PGE 2 suppresss intestinal tumor growth by altering the expression of tumor suppressing and DNA repair genes.

CONTEXT Some studies have inferred that an epidemic of Kaposi sarcoma-associated herpesvirus (KSHV) infection in homosexual men in the United States occurred concurrently with that of human immunodeficiency virus (HIV), but there have been no direct measurements of KSHV prevalence at the beginning of the HIV epidemic. OBJECTIVES To determine the prevalence of KSHV infection in homosexual men in San Francisco, Calif, at the beginning of the HIV epidemic in 1978 and 1979 and to examine changes in prevalence of KSHV at time points from 1978 through 1996 in light of changes in sexual behavior. DESIGN, SETTING, AND PARTICIPANTS Analysis of a clinic-based sample (n = 398) derived from the San Francisco City Clinic Cohort (ages 18-66 years) (n = 2666 for analyses herein) and from population-based samples from the San Francisco Men's Health Study (MHS) (ages 25-54 years) (n = 825 and 252) and the San Francisco Young Men's Health Study (YMHS) (ages 18-29 years) (n = 428-976, and 557); behavioral studies were longitudinal and KSHV prevalence studies were cross-sectional. MAIN OUTCOME MEASURES Antibodies against KSHV and HIV; sexual behaviors. RESULTS The prevalence of KSHV infection in 1978 and 1979 was 26.5% of 235 (a random sample) overall (weighted for HIV infection) vs 6.9% (128/1842) for HIV in the San Francisco City Clinic Cohort sample. The prevalence of KSHV infection remained essentially unchanged between an MHS sample of 252 in 1984 and 1985 (29.6%) and a YMHS sample of 557 in 1995 and 1996 (26.4%), while HIV prevalence dropped from 49.5% of 825 in 1984 and 1985 (MHS) to 17.6% of 428 in 1992 and 1993 (YMHS). The proportion of men practicing unprotected receptive anal intercourse with 1 or more partners declined from 54% to 11% during the 1984 through 1993 period (MHS) with similar though slightly higher values in the YMHS in 1992 and 1993; whereas for unprotected oral intercourse it ranged between 60% and 90% in the 1984 through 1996 period (MHS and YMHS). CONCLUSIONS Infection with KSHV was already highly prevalent in homosexual men when the HIV epidemic began in San Francisco, and its prevalence has been maintained at a nearly constant level. Any declines in the incidence of Kaposi sarcoma do not appear to be caused by a decline in KSHV transmission.

IMPORTANCE Identification of the bacterium responsible for an outbreak can aid in disease management. However, traditional culture-based diagnosis can be difficult, particularly if no specific diagnostic test is available for an outbreak strain. OBJECTIVE To explore the potential of metagenomics, which is the direct sequencing of DNA extracted from microbiologically complex samples, as an open-ended clinical discovery platform capable of identifying and characterizing bacterial strains from an outbreak without laboratory culture. DESIGN, SETTING, AND PATIENTS In a retrospective investigation, 45 samples were selected from fecal specimens obtained from patients with diarrhea during the 2011 outbreak of Shiga-toxigenic Escherichia coli (STEC) O104:H4 in Germany. Samples were subjected to high-throughput sequencing (August-September 2012), followed by a 3-phase analysis (November 2012-February 2013). In phase 1, a de novo assembly approach was developed to obtain a draft genome of the outbreak strain. In phase 2, the depth of coverage of the outbreak strain genome was determined in each sample. In phase 3, sequences from each sample were compared with sequences from known bacteria to identify pathogens other than the outbreak strain. MAIN OUTCOMES AND MEASURES The recovery of genome sequence data for the purposes of identification and characterization of the outbreak strain and other pathogens from fecal samples. RESULTS During phase 1, a draft genome of the STEC outbreak strain was obtained. During phase 2, the outbreak strain genome was recovered from 10 samples at greater than 10-fold coverage and from 26 samples at greater than 1-fold coverage. Sequences from the Shiga-toxin genes were detected in 27 of 40 STEC-positive samples (67%). In phase 3, sequences from Clostridium difficile, Campylobacter jejuni, Campylobacter concisus, and Salmonella enterica were recovered. CONCLUSIONS AND RELEVANCE These results suggest the potential of metagenomics as a culture-independent approach for the identification of bacterial pathogens during an outbreak of diarrheal disease. Challenges include improving diagnostic sensitivity, speeding up and simplifying workflows, and reducing costs.

BACKGROUND Alirocumab, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), lowers plasma low-density lipoprotein (LDL) cholesterol and apolipoprotein B100 (apoB). Although studies in mice and cells have identified increased hepatic LDL receptors as the basis for LDL lowering by PCSK9 inhibitors, there have been no human studies characterizing the effects of PCSK9 inhibitors on lipoprotein metabolism. In particular, it is not known whether inhibition of PCSK9 has any effects on very low-density lipoprotein or intermediate-density lipoprotein (IDL) metabolism. Inhibition of PCSK9 also results in reductions of plasma lipoprotein (a) levels. The regulation of plasma Lp(a) levels, including the role of LDL receptors in the clearance of Lp(a), is poorly defined, and no mechanistic studies of the Lp(a) lowering by alirocumab in humans have been published to date. METHODS Eighteen (10 F, 8 mol/L) participants completed a placebo-controlled, 2-period study. They received 2 doses of placebo, 2 weeks apart, followed by 5 doses of 150 mg of alirocumab, 2 weeks apart. At the end of each period, fractional clearance rates (FCRs) and production rates (PRs) of apoB and apo(a) were determined. In 10 participants, postprandial triglycerides and apoB48 levels were measured. RESULTS Alirocumab reduced ultracentrifugally isolated LDL-C by 55.1%, LDL-apoB by 56.3%, and plasma Lp(a) by 18.7%. The fall in LDL-apoB was caused by an 80.4% increase in LDL-apoB FCR and a 23.9% reduction in LDL-apoB PR. The latter was due to a 46.1% increase in IDL-apoB FCR coupled with a 27.2% decrease in conversion of IDL to LDL. The FCR of apo(a) tended to increase (24.6%) without any change in apo(a) PR. Alirocumab had no effects on FCRs or PRs of very low-density lipoproteins-apoB and very low-density lipoproteins triglycerides or on postprandial plasma triglycerides or apoB48 concentrations. CONCLUSIONS Alirocumab decreased LDL-C and LDL-apoB by increasing IDL- and LDL-apoB FCRs and decreasing LDL-apoB PR. These results are consistent with increases in LDL receptors available to clear IDL and LDL from blood during PCSK9 inhibition. The increase in apo(a) FCR during alirocumab treatment suggests that increased LDL receptors may also play a role in the reduction of plasma Lp(a). CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01959971.

The androgen receptor (AR) is overexpressed and hyperactivated in human castration-resistant prostate cancer (CRPC). However, the determinants of AR overexpression in CRPC are poorly defined. Here we show that retinoic acid receptor-related orphan receptor γ (ROR-γ) is overexpressed and amplified in metastatic CRPC tumors, and that ROR-γ drives AR expression in the tumors. ROR-γ recruits nuclear receptor coactivator 1 and 3 (NCOA1 and NCOA3, also known as SRC-1 and SRC-3) to an AR-ROR response element (RORE) to stimulate AR gene transcription. ROR-γ antagonists suppress the expression of both AR and its variant AR-V7 in prostate cancer (PCa) cell lines and tumors. ROR-γ antagonists also markedly diminish genome-wide AR binding, H3K27ac abundance and expression of the AR target gene network. Finally, ROR-γ antagonists suppressed tumor growth in multiple AR-expressing, but not AR-negative, xenograft PCa models, and they effectively sensitized CRPC tumors to enzalutamide, without overt toxicity, in mice. Taken together, these results establish ROR-γ as a key player in CRPC by acting upstream of AR and as a potential therapeutic target for advanced PCa.

OBJECTIVE To determine whether individual fruits are differentially associated with risk of type 2 diabetes. DESIGN Prospective longitudinal cohort study. SETTING Health professionals in the United States. PARTICIPANTS 66,105 women from the Nurses' Health Study (1984-2008), 85,104 women from the Nurses' Health Study II (1991-2009), and 36,173 men from the Health Professionals Follow-up Study (1986-2008) who were free of major chronic diseases at baseline in these studies. MAIN OUTCOME MEASURE Incident cases of type 2 diabetes, identified through self report and confirmed by supplementary questionnaires. RESULTS During 3,464,641 person years of follow-up, 12,198 participants developed type 2 diabetes. After adjustment for personal, lifestyle, and dietary risk factors of diabetes, the pooled hazard ratio of type 2 diabetes for every three servings/week of total whole fruit consumption was 0.98 (95% confidence interval 0.97 [corrected] to 0.99). With mutual adjustment of individual fruits, the pooled hazard ratios of type 2 diabetes for every three servings/week were 0.74 (0.66 to 0.83) for blueberries, 0.88 (0.83 to 0.93) for grapes and raisins, 0.89 (0.79 to 1.01) for prunes, 0.93 (0.90 to 0.96) for apples and pears, 0.95 (0.91 to 0.98) for bananas, 0.95 (0.91 to 0.99) for grapefruit, 0.97 (0.92 to 1.02) for peaches, plums, and apricots, 0.99 (0.95 to 1.03) for oranges, 1.03 (0.96 to 1.10) for strawberries, and 1.10 (1.02 to 1.18) for cantaloupe. The pooled hazard ratio for the same increment in fruit juice consumption was 1.08 (1.05 to 1.11). The associations with risk of type 2 diabetes differed significantly among individual fruits (P<0.001 in all cohorts). CONCLUSION Our findings suggest the presence of heterogeneity in the associations between individual fruit consumption and risk of type 2 diabetes. Greater consumption of specific whole fruits, particularly blueberries, grapes, and apples, is significantly associated with a lower risk of type 2 diabetes, whereas greater consumption of fruit juice is associated with a higher risk.

Importance Although non–vitamin K antagonist oral anticoagulants (NOACs) are increasingly used to prevent thromboembolic disease, there are limited data on NOAC-related intracerebral hemorrhage (ICH). Objective To assess the association between preceding oral anticoagulant use (warfarin, NOACs, and no oral anticoagulants [OACs]) and in-hospital mortality among patients with ICH. Design, Setting, and Participants Retrospective cohort study of 141 311 patients with ICH admitted from October 2013 to December 2016 to 1662 Get With The Guidelines–Stroke hospitals. Exposures Anticoagulation therapy before ICH, defined as any use of OACs within 7 days prior to hospital arrival. Main Outcomes and Measures In-hospital mortality. Results Among 141 311 patients with ICH (mean [SD] age, 68.3 [15.3] years; 48.1% women), 15 036 (10.6%) were taking warfarin and 4918 (3.5%) were taking NOACs preceding ICH, and 39 585 (28.0%) and 5783 (4.1%) were taking concomitant single and dual antiplatelet agents, respectively. Patients with prior use of warfarin or NOACs were older and had higher prevalence of atrial fibrillation and prior stroke. Acute ICH stroke severity (measured by the National Institutes of Health Stroke Scale) was not significantly different across the 3 groups (median, 9 [interquartile range, 2-21] for warfarin, 8 [2-20] for NOACs, and 8 [2-19] for no OACs). The unadjusted in-hospital mortality rates were 32.6% for warfarin, 26.5% for NOACs, and 22.5% for no OACs. Compared with patients without prior use of OACs, the risk of in-hospital mortality was higher among patients with prior use of warfarin (adjusted risk difference [ARD], 9.0% [97.5% CI, 7.9% to 10.1%]; adjusted odds ratio [AOR], 1.62 [97.5% CI, 1.53 to 1.71]) and higher among patients with prior use of NOACs (ARD, 3.3% [97.5% CI, 1.7% to 4.8%]; AOR, 1.21 [97.5% CI, 1.11-1.32]). Compared with patients with prior use of warfarin, patients with prior use of NOACs had a lower risk of in-hospital mortality (ARD, −5.7% [97.5% CI, −7.3% to −4.2%]; AOR, 0.75 [97.5% CI, 0.69 to 0.81]). The difference in mortality between NOAC-treated patients and warfarin-treated patients was numerically greater among patients with prior use of dual antiplatelet agents (32.7% vs 47.1%; ARD, −15.0% [95.5% CI, −26.3% to −3.8%]; AOR, 0.50 [97.5% CI, 0.29 to 0.86]) than among those taking these agents without prior antiplatelet therapy (26.4% vs 31.7%; ARD, −5.0% [97.5% CI, −6.8% to −3.2%]; AOR, 0.77 [97.5% CI, 0.70 to 0.85]), although the interaction P value (.07) was not statistically significant. Conclusions and Relevance Among patients with ICH, prior use of NOACs or warfarin was associated with higher in-hospital mortality compared with no OACs. Prior use of NOACs, compared with prior use of warfarin, was associated with lower risk of in-hospital mortality.


Genetic deficiency of mast cells leads to decreased weight gain in a mouse model of diet-induced obesity.

Although mast cell functions have classically been related to allergic responses, recent studies indicate that these cells contribute to other common diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis, aortic aneurysm and cancer. This study presents evidence that mast cells also contribute to diet-induced obesity and diabetes. For example, white adipose tissue (WAT) from obese humans and mice contain more mast cells than WAT from their lean counterparts. Furthermore, in the context of mice on a Western diet, genetically induced deficiency of mast cells, or their pharmacological stabilization, reduces body weight gain and levels of inflammatory cytokines, chemokines and proteases in serum and WAT, in concert with improved glucose homeostasis and energy expenditure. Mechanistic studies reveal that mast cells contribute to WAT and muscle angiogenesis and associated cell apoptosis and cathepsin activity. Adoptive transfer experiments of cytokine-deficient mast cells show that these cells, by producing interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), contribute to mouse adipose tissue cysteine protease cathepsin expression, apoptosis and angiogenesis, thereby promoting diet-induced obesity and glucose intolerance. Our results showing reduced obesity and diabetes in mice treated with clinically available mast cell-stabilizing agents suggest the potential of developing new therapies for these common human metabolic disorders.


N348I mutations reduce resistance to nevirapine.

BACKGROUND Homocysteine is a risk factor for coronary artery disease (CAD), although a causal relation remains to be proven. The importance of determining direct causality rests in the fact that plasma homocysteine can be safely and inexpensively reduced by 25% with folic acid. This reduction is maximally achieved by doses of 0.4 mg/d. High-dose folic acid (5 mg/d) improves endothelial function in CAD, although the mechanism is controversial. It has been proposed that improvement occurs through reduction in total (tHcy) or free (non-protein bound) homocysteine (fHcy). We investigated the effects of folic acid on endothelial function before a change in homocysteine in patients with CAD. METHODS AND RESULTS A randomized, placebo-controlled study of folic acid (5 mg/d) for 6 weeks was undertaken in 33 patients. Endothelial function, assessed by flow-mediated dilatation (FMD), was measured before, at 2 and 4 hours after the first dose of folic acid, and after 6 weeks of treatment. Plasma folate increased markedly by 1 hour (200 compared with 25.8 nmol/L; P<0.001). FMD improved at 2 hours (83 compared with 47 microm; P<0.001) and was largely complete by 4 hours (101 compared with 51 microm; P<0.001). tHcy did not significantly differ acutely (4-hour tHcy, 9.56 compared with 9.79 micromol/L; P=NS). fHcy did not differ at 3 hours but was slightly reduced at 4 hours (1.55 compared with 1.78 micromol/L; P=0.02). FMD improvement did not correlate with reductions in either fHcy or tHcy at any time. CONCLUSIONS These data suggest that folic acid improves endothelial function in CAD acutely by a mechanism largely independent of homocysteine.

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.

Background The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre’s database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance. Methods and Findings The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre’s database. N348I increased in prevalence from below 1% in 368 treatmentnao ¨ve individuals to 12.1% in 1,009 treatment-experienced patients (p ¼ 7.7 3 10 � 12 ). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p , 0.001), the lamivudine resistance mutations M184V/I (p , 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p , 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43–4.81). The appearance of N348I was associated with a significant increase in viral load (p , 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wildtype HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance. Conclusions

BACKGROUND Organophosphorus pesticide (OP) self-poisoning is a major problem in the developing rural world. There is little clinical trial data to guide therapy, hindering the identification of best therapy. Despite the recognition of adverse effects, gastric lavage is commonly done in Asia. We aimed to identify studies assessing its effectiveness. METHOD We systematically searched the literature for controlled clinical studies that assessed the effect of gastric lavage in OP pesticide self-poisoning. RESULTS All 56 studies identified were Chinese and reported benefit from the intervention studied, including multiple gastric lavages, use of norepinephrine or pralidoxime in the lavage fluid, concurrent treatment with naloxone or scopolamine, insertion of the gastric tube via a laparotomy incision, and lavage later than 12 h post-ingestion. However, only 23 were RCTs and none presented adequate methodology for their quality to be assessed. The patient population and study treatment protocol were not defined - large variation in case fatality in the control arm of the studies (from 4.5 to 93%) suggests marked variation between studies and likely between study arms. No study compared an intervention against a control group receiving no gastric lavage or provided any data to indicate whether a significant quantity of poison was removed. CONCLUSION Despite widespread use of multiple gastric lavages for OP pesticide poisoning across Asia, there is currently no high-quality evidence to support its clinical effectiveness. There is a need for studies to identify in which patients and for what duration gastric lavage is able to remove significant quantities of poison. Following these studies, large clinical trials will be required to address the effectiveness and safety of gastric lavage (either single or multiple) in acute OP pesticide poisoning.


Question:
Inhibiting glucose-6-phospate dehydrogenase improves lipogenesis by deactivating LKB1-AMPK signaling.

BACKGROUND Interferon-gamma (IFN-gamma) is an essential cytokine in the regulation of inflammatory responses in autoimmune diseases. Little is known about its role in inflammatory heart disease. METHODS AND RESULTS We showed that IFN-gamma receptor-deficient mice (IFN-gammaR(-/-)) on a BALB/c background immunized with a peptide derived from cardiac alpha-myosin heavy chain develop severe myocarditis with high mortality. Although myocarditis subsided in wild-type mice after 3 weeks, IFN-gammaR(-/-) mice showed persistent disease. The persistent inflammation was accompanied by vigorous in vitro CD4 T-cell responses and impaired inducible nitric oxide synthase expression, together with evidence of impaired nitric oxide production in IFN-gammaR(-/-) hearts. Treatment of wild-type mice with the nitric oxide synthetase inhibitor N:-nitro-l-arginine-methyl-ester enhanced in vitro CD4 T-cell proliferation and prevented healing of myocarditis. CONCLUSIONS Our data provide evidence that IFN-gamma protects mice from lethal autoimmune myocarditis by inducing the expression of inducible nitric oxide synthase followed by the downregulation of T-cell responses.

The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.

It is unclear how the immune response in early life becomes appropriately stimulated to provide protection while also avoiding excessive activation as a result of diverse new antigens. T cells are integral to adaptive immunity; mouse studies indicate that tissue localization of T cell subsets is important for both protective immunity and immunoregulation. In humans, however, the early development and function of T cells in tissues remain unexplored. We present here an analysis of lymphoid and mucosal tissue T cells derived from pediatric organ donors in the first two years of life, as compared to adult organ donors, revealing early compartmentalization of T cell differentiation and regulation. Whereas adult tissues contain a predominance of memory T cells, in pediatric blood and tissues the main subset consists of naive recent thymic emigrants, with effector memory T cells (T(EM)) found only in the lungs and small intestine. Additionally, regulatory T (T(reg)) cells comprise a high proportion (30-40%) of CD4(+) T cells in pediatric tissues but are present at much lower frequencies (1-10%) in adult tissues. Pediatric tissue T(reg) cells suppress endogenous T cell activation, and early T cell functionality is confined to the mucosal sites that have the lowest T(reg):T(EM) cell ratios, which suggests control in situ of immune responses in early life.

The overall size and structure of a synaptic terminal is an important determinant of its function. In a large-scale mutagenesis screen, designed to identify Drosophila mutants with abnormally structured neuromuscular junctions (NMJs), we discovered mutations in Drosophila mical, a conserved gene encoding a multi-domain protein with a N-terminal monooxygenase domain. In mical mutants, synaptic boutons do not sprout normally over the muscle surface and tend to form clusters along synaptic branches and at nerve entry sites. Consistent with high expression of MICAL in somatic muscles, immunohistochemical stainings reveal that the subcellular localization and architecture of contractile muscle filaments are dramatically disturbed in mical mutants. Instead of being integrated into a regular sarcomeric pattern, actin and myosin filaments are disorganized and accumulate beneath the plasmamembrane. Whereas contractile elements are strongly deranged, the proposed organizer of sarcomeric structure, D-Titin, is much less affected. Transgenic expression of interfering RNA molecules demonstrates that MICAL is required in muscles for the higher order arrangement of myofilaments. Ultrastructural analysis confirms that myosin-rich thick filaments enter submembranous regions and interfere with synaptic development, indicating that the disorganized myofilaments may cause the synaptic growth phenotype. As a model, we suggest that the filamentous network around synaptic boutons restrains the spreading of synaptic branches.

Background  Celiac disease is a small intestinal inflammatory disorder characterized by malabsorption, nutrient deficiency, and a range of clinical manifestations. It is caused by an inappropriate immune response to dietary gluten and is treated with a gluten-free diet. Recent feeding studies have indicated oats to be safe for celiac disease patients, and oats are now often included in the celiac disease diet. This study aimed to investigate whether oat intolerance exists in celiac disease and to characterize the cells and processes underlying this intolerance. Methods and Findings  We selected for study nine adults with celiac disease who had a history of oats exposure. Four of the patients had clinical symptoms on an oats-containing diet, and three of these four patients had intestinal inflammation typical of celiac disease at the time of oats exposure. We established oats-avenin-specific and -reactive intestinal T-cell lines from these three patients, as well as from two other patients who appeared to tolerate oats. The avenin-reactive T-cell lines recognized avenin peptides in the context of HLA-DQ2. These peptides have sequences rich in proline and glutamine residues closely resembling wheat gluten epitopes. Deamidation (glutamine→glutamic acid conversion) by tissue transglutaminase was involved in the avenin epitope formation. Conclusions  We conclude that some celiac disease patients have avenin-reactive mucosal T-cells that can cause mucosal inflammation. Oat intolerance may be a reason for villous atrophy and inflammation in patients with celiac disease who are eating oats but otherwise are adhering to a strict gluten-free diet. Clinical follow-up of celiac disease patients eating oats is advisable.

IMPORTANCE The programmed death 1 (PD-1) pathway limits immune responses to melanoma and can be blocked with the humanized anti-PD-1 monoclonal antibody pembrolizumab. OBJECTIVE To characterize the association of pembrolizumab with tumor response and overall survival among patients with advanced melanoma. DESIGN, SETTINGS, AND PARTICIPANTS Open-label, multicohort, phase 1b clinical trials (enrollment, December 2011-September 2013). Median duration of follow-up was 21 months. The study was performed in academic medical centers in Australia, Canada, France, and the United States. Eligible patients were aged 18 years and older and had advanced or metastatic melanoma. Data were pooled from 655 enrolled patients (135 from a nonrandomized cohort [n = 87 ipilimumab naive; n = 48 ipilimumab treated] and 520 from randomized cohorts [n = 226 ipilimumab naive; n = 294 ipilimumab treated]). Cutoff dates were April 18, 2014, for safety analyses and October 18, 2014, for efficacy analyses. EXPOSURES Pembrolizumab 10 mg/kg every 2 weeks, 10 mg/kg every 3 weeks, or 2 mg/kg every 3 weeks continued until disease progression, intolerable toxicity, or investigator decision. MAIN OUTCOMES AND MEASURES The primary end point was confirmed objective response rate (best overall response of complete response or partial response) in patients with measurable disease at baseline per independent central review. Secondary end points included toxicity, duration of response, progression-free survival, and overall survival. RESULTS Among the 655 patients (median [range] age, 61 [18-94] years; 405 [62%] men), 581 had measurable disease at baseline. An objective response was reported in 194 of 581 patients (33% [95% CI, 30%-37%]) and in 60 of 133 treatment-naive patients (45% [95% CI, 36% to 54%]). Overall, 74% (152/205) of responses were ongoing at the time of data cutoff; 44% (90/205) of patients had response duration for at least 1 year and 79% (162/205) had response duration for at least 6 months. Twelve-month progression-free survival rates were 35% (95% CI, 31%-39%) in the total population and 52% (95% CI, 43%-60%) among treatment-naive patients. Median overall survival in the total population was 23 months (95% CI, 20-29) with a 12-month survival rate of 66% (95% CI, 62%-69%) and a 24-month survival rate of 49% (95% CI, 44%-53%). In treatment-naive patients, median overall survival was 31 months (95% CI, 24 to not reached) with a 12-month survival rate of 73% (95% CI, 65%-79%) and a 24-month survival rate of 60% (95% CI, 51%-68%). Ninety-two of 655 patients (14%) experienced at least 1 treatment-related grade 3 or 4 adverse event (AE) and 27 of 655 (4%) patients discontinued treatment because of a treatment-related AE. Treatment-related serious AEs were reported in 59 patients (9%). There were no drug-related deaths. CONCLUSIONS AND RELEVANCE Among patients with advanced melanoma, pembrolizumab administration was associated with an overall objective response rate of 33%, 12-month progression-free survival rate of 35%, and median overall survival of 23 months; grade 3 or 4 treatment-related AEs occurred in 14%. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01295827.


Persistor cells are one reason for incomplete responses to Tyrosine kinase inhibitor (TKI) therapy in cancer patients.

Molecular targeted therapy has the potential to dramatically improve survival in patients with cancer. However, complete and durable responses to targeted therapy are rare in individuals with advanced-stage solid cancers. Even the most effective targeted therapies generally do not induce a complete tumor response, resulting in residual disease and tumor progression that limits patient survival. We discuss the emerging need to more fully understand the molecular basis of residual disease as a prelude to designing therapeutic strategies to minimize or eliminate residual disease so that we can move from temporary to chronic control of disease, or a cure, for patients with advanced-stage solid cancers. Ultimately, we propose a shift from the current reactive paradigm of analyzing and treating acquired drug resistance to a pre-emptive paradigm of defining the mechanisms that result in residual disease, to target and limit this disease reservoir.

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN− dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.

Previous studies investigating the role of smooth muscle cells (SMCs) and macrophages in the pathogenesis of atherosclerosis have provided controversial results owing to the use of unreliable methods for clearly identifying each of these cell types. Here, using Myh11-CreERT2 ROSA floxed STOP eYFP Apoe−/− mice to perform SMC lineage tracing, we find that traditional methods for detecting SMCs based on immunostaining for SMC markers fail to detect >80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages, including macrophages and mesenchymal stem cells (MSCs). SMC-specific conditional knockout of Krüppel-like factor 4 (Klf4) resulted in reduced numbers of SMC-derived MSC- and macrophage-like cells, a marked reduction in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness as compared to wild-type controls. On the basis of in vivo KLF4 chromatin immunoprecipitation–sequencing (ChIP-seq) analyses and studies of cholesterol-treated cultured SMCs, we identified >800 KLF4 target genes, including many that regulate pro-inflammatory responses of SMCs. Our findings indicate that the contribution of SMCs to atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.

CONTEXT The epidemic of heart failure has yet to be fully investigated, and data on incidence, survival, and sex-specific temporal trends in community-based populations are limited. OBJECTIVE To test the hypothesis that the incidence of heart failure has declined and survival after heart failure diagnosis has improved over time but that secular trends have diverged by sex. DESIGN, SETTING, AND PARTICIPANTS Population-based cohort study using the resources of the Rochester Epidemiology Project conducted in Olmsted County, Minnesota. Patients were 4537 Olmsted County residents (57% women; mean [SD] age, 74 [14] years) with a diagnosis of heart failure between 1979 and 2000. Framingham criteria and clinical criteria were used to validate the diagnosis   MAIN OUTCOME MEASURES Incidence of heart failure and survival after heart failure diagnosis. RESULTS The incidence of heart failure was higher among men (378/100 000 persons; 95% confidence interval [CI], 361-395 for men; 289/100 000 persons; 95% CI, 277-300 for women) and did not change over time among men or women. After a mean follow-up of 4.2 years (range, 0-23.8 years), 3347 deaths occurred, including 1930 among women and 1417 among men. Survival after heart failure diagnosis was worse among men than women (relative risk, 1.33; 95% CI, 1.24-1.43) but overall improved over time (5-year age-adjusted survival, 43% in 1979-1984 vs 52% in 1996-2000, P<.001). However, men and younger persons experienced larger survival gains, contrasting with less or no improvement for women and elderly persons. CONCLUSION In this community-based cohort, the incidence of heart failure has not declined during 2 decades, but survival after onset of heart failure has increased overall, with less improvement among women and elderly persons.

BACKGROUND Mitochondrial DNA (mtDNA) is a critical activator of inflammation and the innate immune system. However, mtDNA level has not been tested for its role as a biomarker in the intensive care unit (ICU). We hypothesized that circulating cell-free mtDNA levels would be associated with mortality and improve risk prediction in ICU patients. METHODS AND FINDINGS Analyses of mtDNA levels were performed on blood samples obtained from two prospective observational cohort studies of ICU patients (the Brigham and Women's Hospital Registry of Critical Illness [BWH RoCI, n = 200] and Molecular Epidemiology of Acute Respiratory Distress Syndrome [ME ARDS, n = 243]). mtDNA levels in plasma were assessed by measuring the copy number of the NADH dehydrogenase 1 gene using quantitative real-time PCR. Medical ICU patients with an elevated mtDNA level (≥3,200 copies/µl plasma) had increased odds of dying within 28 d of ICU admission in both the BWH RoCI (odds ratio [OR] 7.5, 95% CI 3.6-15.8, p = 1×10(-7)) and ME ARDS (OR 8.4, 95% CI 2.9-24.2, p = 9×10(-5)) cohorts, while no evidence for association was noted in non-medical ICU patients. The addition of an elevated mtDNA level improved the net reclassification index (NRI) of 28-d mortality among medical ICU patients when added to clinical models in both the BWH RoCI (NRI 79%, standard error 14%, p<1×10(-4)) and ME ARDS (NRI 55%, standard error 20%, p = 0.007) cohorts. In the BWH RoCI cohort, those with an elevated mtDNA level had an increased risk of death, even in analyses limited to patients with sepsis or acute respiratory distress syndrome. Study limitations include the lack of data elucidating the concise pathological roles of mtDNA in the patients, and the limited numbers of measurements for some of biomarkers. CONCLUSIONS Increased mtDNA levels are associated with ICU mortality, and inclusion of mtDNA level improves risk prediction in medical ICU patients. Our data suggest that mtDNA could serve as a viable plasma biomarker in medical ICU patients.


Pyridostatin decreases telomere fragility in BRCA2-deficient cells.

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

All patients with metastatic lung, colorectal, pancreatic or head and neck cancers who initially benefit from epidermal growth factor receptor (EGFR)-targeted therapies eventually develop resistance. An increasing understanding of the number and complexity of resistance mechanisms highlights the Herculean challenge of killing tumors that are resistant to EGFR inhibitors. Our growing knowledge of resistance pathways provides an opportunity to develop new mechanism-based inhibitors and combination therapies to prevent or overcome therapeutic resistance in tumors. We present a comprehensive review of resistance pathways to EGFR-targeted therapies in lung, colorectal and head and neck cancers and discuss therapeutic strategies that are designed to circumvent resistance.

Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP.

Studying how protein transmembrane domains transmit signals across membranes is beset by unique challenges. Here, we discuss the circumstances that have led to success and reflect on what has been learned from these examples. Such efforts suggest that some of the most interesting properties of transmembrane helix interactions may be the least amenable to study by current techniques.

Recognition of modified histones by ‘reader’ proteins plays a critical role in the regulation of chromatin. H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions after RNA polymerase II elongation. In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin to a relatively repressive state, thus suppressing cryptic transcription. However, much less is known about the role of H3K36me3 in transcription regulation in mammals. This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies. Here we show that the candidate tumour suppressor ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates RNA polymerase II elongation. Structural studies show that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific ‘Ser 31’ residue in a composite pocket formed by the tandem bromo–PWWP domains of ZMYND11. Chromatin immunoprecipitation followed by sequencing shows a genome-wide co-localization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3. Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription co-repressor by modulating RNA polymerase II at the elongation stage. ZMYND11 is critical for the repression of a transcriptional program that is essential for tumour cell growth; low expression levels of ZMYND11 in breast cancer patients correlate with worse prognosis. Consistently, overexpression of ZMYND11 suppresses cancer cell growth in vitro and tumour formation in mice. Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone-variant-mediated transcription elongation control to tumour suppression.


Question:
Mice that lack Interferon-γ or its receptor are highly susceptible to experimental autoimmune myocarditis.

&NA; Immune cells communicate by exchanging cytokines to achieve a context‐appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self‐assembled niches scaled with the density of cytokine‐consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80–120 &mgr;m, which resulted in a high degree of cell‐to‐cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non‐genetic cell‐to‐cell variability that is often overlooked in single‐cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors. Graphical Abstract Figure. No caption available. HighlightsCytokine penetration in tissues is governed by a diffusion‐consumption mechanismSpherical cytokine niches are generated around cytokine‐producing cellsThe characteristic niche size depends on the density of cytokine consumersCytokine niches are a source of variability in otherwise identical cells &NA; Cytokine‐mediated communication allows immune cells to achieve a context‐appropriate response, but the distance over which this communication happens is unclear. Oyler‐Yaniv et al. (2017) show that a simple diffusion‐consumption mechanism quantitatively describes the spatial spread of cytokines in vivo and results in localized niches of high cytokine concentrations that contribute to cell‐to‐cell variability.

BACKGROUND Interferon-gamma (IFN-gamma) is an essential cytokine in the regulation of inflammatory responses in autoimmune diseases. Little is known about its role in inflammatory heart disease. METHODS AND RESULTS We showed that IFN-gamma receptor-deficient mice (IFN-gammaR(-/-)) on a BALB/c background immunized with a peptide derived from cardiac alpha-myosin heavy chain develop severe myocarditis with high mortality. Although myocarditis subsided in wild-type mice after 3 weeks, IFN-gammaR(-/-) mice showed persistent disease. The persistent inflammation was accompanied by vigorous in vitro CD4 T-cell responses and impaired inducible nitric oxide synthase expression, together with evidence of impaired nitric oxide production in IFN-gammaR(-/-) hearts. Treatment of wild-type mice with the nitric oxide synthetase inhibitor N:-nitro-l-arginine-methyl-ester enhanced in vitro CD4 T-cell proliferation and prevented healing of myocarditis. CONCLUSIONS Our data provide evidence that IFN-gamma protects mice from lethal autoimmune myocarditis by inducing the expression of inducible nitric oxide synthase followed by the downregulation of T-cell responses.

Satellite cells are skeletal muscle stem cells capable of self-renewal and differentiation after transplantation, but whether they contribute to endogenous muscle fiber repair has been unclear. The transcription factor Pax7 marks satellite cells and is critical for establishing the adult satellite cell pool. By using a lineage tracing approach, we show that after injury, quiescent adult Pax7(+) cells enter the cell cycle; a subpopulation returns to quiescence to replenish the satellite cell compartment, while others contribute to muscle fiber formation. We demonstrate that Sprouty1 (Spry1), a receptor tyrosine kinase signaling inhibitor, is expressed in quiescent Pax7(+) satellite cells in uninjured muscle, downregulated in proliferating myogenic cells after injury, and reinduced as Pax7(+) cells re-enter quiescence. We show that Spry1 is required for the return to quiescence and homeostasis of the satellite cell pool during repair. Our results therefore define a role for Spry1 in adult muscle stem cell biology and tissue repair.


Question:
Cytosolic proteins bind to iron-responsive elements on mRNAs coding for TFRC1.

Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element-binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.


Question:
Cis-acting lncRNAs control the expression of genes that are positioned in the vicinity of their transcription sites.

Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.


Question:
The morphology change from large white adipocytes to small brown adipocytes is a sign of decreased energy expenditure potential in white adipose tissue.

OBJECTIVE To determine the rate of early discontinuation and non-publication of randomised controlled trials involving patients undergoing surgery. DESIGN Cross sectional observational study of registered and published trials. SETTING Randomised controlled trials of interventions in patients undergoing a surgical procedure. DATA SOURCES The ClinicalTrials.gov database was searched for interventional trials registered between January 2008 and December 2009 using the keyword "surgery". Recruitment status was extracted from the ClinicalTrials.gov database. A systematic search for studies published in peer reviewed journals was performed; if they were not found, results posted on the ClinicalTrials.gov results database were sought. Email queries were sent to trial investigators of discontinued and unpublished completed trials if no reason for the respective status was disclosed. MAIN OUTCOME MEASURES Trial discontinuation before completion and non-publication after completion. Logistic regression was used to determine the effect of funding source on publication status, with adjustment for intervention type and trial size. RESULTS Of 818 registered trials found using the keyword "surgery", 395 met the inclusion criteria. Of these, 21% (81/395) were discontinued early, most commonly owing to poor recruitment (44%, 36/81). The remaining 314 (79%) trials proceeded to completion, with a publication rate of 66% (208/314) at a median time of 4.9 (interquartile range 4.0-6.0) years from study completion to publication search. A further 6% (20/314) of studies presented results on ClinicalTrials.gov without a corresponding peer reviewed publication. Industry funding did not affect the rate of discontinuation (adjusted odds ratio 0.91, 95% confidence interval 0.54 to 1.55) but was associated with a lower odds of publication for completed trials (0.43, 0.26 to 0.72). Investigators' email addresses for trials with an uncertain fate were identified for 71.4% (10/14) of discontinued trials and 83% (101/122) of unpublished studies. Only 43% (6/14) and 20% (25/122) replies were received. Email responses for completed trials indicated 11 trials in press, five published studies (four in non-indexed peer reviewed journals), and nine trials remaining unpublished. CONCLUSIONS One in five surgical randomised controlled trials are discontinued early, one in three completed trials remain unpublished, and investigators of unpublished studies are frequently not contactable. This represents a waste of research resources and raises ethical concerns regarding hidden clinical data and futile participation by patients with its attendant risks. To promote future efficiency and transparency, changes are proposed to research governance frameworks to overcome these concerns.

BACKGROUND Although unstable coronary artery disease is the most common reason for admission to a coronary care unit, the long-term prognosis of patients with this diagnosis is unknown. This is particularly true for patients with diabetes mellitus, who are known to have a high morbidity and mortality after an acute myocardial infarction. METHODS AND RESULTS Prospectively collected data from 6 different countries in the Organization to Assess Strategies for Ischemic Syndromes (OASIS) registry were analyzed to determine the 2-year prognosis of diabetic and nondiabetic patients who were hospitalized with unstable angina or non-Q-wave myocardial infarction. Overall, 1718 of 8013 registry patients (21%) had diabetes. Diabetic patients had a higher rate of coronary bypass surgery than nondiabetic patients (23% versus 20%, P:<0.001) but had similar rates of catheterization and angioplasty. Diabetes independently predicted mortality (relative risk [RR], 1.57; 95% CI, 1.38 to 1.81; P:<0.001), as well as cardiovascular death, new myocardial infarction, stroke, and new congestive heart failure. Moreover, compared with their nondiabetic counterparts, women had a significantly higher risk than men (RR, 1.98; 95% CI, 1.60 to 2.44; and RR, 1.28; 95% CI, 1.06 to 1.56, respectively). Interestingly, diabetic patients without prior cardiovascular disease had the same event rates for all outcomes as nondiabetic patients with previous vascular disease. CONCLUSIONS Hospitalization for unstable angina or non-Q-wave myocardial infarction predicts a high 2-year morbidity and mortality; this is especially evident for patients with diabetes. Diabetic patients with no previous cardiovascular disease have the same long-term morbidity and mortality as nondiabetic patients with established cardiovascular disease after hospitalization for unstable coronary artery disease.

The overall size and structure of a synaptic terminal is an important determinant of its function. In a large-scale mutagenesis screen, designed to identify Drosophila mutants with abnormally structured neuromuscular junctions (NMJs), we discovered mutations in Drosophila mical, a conserved gene encoding a multi-domain protein with a N-terminal monooxygenase domain. In mical mutants, synaptic boutons do not sprout normally over the muscle surface and tend to form clusters along synaptic branches and at nerve entry sites. Consistent with high expression of MICAL in somatic muscles, immunohistochemical stainings reveal that the subcellular localization and architecture of contractile muscle filaments are dramatically disturbed in mical mutants. Instead of being integrated into a regular sarcomeric pattern, actin and myosin filaments are disorganized and accumulate beneath the plasmamembrane. Whereas contractile elements are strongly deranged, the proposed organizer of sarcomeric structure, D-Titin, is much less affected. Transgenic expression of interfering RNA molecules demonstrates that MICAL is required in muscles for the higher order arrangement of myofilaments. Ultrastructural analysis confirms that myosin-rich thick filaments enter submembranous regions and interfere with synaptic development, indicating that the disorganized myofilaments may cause the synaptic growth phenotype. As a model, we suggest that the filamentous network around synaptic boutons restrains the spreading of synaptic branches.

Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4+3− inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4+3− cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire+ mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80−Aire− mTEC progenitors into CD80+Aire+ mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire+ mTECs and highlight a previously unrecognized role for CD4+3−RANKL+ inducer cells in intrathymic self-tolerance.

Uncoupling protein 1 (UCP1) is highly expressed in brown adipose tissue, where it generates heat by uncoupling electron transport from ATP production. UCP1 is also found outside classical brown adipose tissue depots, in adipocytes that are termed 'brite' (brown-in-white) or 'beige'. In humans, the presence of brite or beige (brite/beige) adipocytes is correlated with a lean, metabolically healthy phenotype, but whether a causal relationship exists is not clear. Here we report that human brite/beige adipocyte progenitors proliferate in response to pro-angiogenic factors, in association with expanding capillary networks. Adipocytes formed from these progenitors transform in response to adenylate cyclase activation from being UCP1 negative to being UCP1 positive, which is a defining feature of the beige/brite phenotype, while displaying uncoupled respiration. When implanted into normal chow-fed, or into high-fat diet (HFD)-fed, glucose-intolerant NOD-scid IL2rg(null) (NSG) mice, brite/beige adipocytes activated in vitro enhance systemic glucose tolerance. These adipocytes express neuroendocrine and secreted factors, including the pro-protein convertase PCSK1, which is strongly associated with human obesity. Pro-angiogenic conditions therefore drive the proliferation of human beige/brite adipocyte progenitors, and activated beige/brite adipocytes can affect systemic glucose homeostasis, potentially through a neuroendocrine mechanism.


Question:
DMS3 protein enables Pol V transcription in vivo.

Nuclear transcription is not restricted to genes but occurs throughout the intergenic and noncoding space of eukaryotic genomes. The functional significance of this widespread noncoding transcription is mostly unknown. We show that Arabidopsis RNA polymerase IVb/Pol V, a multisubunit nuclear enzyme required for siRNA-mediated gene silencing of transposons and other repeats, transcribes intergenic and noncoding sequences, thereby facilitating heterochromatin formation and silencing of overlapping and adjacent genes. Pol IVb/Pol V transcription requires the chromatin-remodeling protein DRD1 but is independent of siRNA biogenesis. However, Pol IVb/Pol V transcription and siRNA production are both required to silence transposons, suggesting that Pol IVb/Pol V generates RNAs or chromatin structures that serve as scaffolds for siRNA-mediated heterochromatin-forming complexes. Pol IVb/Pol V function provides a solution to a paradox of epigenetic control: the need for transcription in order to transcriptionally silence the same region.


Human embryonic stem cells give rise to cell types from all three embryonic germ layers.

Despite growing interest and a recent surge in papers, the role of autophagy in glucose and lipid metabolism is unclear. We produced mice with skeletal muscle–specific deletion of Atg7 (encoding autophagy-related 7). Unexpectedly, these mice showed decreased fat mass and were protected from diet-induced obesity and insulin resistance; this phenotype was accompanied by increased fatty acid oxidation and browning of white adipose tissue (WAT) owing to induction of fibroblast growth factor 21 (Fgf21). Mitochondrial dysfunction induced by autophagy deficiency increased Fgf21 expression through induction of Atf4, a master regulator of the integrated stress response. Mitochondrial respiratory chain inhibitors also induced Fgf21 in an Atf4-dependent manner. We also observed induction of Fgf21, resistance to diet-induced obesity and amelioration of insulin resistance in mice with autophagy deficiency in the liver, another insulin target tissue. These findings suggest that autophagy deficiency and subsequent mitochondrial dysfunction promote Fgf21 expression, a hormone we consequently term a 'mitokine', and together these processes promote protection from diet-induced obesity and insulin resistance.

CONTEXT Vaso-occlusion is central to the painful crises and acute and chronic organ damage in sickle cell disease. Abnormal nitric oxide-dependent regulation of vascular tone, adhesion, platelet activation, and inflammation contributes to the pathophysiology of vaso-occlusion. Nitric oxide may have promise as a mechanism-of-disease-based therapy for treatment of vaso-occlusion. OBJECTIVE To explore the efficacy and safety of inhaled nitric oxide (INO) for treatment of vaso-occlusive crisis in pediatric patients. DESIGN Prospective, double-blind, placebo-controlled, randomized clinical trial with enrollment between September 1999 and October 2001. SETTING Urban, tertiary care children's hospital in the United States. PARTICIPANTS Twenty patients aged 10 to 21 years with sickle cell disease and severe acute vaso-occlusive crisis. INTERVENTION Patients were randomly assigned to receive INO (80 ppm with 21% final concentration of inspired oxygen; n = 10), or placebo (21% inspired oxygen; n = 10) for 4 hours. MAIN OUTCOME MEASURES Change in pain at 4 hours of inhalation compared with preinhalation pain, measured on a 10-cm visual analog scale (VAS); secondary outcome measures were pain over 6 hours, parenteral narcotic use over 24 hours, duration of hospitalization, blood pressure, oxygen saturation, and methemoglobin concentration. RESULTS Preinhalation VAS pain scores were similar in the INO and placebo groups (P =.80). The decrease in VAS pain scores at 4 hours was 2.0 cm in the INO group and 1.2 cm in the placebo group (P =.37). Repeated-measures analysis of variance for hourly pain scores showed a 1-cm/h greater reduction in the INO group than the placebo group (P =.02). Morphine use over 6 hours was significantly less in the INO group (mean cumulative use, 0.29 vs 0.44 mg/kg; P =.03) but was not different over 4 hours (0.26 vs 0.32 mg/kg; P =.21) or 24 hours (0.63 vs 0.91 mg/kg; P =.15). Duration of hospitalization was 78 and 100 hours in the INO and placebo groups, respectively (P =.19). No INO toxicity was observed. CONCLUSIONS Results of this exploratory study suggest that INO may be beneficial for acute vaso-occlusive crisis. These preliminary results warrant further investigation.

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. These cells have, therefore, potential for in vitro differentiation studies, gene function, and so on. The aim of this study was to produce a human embryonic stem cell line. An inner cell mass of a human blastocyst was separated and cultured on mouse embryonic fibroblasts in embryonic stem cell medium with related additives. The established line was evaluated by morphology; passaging; freezing and thawing; alkaline phosphatase; Oct-4 expression; anti-surface markers including Tra-1-60 and Tra-1-81; and karyotype and spontaneous differentiation. Differentiated cardiomyocytes and neurons were evaluated by transmission electron microscopy and immunocytochemistry. Here, we report the derivation of a new embryonic stem cell line (Royan H1) from a human blastocyst that remains undifferentiated in morphology during continuous passaging for more than 30 passages, maintains a normal XX karyotype, is viable after freezing and thawing, and expresses alkaline phosphatase, Oct-4, Tra-1-60, and Tra-1-81. These cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers in the presence or absence of recombinant human leukemia inhibitory factor. Royan H1 cells can differentiate in vitro in the absence of feeder cells and can produce embryoid bodies that can further differentiate into beating cardiomyocytes as well as neurons. These results define Royan H1 cells as a new human embryonic stem cell line.

The unique metabolic demands of cancer cells underscore potentially fruitful opportunities for drug discovery in the era of precision medicine. However, therapeutic targeting of cancer metabolism has led to surprisingly few new drugs to date. The neutral amino acid glutamine serves as a key intermediate in numerous metabolic processes leveraged by cancer cells, including biosynthesis, cell signaling, and oxidative protection. Herein we report the preclinical development of V-9302, a competitive small molecule antagonist of transmembrane glutamine flux that selectively and potently targets the amino acid transporter ASCT2. Pharmacological blockade of ASCT2 with V-9302 resulted in attenuated cancer cell growth and proliferation, increased cell death, and increased oxidative stress, which collectively contributed to antitumor responses in vitro and in vivo. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in oncology, representing a new class of targeted therapy and laying a framework for paradigm-shifting therapies targeting cancer cell metabolism.


Patients in stable partnerships have a slower progression from HIV to AIDS.

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-γ (IFN-γ)–producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X7 purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1β (IL-1β). The priming of IFN-γ–producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3−/−) or caspase-1–deficient (Casp-1−/−) mice unless exogenous IL-1β is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7−/− or Nlrp3−/− or Casp1−/− hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.

Defective brain hormonal signaling has been associated with Alzheimer's disease (AD), a disorder characterized by synapse and memory failure. Irisin is an exercise-induced myokine released on cleavage of the membrane-bound precursor protein fibronectin type III domain-containing protein 5 (FNDC5), also expressed in the hippocampus. Here we show that FNDC5/irisin levels are reduced in AD hippocampi and cerebrospinal fluid, and in experimental AD models. Knockdown of brain FNDC5/irisin impairs long-term potentiation and novel object recognition memory in mice. Conversely, boosting brain levels of FNDC5/irisin rescues synaptic plasticity and memory in AD mouse models. Peripheral overexpression of FNDC5/irisin rescues memory impairment, whereas blockade of either peripheral or brain FNDC5/irisin attenuates the neuroprotective actions of physical exercise on synaptic plasticity and memory in AD mice. By showing that FNDC5/irisin is an important mediator of the beneficial effects of exercise in AD models, our findings place FNDC5/irisin as a novel agent capable of opposing synapse failure and memory impairment in AD.

Neutrophils rapidly undergo polarization and directional movement to infiltrate the sites of infection and inflammation. Here, we show that an inhibitory MHC I receptor, Ly49Q, was crucial for the swift polarization of and tissue infiltration by neutrophils. During the steady state, Ly49Q inhibited neutrophil adhesion by preventing focal-complex formation, likely by inhibiting Src and PI3 kinases. However, in the presence of inflammatory stimuli, Ly49Q mediated rapid neutrophil polarization and tissue infiltration in an ITIM-domain-dependent manner. These opposite functions appeared to be mediated by distinct use of effector phosphatase SHP-1 and SHP-2. Ly49Q-dependent polarization and migration were affected by Ly49Q regulation of membrane raft functions. We propose that Ly49Q is pivotal in switching neutrophils to their polarized morphology and rapid migration upon inflammation, through its spatiotemporal regulation of membrane rafts and raft-associated signaling molecules.

Experience gained from the Global Malaria Eradication Program (1955-72) identified a set of shared technical and operational factors that enabled some countries to successfully eliminate malaria. Spatial data for these factors were assembled for all malaria-endemic countries and combined to provide an objective, relative ranking of countries by technical, operational, and combined elimination feasibility. The analysis was done separately for Plasmodium falciparum and Plasmodium vivax, and the limitations of the approach were discussed. The relative rankings suggested that malaria elimination would be most feasible in countries in the Americas and Asia, and least feasible in countries in central and west Africa. The results differed when feasibility was measured by technical or operational factors, highlighting the different types of challenge faced by each country. The results are not intended to be prescriptive, predictive, or to provide absolute assessments of feasibility, but they do show that spatial information is available to facilitate evidence-based assessments of the relative feasibility of malaria elimination by country that can be rapidly updated.


Question:
There was an estimated 30 million cases of pneumonia in young children worldwide in 2010.

BACKGROUND The annual number of hospital admissions and in-hospital deaths due to severe acute lower respiratory infections (ALRI) in young children worldwide is unknown. We aimed to estimate the incidence of admissions and deaths for such infections in children younger than 5 years in 2010. METHODS We estimated the incidence of admissions for severe and very severe ALRI in children younger than 5 years, stratified by age and region, with data from a systematic review of studies published between Jan 1, 1990, and March 31, 2012, and from 28 unpublished population-based studies. We applied these incidence estimates to population estimates for 2010, to calculate the global and regional burden in children admitted with severe ALRI in that year. We estimated in-hospital mortality due to severe and very severe ALRI by combining incidence estimates with case fatality ratios from hospital-based studies. FINDINGS We identified 89 eligible studies and estimated that in 2010, 11·9 million (95% CI 10·3-13·9 million) episodes of severe and 3·0 million (2·1-4·2 million) episodes of very severe ALRI resulted in hospital admissions in young children worldwide. Incidence was higher in boys than in girls, the sex disparity being greatest in South Asian studies. On the basis of data from 37 hospital studies reporting case fatality ratios for severe ALRI, we estimated that roughly 265,000 (95% CI 160,000-450,000) in-hospital deaths took place in young children, with 99% of these deaths in developing countries. Therefore, the data suggest that although 62% of children with severe ALRI are treated in hospitals, 81% of deaths happen outside hospitals. INTERPRETATION Severe ALRI is a substantial burden on health services worldwide and a major cause of hospital referral and admission in young children. Improved hospital access and reduced inequities, such as those related to sex and rural status, could substantially decrease mortality related to such infection. Community-based management of severe disease could be an important complementary strategy to reduce pneumonia mortality and health inequities. FUNDING WHO.


For every 1,000 children with cerebral palsy, more than 300 of them are premature or underweight at birth.

CONTEXT Although cerebral palsy (CP) among extremely premature infants has been reported as a major morbidity outcome, there are difficulties comparing published CP rates from many sites over various birth years. OBJECTIVE To assess the changes in population-based, gestational age-specific prevalence rates of CP among extremely premature infants over 30 years. DESIGN Prospective population-based longitudinal outcome study. SETTING AND PARTICIPANTS In Northern Alberta, 2318 infants 20 to 27 weeks' gestational age with birth weights of 500 to 1249 g were liveborn from 1974 through 2003. By 2 years of age, 1437 (62%) had died, 23 (1%) were lost to follow-up, and 858 (37%) had received multidisciplinary neurodevelopmental assessment. MAIN OUTCOME MEASURE Population-based prevalence rates of CP were determined. Logistic regression with linear spline was used to assess changes in CP prevalence over time. RESULTS At age 2 years, 122 (14.2%) of 858 survivors had CP. This diagnosis was confirmed for each child by age 3 years or older. Among those whose gestational age was 20 to 25 weeks, population-based survival increased from 4% to 31% (P<.001), while CP prevalence per 1000 live births increased monotonically from 0 to 110 until the years 1992-1994 (P<.001) and decreased thereafter to 22 in the years 2001-2003 (P<.001). Among those whose gestational age was 26 to 27 weeks, population-based survival increased from 23% to between 75% and 80% (P<.001), while CP prevalence per 1000 live births increased monotonically from 15 to 155 until the years 1992-1994 (P<.001) and then decreased to 16 in the years 2001-2003 (P<.001). For all survivors born in the years 2001-2003, CP prevalence was 19 per 1000 live births. CONCLUSION Population-based CP prevalence rates for children whose gestational age was 20 to 27 weeks and whose birth weight ranged from 500 to 1249 g show steady reductions in the last decade with stable or reducing mortality, reversing trends prior to 1992-1994.


Pyridostatin reduces proliferation of homologous recombination - defective cells.

Panic disorder is a severe anxiety disorder with recurrent, debilitating panic attacks. In individuals with panic disorder there is evidence of decreased central gamma-aminobutyric acid (GABA) activity as well as marked increases in autonomic and respiratory responses after intravenous infusions of hypertonic sodium lactate. In a rat model of panic disorder, chronic inhibition of GABA synthesis in the dorsomedial-perifornical hypothalamus of rats produces anxiety-like states and a similar vulnerability to sodium lactate-induced cardioexcitatory responses. The dorsomedial-perifornical hypothalamus is enriched in neurons containing orexin (ORX, also known as hypocretin), which have a crucial role in arousal, vigilance and central autonomic mobilization, all of which are key components of panic. Here we show that activation of ORX-synthesizing neurons is necessary for developing a panic-prone state in the rat panic model, and either silencing of the hypothalamic gene encoding ORX (Hcrt) with RNAi or systemic ORX-1 receptor antagonists blocks the panic responses. Moreover, we show that human subjects with panic anxiety have elevated levels of ORX in the cerebrospinal fluid compared to subjects without panic anxiety. Taken together, our results suggest that the ORX system may be involved in the pathophysiology of panic anxiety and that ORX antagonists constitute a potential new treatment strategy for panic disorder.

BACKGROUND Although cigarette smoking, excessive alcohol drinking, obesity, and several other well-studied unhealthy lifestyle-related factors each have been linked to the risk of multiple chronic diseases and premature death, little is known about the combined impact on mortality outcomes, in particular among Chinese and other non-Western populations. The objective of this study was to quantify the overall impact of lifestyle-related factors beyond that of active cigarette smoking and alcohol consumption on all-cause and cause-specific mortality in Chinese women. METHODS AND FINDINGS We used data from the Shanghai Women's Health Study, an ongoing population-based prospective cohort study in China. Participants included 71,243 women aged 40 to 70 years enrolled during 1996-2000 who never smoked or drank alcohol regularly. A healthy lifestyle score was created on the basis of five lifestyle-related factors shown to be independently associated with mortality outcomes (normal weight, lower waist-hip ratio, daily exercise, never exposed to spouse's smoking, higher daily fruit and vegetable intake). The score ranged from zero (least healthy) to five (most healthy) points. During an average follow-up of 9 years, 2,860 deaths occurred, including 775 from cardiovascular disease (CVD) and 1,351 from cancer. Adjusted hazard ratios for mortality decreased progressively with an increasing number of healthy lifestyle factors. Compared to women with a score of zero, hazard ratios (95% confidence intervals) for women with four to five factors were 0.57 (0.44-0.74) for total mortality, 0.29 (0.16-0.54) for CVD mortality, and 0.76 (0.54-1.06) for cancer mortality. The inverse association between the healthy lifestyle score and mortality was seen consistently regardless of chronic disease status at baseline. The population attributable risks for not having 4-5 healthy lifestyle factors were 33% for total deaths, 59% for CVD deaths, and 19% for cancer deaths. CONCLUSIONS In this first study, to our knowledge, to quantify the combined impact of lifestyle-related factors on mortality outcomes in Chinese women, a healthier lifestyle pattern-including being of normal weight, lower central adiposity, participation in physical activity, nonexposure to spousal smoking, and higher fruit and vegetable intake-was associated with reductions in total and cause-specific mortality among lifetime nonsmoking and nondrinking women, supporting the importance of overall lifestyle modification in disease prevention. Please see later in the article for the Editors' Summary.

Human skin copes with harmful environmental factors that are circadian in nature, yet how circadian rhythms modulate the function of human epidermal stem cells is mostly unknown. Here we show that in human epidermal stem cells and their differentiated counterparts, core clock genes peak in a successive and phased manner, establishing distinct temporal intervals during the 24 hr day period. Each of these successive clock waves is associated with a peak in the expression of subsets of transcripts that temporally segregate the predisposition of epidermal stem cells to respond to cues that regulate their proliferation or differentiation, such as TGFβ and calcium. Accordingly, circadian arrhythmia profoundly affects stem cell function in culture and in vivo. We hypothesize that this intricate mechanism ensures homeostasis by providing epidermal stem cells with environmentally relevant temporal functional cues during the course of the day and that its perturbation may contribute to aging and carcinogenesis.

The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

Serotonin signaling suppresses generation of amyloid-β (Aβ) in vitro and in animal models of Alzheimer’s disease (AD). We show that in an aged transgenic AD mouse model (APP/PS1 plaque-bearing mice), the antidepressant citalopram, a selective serotonin reuptake inhibitor, decreased Aβ in brain interstitial fluid in a dose-dependent manner. Growth of individual amyloid plaques was assessed in plaque-bearing mice that were chronically administered citalopram. Citalopram arrested the growth of preexisting plaques and reduced the appearance of new plaques by 78%. In healthy human volunteers, citalopram’s effects on Aβ production and Aβ concentrations in cerebrospinal fluid (CSF) were measured prospectively using stable isotope labeling kinetics, with CSF sampling during acute dosing of citalopram. Aβ production in CSF was slowed by 37% in the citalopram group compared to placebo. This change was associated with a 38% decrease in total CSF Aβ concentrations in the drug-treated group. The ability to safely decrease Aβ concentrations is potentially important as a preventive strategy for AD. This study demonstrates key target engagement for future AD prevention trials.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (encoded by Cftr) that impair its role as an apical chloride channel that supports bicarbonate transport. Individuals with cystic fibrosis show retained, thickened mucus that plugs airways and obstructs luminal organs as well as numerous other abnormalities that include inflammation of affected organs, alterations in lipid metabolism and insulin resistance. Here we show that colonic epithelial cells and whole lung tissue from Cftr-deficient mice show a defect in peroxisome proliferator-activated receptor-gamma (PPAR-gamma, encoded by Pparg) function that contributes to a pathological program of gene expression. Lipidomic analysis of colonic epithelial cells suggests that this defect results in part from reduced amounts of the endogenous PPAR-gamma ligand 15-keto-prostaglandin E(2) (15-keto-PGE(2)). Treatment of Cftr-deficient mice with the synthetic PPAR-gamma ligand rosiglitazone partially normalizes the altered gene expression pattern associated with Cftr deficiency and reduces disease severity. Rosiglitazone has no effect on chloride secretion in the colon, but it increases expression of the genes encoding carbonic anhydrases 4 and 2 (Car4 and Car2), increases bicarbonate secretion and reduces mucus retention. These studies reveal a reversible defect in PPAR-gamma signaling in Cftr-deficient cells that can be pharmacologically corrected to ameliorate the severity of the cystic fibrosis phenotype in mice.


Question:
Decrease of p62 in prostate tumor stroma results in defective autophagy.

CONTEXT Delayed cerebral vasospasm causes permanent neurological deficits or death in at least 15% of patients following otherwise successful treatment for ruptured intracranial aneurysm. Decreased bioavailability of nitric oxide has been associated with the development of cerebral vasospasm. OBJECTIVE To determine whether infusions of nitrite will prevent delayed cerebral vasospasm. DESIGN, SETTING, AND SUBJECTS A total of 14 anesthetized cynomolgus monkeys had an autologous blood clot placed around the right middle cerebral artery. Cerebral arteriography was performed before clot placement and on days 7 and 14 to assess vasospasm. The study was conducted from August 2003 to February 2004. INTERVENTIONS A 90-mg sodium nitrite intravenous solution infused over 24 hours plus a 45-mg sodium nitrite bolus daily (n = 3); a 180-mg sodium nitrite intravenous solution infused over 24 hours (n = 3); or a control saline solution infusion (n = 8). Each was infused continuously for 14 days. MAIN OUTCOME MEASURES Nitrite, S-nitrosothiol, and methemoglobin levels in blood and cerebrospinal fluid and degree of arteriographic vasospasm. RESULTS In control monkeys, mean (SD) cerebrospinal fluid nitrite levels decreased from 3.1 (1.5) micromol/L to 0.4 (0.1) micromol/L at day 7 and to 0.4 (0.4) micromol/L at day 14 (P = .03). All 8 control monkeys developed significant vasospasm of the right middle cerebral artery, which was complicated by stroke and death in 1 animal. Sodium nitrite infusions increased the nitrite and methemoglobin levels (<2.1% of total hemoglobin) in the blood and cerebrospinal fluid without evoking systemic hypotension. Nitrite infusion prevented development of vasospasm (no animals developed significant vasospasm; mean [SD] reduction in right middle cerebral artery area on day 7 after subarachnoid hemorrhage of 8% [9%] in nitrite-treated monkeys vs 47% [5%] in saline-treated controls; P<.001). There was a negative correlation between the concentration of nitrite in cerebrospinal fluid and the degree of cerebral vasospasm (P<.001). Pharmacological effects of nitrite infusion were also associated with the formation of S-nitrosothiol in cerebrospinal fluid. There was no clinical or pathological evidence of nitrite toxicity. CONCLUSION Subacute sodium nitrite infusions prevented delayed cerebral vasospasm in a primate model of subarachnoid hemorrhage.

Hepatic stellate cells (HSCs) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). Vitamin D receptor (VDR) activation in HSCs inhibits liver inflammation and fibrosis. We found that p62/SQSTM1, a protein upregulated in liver parenchymal cells but downregulated in HCC-associated HSCs, negatively controls HSC activation. Total body or HSC-specific p62 ablation potentiates HSCs and enhances inflammation, fibrosis, and HCC progression. p62 directly interacts with VDR and RXR promoting their heterodimerization, which is critical for VDR:RXR target gene recruitment. Loss of p62 in HSCs impairs the repression of fibrosis and inflammation by VDR agonists. This demonstrates that p62 is a negative regulator of liver inflammation and fibrosis through its ability to promote VDR signaling in HSCs, whose activation supports HCC.

BACKGROUND Results of the few cohort studies from countries with low incomes or middle incomes suggest a lower incidence of dementia than in high-income countries. We assessed incidence of dementia according to criteria from the 10/66 Dementia Research Group and Diagnostic and Statistical Manual of Mental Disorders (DSM) IV, the effect of dementia at baseline on mortality, and the independent effects of age, sex, socioeconomic position, and indicators of cognitive reserve. METHODS We did a population-based cohort study of all people aged 65 years and older living in urban sites in Cuba, the Dominican Republic, and Venezuela, and rural and urban sites in Peru, Mexico, and China, with ascertainment of incident 10/66 and DSM-IV dementia 3-5 years after cohort inception. We used questionnaires to obtain information about age in years, sex, educational level, literacy, occupational attainment, and number of household assets. We obtained information about mortality from all sites. For participants who had died, we interviewed a friend or relative to ascertain the likelihood that they had dementia before death. FINDINGS 12,887 participants were interviewed at baseline. 11,718 were free of dementia, of whom 8137 (69%) were reinterviewed, contributing 34,718 person-years of follow-up. Incidence for 10/66 dementia varied between 18·2 and 30·4 per 1000 person-years, and were 1·4-2·7 times higher than were those for DSM-IV dementia (9·9-15·7 per 1000 person-years). Mortality hazards were 1·56-5·69 times higher in individuals with dementia at baseline than in those who were dementia-free. Informant reports suggested a high incidence of dementia before death; overall incidence might be 4-19% higher if these data were included. 10/66 dementia incidence was independently associated with increased age (HR 1·67; 95% CI 1·56-1·79), female sex (0·72; 0·61-0·84), and low education (0·89; 0·81-0·97), but not with occupational attainment (1·04; 0·95-1·13). INTERPRETATION Our results provide supportive evidence for the cognitive reserve hypothesis, showing that in middle-income countries as in high-income countries, education, literacy, verbal fluency, and motor sequencing confer substantial protection against the onset of dementia. FUNDING Wellcome Trust Health Consequences of Population Change Programme, WHO, US Alzheimer's Association, FONACIT/ CDCH/ UCV.

The role of primate frontal cortex in vocal communication and its significance in language evolution have a controversial history. While evidence indicates that vocalization processing occurs in ventrolateral prefrontal cortex neurons, vocal-motor activity has been conjectured to be primarily subcortical and suggestive of a distinctly different neural architecture from humans. Direct evidence of neural activity during natural vocal communication is limited, as previous studies were performed in chair-restrained animals. Here we recorded the activity of single neurons across multiple regions of prefrontal and premotor cortex while freely moving marmosets engaged in a natural vocal behavior known as antiphonal calling. Our aim was to test whether neurons in marmoset frontal cortex exhibited responses during vocal-signal processing and/or vocal-motor production in the context of active, natural communication. We observed motor-related changes in single neuron activity during vocal production, but relatively weak sensory responses for vocalization processing during this natural behavior. Vocal-motor responses occurred both prior to and during call production and were typically coupled to the timing of each vocalization pulse. Despite the relatively weak sensory responses a population classifier was able to distinguish between neural activity that occurred during presentations of vocalization stimuli that elicited an antiphonal response and those that did not. These findings are suggestive of the role that nonhuman primate frontal cortex neurons play in natural communication and provide an important foundation for more explicit tests of the functional contributions of these neocortical areas during vocal behaviors.

Viral replication and microbial translocation from the gut to the blood during HIV infection lead to hyperimmune activation, which contributes to the decline in CD4+ T cell numbers during HIV infection. Programmed death-1 (PD-1) and interleukin-10 (IL-10) are both upregulated during HIV infection. Blocking interactions between PD-1 and programmed death ligand-1 (PD-L1) and between IL-10 and IL-10 receptor (IL-10R) results in viral clearance and improves T cell function in animal models of chronic viral infections. Here we show that high amounts of microbial products and inflammatory cytokines in the plasma of HIV-infected subjects lead to upregulation of PD-1 expression on monocytes that correlates with high plasma concentrations of IL-10. Triggering of PD-1 expressed on monocytes by PD-L1 expressed on various cell types induced IL-10 production and led to reversible CD4+ T cell dysfunction. We describe a new function for PD-1 whereby microbial products inhibit T cell expansion and function by upregulating PD-1 levels and IL-10 production by monocytes after binding of PD-1 by PD-L1.