIV acquisition among heterosexual men in Africa, but it is unclear if circumcision may reduce HIV acquisition among men who have sex with men (MSM) in the United States, or whether MSM would be willing to be circumcised if recommended.

Methods

We interviewed presumed-HIV negative MSM at gay pride events in 2006. We asked uncircumcised respondents about willingness to be circumcised if it were proven to reduce risk of HIV among MSM and perceived barriers to circumcision. Multivariate logistic regression was used to identify covariates associated with willingness to be circumcised.

Results

Of 780 MSM, 133 (17%) were uncircumcised. Of these, 71 (53%) were willing to be circumcised. Willingness was associated with black race (exact odds ratio [OR]: 3.4, 95% confidence interval [CI]: 1.3–9.8), non-injection drug use (OR: 6.1, 95% CI: 1.8–23.7) and perceived reduced risk of penile cancer (OR: 4.7, 95% CI: 2.0–11.9). The most commonly endorsed concerns about circumcision were post-surgical pain and wound infection.

Conclusions

Over half of uncircumcised MSM, especially black MSM, expressed willingness to be circumcised. Perceived risks and benefits of circumcision should be a part of educational materials if circumcision is recommended for MSM in the United States.     

Methylation and loss of Secreted Frizzled-Related Protein 3 enhances melanoma cell migration and invasion

Background

Wnt signaling is important in development and can also contribute to the initiation and progression of cancer. The Secreted Frizzled Related Proteins (SFRPs) constitute a family of Wnt modulators, crucial for controlling Wnt signaling. Here we investigate the expression and role of SFRP3 in melanoma.

Methodology/Principal Findings

We show that SFRP3 mRNA is down-regulated in malignant melanoma tumors as compared to normal/benign tissue. Furthermore, we found that SFRP3 expression was lost in the malignant melanoma cell lines, A2058, HTB63 and A375, but not in the non-transformed melanocyte cell line, Hermes 3A. Methylated CpG rich areas were detected in the SFRP3 gene in melanoma cell lines and their SFRP3 expression could be restored using the demethylating agent, 5′aza-deoxycytidine. Addition of recombinant SFRP3 to melanoma cells had no effect on viable cell numbers, but decreased cell migration and invasion. Wnt5a signaling has been shown to increase the migration and invasion of malignant melanoma cells, and high expression of Wnt5a in melanoma tumors has been connected to a poor prognosis. We found that recombinant SFRP3 could inhibit Wnt5a signaling, and that it inhibited melanoma cell migration and invasion in a Wnt5a-dependent manner.

Conclusion/Significance

We conclude that SFRP3 functions as a melanoma migration and invasion suppressor by interfering with Wnt5a signaling.     

Size-Based Enrichment of Exfoliated Tumor Cells in Urine Increases the Sensitivity for DNA-Based Detection of Bladder Cancer

Bladder cancer is diagnosed by cystoscopy, a costly and invasive procedure that is associated with patient discomfort. Analysis of tumor-specific markers in DNA from sediments of voided urine has the potential for non-invasive detection of bladder cancer; however, the sensitivity is limited by low fractions and small numbers of tumor cells exfoliated into the urine from low-grade tumors. The purpose of this study was to improve the sensitivity for non-invasive detection of bladder cancer by size-based capture and enrichment of tumor cells in urine. In a split-sample set-up, urine from a consecutive series of patients with primary or recurrent bladder tumors (N = 189) was processed by microfiltration using a membrane filter with a defined pore-size, and sedimentation by centrifugation, respectively. DNA from the samples was analyzed for seven bladder tumor-associated methylation markers using MethyLight and pyrosequencing assays. The fraction of tumor-derived DNA was higher in the filter samples than in the corresponding sediments for all markers (p<0.000001). Across all tumor stages, the number of cases positive for one or more markers was 87% in filter samples compared to 80% in the corresponding sediments. The largest increase in sensitivity was achieved in low-grade Ta tumors, with 82 out of 98 cases positive in the filter samples (84%) versus 74 out of 98 in the sediments (75%). Our results show that pre-analytic processing of voided urine by size-based filtration can increase the sensitivity for DNA-based detection of bladder cancer.     

Systematic analysis of a wasp parasitism arsenal

Parasitoid wasps are among the most diverse insects on earth with many species causing major mortality in host populations. Parasitoids introduce a variety of factors into hosts to promote parasitism, including symbiotic viruses, venom, teratocytes and wasp larvae. Polydnavirus‐carrying wasps use viruses to globally suppress host immunity and prevent rejection of developing parasites. Although prior results provide detailed insights into the genes viruses deliver to hosts, little is known about other products. RNAseq and proteomics were used to characterize the proteins secreted by venom glands, teratocytes and larvae from Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). These data revealed that venom glands and teratocytes secrete large amounts of a small number of products relative to ovaries and larvae. Venom and teratocyte products exhibited almost no overlap with one another or MdBV genes, which suggested that M. demolitor effector molecules are functionally partitioned according to their source. This finding was well illustrated in the case of MdBV and teratocytes. Many viral proteins have immunosuppressive functions that include disruption of antimicrobial peptide production, yet this study showed that teratocytes express high levels of the antimicrobial peptide hymenoptaecin, which likely compensates for MdBV‐mediated immunosuppression. A second key finding was the prevalence of duplications among genes encoding venom and teratocyte molecules. Several of these gene families share similarities with proteins from other species, while also showing specificity of expression in venom glands or teratocytes. Overall, these results provide the first comprehensive analysis of the proteins a polydnavirus‐carrying wasp introduces into its host.     

Bacteriological Survey of Raw Beef Patties Produced at Establishments Under Federal Inspection

At the time of manufacture, 76% of 74 sets of raw beef patties collected in 42 federally inspected establishments had aerobic plate counts of 1,000,000 or fewer/g; 84% contained 100 or fewer coliforms/g; 92% contained 100 or fewer Escherichia coli/g; and 85% contained 100 or fewer Staphylococcus aureus/g (geometric means of 10 patties/set). Salmonellae were isolated from only three (0.4%) of 735 beef patties.     

Model-Based Quantification of the Systemic Interplay between Glucose and Fatty Acids in the Postprandial State

In metabolic diseases such as Type 2 Diabetes and Non-Alcoholic Fatty Liver Disease, the systemic regulation of postprandial metabolite concentrations is disturbed. To understand this dysregulation, a quantitative and temporal understanding of systemic postprandial metabolite handling is needed. Of particular interest is the intertwined regulation of glucose and non-esterified fatty acids (NEFA), due to the association between disturbed NEFA metabolism and insulin resistance. However, postprandial glucose metabolism is characterized by a dynamic interplay of simultaneously responding regulatory mechanisms, which have proven difficult to measure directly. Therefore, we propose a mathematical modelling approach to untangle the systemic interplay between glucose and NEFA in the postprandial period. The developed model integrates data of both the perturbation of glucose metabolism by NEFA as measured under clamp conditions, and postprandial time-series of glucose, insulin, and NEFA. The model can describe independent data not used for fitting, and perturbations of NEFA metabolism result in an increased insulin, but not glucose, response, demonstrating that glucose homeostasis is maintained. Finally, the model is used to show that NEFA may mediate up to 30–45% of the postprandial increase in insulin-dependent glucose uptake at two hours after a glucose meal. In conclusion, the presented model can quantify the systemic interactions of glucose and NEFA in the postprandial state, and may therefore provide a new method to evaluate the disturbance of this interplay in metabolic disease.     

The Sub-Cellular Localization of WRAP53 Has Prognostic Impact in Breast Cancer

WRAP53 protein controls intracellular trafficking of DNA repair proteins, the telomerase enzyme, and splicing factors. Functional loss of the protein has been linked to carcinogenesis, premature aging and neurodegeneration. The aim of this study was to investigate the prognostic significance of WRAP53 protein expression in breast cancer. A tissue microarray was constructed from primary breast tumors and immunostained by a polyclonal WRAP53 antibody to assess the protein expression pattern. Two different patient cohorts with long term follow-up were studied; a test- and a validation set of 154 and 668 breast tumor samples respectively. Breast cancer patients with tumor cells lacking the expression of WRAP53 in the nucleus had a significantly poorer outcome compared to patients with tumor cells expressing this protein in the nuclei (HR = 1.95, 95%CI = 1.09–3.51, p = 0.025). Nuclear localization of WRAP53 was further shown to be an independent marker of prognosis in multivariate analysis (HR = 2.57, 95%CI = 1.27–5.19, p = 0.008), and also significantly associated with better outcome in patients with TP53 mutation. Here we show that the sub-cellular localization of the WRAP53 protein has a significant impact on breast cancer survival, and thus has a potential as a clinical marker in diagnostics and treatment.     

Weight Loss and Premature Death: The 1946 British Birth Cohort Study

Objective

The relationship between weight loss and mortality has important clinical and public health significance but has proved to be complex. Evidence is mixed and particularly limited on the association between weight loss in mid-life and premature death (i.e. before 65 years of age), a small albeit important segment of total mortality. We aimed to study the association between midlife weight change and mortality accounting for health and lifestyle characteristics, and also considering potential bias due to preexisting chronic diseases and smoking status.

Design

Longitudinal, population-based, ‘the 1946 British’ birth cohort study.

Subjects and Measures

In 2750 men and women, mortality from age 53 through 65 years was analyzed according to categories of measured 10 year weight change between 43 and 53 years. Cox''s hazard ratios (HR) were progressively adjusted for socio-demographic, lifestyle and health characteristics.

Results

Nearly 20% of participants lost weight and over 50% gained 5 kg or more in midlife. There were 164 deaths. Compared to those who gained between 2 and 5 kg, those who lost 5 kg or more had an increased risk of premature death independently of midlife physical activity, socio-economic circumstances and educational attainment. This association was unaltered when highest weight loss (lost more than 15 Kg) (p = 0.04) and early deaths were excluded (p<0.001), but was no longer significant after adjustment for cardiovascular risk factors and health status (HR = 1.8; 95% CI: 0.9 to 3.5).

Conclusion

The inverse association between weight loss in midlife and higher risk of premature death may be explained by vascular risk factors and ill health. In consideration of the burden of premature death, closer monitoring of weight loss in mid-life is warranted.     

In vitro culture of Trichogramma pretiosum on an artificial medium

The composition of an artificial medium and environmental conditions are described for the in vitro rearing of the egg parasite Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). The medium was composed of defined amounts of protein, carbohydrates, lipid, salts, and vitamins, but also contained up to 40% insect hemolymph. The hemolymph was necessary to induce pupation. T. pretiosum eggs were obtained by dissection of Heliothis virescens (F.) (Lepidoptera: Noctuidae) eggs. In vitro reared T. pretiosum were similar in size to H. virescens reared T. pretiosum, and females were fecund.

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Résumé Les oeufs de Trichogramma pretiosum ont été obtenus par dissection d'oeufs d'Heliothis virescens. T. pretiosum Riley (Hymenoptère, Trichogrammatidae) a été élevé avec succès sur un substrat synthétique. Outre des quantités définies de protéines, glucides, lipides, éléments minéraux et vitamines, la ration contenait aussi jusqu'à 40% d'hémolymphe de Manduca sexta. L'hémolymphe était nécessaire pour induire la nymphose. En plus de la nourriture, les conditions d'environnement sont apparues extrêmement importantes pour élever T. pretiosum dans des conditions satisfaisantes. Le contrôle de l'humidité relative, en particulier, était le facteur le plus important. Les adultes produits au cours de cette étude étaient d'apparence normale; ils se sont accouplés sans problèmes, les femelles étaient fécondes et leur taille ne différait pas de celle d'individus élevés sur H. virescens.