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Spermatogonial stem cells functionality reside in the slow-cycling and heterogeneous undifferentiated spermatogonia cell population. This pool of cells supports lifelong fertility in adult males by balancing self-renewal and differentiation to produce haploid gametes. However, the molecular mechanisms underpinning long-term stemness of undifferentiated spermatogonia during adulthood remain unclear. Here, we discover that an epigenetic regulator, Polycomb repressive complex 1 (PRC1), shields adult undifferentiated spermatogonia from differentiation, maintains slow cycling, and directs commitment to differentiation during steady-state spermatogenesis in adults. We show that PRC2-mediated H3K27me3 is an epigenetic hallmark of adult undifferentiated spermatogonia. Indeed, spermatogonial differentiation is accompanied by a global loss of H3K27me3. Disruption of PRC1 impairs global H3K27me3 deposition, leading to precocious spermatogonial differentiation. Therefore, PRC1 directs PRC2-H3K27me3 deposition to maintain the self-renewing state of undifferentiated spermatogonia. Importantly, in contrast to its role in other tissue stem cells, PRC1 negatively regulates the cell cycle to maintain slow cycling of undifferentiated spermatogonia. Our findings have implications for how epigenetic regulators can be tuned to regulate the stem cell potential, cell cycle and differentiation to ensure lifelong fertility in adult males.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by DMD gene mutations, resulting in absence of functional dystrophin protein. Viltolarsen, an exon 53 skipping therapy, significantly increased dystrophin levels in patients with DMD. Presented here are completed study results of > 4 years of functional outcomes in viltolarsen-treated patients compared to a historical control group (Cooperative International Neuromuscular Research Group Duchenne Natural History Study [CINRG DNHS]). OBJECTIVE: To evaluate the efficacy and safety of viltolarsen for an additional 192 weeks in boys with DMD. METHODS: This phase 2, open-label, 192-week long-term extension (LTE) study (NCT03167255) evaluated the efficacy and safety of viltolarsen in participants aged 4 to

BackgroundHuman epidermal growth factor receptor 2 (HER2)-targeted therapies have not been approved for patients with non-small-cell lung cancer (NSCLC). The efficacy and safety of trastuzumab deruxtecan (formerly DS-8201), a HER2 antibody-drug conjugate, in patients with HER2-mutant NSCLC have not been investigated extensively.MethodsWe conducted a multicenter, international, phase 2 study in which trastuzumab deruxtecan (6.4 mg per kilogram of body weight) was administered to patients who had metastatic HER2-mutant NSCLC that was refractory to standard treatment. The primary outcome was objective response as assessed by independent central review. Secondary outcomes included the duration of response, progression-free survival, overall survival, and safety. Biomarkers of HER2 alterations were assessed.ResultsA total of 91 patients were enrolled. The median duration of follow-up was 13.1 months (range, 0.7 to 29.1). Centrally confirmed objective response occurred in 55% of the patients (95% confidence interval [CI], 44 to 65). The median duration of response was 9.3 months (95% CI, 5.7 to 14.7). Median progression-free survival was 8.2 months (95% CI, 6.0 to 11.9), and median overall survival was 17.8 months (95% CI, 13.8 to 22.1). The safety profile was generally consistent with those from previous studies; grade 3 or higher drug-related adverse events occurred in 46% of patients, the most common event being neutropenia (in 19%). Adjudicated drug-related interstitial lung disease occurred in 26% of patients and resulted in death in 2 patients. Responses were observed across different HER2 mutation subtypes, as well as in patients with no detectable HER2 expression or HER2 amplification.ConclusionsTrastuzumab deruxtecan showed durable anticancer activity in patients with previously treated HER2-mutant NSCLC. The safety profile included interstitial lung disease that was fatal in two cases. Observed toxic effects were generally consistent with those in previously reported studies. (Funded by Daiichi Sankyo and AstraZeneca; DESTINY-Lung01 ClinicalTrials.gov number, NCT03505710.).

Corticosteroids are most commonly used to treat HTLV-1-associated myelopathy (HAM); however, their clinical efficacy has not been tested in randomized clinical trials. This randomized controlled trial included 8 and 30 HAM patients with rapidly and slowly progressing walking disabilities, respectively. Rapid progressors were assigned (1:1) to receive or not receive a 3-day course of intravenous methylprednisolone in addition to oral prednisolone therapy. Meanwhile, slow progressors were assigned (1:1) to receive oral prednisolone or placebo. The primary outcomes were a composite of ≥1-grade improvement in the Osame Motor Disability Score or ≥30% improvement in the 10 m walking time (10 mWT) at week 2 for rapid progressors and changes from baseline in 10 mWT at week 24 for slow progressors. In the rapid progressor trial, all four patients with but only one of four without intravenous methylprednisolone achieved the primary outcome (p = 0.14). In the slow progressor trial, the median changes in 10 mWT were -13.8% (95% CI: -20.1--7.1; p < 0.001) and -6.0% (95% CI: -12.8-1.3; p = 0.10) with prednisolone and placebo, respectively (p for between-group difference = 0.12). Whereas statistical significance was not reached for the primary endpoints, the overall data indicated the benefit of corticosteroid therapy. (Registration number: UMIN000023798, UMIN000024085).

BACKGROUND: Duchenne muscular dystrophy (DMD) is a rare, genetic disease caused by mutations in the DMD gene resulting in an absence of functional dystrophin protein. Viltolarsen, an exon 53 skipping therapy, has been shown to increase endogenous dystrophin levels. Herein, long-term (>2 years) functional outcomes in viltolarsen treated patients were compared to a matched historical control group. OBJECTIVE: To evaluate long-term efficacy and safety of the anti-sense oligonucleotide viltolarsen in the treatment of patients with DMD amenable to exon 53 skipping therapy. METHODS: This trial (NCT03167255) is the extension of a previously published 24-week trial in North America (NCT02740972) that examined dystrophin levels, timed function tests compared to a matched historical control group (Cooperative International Neuromuscular Research Group Duchenne Natural History Study, CINRG DNHS), and safety in boys 4 to

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Therapeutic innovation for human papilloma virus-related (HPV+) head and neck squamous cell carcinomas (HNSCCs) is impaired by inadequate preclinical models and the absence of accurate biomarkers. Our study establishes the first well-characterized panel of patient-derived xenografts (PDXs) and organoids from HPV+ HNSCCs while determining fidelity of the models to the distinguishing genetic features of this cancer type. Despite low engraftment rates, whole exome sequencing showed that PDXs retain multiple distinguishing features of HPV+ HNSCC lost in existing cell lines, including PIK3CA mutations, TRAF3 deletion and the absence of EGFR amplifications. Engrafted HPV+ tumors frequently contained NOTCH1 mutations, thus providing new models for a negatively prognostic alteration in this disease. Genotype-phenotype associations in the models were then tested for prediction of tumor progression and survival in published clinical cohorts. Observation of high tumor mutational burdens (TMBs) in the faster-growing models facilitated identification of a novel association between TMB and local progression in both HPV+ and HPV- patients that was prognostic in HPV- cases. In addition, reduced E7 and p16INK4A levels found in a PDX from an outlier case with lethal outcome led to detection of similar profiles among recurrent HPV+ HNSCCs. Transcriptional data from the Cancer Genome Atlas was used to demonstrate that the lower E2F target gene expression predicted by reduced E7 levels has potential as a biomarker of disease recurrence risk. Our findings bridge a critical gap in preclinical models for HPV+ HNSCCs and simultaneously reveal novel potential applications of quantifying mutational burden and viral oncogene functions for biomarker development.

Prostate cancer (PCa) is characterized by a unique dependence on optimal androgen receptor (AR) activity where physiological androgen concentrations induce proliferation but castrate and supraphysiological levels suppress growth. This feature has been exploited in bipolar androgen therapy (BAT) for castrate resistant malignancies. Here, we investigated the role of the tumor suppressor protein p14ARF in maintaining optimal AR activity and the function of the AR itself in regulating p14ARF levels. We used a tumor tissue array of differing stages and grades to define the relationships between these components and identified a strong positive correlation between p14ARF and AR expression. Mechanistic studies utilizing CWR22 xenograft and cell culture models revealed that a decrease in AR reduced p14ARF expression and deregulated E2F factors, which are linked to p14ARF and AR regulation. Chromatin immunoprecipitation studies identified AR binding sites upstream of p14ARF. p14ARF depletion enhanced AR-dependent PSA and TMPRSS2 transcription, hence p14ARF constrains AR activity. However, p14ARF depletion ultimately results in apoptosis. In PCa cells, AR co-ops p14ARF as part of a feedback mechanism to ensure optimal AR activity for maximal prostate cancer cell survival and proliferation.

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale1-3. Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter4; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation5,6; analyses timings and patterns of tumour evolution7; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity8,9; and evaluates a range of more-specialized features of cancer genomes8,10-18.

Identification of the precise molecular pathways involved in oncogene-induced transformation may help us gain a better understanding of tumor initiation and promotion. Here, we demonstrate that SOX2+ foregut epithelial cells are prone to oncogenic transformation upon mutagenic insults, such as KrasG12D and p53 deletion. GFP-based lineage-tracing experiments indicate that SOX2+ cells are the cells-of-origin of esophagus and stomach hyperplasia. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion. p53 homozygous deletion is required for the acquisition of an invasive potential, and KrasG12D expression, but not p53 deletion, suffices for tumor formation. Global gene expression analysis reveals secreting factors upregulated in the hyperplasia induced by oncogenic KRAS and highlights a crucial role for the CXCR2 pathway in driving hyperplasia. Collectively, the array of genetic models presented here demonstrate that stratified epithelial cells are susceptible to oncogenic insults, which may lead to a better understanding of tumor initiation and aid in the design of new cancer therapeutics.

Excessive MMP activity may impair tendon-to-bone healing. However, little is known about the effect of joint motion on MMP activity after ACL reconstruction. The aim of this study was to determine the effect of different durations of knee immobilization on MMP activity in a mouse ACL reconstruction model using a fluorescent MMP probe which detects MMP 2, 3, 9, and 13 and near-infra red in vivo imaging. Sixty C57BL male mice underwent ACL reconstruction. Post-operatively, the animals were treated with free cage activity (Group 1), or with the use of an external fixator to restrict knee motion and weight bearing for 5 days (Group 2), 14 days (Group 3), and 28 days (Group 4). At days 3, 7, 16, 23, and 30, five mice underwent IVIS imaging. At days 3, 7, 16, and 30, histological analysis was also performed. Probe signal intensity in the whole limb peaked at day 7, followed by a decrease at day 16, and maintenance up to day 30. There was no significant difference among groups at any time point based on IVIS, but histologic localization of MMP probe signal showed significantly less activity in Group 2 and Group 3 compared to Group 4 in the bone tunnel at day 30. We demonstrated that short-term immobilization led to less MMP activity around the bone tunnel compared with prolonged immobilization. A short period of immobilization after ACL reconstruction might enhance graft-bone interface healing by mitigating excess MMP expression. These findings have implications for post-operative rehabilitation protocols following ACL reconstruction. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:325-334, 2019.

BackgroundThe integrase inhibitor dolutegravir could have a major role in future antiretroviral therapy (ART) regimens in sub-Saharan Africa because of its high potency and barrier to resistance, good tolerability, and low cost, but there is uncertainty over appropriate policies for use relating to the potential for drug resistance spread and a possible increased risk of neural tube defects in infants if used in women at the time of conception. We used an existing individual-based model of HIV transmission, progression, and the effect of ART with the aim of informing policy makers on approaches to the use of dolutegravir that are likely to lead to the highest population health gains.MethodsWe used an existing individual-based model of HIV transmission and progression in adults, which takes into account the effects of drug resistance and differential drug potency in determining viral suppression and clinical outcomes to compare predicted outcomes of alternative ART regimen policies. We calculated disability adjusted life-years (DALYs) for each policy, assuming that a woman having a child with a neural tube defect incurs an extra DALY per year for the remainder of the time horizon and accounting for mother-to-child transmission. We used a 20 year time horizon, a 3% discount rate, and a cost-effectiveness threshold of US$500 per DALY averted.FindingsThe greatest number of DALYs is predicted to be averted with use of a policy in which tenofovir, lamivudine, and dolutegravir is used in all people on ART, including switching to tenofovir, lamivudine, and dolutegravir in those currently on ART, regardless of current viral load suppression and intention to have (more) children. This result was consistent in several sensitivity analyses. We predict that this policy would be cost-saving.InterpretationUsing a standard DALY framework to compare health outcomes from a public health perspective, the benefits of transition to tenofovir, lamivudine, and dolutegravir for all substantially outweighed the risks.FundingBill & Melinda Gates Foundation.

PurposeTo investigate the use of kartogenin (KGN) in augmenting healing of the repaired enthesis after rotator cuff repair in a murine model.MethodsSeventy-two C57BL/6 wild-type mice underwent unilateral detachment and transosseous repair of the supraspinatus tendon augmented with either fibrin sealant (control group; n = 36) or fibrin sealant containing 100 μmol/L of KGN (experimental group; n = 36) applied at the repair site. Postoperatively, mice were allowed free cage activity without immobilization. Mice were humanely killed at 2 and 4 weeks postoperatively. Repair site integrity was evaluated histologically through fibrocartilage formation and collagen fiber organization and biomechanically through load-to-failure testing of the supraspinatus tendon-bone construct.ResultsAt 2 weeks, no differences were noted in percent area of fibrocartilage, collagen organization, or ultimate strength between groups. At 4 weeks, superior collagen fiber organization (based on collagen birefringence [17.3 ± 2.0 vs 7.0 ± 6.5 integrated density/μm2; P

How fully differentiated cells that experience carcinogenic insults become proliferative cancer progenitors that acquire multiple initiating mutations is not clear. This question is of particular relevance to hepatocellular carcinoma (HCC), which arises from differentiated hepatocytes. Here we show that one solution to this problem is provided by CD44, a hyaluronic acid receptor whose expression is rapidly induced in carcinogen-exposed hepatocytes in a STAT3-dependent manner. Once expressed, CD44 potentiates AKT activation to induce the phosphorylation and nuclear translocation of Mdm2, which terminates the p53 genomic surveillance response. This allows DNA-damaged hepatocytes to escape p53-induced death and senescence and respond to proliferative signals that promote fixation of mutations and their transmission to daughter cells that go on to become HCC progenitors.

BackgroundWe compared Kaposi sarcoma (KS) risk in adults who started antiretroviral therapy (ART) across the Asia-Pacific, South Africa, Europe, Latin, and North America.MethodsWe included cohort data of human immunodeficiency virus (HIV)-positive adults who started ART after 1995 within the framework of 2 large collaborations of observational HIV cohorts. We present incidence rates and adjusted hazard ratios (aHRs).ResultsWe included 208140 patients from 57 countries. Over a period of 1066572 person-years, 2046 KS cases were diagnosed. KS incidence rates per 100000 person-years were 52 in the Asia-Pacific and ranged between 180 and 280 in the other regions. KS risk was 5 times higher in South African women (aHR, 4.56; 95% confidence intervals [CI], 2.73-7.62) than in their European counterparts, and 2 times higher in South African men (2.21; 1.34-3.63). In Europe, Latin, and North America KS risk was 6 times higher in men who have sex with men (aHR, 5.95; 95% CI, 5.09-6.96) than in women. Comparing patients with current CD4 cell counts ≥700 cells/µL with those whose counts were

Disturbances in peripheral and brain lipid metabolism, including the omega-3 fatty acid docosahexaenoic acid (DHA), have been reported in major depressive disorder (MDD). However, these changes have yet to be confirmed in MDD with seasonal pattern (MDD-s), a subtype of recurrent MDD. The present exploratory study quantified plasma plasmalogen and diacyl-phospholipid species, and fatty acids within total phospholipids, cholesteryl esters, triacylglycerols and free fatty acids in non-medicated MDD-s participants (n = 9) during euthymia in summer or fall, and during depression in winter in order to screen for potential high sensitivity lipid biomarkers. Triacylglycerol alpha-linolenic acid concentration was significantly decreased, and myristoleic acid concentration was significantly increased, during winter depression compared to summer-fall euthymia. 1-stearyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, a diacyl-phospholipid containing stearic acid and DHA, was significantly decreased in winter depression. Concentrations of cholesteryl ester oleic acid and several polyunsaturated fatty acids between summer/fall and winter increased in proportion to the increase in depressive symptoms. The observed changes in lipid metabolic pathways in winter-type MDD-s offer new promise for lipid biomarker development.

The demand for wearable sensors has grown rapidly in recent years, with increasing attention being given to epidermal chemical sensing. Here, we present the first example of a fully integrated eyeglasses wireless multiplexed chemical sensing platform capable of real-time monitoring of sweat electrolytes and metabolites. The new concept has been realized by integrating an amperometric lactate biosensor and a potentiometric potassium ion-selective electrode into the two nose-bridge pads of the glasses and interfacing them with a wireless electronic backbone placed on the glasses' arms. Simultaneous real-time monitoring of sweat lactate and potassium levels with no apparent cross-talk is demonstrated along with wireless signal transduction. The electrochemical sensors were screen-printed on polyethylene terephthalate (PET) stickers and placed on each side of the glasses' nose pads in order to monitor sweat metabolites and electrolytes. The electronic backbone on the arms of the glasses' frame offers control of the amperometric and potentiometric transducers and enables Bluetooth wireless data transmission to the host device. The new eyeglasses system offers an interchangeable-sensor feature in connection with a variety of different nose-bridge amperometric and potentiometric sensor stickers. For example, the lactate bridge-pad sensor was replaced with a glucose one to offer convenient monitoring of sweat glucose. Such a fully integrated wireless "Lab-on-a-Glass" multiplexed biosensor platform can be readily expanded for the simultaneous monitoring of additional sweat electrolytes and metabolites.

BackgroundIt is unknown what properties would be required to make an intervention in low income countries that can eradicate or control human immunodeficiency virus (HIV) without antiretroviral therapy (ART) cost-effective.MethodsWe used a model of HIV and ART to investigate the effect of introducing an ART-free viral suppression intervention in 2022 using Zimbabwe as an example country. We assumed that the intervention (cost: $500) would be accessible for 90% of the population, be given to those receiving effective ART, have sufficient efficacy to allow ART interruption in 95%, with a rate of viral rebound of 5% per year in the first 3 months, and a 50% decline in rate with each successive year.ResultsAn ART-free viral suppression intervention with these properties would result in >0.53 million disability-adjusted-life-years averted over 2022-2042, with a reduction in HIV program costs of $300 million (8.7% saving). An intervention of this efficacy costing anything up to $1400 is likely to be cost-effective in this setting.ConclusionsInterventions aimed at curing HIV infection have the potential to improve overall disease burden and to reduce costs. Given the effectiveness and cost of ART, such interventions would have to be inexpensive and highly effective.

BackgroundParker et al. (2006) proposed a new approach to classify specific sub-types of non-melancholic depression caused by various stress factors and premorbid personality styles: the Temperament and Personality Questionnaire (T&P). The current study aim was to develop the Japanese version of the T&P and evaluate its reliability and validity.MethodsWe studied 114 patients with non-melancholic depression. Reliability was assessed using the test-retest method. Convergent validity of the T&P was compared with the clinician ratings of each patient for the eight personality traits. We also assessed the impact of depressive state on the T&P.ResultsThe test-retest intraclass correlation coefficients among eight constructs of the T&P ranged from 0.77 to 0.89, indicating good-to-excellent reliability. Anxious Worrying (rho=0.29), Perfectionism (rho=0.17), Personal Reserve (rho=0.18), Irritability (rho=0.38), and Social Avoidance (rho=0.32) showed adequate levels of convergent validity; Rejection Sensitivity (rho=0.16), Self-criticism (rho=-0.02), and Self-focus (rho=0.07) showed relatively weak convergent validity. Perfectionism (rho=-0.06), Social Avoidance (rho=0.17), Anxious Worrying (rho=0.40), Personal Reserve (rho=0.30), Irritability (rho=0.28), Rejection Sensitivity (rho=0.35), Self-criticism (rho=0.49), and Self-focus (rho=0.24) showed minimal sensitivity to mood state effects.LimitationsOnly one site was used. While a Likert scale was used, the clinician-rated personality trait measure had not been validated.ConclusionsThe J-T&P is a reliable and valid measure for assessing temperament and personality in Japanese patients with non-melancholic depression.

ObjectiveThe authors compared early-career psychiatrists' selection of psychological treatments for patients with mild to moderate major depressive disorder (MDD) in the US and Japan.MethodsA total of 120 early-career psychiatrists from two residency programs in the US and Japan participated in web-based surveys. The psychiatrists selected first- and second-line psychological treatments in response to two case vignettes of patients with mild and moderate MDD.ResultsEighty-one psychiatrists (68%) returned the surveys, of whom 39 (48%) were American and 42 (52%) Japanese. In response to the mild MDD case, more US psychiatrists selected high-intensity psychological treatments (P

Interpretation of biological mechanisms underlying genetic risk associations for prostate cancer is complicated by the relatively large number of risk variants (n = 100) and the thousands of surrogate SNPs in linkage disequilibrium. Here, we combined three distinct approaches: multiethnic fine-mapping, putative functional annotation (based upon epigenetic data and genome-encoded features), and expression quantitative trait loci (eQTL) analyses, in an attempt to reduce this complexity. We examined 67 risk regions using genotyping and imputation-based fine-mapping in populations of European (cases/controls: 8600/6946), African (cases/controls: 5327/5136), Japanese (cases/controls: 2563/4391) and Latino (cases/controls: 1034/1046) ancestry. Markers at 55 regions passed a region-specific significance threshold (P-value cutoff range: 3.9 × 10(-4)-5.6 × 10(-3)) and in 30 regions we identified markers that were more significantly associated with risk than the previously reported variants in the multiethnic sample. Novel secondary signals (P < 5.0 × 10(-6)) were also detected in two regions (rs13062436/3q21 and rs17181170/3p12). Among 666 variants in the 55 regions with P-values within one order of magnitude of the most-associated marker, 193 variants (29%) in 48 regions overlapped with epigenetic or other putative functional marks. In 11 of the 55 regions, cis-eQTLs were detected with nearby genes. For 12 of the 55 regions (22%), the most significant region-specific, prostate-cancer associated variant represented the strongest candidate functional variant based on our annotations; the number of regions increased to 20 (36%) and 27 (49%) when examining the 2 and 3 most significantly associated variants in each region, respectively. These results have prioritized subsets of candidate variants for downstream functional evaluation.

Variants in cullin 4B (CUL4B) are a known cause of syndromic X-linked intellectual disability. Here, we describe an additional 25 patients from 11 families with variants in CUL4B. We identified nine different novel variants in these families and confirmed the pathogenicity of all nontruncating variants. Neuroimaging data, available for 15 patients, showed the presence of cerebral malformations in ten patients. The cerebral anomalies comprised malformations of cortical development (MCD), ventriculomegaly, and diminished white matter volume. The phenotypic heterogeneity of the cerebral malformations might result from the involvement of CUL-4B in various cellular pathways essential for normal brain development. Accordingly, we show that CUL-4B interacts with WDR62, a protein in which variants were previously identified in patients with microcephaly and a wide range of MCD. This interaction might contribute to the development of cerebral malformations in patients with variants in CUL4B.

Background and purposeStudies show that women are more likely to receive do-not-resuscitate (DNR) orders after acute medical illnesses than men. However, the sex differences in the use of DNR orders after acute intracerebral hemorrhage (ICH) have not been described.MethodsWe conducted a retrospective study of consecutive patients hospitalized for acute ICH at a tertiary stroke center between 2006 and 2010. Unadjusted and multivariable logistic regression analyses were performed to test for associations between female sex and early (

Female mosquitoes of some species are generalists and will blood-feed on a variety of vertebrate hosts, whereas others display marked host preference. Anopheles gambiae and Aedes aegypti have evolved a strong preference for humans, making them dangerously efficient vectors of malaria and Dengue haemorrhagic fever. Specific host odours probably drive this strong preference because other attractive cues, including body heat and exhaled carbon dioxide (CO2), are common to all warm-blooded hosts. Insects sense odours via several chemosensory receptor families, including the odorant receptors (ORs), membrane proteins that form heteromeric odour-gated ion channels comprising a variable ligand-selective subunit and an obligate co-receptor called Orco (ref. 6). Here we use zinc-finger nucleases to generate targeted mutations in the orco gene of A. aegypti to examine the contribution of Orco and the odorant receptor pathway to mosquito host selection and sensitivity to the insect repellent DEET (N,N-diethyl-meta-toluamide). orco mutant olfactory sensory neurons have greatly reduced spontaneous activity and lack odour-evoked responses. Behaviourally, orco mutant mosquitoes have severely reduced attraction to honey, an odour cue related to floral nectar, and do not respond to human scent in the absence of CO2. However, in the presence of CO2, female orco mutant mosquitoes retain strong attraction to both human and animal hosts, but no longer strongly prefer humans. orco mutant females are attracted to human hosts even in the presence of DEET, but are repelled upon contact, indicating that olfactory- and contact-mediated effects of DEET are mechanistically distinct. We conclude that the odorant receptor pathway is crucial for an anthropophilic vector mosquito to discriminate human from non-human hosts and to be effectively repelled by volatile DEET.

Genistein has been shown to inhibit cancers both in vitro and in vivo, by altering the expression of several microRNAs (miRNAs). In this study, we focused on tumor suppressor miRNAs regulated by genistein and investigated their function in prostate cancer (PCa) and target pathways. Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion in vitro and in vivo. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as Pathways in cancer, Jak-STAT signaling pathway, and Wnt signaling pathway. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3 UTR of several target genes (such as RAC1, EGFR and EP300) that are components of Pathways in cancer. Quantitative real-time PCR and Western analysis showed that the mRNA and protein expression levels of the three target genes in PCa cells were markedly down-regulated with miR-574-3p. Loss-of-function studies demonstrated that the three target genes significantly affect cell proliferation, migration and invasion in PCa cell lines. Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa.

BackgroundCurrent guidelines recommend offering genetic testing for Lynch syndrome to individuals whose tumors suggest this condition and to relatives of affected individuals. Little is known, however, regarding how patients view the prospect of such testing. In addition, data on preferences (utilities) for the potential outcomes of testing decisions for use in cost-effectiveness analyses are lacking.MethodsTime tradeoff utilities were elicited for 10 potential outcomes of Lynch syndrome testing decisions and 3 associated cancers from 70 participants, representing a range of knowledge about and experiences with Lynch syndrome.ResultsHighest mean utilities were assigned to scenarios in which only the assessor's sibling had Lynch-associated colorectal cancer (ranging from 0.669 ± 0.231 to 0.760 ± 0.220). Utilities assigned to scenarios in which the assessor had Lynch-associated colorectal cancer ranged from 0.605 ± 0.252 to 0.682 ± 0.246, whereas the lowest mean utilities were assigned to 2 of the general cancer states (0.601 ± 0.238 and 0.593 ± 0.272 for colorectal and ovarian cancer respectively). Only 43% of the sample assigned higher values to undergoing Lynch testing and receiving negative results versus forgoing Lynch testing, whereas 50% assigned higher values to undergoing rather than forgoing surgery to prevent a subsequent cancer.ConclusionsGenetic testing for Lynch syndrome, regardless of results, can have profound effects on quality of life; the utilities we collected can be used to incorporate these effects into cost-effectiveness analyses. Importantly, preferences for the potential outcomes of testing vary substantially, calling into question the extent to which patients would avail themselves of such testing if it were offered to them.

Genistein has been shown to suppress the growth of several cancers through modulation of various pathways. However, the effects of genistein on the regulation of oncogenic microRNA-151 (miR-151) have not been reported. In this study, we investigated whether genistein could alter the expression of oncogenic miR-151 and its target genes that are involved in the progression and metastasis of prostate cancer (PCa). Real-time RT-PCR showed that the expression of miR-151 was higher in PC3 and DU145 cells compared with RWPE-1 cells. Treatment of PC3 and DU145 cells with 25 µM genistein down-regulated the expression of miR-151 compared with vehicle control. Inhibition of miR-151 in PCa cells by genistein significantly inhibited cell migration and invasion. In-silico analysis showed that several genes (CASZ1, IL1RAPL1, SOX17, N4BP1 and ARHGDIA) suggested to have tumor suppressive functions were target genes of miR-151. Luciferase reporter assays indicated that miR-151 directly binds to specific sites on the 3UTR of target genes. Quantitative real-time PCR analysis showed that the mRNA expression levels of the five target genes in PC3 and DU145 were markedly changed with miR-151 mimics and inhibitor. Kaplan-Meier curves and log-rank tests revealed that high expression levels of miR-151 had an adverse effect on survival rate. This study suggests that genistein mediated suppression of oncogenic miRNAs can be an important dietary therapeutic strategy for the treatment of PCa.

ObjectiveTo characterize Alexander disease (AxD) phenotypes and determine correlations with age at onset (AAO) and genetic mutation. AxD is an astrogliopathy usually characterized on MRI by leukodystrophy and caused by glial fibrillary acidic protein (GFAP) mutations.MethodsWe present 30 new cases of AxD and reviewed 185 previously reported cases. We conducted Wilcoxon rank sum tests to identify variables scaling with AAO, survival analysis to identify predictors of mortality, and χ(2) tests to assess the effects of common GFAP mutations. Finally, we performed latent class analysis (LCA) to statistically define AxD subtypes.ResultsLCA identified 2 classes of AxD. Type I is characterized by early onset, seizures, macrocephaly, motor delay, encephalopathy, failure to thrive, paroxysmal deterioration, and typical MRI features. Type II is characterized by later onset, autonomic dysfunction, ocular movement abnormalities, bulbar symptoms, and atypical MRI features. Survival analysis predicted a nearly 2-fold increase in mortality among patients with type I AxD relative to those with type II. R79 and R239 GFAP mutations were most common (16.6% and 20.3% of all cases, respectively). These common mutations predicted distinct clinical outcomes, with R239 predicting the most aggressive course.ConclusionsAAO and the GFAP mutation site are important clinical predictors in AxD, with clear correlations to defined patterns of phenotypic expression. We propose revised AxD subtypes, type I and type II, based on analysis of statistically defined patient groups.