Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse.

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ(2)=1022.00, p<0.0005), and CSNB, testing 43 horses (χ(2)=43, p<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.

Accumulating mutations in series of haplotypes at the KIT and MITF loci are major determinants of white markings in Franches-Montagnes horses.

Coat color and pattern variations in domestic animals are frequently inherited as simple monogenic traits, but a number are known to have a complex genetic basis. While the analysis of complex trait data remains a challenge in all species, we can use the reduced haplotypic diversity in domestic animal populations to gain insight into the genomic interactions underlying complex phenotypes. White face and leg markings are examples of complex traits in horses where little is known of the underlying genetics. In this study, Franches-Montagnes (FM) horses were scored for the occurrence of white facial and leg markings using a standardized scoring system. A genome-wide association study (GWAS) was performed for several white patterning traits in 1,077 FM horses. Seven quantitative trait loci (QTL) affecting the white marking score with p-values p≤10(-4) were identified. Three loci, MC1R and the known white spotting genes, KIT and MITF, were identified as the major loci underlying the extent of white patterning in this breed. Together, the seven loci explain 54% of the genetic variance in total white marking score, while MITF and KIT alone account for 26%. Although MITF and KIT are the major loci controlling white patterning, their influence varies according to the basic coat color of the horse and the specific body location of the white patterning. Fine mapping across the MITF and KIT loci was used to characterize haplotypes present. Phylogenetic relationships among haplotypes were calculated to assess their selective and evolutionary influences on the extent of white patterning. This novel approach shows that KIT and MITF act in an additive manner and that accumulating mutations at these loci progressively increase the extent of white markings.

Novel variants in the KIT and PAX3 genes in horses with white-spotted coat colour phenotypes.

Variants in the EDNRB, KIT, MITF, PAX3 and TRPM1 genes are known to cause white spotting phenotypes in horses, which can range from the common white markings up to completely white horses. In this study, we investigated these candidate genes in 169 horses with white spotting phenotypes not explained by the previously described variants. We identified a novel missense variant, PAX3:p.Pro32Arg, in Appaloosa horses with a splashed white phenotype in addition to their leopard complex spotting patterns. We also found three novel variants in the KIT gene. The splice site variant c.1346+1G>A occurred in a Swiss Warmblood horse with a pronounced depigmentation phenotype. The missense variant p.Tyr441Cys was present in several part-bred Arabians with sabino-like depigmentation phenotypes. Finally, we provide evidence suggesting that the common and widely distributed KIT:p.Arg682His variant has a very subtle white-increasing effect, which is much less pronounced than the effect of the other described KIT variants. We termed the new KIT variants W18-W20 to provide a simple and unambiguous nomenclature for future genetic testing applications.

A Gly98Val mutation in the N-Myc downstream regulated gene 1 (NDRG1) in Alaskan Malamutes with polyneuropathy.

The first cases of early-onset progressive polyneuropathy appeared in the Alaskan Malamute population in Norway in the late 1970s. Affected dogs were of both sexes and were ambulatory paraparetic, progressing to non-ambulatory tetraparesis. On neurologic examination, affected dogs displayed predominantly laryngeal paresis, decreased postural reactions, decreased spinal reflexes and muscle atrophy. The disease was considered eradicated through breeding programmes but recently new cases have occurred in the Nordic countries and the USA. The N-myc downstream-regulated gene (NDRG1) is implicated in neuropathies with comparable symptoms or clinical signs both in humans and in Greyhound dogs. This gene was therefore considered a candidate gene for the polyneuropathy in Alaskan Malamutes. The coding sequence of the NDRG1 gene derived from one healthy and one affected Alaskan Malamute revealed a non-synonymous G>T mutation in exon 4 in the affected dog that causes a Gly98Val amino acid substitution. This substitution was categorized to be "probably damaging" to the protein function by PolyPhen2 (score: 1.000). Subsequently, 102 Alaskan Malamutes from the Nordic countries and the USA known to be either affected (n = 22), obligate carriers (n = 7) or healthy (n = 73) were genotyped for the SNP using TaqMan. All affected dogs had the T/T genotype, the obligate carriers had the G/T genotype and the healthy dogs had the G/G genotype except for 13 who had the G/T genotype. A protein alignment showed that residue 98 is conserved in mammals and also that the entire NDRG1 protein is highly conserved (94.7%) in mammals. We conclude that the G>T substitution is most likely the mutation that causes polyneuropathy in Alaskan Malamutes. Our characterization of a novel candidate causative mutation for polyneuropathy offers a new canine model that can provide further insight into pathobiology and therapy of human polyneuropathy. Furthermore, selection against this mutation can now be used to eliminate the disease in Alaskan Malamutes.

Chlamydia prevalence in young attenders of rural and regional primary care services in Australia: a cross-sectional survey.

OBJECTIVE To estimate chlamydia prevalence among 16-29-year-olds attending general practice clinics in Australia. DESIGN, PARTICIPANTS AND SETTING A cross-sectional survey was conducted from May 2010 to December 2012. Sexually experienced 16-29-year-olds were recruited from 134 general practice clinics in 54 rural and regional towns in four states and in nine metropolitan clinics (consecutive patients were invited to participate). Participants completed a questionnaire and were tested for chlamydia. MAIN OUTCOME MEASURE Chlamydia prevalence. RESULTS Of 4284 participants, 197 tested positive for chlamydia (4.6%; 95% CI, 3.9%-5.3%). Prevalence was similar in men (5.2% [65/1257]; 95% CI, 3.9%-6.4%) and women (4.4% [132/3027]; 95% CI, 3.5%-5.2%) (P = 0.25) and high in those reporting genital symptoms or a partner with a sexually transmissible infection (STI) - 17.0% in men (8/47; 95% CI, 2.8%-31.2%); 9.5% in women (16/169; 95% CI, 5.1%-13.8%). Nearly three-quarters of cases (73.4% [130/177]) were diagnosed in asymptomatic patients attending for non-sexual health reasons, and 83.8% of all participants (3258/3890) had attended for non-sexual health reasons. Prevalence was slightly higher in participants from rural and regional areas (4.8% [179/3724]; 95% CI, 4.0%-5.6%) than those from metropolitan areas (3.1% [17/548]; 95% CI, 1.5%-4.7%) (P = 0.08). In multivariable analysis, increasing partner numbers in previous 12 months (adjusted odds ratio [AOR] for three or more partners, 5.11 [95% CI, 2.35-11.08]), chlamydia diagnosis in previous 12 months (AOR, 4.35 [95% CI, 1.52-12.41]) and inconsistent condom use with most recent partner (AOR, 2.90 [95% CI, 1.31-6.40]) were significantly associated with chlamydia in men. In women, increasing partner numbers in previous 12 months (AOR for two partners, 2.59 [95% CI, 1.59-4.23]; AOR for three or more partners, 3.58 [95% CI, 2.26-5.68]), chlamydia diagnosis in previous 12 months (AOR, 3.13 [95% CI, 1.62-6.06]) and age (AOR for 25-29-year-olds, 0.23 [95% CI, 0.12-0.44]) were associated with chlamydia. CONCLUSIONS Chlamydia prevalence is similar in young men and women attending general practice. Testing only those with genital symptoms or a partner with an STI would have missed three-quarters of cases. Most men and women are amenable to being tested in general practice, even in rural and regional areas.

Plasticity, plasticity, plasticity…and the rigid problem of sex

Why is popular understanding of female-male differences still based on rigid models of development, even though contemporary developmental sciences emphasize plasticity? Is it because the science of sex differences still works from the same rigid models?

The need for transparency and good practices in the qPCR literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide receptor antagonist is effective as monotherapy and in combination with rapamycin

UNLABELLED The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. METHODS To determine the effect of treatment with rapamycin and radiotherapy with a novel (177)Lu-labeled GRPr antagonist ((177)Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and (177)Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on (177)Lu-RM2 tumor uptake, in vivo small-animal PET studies with (68)Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of (177)Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with (177)Lu-RM2 alone or after pretreatment with rapamycin. RESULTS Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with (177)Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of (177)Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. CONCLUSION Radiotherapy using a (177)Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

No evidence for an association of posttraumatic stress disorder with circulating levels of CRP and IL-18 in a population-based study.

Several studies have shown associations of posttraumatic stress disorder (PTSD) with the development of cardiometabolic diseases. The underlying psychopathological mechanisms, including potential links to inflammatory processes, have been discussed but remain elusive. Therefore, the aim of the present study was to evaluate the association of PTSD symptoms with the inflammatory biomarkers C-reactive protein (CRP) and interleukin-18 (IL-18). The study population consisted of 3012 participants aged 32-81years drawn from the population-based KORA F4 study conducted in 2006-08 in the Augsburg region (Southern Germany). PTSD symptoms were measured by the Impact of Event Scale, the Posttraumatic Diagnostic Scale and interview data and classified as no, partial or full PTSD. The associations of PTSD with CRP and IL-18 concentrations were estimated by multiple regression analyses with adjustments for age, sex and cardiometabolic risk factors. Linear regression analyses showed no significant association between PTSD and CRP or IL-18 concentration: adjusted for age and sex, the geometric mean concentrations in participants with full PTSD was for CRP 9% lower and for IL-18 1% higher than in participants with no PTSD (p values 0.53 and 0.89). However, further analyses indicated that individuals with partial PTSD had an increased chance of belonging to the highest quartile of the IL-18 concentration. No significant association was observed for any of the three subscales intrusion, avoidance or hyperarousal with CRP or IL-18 concentration. This large, population-based study could not find an association of full PTSD with CRP and IL-18 concentrations. Further research is needed to analyse these relationships.

THE ROLE OF PLATELETS IN OVARIAN CARCINOMA

Platelets represent one of the largest storage pools of angiogenic and oncogenic growth factors in the human body. The observation that thrombocytosis (platelet count >450,000/uL) occurs in patients with solid malignancies was made over 100 years ago. However, the clinical and biological implications as well as the underlying mechanism of paraneoplastic thrombocytosis associated with ovarian carcinoma remains unknown and were the focus of the current study. Following IRB approval, patient data were collected on 619 patients from 4 U.S. centers and used to test associations between platelet count at initial diagnosis, clinicopathologic factors, and outcome. In vitro effects of plasma-purified platelets on ovarian cancer cell proliferation, docetaxel-induced apoptosis, and migration were evaluated using BrdU-PI flow cytometric and two-chamber chemotaxis assays. In vivo effects of platelet depletion on tumor growth, proliferation, apoptosis, and angiogenesis were examined using an anti-platelet antibody (anti-mouse glycoprotein 1ba, Emfret) to reduce platelets by 50%. Complete blood counts and number of mature megakaryocytes in the spleen and bone marrow were compared between control mice and ovarian cancer-bearing mice. Plasma levels of key megakaryo- and thrombopoietic factors including thrombopoietin (TPO), IL-1a, IL-3, IL-4, IL-6, IL-11, G-CSF, GM-CSF, stem cell factor, and FLT-3 ligand were assayed in a subset of 150 patients at the time of initial diagnosis with advanced stage, high grade epithelial ovarian cancer using immunobead-based cytokine profiling coupled with the Luminex® xMAP platform. Plasma cytokines significantly associated with thrombocytosis in ovarian cancer patients were subsequently evaluated in mouse models of ovarian cancer using ELISA immunoassays. The results of human and mouse plasma cytokine profiling were used to inform subsequent in vivo studies evaluating the effect of siRNA-induced silencing of select megakaryo- and thrombopoietic cytokines on paraneoplastic thrombocytosis. Thirty-one percent of patients had thrombocytosis at initial diagnosis. Compared to patients with normal platelet counts, women with thrombocytosis were significantly more likely to have advanced stage disease (p<0.001) and poor median progression-free (0.94 vs 1.35 years, p<0.001) and overall survival (2.62 vs 4.65 years, p<0.001). On multivariate analysis, thrombocytosis remained an independent predictor of decreased overall survival. Our analysis revealed that thrombocytosis significantly increases the risk of VTE in ovarian cancer patients and that thrombocytosis is an independent predictor of increased mortality in women who do develop a blood clot. Platelets increased ovarian cancer cell proliferation and migration by 4.1- and 2.8-fold (p<0.01), respectively. Platelets reduced docetaxel-induced apoptosis in ovarian cancer cells by 2-fold (p<0.001). In vivo, platelet depletion reduced tumor growth by 50%. Staining of in vivo specimens revealed decreased tumor cell proliferation (p<0.001) and increased tumor and endothelial cell apoptosis (p<0.01). Platelet depletion also significantly decreased microvessel density and pericyte coverage (p<0.001). Platelet counts increase by 31-130% in mice with invasive ovarian cancer compared to controls (p<0.01) and strongly correlate with mean megakaryocyte counts in the spleen and bone marrow (r=0.95, p<0.05). Plasma levels of TPO, IL-6, and G-CSF were significantly increased in ovarian cancer patients with thrombocytosis. Plasma levels of the same cytokines were found to be significantly elevated in orthotopic mouse models of ovarian cancer, which consistently develop paraneoplastic thromocytosis. Silencing TPO, IL-6, and G-CSF significantly abrogated paraneoplastic thrombocytosis in vivo. This study provides new understanding of the clinical and biological significance of paraneoplastic thrombocytosis in ovarian cancer and uncovers key humoral factors driving this process. Blocking the development of paraneoplastic thrombocytosis and interfering with platelet-cancer cell interactions could represent novel therapeutic strategies.

Myocardial Infarction - Stress PRevention INTervention (MI-SPRINT) to reduce the incidence of posttraumatic stress after acute myocardial infarction through trauma-focused psychological counseling: study protocol for a randomized controlled trial

BACKGROUND Posttraumatic Stress Disorder (PTSD) may occur in patients after exposure to a life-threatening illness. About one out of six patients develop clinically relevant levels of PTSD symptoms after acute myocardial infarction (MI). Symptoms of PTSD are associated with impaired quality of life and increase the risk of recurrent cardiovascular events. The main hypothesis of the MI-SPRINT study is that trauma-focused psychological counseling is more effective than non-trauma focused counseling in preventing posttraumatic stress after acute MI. METHODS/DESIGN The study is a single-center, randomized controlled psychological trial with two active intervention arms. The sample consists of 426 patients aged 18 years or older who are at 'high risk' to develop clinically relevant posttraumatic stress symptoms. 'High risk' patients are identified with three single-item questions with a numeric rating scale (0 to 10) asking about 'pain during MI', 'fear of dying until admission' and/or 'worrying and feeling helpless when being told about having MI'. Exclusion criteria are emergency heart surgery, severe comorbidities, current severe depression, disorientation, cognitive impairment and suicidal ideation. Patients will be randomly allocated to a single 45-minute counseling session targeting either specific MI-triggered traumatic reactions (that is, the verum intervention) or the general role of psychosocial stress in coronary heart disease (that is, the control intervention). The session will take place in the coronary care unit within 48 hours, by the bedside, after patients have reached stable circulatory conditions. Each patient will additionally receive an illustrated information booklet as study material. Sociodemographic factors, psychosocial and medical data, and cardiometabolic risk factors will be assessed during hospitalization. The primary outcome is the interviewer-rated posttraumatic stress level at three-month follow-up, which is hypothesized to be at least 20% lower in the verum group than in the control group using the t-test. Secondary outcomes are posttraumatic stress levels at 12-month follow-up, and psychosocial functioning and cardiometabolic risk factors at both follow-up assessments. DISCUSSION If the verum intervention proves to be effective, the study will be the first to show that a brief trauma-focused psychological intervention delivered within a somatic health care setting can reduce the incidence of posttraumatic stress in acute MI patients. TRIAL REGISTRATION ClinicalTrials.gov: NCT01781247.

rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.