Question: A deficiency of vitamin B6 decreases blood levels of homocysteine.

Folic acid improves endothelial function in coronary artery disease via mechanisms largely independent of homocysteine lowering.. 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.

Dual role of interleukin-10 in the regulation of respiratory syncitial virus (RSV)-induced lung inflammation.. RSV lower respiratory tract infections (LRTI) are among the most common diseases necessitating hospital admission in children. In addition to causing acute respiratory failure, RSV infections are associated with sequelae such as secondary bacterial infections and reactive airway disease. One characteristic host response observed in severe RSV-induced LRTI and/or subsequent development of asthma is increased expression of interleukin (IL)-10. However, contradictory results have been reported regarding whether IL-10 inhibits asthmatic responses or intensifies the disease. We aimed to reconcile these discordant observations by elucidating the role of IL-10 in regulating the host response to RSV LRTI. In this study, we used a lung-specific, inducible IL-10 over-expression (OE) transgenic mouse model to address this question. Our results showed that the presence of IL-10 at the time of RSV infection not only attenuated acute inflammatory process (i.e. 24 h post-infection), but also late inflammatory changes [characterized by T helper type 2 (Th2) cytokine and chemokine expression]. While this result appears contradictory to some clinical observations where elevated IL-10 levels are observed in asthmatic patients, we also found that delaying IL-10 OE until the late immune response to RSV infection, additive effects rather than inhibitory effects were observed. Importantly, in non-infected, IL-10 OE mice, IL-10 OE alone induced up-regulation of Th2 cytokine (IL-13 and IL-5) and Th2-related chemokine [monocyte chemoattractant protein 1 (MCP-1), chemokine (C-C motif) ligand 3 (CCL3) and regulated upon activation normal T cell expressed and secreted (RANTES)] expression. We identified a subset of CD11b(+)CD11c(+)CD49b(+)F4/80(-)Gr-1(-) myeloid cells as a prinicipal source of IL-10-induced IL-13 production. Therefore, the augmented pathological responses observed in our 'delayed' IL-10 over-expression model could be attributed to IL-10 OE alone. Taken together, our study indicated dual roles of IL-10 on RSV-induced lung inflammation which appear to depend upon the timing of when elevated IL-10 is expressed in the lung.


Question: Primary cervical cytology screening with HPV detection has higher longitudinal sensitivity to detect severe cervical intraepithelial neoplasia than conventional cytology.

Human papillomavirus testing for the detection of high-grade cervical intraepithelial neoplasia and cancer: final results of the POBASCAM randomised controlled trial.. BACKGROUND Human papillomavirus (HPV) testing is more sensitive for the detection of high-grade cervical lesions than is cytology, but detection of HPV by DNA screening in two screening rounds 5 years apart has not been assessed. The aim of this study was to assess whether HPV DNA testing in the first screen decreases detection of cervical intraepithelial neoplasia (CIN) grade 3 or worse, CIN grade 2 or worse, and cervical cancer in the second screening. METHODS In this randomised trial, women aged 29-56 years participating in the cervical screening programme in the Netherlands were randomly assigned to receive HPV DNA (GP5+/6+-PCR method) and cytology co-testing or cytology testing alone, from January, 1999, to September, 2002. Randomisation (in a 1:1 ratio) was done with computer-generated random numbers after the cervical specimen had been taken. At the second screening 5 years later, HPV DNA and cytology co-testing was done in both groups; researchers were masked to the patient's assignment. The primary endpoint was the number of CIN grade 3 or worse detected. Analysis was done by intention to screen. The trial is now finished and is registered, number ISRCTN20781131. FINDINGS 22,420 women were randomly assigned to the intervention group and 22 518 to the control group; 19 999 in the intervention group and 20,106 in the control group were eligible for analysis at the first screen. At the second screen, 19 579 women in the intervention group and 19,731 in the control group were eligible, of whom 16,750 and 16,743, respectively, attended the second screen. In the second round, CIN grade 3 or worse was less common in the intervention group than in the control group (88 of 19 579 in the intervention group vs 122 of 19,731 in the control group; relative risk 0·73, 95% CI 0·55-0·96; p=0·023). Cervical cancer was also less common in the intervention group than in the control group (four of 19 579 in the intervention group vs 14 of 19,731; 0·29, 0·10-0·87; p=0·031). In the baseline round, detection of CIN grade 3 or worse did not differ significantly between groups (171 of 19 999 vs 150 of 20,106; 1·15, 0·92-1·43; p=0·239) but was significantly more common in women with normal cytology (34 of 19,286 vs 12 of 19,373; 2·85, 1·47-5·49; p=0·001). Furthermore, significantly more cases of CIN grade 2 or worse were detected in the intervention group than in the control group (267 of 19 999 vs 215 of 20,106; 1·25, 1·05-1·50; p=0·015). In the second screen, fewer HPV16-positive CIN grade 3 or worse were detected in the intervention group than in the control group (17 of 9481 vs 35 of 9354; 0·48, 0·27-0·85; p=0·012); detection of non-HPV16-positive CIN grade 3 or worse did not differ between groups (25 of 9481 vs 25 of 9354; 0·99, 0·57-1·72; p=1·00). The cumulative detection of CIN grade 3 or worse and CIN grade 2 or worse did not differ significantly between study arms, neither for the whole study group (CIN grade 3 or worse: 259 of 19 999 vs 272 of 20,106; 0·96, 0·81-1·14, p=0·631; CIN grade 2 or worse: 427 of 19 999 vs 399 of 20,106; 1·08, 0·94-1·24; p=0·292), nor for subgroups of women invited for the first time (CIN grade 3 or worse in women aged 29-33 years: 102 of 3139 vs 105 of 3128; 0·97, 0·74-1·27; CIN grade 2 or worse in women aged 29-33 years: 153 of 3139 vs 151 of 3128; 1·01, 0·81-1·26; CIN grade 3 or worse in women aged 34-56 years: 157 of 16,860 vs 167 of 16 978; 0·95, 0·76-1·18; CIN grade 2 or worse in women aged 34-56 years: 274 of 16,860 vs 248 of 16 978; 1·11, 0·94-1·32). INTERPRETATION Implementation of HPV DNA testing in cervical screening leads to earlier detection of clinically relevant CIN grade 2 or worse, which when adequately treated, improves protection against CIN grade 3 or worse and cervical cancer. Early detection of high-grade cervical legions caused by HPV16 was a major component of this benefit. Our results lend support to the use of HPV DNA testing for all women aged 29 years and older. FUNDING Zorg Onderzoek Nederland (Netherlands Organisation for Health Research and Development).

Phase 2 safety trial targeting amyloid beta production with a gamma-secretase inhibitor in Alzheimer disease.. OBJECTIVE To evaluate the safety, tolerability, and amyloid beta (Abeta) response to the gamma-secretase inhibitor LY450139 in Alzheimer disease. DESIGN Multicenter, randomized, double-blind, dose-escalation, placebo-controlled trial. SETTING Community-based clinical research centers. Patients Fifty-one individuals with mild to moderate Alzheimer disease were randomized to receive placebo (n=15) or LY450139 (100 mg [n=22] or 140 mg [n=14]), with 43 completing the treatment phase. Intervention The LY450139 groups received 60 mg/d for 2 weeks, then 100 mg/d for 6 weeks, and then either 100 or 140 mg/d for 6 additional weeks. MAIN OUTCOME MEASURES Primary outcome measures were adverse events, plasma and cerebrospinal fluid Abeta levels, vital signs, electrocardiographic data, and laboratory safety test results. Secondary outcome measures included the Alzheimer's Disease Assessment Scale cognitive subscale and the Alzheimer's Disease Cooperative Study Activities of Daily Living Scale. RESULTS Group differences were seen in skin and subcutaneous tissue concerns (P=.05), including 3 possible drug rashes and 3 reports of hair color change in the treatment groups. There were 3 adverse event-related discontinuations, including 1 transient bowel obstruction. The plasma Abeta(40) concentration was reduced by 58.2% for the 100-mg group and 64.6% for the 140-mg group (P<.001). No significant reduction was seen in cerebrospinal fluid Abeta levels. No group differences were seen in cognitive or functional measures. CONCLUSIONS LY450139 was generally well tolerated at doses of up to 140 mg/d for 14 weeks, with several findings indicating the need for close clinical monitoring in future studies. Decreases in plasma Abeta concentrations were consistent with inhibition of gamma-secretase. Trial Registration clinicaltrials.gov Identifier: NCT00244322.


Question: Bariatric surgery increases rates of postmenopausal breast cancer.

Bariatric Surgery in the United Kingdom: A Cohort Study of Weight Loss and Clinical Outcomes in Routine Clinical Care.. BACKGROUND Bariatric surgery is becoming a more widespread treatment for obesity. Comprehensive evidence of the long-term effects of contemporary surgery on a broad range of clinical outcomes in large populations treated in routine clinical practice is lacking. The objective of this study was to measure the association between bariatric surgery, weight, body mass index, and obesity-related co-morbidities. METHODS AND FINDINGS This was an observational retrospective cohort study using data from the United Kingdom Clinical Practice Research Datalink. All 3,882 patients registered in the database and with bariatric surgery on or before 31 December 2014 were included and matched by propensity score to 3,882 obese patients without surgery. The main outcome measures were change in weight and body mass index over 4 y; incident diagnoses of type 2 diabetes mellitus (T2DM), hypertension, angina, myocardial infarction (MI), stroke, fractures, obstructive sleep apnoea, and cancer; mortality; and resolution of hypertension and T2DM. Weight measures were available for 3,847 patients between 1 and 4 mo, 2,884 patients between 5 and 12 mo, and 2,258 patients between 13 and 48 mo post-procedure. Bariatric surgery patients exhibited rapid weight loss for the first four postoperative months, at a rate of 4.98 kg/mo (95% CI 4.88-5.08). Slower weight loss was sustained to the end of 4 y. Gastric bypass (6.56 kg/mo) and sleeve gastrectomy (6.29 kg/mo) were associated with greater initial weight reduction than gastric banding (2.77 kg/mo). Protective hazard ratios (HRs) were detected for bariatric surgery for incident T2DM, 0.68 (95% CI 0.55-0.83); hypertension, 0.35 (95% CI 0.27-0.45); angina, 0.59 (95% CI 0.40-0.87);MI, 0.28 (95% CI 0.10-0.74); and obstructive sleep apnoea, 0.55 (95% CI 0.40-0.87). Strong associations were found between bariatric surgery and the resolution of T2DM, with a HR of 9.29 (95% CI 6.84-12.62), and between bariatric surgery and the resolution of hypertension, with a HR of 5.64 (95% CI 2.65-11.99). No association was detected between bariatric surgery and fractures, cancer, or stroke. Effect estimates for mortality found no protective association with bariatric surgery overall, with a HR of 0.97 (95% CI 0.66-1.43). The data used were recorded for the management of patients in primary care and may be subject to inaccuracy, which would tend to lead to underestimates of true relative effect sizes. CONCLUSIONS Bariatric surgery as delivered in the UK healthcare system is associated with dramatic weight loss, sustained at least 4 y after surgery. This weight loss is accompanied by substantial improvements in pre-existing T2DM and hypertension, as well as a reduced risk of incident T2DM, hypertension, angina, MI, and obstructive sleep apnoea. Widening the availability of bariatric surgery could lead to substantial health benefits for many people who are morbidly obese.

Potent, Selective, and Orally Bioavailable Inhibitors of VPS34 Provide Chemical Tools to Modulate Autophagy in Vivo.. Autophagy is a dynamic process that regulates lysosomal-dependent degradation of cellular components. Until recently the study of autophagy has been hampered by the lack of reliable pharmacological tools, but selective inhibitors are now available to modulate the PI 3-kinase VPS34, which is required for autophagy. Here we describe the discovery of potent and selective VPS34 inhibitors, their pharmacokinetic (PK) properties, and ability to inhibit autophagy in cellular and mouse models.


Question: Misunderstandings between doctors and patients can lead to non-adherence.

Misunderstandings in prescribing decisions in general practice: qualitative study.. OBJECTIVES To identify and describe misunderstandings between patients and doctors associated with prescribing decisions in general practice. DESIGN Qualitative study. SETTING 20 general practices in the West Midlands and south east England. PARTICIPANTS 20 general practitioners and 35 consulting patients. MAIN OUTCOME MEASURES Misunderstandings between patients and doctors that have potential or actual adverse consequences for taking medicine. RESULTS 14 categories of misunderstanding were identified relating to patient information unknown to the doctor, doctor information unknown to the patient, conflicting information, disagreement about attribution of side effects, failure of communication about doctor's decision, and relationship factors. All the misunderstandings were associated with lack of patients' participation in the consultation in terms of the voicing of expectations and preferences or the voicing of responses to doctors' decisions and actions. They were all associated with potential or actual adverse outcomes such as non-adherence to treatment. Many were based on inaccurate guesses and assumptions. In particular doctors seemed unaware of the relevance of patients' ideas about medicines for successful prescribing. CONCLUSIONS Patients' participation in the consultation and the adverse consequences of lack of participation are important. The authors are developing an educational intervention that builds on these findings.

Prevention of Rectal SHIV Transmission in Macaques by Daily or Intermittent Prophylaxis with Emtricitabine and Tenofovir . BACKGROUND In the absence of an effective vaccine, HIV continues to spread globally, emphasizing the need for novel strategies to limit its transmission. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs could prove to be an effective intervention strategy if highly efficacious and cost-effective PrEP modalities are identified. We evaluated daily and intermittent PrEP regimens of increasing antiviral activity in a macaque model that closely resembles human transmission. METHODS AND FINDINGS We used a repeat-exposure macaque model with 14 weekly rectal virus challenges. Three drug treatments were given once daily, each to a different group of six rhesus macaques. Group 1 was treated subcutaneously with a human-equivalent dose of emtricitabine (FTC), group 2 received orally the human-equivalent dosing of both FTC and tenofovir-disoproxil fumarate (TDF), and group 3 received subcutaneously a similar dosing of FTC and a higher dose of tenofovir. A fourth group of six rhesus macaques (group 4) received intermittently a PrEP regimen similar to group 3 only 2 h before and 24 h after each weekly virus challenge. Results were compared to 18 control macaques that did not receive any drug treatment. The risk of infection in macaques treated in groups 1 and 2 was 3.8- and 7.8-fold lower than in untreated macaques (p = 0.02 and p = 0.008, respectively). All six macaques in group 3 were protected. Breakthrough infections had blunted acute viremias; drug resistance was seen in two of six animals. All six animals in group 4 that received intermittent PrEP were protected. CONCLUSIONS This model suggests that single drugs for daily PrEP can be protective but a combination of antiretroviral drugs may be required to increase the level of protection. Short but potent intermittent PrEP can provide protection comparable to that of daily PrEP in this SHIV/macaque model. These findings support PrEP trials for HIV prevention in humans and identify promising PrEP modalities.


Question: Pyridostatin induces double-strand breaks accumulation.

Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds. G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.

The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of Pre-rRNA processing factors.. Mature ribosomal RNAs (rRNAs) are produced from polycistronic precursors following complex processing. Precursor (pre)-rRNA processing has been extensively characterized in yeast and was assumed to be conserved in humans. We functionally characterized 625 nucleolar proteins in HeLa cells and identified 286 required for processing, including 74 without a yeast homolog. For selected candidates, we demonstrated that pre-rRNA processing defects are conserved in different cell types (including primary cells), defects are not due to activation of a p53-dependent nucleolar tumor surveillance pathway, and they precede cell-cycle arrest and apoptosis. We also investigated the exosome's role in processing internal transcribed spacers (ITSs) and report that 3' end maturation of 18S rRNA involves EXOSC10/Rrp6, a yeast ITS2 processing factor. We conclude that human cells adopt unique strategies and recruit distinct trans-acting factors to carry out essential processing steps, posing fundamental implications for understanding ribosomopathies at the molecular level and developing effective therapeutic agents.


Question: Systemic immunosuppressive (IS) therapy increases the chance of cancer mortality in patients with inflammatory eye disease (IED)

Overall and cancer related mortality among patients with ocular inflammation treated with immunosuppressive drugs: retrospective cohort study.. CONTEXT Whether immunosuppressive treatment adversely affects survival is unclear. OBJECTIVE To assess whether immunosuppressive drugs increase mortality. DESIGN Retrospective cohort study evaluating overall and cancer mortality in relation to immunosuppressive drug exposure among patients with ocular inflammatory diseases. Demographic, clinical, and treatment data derived from medical records, and mortality results from United States National Death Index linkage. The cohort's mortality risk was compared with US vital statistics using standardised mortality ratios. Overall and cancer mortality in relation to use or non-use of immunosuppressive drugs within the cohort was studied with survival analysis. SETTING Five tertiary ocular inflammation clinics. Patients 7957 US residents with non-infectious ocular inflammation, 2340 of whom received immunosuppressive drugs during follow up. Exposures Use of antimetabolites, T cell inhibitors, alkylating agents, and tumour necrosis factor inhibitors. MAIN OUTCOME MEASURES Overall mortality, cancer mortality. RESULTS Over 66 802 person years (17 316 after exposure to immunosuppressive drugs), 936 patients died (1.4/100 person years), 230 (24.6%) from cancer. For patients unexposed to immunosuppressive treatment, risks of death overall (standardised mortality ratio 1.02, 95% confidence interval [CI] 0.94 to 1.11) and from cancer (1.10, 0.93 to 1.29) were similar to those of the US population. Patients who used azathioprine, methotrexate, mycophenolate mofetil, ciclosporin, systemic corticosteroids, or dapsone had overall and cancer mortality similar to that of patients who never took immunosuppressive drugs. In patients who used cyclophosphamide, overall mortality was not increased and cancer mortality was non-significantly increased. Tumour necrosis factor inhibitors were associated with increased overall (adjusted hazard ratio [HR] 1.99, 95% CI 1.00 to 3.98) and cancer mortality (adjusted HR 3.83, 1.13 to 13.01). CONCLUSIONS Most commonly used immunosuppressive drugs do not seem to increase overall or cancer mortality. Our results suggesting that tumour necrosis factor inhibitors might increase mortality are less robust than the other findings; additional evidence is needed.

Tammarniit (Mistakes): Inuit Relocation in the Eastern Arctic, 1939-63. Preface Introduction 1. Are Inuit Indians?: Relief, Jurisdiction, and Government Responsibility 2. Social Welfare and Social Crisis in the Eastern Arctic 3. Planning for Relocation in the Eastern Arctic 4. Recolonizing the Arctic Islands: The 1953 Relocations to Resolute Bay and Craig Harbour 5. The Ennadai Lake Relocations, 1950-60 6. The Garry Lake Famine 7. The Whale Cove Relocation 8. Relocation and Responsibility, 1955-63 Notes Bibliography Index


Question: Pioglitazone use is not associated with an increased risk of prostate cancer.

Pioglitazone Use and Risk of Bladder Cancer and Other Common Cancers in Persons With Diabetes.. IMPORTANCE Studies suggest pioglitazone use may increase risk of cancers. OBJECTIVE To examine whether pioglitazone use for diabetes is associated with risk of bladder and 10 additional cancers. DESIGN, SETTING, AND PARTICIPANTS Cohort and nested case-control analyses among persons with diabetes. A bladder cancer cohort followed 193,099 persons aged 40 years or older in 1997-2002 until December 2012; 464 case patients and 464 matched controls were surveyed about additional confounders. A cohort analysis of 10 additional cancers included 236,507 persons aged 40 years or older in 1997-2005 and followed until June 2012. Cohorts were from Kaiser Permanente Northern California. EXPOSURES Ever use, duration, cumulative dose, and time since initiation of pioglitazone as time dependent. MAIN OUTCOMES AND MEASURES Incident cancer, including bladder, prostate, female breast, lung/bronchus, endometrial, colon, non-Hodgkin lymphoma, pancreas, kidney/renal pelvis, rectum, and melanoma. RESULTS Among 193,099 persons in the bladder cancer cohort, 34,181 (18%) received pioglitazone (median duration, 2.8 years; range, 0.2-13.2 years) and 1261 had incident bladder cancer. Crude incidences of bladder cancer in pioglitazone users and nonusers were 89.8 and 75.9 per 100,000 person-years, respectively. Ever use of pioglitazone was not associated with bladder cancer risk (adjusted hazard ratio [HR], 1.06; 95% CI, 0.89-1.26). Results were similar in case-control analyses (pioglitazone use: 19.6% among case patients and 17.5% among controls; adjusted odds ratio, 1.18; 95% CI, 0.78-1.80). In adjusted analyses, there was no association with 8 of the 10 additional cancers; ever use of pioglitazone was associated with increased risk of prostate cancer (HR, 1.13; 95% CI, 1.02-1.26) and pancreatic cancer (HR, 1.41; 95% CI, 1.16-1.71). Crude incidences of prostate and pancreatic cancer in pioglitazone users vs nonusers were 453.3 vs 449.3 and 81.1 vs 48.4 per 100,000 person-years, respectively. No clear patterns of risk for any cancer were observed for time since initiation, duration, or dose. CONCLUSIONS AND RELEVANCE Pioglitazone use was not associated with a statistically significant increased risk of bladder cancer, although an increased risk, as previously observed, could not be excluded. The increased prostate and pancreatic cancer risks associated with ever use of pioglitazone merit further investigation to assess whether they are causal or are due to chance, residual confounding, or reverse causality.

A Teratocarcinoma-Like Human Embryonic Stem Cell (hESC) Line and Four hESC Lines Reveal Potentially Oncogenic Genomic Changes. The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis.


Question: siRNA knockdown of A20 slows tumor progression in an in vivo murine xenograft model.

Targeting A20 Decreases Glioma Stem Cell Survival and Tumor Growth. Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-kappaB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFalpha-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFalpha-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.

Dissociation of β1- and β2-adrenergic receptor subtypes in the retrieval of cocaine-associated memory.. Drug seeking is maintained by encounters with drug-associated cues, and disrupting retrieval of these drug-cue associations would reduce the risk of relapse. Retrieval of cocaine-associated memories is dependent on β-adrenergic receptor (β-AR) activation, and blockade of these receptors induces a persistent retrieval deficit. Whether retrieval of cocaine-associated memory is mediated by a specific β-AR subtype, however, remains unclear. Using a cocaine conditioned place preference (CPP) procedure, we examined whether retrieval of a cocaine CPP memory is mediated collectively by β1- and β2-ARs, or by one of these β-AR subtypes alone. We show that co-blockade of β1- and β2-ARs abolished CPP expression on that and subsequent drug-free CPP tests, resulting in a long-lasting retrieval deficit that prevented subsequent cocaine-induced reinstatement. To dissociate the necessity of either β1- or β2-ARs alone, we administered subtype-specific antagonists prior to retrieval. Administration of a β1-AR antagonist before the initial CPP trial dose-dependently reduced expression of a CPP on that and subsequent drug-free trials as compared to vehicle administration. In contrast, administration of a β2-AR antagonist had no effect on initial CPP expression, although the highest dose reduced subsequent CPP expression. Importantly, either β1- or β2-AR blockade prior to an initial retrieval trial prevented subsequent cocaine-induced reinstatement. Our findings indicate that the β1-AR subtype mediates retrieval of a cocaine CPP, and that acutely blocking either β1- or β2-ARs can prevent subsequent cocaine-induced reinstatement. Thus, β-AR antagonists, particularly β1-ARs antagonists, could serve as adjuncts for addiction therapies to prevent retrieval of drug-associated memories and provide protection against relapse.


Question: Improvements in OER catalysts show stable activity over several hundred hours.

Homogeneously dispersed multimetal oxygen-evolving catalysts. Earth-abundant first-row (3d) transition metal–based catalysts have been developed for the oxygen-evolution reaction (OER); however, they operate at overpotentials substantially above thermodynamic requirements. Density functional theory suggested that non-3d high-valency metals such as tungsten can modulate 3d metal oxides, providing near-optimal adsorption energies for OER intermediates. We developed a room-temperature synthesis to produce gelled oxyhydroxides materials with an atomically homogeneous metal distribution. These gelled FeCoW oxyhydroxides exhibit the lowest overpotential (191 millivolts) reported at 10 milliamperes per square centimeter in alkaline electrolyte. The catalyst shows no evidence of degradation after more than 500 hours of operation. X-ray absorption and computational studies reveal a synergistic interplay between tungsten, iron, and cobalt in producing a favorable local coordination environment and electronic structure that enhance the energetics for OER.

Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses. BACKGROUND Avian influenza (AI) caused by H7 AI viruses (AIVs) of both low pathogenicity (LP) and high pathogenicity (HP) are notifiable poultry diseases. OBJECTIVES Design and validate two RealTime reverse transcriptase polymerase chain reactions (RRT PCRs) for Eurasian H7 AIV detection and pathotyping. METHODS The H7 RRT PCRs amplified within the (i) HA2 and (ii) cleavage site CS regions of the haemagglutinin gene. Both were validated against 65 H7 AIVs, 57 non-H7 AIVs and 259 poultry swabs in comparison to M gene (AI generic) RRT PCR and virus isolation (VI). An additional 38 swabs and 20 tissue specimens extended validation against M gene RRT PCR. RESULTS Both H7 RRT PCRs amplified all 61 Eurasian lineage H7 AIVs and none of 57 non-H7 AIVs. A total of 297 poultry swabs were used to determine diagnostic sensitivity and specificity relative to M gene RRT PCR, sensitivity was 95.4% and 64.6% for the HA2 and CS RRT PCRs respectively, and specificity 97.9% and 99.6% respectively. The H7 HA2 RRT PCR was more sensitive than VI. This was emphasized by analysis of 37 swabs from turkeys infected experimentally with HPAI H7N1 virus sampled at 24 hours post-inoculation and LPAI H7N1 chicken infections sampled at 40-64 hours. Although less sensitive, usefulness of the H7 CS RRT PCR was confirmed by the correct molecular pathotyping for all 61 Eurasian lineage H7 AIVs tested. CONCLUSIONS The high sensitivity of H7 HA2 RRT PCR confirms its suitability for use in poultry surveillance and disease diagnosis. H7 CS RRT PCR provides an opportunity for rapid pathotyping of H7 AIVs.


Question: The deregulated and prolonged activation of monocytes has deleterious effects in chronic infectious conditions.

The myeloid transcription factor KLF2 regulates the host response to polymicrobial infection and endotoxic shock.. Precise control of myeloid cell activation is required for optimal host defense. However, this activation process must be under exquisite control to prevent uncontrolled inflammation. Herein, we identify the Kruppel-like transcription factor 2 (KLF2) as a potent regulator of myeloid cell activation in vivo. Exposure of myeloid cells to hypoxia and/or bacterial products reduced KLF2 expression while inducing hypoxia inducible factor-1α (HIF-1α), findings that were recapitulated in human septic patients. Myeloid KLF2 was found to be a potent inhibitor of nuclear factor-kappaB (NF-κB)-dependent HIF-1α transcription and, consequently, a critical determinant of outcome in models of polymicrobial infection and endotoxemia. Collectively, these observations identify KLF2 as a tonic repressor of myeloid cell activation in vivo and an essential regulator of the innate immune system.

Epigenetic Therapy Ties MYC Depletion to Reversing Immune Evasion and Treating Lung Cancer. Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/β-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.


Question: The PPR MDA5 has twenty N-terminal CARD domains.

Immune signaling by RIG-I-like receptors.. The RIG-I-like receptors (RLRs) RIG-I, MDA5, and LGP2 play a major role in pathogen sensing of RNA virus infection to initiate and modulate antiviral immunity. The RLRs detect viral RNA ligands or processed self RNA in the cytoplasm to trigger innate immunity and inflammation and to impart gene expression that serves to control infection. Importantly, RLRs cooperate in signaling crosstalk networks with Toll-like receptors and other factors to impart innate immunity and to modulate the adaptive immune response. RLR regulation occurs at a variety of levels ranging from autoregulation to ligand and cofactor interactions and posttranslational modifications. Abberant RLR signaling or dysregulation of RLR expression is now implicated in the development of autoimmune diseases. Understanding the processes of RLR signaling and response will provide insights to guide RLR-targeted therapeutics for antiviral and immune-modifying applications.

Risk of breast cancer in relation to blood lipids: a prospective study of 31,209 Norwegian women. In this prospective study, the relationship between blood lipids and breast cancer risk was examined. Between 1977 and 1983, 31,209 Norwegian women, 20 to 54 years of age, attended a health screening carried out by the Norwegian National Health Screening Services. The screening consisted of a questionnaire, anthropometric measurements, and nonfasting blood drawn for analysis of total serum cholesterol (TC), triglyceride (TG), and high density lipoprotein (HDL) cholesterol. Low density lipoprotein (LDL) cholesterol was calculated by the Friedewald's formula. During the seven to 13 years of follow-up, 302 breast cancer cases were identified by linkage to the Norwegian Cancer Registry. After adjustment for some of the known risk factors of breast cancer, the relative risk of women in the highest quartile of TC compared with women in the lowest quartile was 0.87 (95 percent confidence interval [CI]=0.61–1.23). The corresponding relative risks and CIs were 0.82 (CI=0.58–1.16) for TG, 1.02 (CI=0.73–1.42) for HDL, and 0.93 (CI=0.67–1.29) for LDL. No association between breast cancer risk and blood lipids was found in the total population, nor when the data were divided into those diagnosed before or after the age of 50 as a dividing line between pre- and postmenopausal diagnosis.


Question: CD28 signals regulate trafficking of murine auto-reactive T cells into target tissues.

CD28 and ITK signals regulate autoreactive T cell trafficking. 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.

Reserves, functional, immunoregulatory, and cytogenetic properties of bone marrow mesenchymal stem cells in patients with myelodysplastic syndromes.. Defective hematopoiesis supporting capacity of bone marrow (BM) stroma has been implicated in the pathophysiology of myelodysplastic syndromes (MDS). The aim of this study is to explore whether the BM stroma progenitors, namely the mesenchymal stem cells (MSCs), are primarily affected in MDS by evaluating the reserves, the functional properties, as well as the cytogenetic characteristics, in comparison to BM hematopoietic cells, in patients with de novo MDS (n = 13). The number, differentiation potential toward adipocytes/chondrocytes/osteoblasts and immunosuppressive function in terms of inhibition of mitogen-induced T-cell proliferation did not differ significantly between patient and normal (n = 20) MSCs. Patient MSCs did not show any aberrations in the production of proinflammatory or growth-promoting cytokines and did not harbor the cytogenetic abnormalities present in hematopoietic cells. Occasional patient and normal MSC cultures, however, developed irrelevant chromosomal alterations (trisomies 5 and 7) with uncertain pathophysiologic significance. Compared to controls, patient MSCs displayed impaired proliferative and clonogenic potential through passages that might represent a nonspecific abnormality associated with the chronic inflammatory process present in patients' BM. These data suggest that BM MSCs from MDS patients do not belong to the abnormal clone and do not represent the main cellular source contributing to the inflammatory marrow microenvironment.


Question: Early patent ductus ateriosus (PDA) screening decreases in-hospital mortality.

Association Between Early Screening for Patent Ductus Arteriosus and In-Hospital Mortality Among Extremely Preterm Infants.. IMPORTANCE There is currently no consensus for the screening and treatment of patent ductus arteriosus (PDA) in extremely preterm infants. Less pharmacological closure and more supportive management have been observed without evidence to support these changes. OBJECTIVE To evaluate the association between early screening echocardiography for PDA and in-hospital mortality. DESIGN, SETTING, AND PARTICIPANTS Comparison of screened and not screened preterm infants enrolled in the EPIPAGE 2 national prospective population-based cohort study that included all preterm infants born at less than 29 weeks of gestation and hospitalized in 68 neonatal intensive care units in France from April through December 2011. Two main analyses were performed to adjust for potential selection bias, one using propensity score matching and one using neonatal unit preference for early screening echocardiography as an instrumental variable. EXPOSURES Early screening echocardiography before day 3 of life. MAIN OUTCOMES AND MEASURES The primary outcome was death between day 3 and discharge. The secondary outcomes were major neonatal morbidities (pulmonary hemorrhage, severe bronchopulmonary dysplasia, severe cerebral lesions, and necrotizing enterocolitis). RESULTS Among the 1513 preterm infants with data available to determine exposure, 847 were screened for PDA and 666 were not; 605 infants from each group could be paired. Exposed infants were treated for PDA more frequently during their hospitalization than nonexposed infants (55.1% vs 43.1%; odds ratio [OR], 1.62 [95% CI, 1.31 to 2.00]; absolute risk reduction [ARR] in events per 100 infants, -12.0 [95% CI, -17.3 to -6.7). Exposed infants had a lower hospital death rate (14.2% vs 18.5% ; OR, 0.73 [95% CI, 0.54 to 0.98]; ARR, 4.3 [95% CI, 0.3 to 8.3]) and a lower rate of pulmonary hemorrhage (5.6% vs 8.9%; OR, 0.60 [95% CI, 0.38 to 0.95]; ARR, 3.3 [95% CI, 0.4 to 6.3]). No differences in rates of necrotizing enterocolitis, severe bronchopulmonary dysplasia, or severe cerebral lesions were observed. In the overall cohort, instrumental variable analysis yielded an adjusted OR for in-hospital mortality of 0.62 [95% CI, 0.37 to 1.04]. CONCLUSIONS AND RELEVANCE In this national population-based cohort of extremely preterm infants, screening echocardiography before day 3 of life was associated with lower in-hospital mortality and likelihood of pulmonary hemorrhage but not with differences in necrotizing enterocolitis, severe bronchopulmonary dysplasia, or severe cerebral lesions. However, results of the instrumental variable analysis leave some ambiguity in the interpretation, and longer-term evaluation is needed to provide clarity.

Exercise-induced asthma screening of elite athletes: field versus laboratory exercise challenge.. PURPOSE The purpose of this study was to compare a laboratory based exercise challenge (LBC) to a field based exercise challenge (FBC) for pulmonary function test (PFT) exercise-induced asthma (EIA) screening of elite athletes. METHODS Twenty-three elite cold weather athletes (14 men, 9 women) PFT positive for EIA (FBC screened) served as subjects. Twenty-three gender and sport matched controls (nonasthmatics) were randomly selected to establish PFT reference values for normal elite athletes. Before FBC, athletes completed a medical history questionnaire for EIA symptoms. FBC evaluations consisted of baseline spirometry, actual or simulated competition, and 5, 10, and 15 min postexercise spirometry. PFT positive athletes were evaluated in the laboratory using an exercise challenge simulating race intensity (ambient conditions: 21 degrees C, 60% relative humidity). PFT procedures were identical to FBC. RESULTS 91% of PFT positive and 48% of PFT normal athletes reported at least one symptom of EIA, with postrace cough most frequent. Baseline spirometry was the same for PFT positives and normal controls. Lower limit reference range (MN - 2 SD) of FEV1 for controls suggests that postexercise decrements of greater than approximately -7% indicate abnormal airway response in this population. Exercise time duration did not effect bronchial reactivity; 78% of FBC PFT positives were PFT normal post-LBC. CONCLUSION Self-reported symptoms by elite athletes are not reliable in identifying EIA. Reference range criterion for FEV1 decrement in the elite athlete postexercise contrasts current recommended guidelines. Moreover, a large number of false negatives may occur in this population if EIA screening is performed with inadequate exercise and environmental stress.


Question: Transcription rates in S. cerevisiae range between 0.7 and 2 kb/min.

Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo.. A number of proteins and drugs have been implicated in the process of transcriptional elongation by RNA polymerase (Pol) II, but the factors that govern the elongation rate (nucleotide additions per min) and processivity (nucleotide additions per initiation event) in vivo are poorly understood. Here, we show that a mutation in the Rpb2 subunit of Pol II reduces both the elongation rate and processivity in vivo. In contrast, none of the putative elongation factors tested affect the elongation rate, although mutations in the THO complex and in Spt4 significantly reduce processivity. The drugs 6-azauracil and mycophenolic acid reduce both the elongation rate and processivity, and this processivity defect is aggravated by mutations in Spt4, TFIIS, and CTDK-1. Our results suggest that, in vivo, a reduced rate of Pol II elongation leads to premature dissociation along the chromatin template and that Pol II processivity can be uncoupled from elongation rate.

Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells. The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.


Question: Smc5/6 engagment halts the activation of SUMO E3 ligase Mms21 by ATP-dependent remolding.

ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair. Modification of proteins by SUMO is essential for the maintenance of genome integrity. During DNA replication, the Mms21-branch of the SUMO pathway counteracts recombination intermediates at damaged replication forks, thus facilitating sister chromatid disjunction. The Mms21 SUMO ligase docks to the arm region of the Smc5 protein in the Smc5/6 complex; together, they cooperate during recombinational DNA repair. Yet how the activity of the SUMO ligase is controlled remains unknown. Here we show that the SUMO ligase and the chromosome disjunction functions of Mms21 depend on its docking to an intact and active Smc5/6 complex, indicating that the Smc5/6-Mms21 complex operates as a large SUMO ligase in vivo. In spite of the physical distance separating the E3 and the nucleotide-binding domains in Smc5/6, Mms21-dependent sumoylation requires binding of ATP to Smc5, a step that is part of the ligase mechanism that assists Ubc9 function. The communication is enabled by the presence of a conserved disruption in the coiled coil domain of Smc5, pointing to potential conformational changes for SUMO ligase activation. In accordance, scanning force microscopy of the Smc5-Mms21 heterodimer shows that the molecule is physically remodeled in an ATP-dependent manner. Our results demonstrate that the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair.

Detection and function of nitric oxide during the hypersensitive response in Arabidopsis thaliana: where there's a will there's a way.. Nitric oxide (NO) was identified as a key player in plant defence responses approximately 20 years ago and a large body of evidence has accumulated since then supporting its role as a signalling molecule. However, there are many discrepancies in current NO detection assays and the enzymatic pathways responsible for its synthesis have yet to be determined. This has provoked strong debates concerning the function of NO in plants, even questioning its existence in planta. Here we gather data obtained using the model pathosystem Arabidopsis/Pseudomonas, which confirms the production of NO during the hypersensitive response and supports is role as a trigger of hypersensitive cell death and a mediator of defence gene expression. Finally, we discuss potential sources of NO synthesis, focusing on the role of nitrite as major substrate for NO production during incompatible interactions.


Question: Patients with microcytosis and higher erythrocyte count are more vulnerable to severe malarial anaemia when infected with Plasmodium falciparum.

Increased Microerythrocyte Count in Homozygous α+-Thalassaemia Contributes to Protection against Severe Malarial Anaemia. BACKGROUND The heritable haemoglobinopathy alpha(+)-thalassaemia is caused by the reduced synthesis of alpha-globin chains that form part of normal adult haemoglobin (Hb). Individuals homozygous for alpha(+)-thalassaemia have microcytosis and an increased erythrocyte count. Alpha(+)-thalassaemia homozygosity confers considerable protection against severe malaria, including severe malarial anaemia (SMA) (Hb concentration < 50 g/l), but does not influence parasite count. We tested the hypothesis that the erythrocyte indices associated with alpha(+)-thalassaemia homozygosity provide a haematological benefit during acute malaria. METHODS AND FINDINGS Data from children living on the north coast of Papua New Guinea who had participated in a case-control study of the protection afforded by alpha(+)-thalassaemia against severe malaria were reanalysed to assess the genotype-specific reduction in erythrocyte count and Hb levels associated with acute malarial disease. We observed a reduction in median erythrocyte count of approximately 1.5 x 10(12)/l in all children with acute falciparum malaria relative to values in community children (p < 0.001). We developed a simple mathematical model of the linear relationship between Hb concentration and erythrocyte count. This model predicted that children homozygous for alpha(+)-thalassaemia lose less Hb than children of normal genotype for a reduction in erythrocyte count of >1.1 x 10(12)/l as a result of the reduced mean cell Hb in homozygous alpha(+)-thalassaemia. In addition, children homozygous for alpha(+)-thalassaemia require a 10% greater reduction in erythrocyte count than children of normal genotype (p = 0.02) for Hb concentration to fall to 50 g/l, the cutoff for SMA. We estimated that the haematological profile in children homozygous for alpha(+)-thalassaemia reduces the risk of SMA during acute malaria compared to children of normal genotype (relative risk 0.52; 95% confidence interval [CI] 0.24-1.12, p = 0.09). CONCLUSIONS The increased erythrocyte count and microcytosis in children homozygous for alpha(+)-thalassaemia may contribute substantially to their protection against SMA. A lower concentration of Hb per erythrocyte and a larger population of erythrocytes may be a biologically advantageous strategy against the significant reduction in erythrocyte count that occurs during acute infection with the malaria parasite Plasmodium falciparum. This haematological profile may reduce the risk of anaemia by other Plasmodium species, as well as other causes of anaemia. Other host polymorphisms that induce an increased erythrocyte count and microcytosis may confer a similar advantage.

Ghrelin's second life: from appetite stimulator to glucose regulator.. Ghrelin, a 28 amino acid peptide hormone produced by the stomach, was the first orexigenic hormone to be discovered from the periphery. The octanoyl modification at Ser³, mediated by ghrelin O-acyltransferase (GOAT), is essential for ghrelin's biological activity. Ghrelin stimulates food intake through binding to its receptor (GRLN-R) on neurons in the arcuate nucleus of the hypothalamus. Ghrelin is widely expressed throughout the body; accordingly, it is implicated in several other physiological functions, which include growth hormone release, gastric emptying, and body weight regulation. Ghrelin and GRLN-R expression are also found in the pancreas, suggesting a local physiological role. Accordingly, several recent studies now point towards an important role for ghrelin and its receptor in the regulation of blood glucose homeostasis, which is the main focus of this review. Several mechanisms of this regulation by ghrelin have been proposed, and one possibility is through the regulation of insulin secretion. Despite some controversy, most studies suggest that ghrelin exerts an inhibitory effect on insulin secretion, resulting in increased circulating glucose levels. Ghrelin may thus be a diabetogenic factor. Obesity-related type 2 diabetes has become an increasingly important health problem, almost reaching epidemic proportions in the world; therefore, antagonists of the ghrelin-GOAT signaling pathway, which will tackle both energy- and glucose homeostasis, may be considered as promising new therapies for this disease.


Question: Microglia are an innate immune cell type of the peripheral nervous system.

Sublime Microglia: Expanding Roles for the Guardians of the CNS. Recent findings challenge the concept that microglia solely function in disease states in the central nervous system (CNS). Rather than simply reacting to CNS injury, infection, or pathology, emerging lines of evidence indicate that microglia sculpt the structure of the CNS, refine neuronal circuitry and network connectivity, and contribute to plasticity. These physiological functions of microglia in the normal CNS begin during development and persist into maturity. Here, we develop a conceptual framework for functions of microglia beyond neuroinflammation and discuss the rich repertoire of signaling and communication motifs in microglia that are critical both in pathology and for the normal physiology of the CNS.

Etiology of type 1 diabetes.. Recent genetic mapping and gene-phenotype studies have revealed the genetic architecture of type 1 diabetes. At least ten genes so far can be singled out as strong causal candidates. The known functions of these genes indicate the primary etiological pathways of this disease, including HLA class II and I molecules binding to preproinsulin peptides and T cell receptors, T and B cell activation, innate pathogen-viral responses, chemokine and cytokine signaling, and T regulatory and antigen-presenting cell functions. This review considers research in the field of type 1 diabetes toward identifying disease mechanisms using genetic approaches. The expression and functions of these pathways, and, therefore, disease susceptibility, will be influenced by epigenetic and environmental factors. Certain inherited immune phenotypes will be early precursors of type 1 diabetes and could be useful in future clinical trials.


Question: Deltex has no known interactions with eIF3f.

The Translation Initiation Factor 3f (eIF3f) Exhibits a Deubiquitinase Activity Regulating Notch Activation. Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.

D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate.. Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by >/=5 microM thrombin receptor-activating peptide (TRAP), which causes rapid and massive wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 microM) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.


Question: CDK6 shows improved binding to loss-of-function variants of p18 INK4C.

Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development. We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18(INK4C) and p16(INK4A) codeletion. Functional reconstitution of p18(INK4C) in GBM cells null for both p16(INK4A) and p18(INK4C) resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18(INK4C) in p16(INK4A)-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16(INK4A) in primary astrocytes induced a concomitant increase in p18(INK4C). Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18(INK4C) in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation.

Microenvironment-dependent growth of pre-neoplastic and malignant plasma cells in humanized mice. Most human cancers, including myeloma, are preceded by a precursor state. There is an unmet need for in vivo models to study the interaction of human preneoplastic cells in the bone marrow microenvironment with non-malignant cells. Here, we genetically humanized mice to permit the growth of primary human preneoplastic and malignant plasma cells together with non-malignant cells in vivo. Growth was largely restricted to the bone marrow, mirroring the pattern in patients with myeloma. Xenografts captured the genomic complexity of parental tumors and revealed additional somatic changes. Moreover, xenografts from patients with preneoplastic gammopathy showed progressive growth, suggesting that the clinical stability of these lesions may in part be due to growth controls extrinsic to tumor cells. These data demonstrate a new approach to investigate the entire spectrum of human plasma cell neoplasia and illustrate the utility of humanized models for understanding the functional diversity of human tumors.


Question: Toll-like receptor (TLR) signaling is involved in the pathogenesis of human MDS.

Induction of myelodysplasia by myeloid-derived suppressor cells.. Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited components to CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppressive cytokines IL-10 and TGF-β by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif–bearing (ITAM-bearing) adapter protein (DAP12) interruption of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.

The Apaf-1 apoptosome induces formation of caspase-9 homo- and heterodimers with distinct activities. According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.


Question: Mutations in G-Beta protein GNB1 are present in many cancers, resulting in loss of interaction with G-alpha subunits and concomitant activation of AKT pathway.

Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance. Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Implanted microvessels progress through distinct neovascularization phenotypes.. We have previously demonstrated that implanted microvessels form a new microcirculation with minimal host-derived vessel investment. Our objective was to define the vascular phenotypes present during neovascularization in these implants and identify post-angiogenesis events. Morphological, functional and transcriptional assessments identified three distinct vascular phenotypes in the implants: sprouting angiogenesis, neovascular remodeling, and network maturation. A sprouting angiogenic phenotype appeared first, characterized by high proliferation and low mural cell coverage. This was followed by a neovascular remodeling phenotype characterized by a perfused, poorly organized neovascular network, reduced proliferation, and re-associated mural cells. The last phenotype included a vascular network organized into a stereotypical tree structure containing vessels with normal perivascular cell associations. In addition, proliferation was low and was restricted to the walls of larger microvessels. The transition from angiogenesis to neovascular remodeling coincided with the appearance of blood flow in the implant neovasculature. Analysis of vascular-specific and global gene expression indicates that the intermediate, neovascular remodeling phenotype is transcriptionally distinct from the other two phenotypes. Therefore, this vascular phenotype likely is not simply a transitional phenotype but a distinct vascular phenotype involving unique cellular and vascular processes. Furthermore, this neovascular remodeling phase may be a normal aspect of the general neovascularization process. Given that this phenotype is arguably dysfunctional, many of the microvasculatures present within compromised or diseased tissues may not represent a failure to progress appropriately through a normally occurring neovascularization phenotype.


Question: Cognitive behavioral therapy is an ineffective treatment for insomnia

Cognitive behavioral therapy vs zopiclone for treatment of chronic primary insomnia in older adults: a randomized controlled trial.. CONTEXT Insomnia is a common condition in older adults and is associated with a number of adverse medical, social, and psychological consequences. Previous research has suggested beneficial outcomes of both psychological and pharmacological treatments, but blinded placebo-controlled trials comparing the effects of these treatments are lacking. OBJECTIVE To examine short- and long-term clinical efficacy of cognitive behavioral therapy (CBT) and pharmacological treatment in older adults experiencing chronic primary insomnia. DESIGN, SETTING, AND PARTICIPANTS A randomized, double-blinded, placebo-controlled trial of 46 adults (mean age, 60.8 y; 22 women) with chronic primary insomnia conducted between January 2004 and December 2005 in a single Norwegian university-based outpatient clinic for adults and elderly patients. INTERVENTION CBT (sleep hygiene, sleep restriction, stimulus control, cognitive therapy, and relaxation; n = 18), sleep medication (7.5-mg zopiclone each night; n = 16), or placebo medication (n = 12). All treatment duration was 6 weeks, and the 2 active treatments were followed up at 6 months. MAIN OUTCOME MEASURES Ambulant clinical polysomnographic data and sleep diaries were used to determine total wake time, total sleep time, sleep efficiency, and slow-wave sleep (only assessed using polysomnography) on all 3 assessment points. RESULTS CBT resulted in improved short- and long-term outcomes compared with zopiclone on 3 out of 4 outcome measures. For most outcomes, zopiclone did not differ from placebo. Participants receiving CBT improved their sleep efficiency from 81.4% at pretreatment to 90.1% at 6-month follow-up compared with a decrease from 82.3% to 81.9% in the zopiclone group. Participants in the CBT group spent much more time in slow-wave sleep (stages 3 and 4) compared with those in other groups, and spent less time awake during the night. Total sleep time was similar in all 3 groups; at 6 months, patients receiving CBT had better sleep efficiency using polysomnography than those taking zopiclone. CONCLUSION These results suggest that interventions based on CBT are superior to zopiclone treatment both in short- and long-term management of insomnia in older adults. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00295386.

Crystal structures of the glutamate receptor ion channel GluK3 and GluK5 amino-terminal domains.. Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic neurotransmission in the central nervous system. The selective assembly of iGluRs into AMPA, kainate, and N-methyl-d-aspartic acid (NMDA) receptor subtypes is regulated by their extracellular amino-terminal domains (ATDs). Kainate receptors are further classified into low-affinity receptor families (GluK1-GluK3) and high-affinity receptor families (GluK4-GluK5) based on their affinity for the neurotoxin kainic acid. These two families share a 42% sequence identity for the intact receptor but only a 27% sequence identity at the level of ATD. We have determined for the first time the high-resolution crystal structures of GluK3 and GluK5 ATDs, both of which crystallize as dimers but with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5, the R2 domain dimer assembly is similar to those reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-GluK5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-GluK3. Our analysis also reveals that the relative orientation of domains R1 and R2 in individual non-NMDA receptor ATDs varies by up to 10°, in contrast to the 50° difference reported for the NMDA receptor GluN2B subunit. This restricted domain movement in non-NMDA receptor ATDs seems to result both from extensive intramolecular contacts between domain R1 and domain R2 and from their assembly as dimers, which interact at both R1 and R2 domains. Our results provide the first insights into the structure and function of GluK4-GluK5, the least understood family of iGluRs.


Question: Cellular clocks are not predictive of mitosis timing in NIH 3T3 cells.

Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle.. Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.

Scaling of Dorsal-Ventral Patterning by Embryo Size-Dependent Degradation of Spemann’s Organizer Signals. Spemann's organizer plays a key role in dorsal-ventral (DV) patterning in the amphibian embryo by secreting diffusible proteins such as Chordin, an antagonist to ventralizing bone morphogenetic proteins (BMPs). The DV patterning is so robust that an amphibian embryo with its ventral half surgically removed can develop into a smaller but proportionally patterned larva. Here, we show that this robust patterning depends on facilitated Chordin degradation and requires the expression of the Chordin-proteinase inhibitor Sizzled on the opposite side. Sizzled, which is stable and diffuses widely along the DV axis, stabilizes Chordin and expands its distribution in the ventral direction. This expanded Chordin distribution, in turn, limits BMP-dependent Sizzled production, forming an axis-wide feedback loop for shaping Chordin's activity. Using bisection assays, we demonstrate that Chordin degradation is dynamically controlled by embryo-size-coupled Sizzled accumulation. We propose a scaling model that enables the DV pattern to adjust proportionally to embryonic axis size.


Question: Inhibiting HDAC6 increases survival of mice with ARID1A mutated tumors.

ARID1A-mutated ovarian cancers depend on HDAC6 activity. ARID1A, encoding a subunit of the SWI/SNF chromatin-remodelling complex, is the most frequently mutated epigenetic regulator across all human cancers. ARID1A and TP53 mutations are typically mutually exclusive. Therapeutic approaches that correlate with this genetic characteristic remain to be explored. Here, we show that HDAC6 activity is essential in ARID1A-mutated ovarian cancers. Inhibition of HDAC6 activity using a clinically applicable small-molecule inhibitor significantly improved the survival of mice bearing ARID1A-mutated tumours. This correlated with the suppression of growth and dissemination of ARID1A-mutated, but not wild-type, tumours. The dependence on HDAC6 activity in ARID1A-mutated cells correlated with a direct transcriptional repression of HDAC6 by ARID1A. HDAC6 inhibition selectively promoted apoptosis of ARID1A-mutated cells. HDAC6 directly deacetylates Lys120 of p53, a pro-apoptotic post-translational modification. Thus, ARID1A mutation inactivates the apoptosis-promoting function of p53 by upregulating HDAC6. Together, these results indicate that pharmacological inhibition of HDAC6 is a therapeutic strategy for ARID1A-mutated cancers.

Amylase, Lipase, and Acute Pancreatitis in People With Type 2 Diabetes Treated With Liraglutide: Results From the LEADER Randomized Trial.. OBJECTIVE To evaluate serum amylase and lipase levels and the rate of acute pancreatitis in patients with type 2 diabetes and high cardiovascular risk randomized to liraglutide or placebo and observed for 3.5-5.0 years. RESEARCH DESIGN AND METHODS A total of 9,340 patients with type 2 diabetes were randomized to either liraglutide or placebo (median observation time 3.84 years). Fasting serum lipase and amylase were monitored. Acute pancreatitis was adjudicated in a blinded manner. RESULTS Compared with the placebo group, liraglutide-treated patients had increases in serum lipase and amylase of 28.0% and 7.0%, respectively. Levels were increased at 6 months and then remained stable. During the study, 18 (0.4% [1.1 events/1,000 patient-years of observation] [PYO]) liraglutide-treated and 23 (0.5% [1.7 events/1,000 PYO]) placebo patients had acute pancreatitis confirmed by adjudication. Most acute pancreatitis cases occurred ≥12 months after randomization. Liraglutide-treated patients with prior history of pancreatitis (n = 147) were not more likely to develop acute pancreatitis than similar patients in the placebo group (n = 120). Elevations of amylase and lipase levels did not predict future risk of acute pancreatitis (positive predictive value <1.0%) in patients treated with liraglutide. CONCLUSIONS In a population with type 2 diabetes at high cardiovascular risk, there were numerically fewer events of acute pancreatitis among liraglutide-treated patients (regardless of previous history of pancreatitis) compared with the placebo group. Liraglutide was associated with increases in serum lipase and amylase, which were not predictive of an event of subsequent acute pancreatitis.


Question: CCL19 is a ligand for CCR7.

Immobilized chemokine fields and soluble chemokine gradients cooperatively shape migration patterns of dendritic cells.. Chemokines orchestrate immune cell trafficking by eliciting either directed or random migration and by activating integrins in order to induce cell adhesion. Analyzing dendritic cell (DC) migration, we showed that these distinct cellular responses depended on the mode of chemokine presentation within tissues. The surface-immobilized form of the chemokine CCL21, the heparan sulfate-anchoring ligand of the CC-chemokine receptor 7 (CCR7), caused random movement of DCs that was confined to the chemokine-presenting surface because it triggered integrin-mediated adhesion. Upon direct contact with CCL21, DCs truncated the anchoring residues of CCL21, thereby releasing it from the solid phase. Soluble CCL21 functionally resembles the second CCR7 ligand, CCL19, which lacks anchoring residues and forms soluble gradients. Both soluble CCR7 ligands triggered chemotactic movement, but not surface adhesion. Adhesive random migration and directional steering cooperate to produce dynamic but spatially restricted locomotion patterns closely resembling the cellular dynamics observed in secondary lymphoid organs.

Successful '9-month Bangladesh regimen' for multidrug-resistant tuberculosis among over 500 consecutive patients.. SETTING Tuberculosis (TB) program, Damien Foundation Projects, Bangladesh. OBJECTIVE To summarize the outcome and its determinants of the first treatment for multidrug-resistant TB using a standardized regimen consisting of a minimum 9 months. DESIGN This was a prospective, observational study of a gatifloxacin (GFX) based directly observed regimen, mainly with initial hospitalization. The 4-month intensive phase was extended until sputum smear conversion. Patients were monitored using culture for up to 2 years after treatment completion. RESULTS Of the 515 patients who met the study inclusion criteria and were successively enrolled from 2005 to 2011, 84.4% had a bacteriologically favorable outcome. Due to extensive disease with delayed sputum conversion, only half of the patients completed treatment within 9 months; however, 95% were able to complete treatment within 12 months. Eleven patients failed or relapsed, and 93.1% of the 435 patients who were successfully treated completed at least 12 months post-treatment follow-up. The strongest risk factor for a bacteriologically unfavorable outcome was high-level fluoroquinolone (FQ) resistance, particularly when compounded by initial pyrazinamide (PZA) resistance. Low-level FQ resistance had no unfavorable effect on treatment outcome. Amplification of drug resistance occurred only once, in a patient strain that was initially only susceptible to kanamycin and clofazimine. CONCLUSION The excellent outcome of the Bangladesh regimen was largely maintained. Bacteriological treatment failures and relapses were rare, except among patients with high-level GFX resistance, notably in the presence of PZA resistance.


Question: A deficiency of vitamin B12 decreases blood levels of homocysteine.

Randomized trial of folic acid supplementation and serum homocysteine levels.. BACKGROUND Lowering serum homocysteine levels with folic acid is expected to reduce mortality from ischemic heart disease. Homocysteine reduction is known to be maximal at a folic acid dosage of 1 mg/d, but the effect of lower doses (relevant to food fortification) is unclear. METHODS We randomized 151 patients with ischemic heart disease to 1 of 5 dosages of folic acid (0.2, 0.4, 0.6, 0.8, and 1.0 mg/d) or placebo. Fasting blood samples for serum homocysteine and serum folate analysis were taken initially, after 3 months of supplementation, and 3 months after folic acid use was discontinued. RESULTS Median serum homocysteine level decreased with increasing folic acid dosage, to a maximum at 0.8 mg of folic acid per day, when the homocysteine reduction (placebo adjusted) was 2.7 micromol/L (23%), similar to the known effect of folic acid dosages of 1 mg/d and above. The higher a person's initial serum homocysteine level, the greater was the response to folic acid, but there were statistically significant reductions regardless of the initial level. Serum folate level increased approximately linearly (5.5 nmol/L for every 0.1 mg of folic acid). Within-person fluctuations over time in serum homocysteine levels, measured in the placebo group, were large compared with the effect of folic acid, indicating that monitoring of the reduction in an individual is impractical. CONCLUSIONS A dosage of folic acid of 0.8 mg/d appears necessary to achieve the maximum reduction in serum homocysteine level across the range of homocysteine levels in the population. Current US food fortification levels will achieve only a small proportion of the achievable homocysteine reduction.

Metabolic phenotyping of human blood plasma: a powerful tool to discriminate between cancer types?. BACKGROUND Accumulating evidence has shown that cancer cell metabolism differs from that of normal cells. However, up to now it is not clear whether different cancer types are characterized by a specific metabolite profile. Therefore, this study aims to evaluate whether the plasma metabolic phenotype allows to discriminate between lung and breast cancer. PATIENTS AND METHODS The proton nuclear magnetic resonance spectrum of plasma is divided into 110 integration regions, representing the metabolic phenotype. These integration regions reflect the relative metabolite concentrations and were used to train a classification model in discriminating between 80 female breast cancer patients and 54 female lung cancer patients, all with an adenocarcinoma. The validity of the model was examined by permutation testing and by classifying an independent validation cohort of 60 female breast cancer patients and 81 male lung cancer patients, all with an adenocarcinoma. RESULTS The model allows to classify 99% of the breast cancer patients and 93% of the lung cancer patients correctly with an area under the curve (AUC) of 0.96 and can be validated in the independent cohort with a sensitivity of 89%, a specificity of 82% and an AUC of 0.94. Decreased levels of sphingomyelin and phosphatidylcholine (phospholipids with choline head group) and phospholipids with short, unsaturated fatty acid chains next to increased levels of phospholipids with long, saturated fatty acid chains seem to indicate that cell membranes of lung tumors are more rigid and less sensitive to lipid peroxidation. The other discriminating metabolites are pointing to a more pronounced response of the body to the Warburg effect for lung cancer. CONCLUSION Metabolic phenotyping of plasma allows to discriminate between lung and breast cancer, indicating that the metabolite profile reflects more than a general cancer marker. CLINICAL TRIAL REGISTRATION NUMBER NCT02362776.


Question: RAD52 is involved in break-induced DNA replication (BIR).

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks. Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.

Foxp3(+) T cells regulate immunoglobulin a selection and facilitate diversification of bacterial species responsible for immune homeostasis.. Foxp3(+) T cells play a critical role for the maintenance of immune tolerance. Here we show that in mice, Foxp3(+) T cells contributed to diversification of gut microbiota, particularly of species belonging to Firmicutes. The control of indigenous bacteria by Foxp3(+) T cells involved regulatory functions both outside and inside germinal centers (GCs), consisting of suppression of inflammation and regulation of immunoglobulin A (IgA) selection in Peyer's patches, respectively. Diversified and selected IgAs contributed to maintenance of diversified and balanced microbiota, which in turn facilitated the expansion of Foxp3(+) T cells, induction of GCs, and IgA responses in the gut through a symbiotic regulatory loop. Thus, the adaptive immune system, through cellular and molecular components that are required for immune tolerance and through the diversification as well as selection of antibody repertoire, mediates host-microbial symbiosis by controlling the richness and balance of bacterial communities required for homeostasis.


Question: Physical activity level is associated with the difference in maximal oxygen consumption between black and white youth.

Relationship of physical activity and television watching with body weight and level of fatness among children: results from the Third National Health and Nutrition Examination Survey.. CONTEXT Physical inactivity contributes to weight gain in adults, but whether this relationship is true for children of different ethnic groups is not well established. OBJECTIVE To assess participation in vigorous activity and television watching habits and their relationship to body weight and fatness in US children. DESIGN Nationally representative cross-sectional survey with an in-person interview and medical examination. SETTING AND PARTICIPANTS Between 1988 and 1994, 4063 children aged 8 through 16 years were examined as part of the National Health and Nutrition Examination Survey III. Mexican Americans and non-Hispanic blacks were oversampled to produce reliable estimates for these groups. MAIN OUTCOME MEASURES Episodes of weekly vigorous activity and daily hours of television watched, and their relationship to body mass index and body fatness. RESULTS Eighty percent of US children reported performing 3 or more bouts of vigorous activity each week. This rate was lower in non-Hispanic black and Mexican American girls (69% and 73%, respectively). Twenty percent of US children participated in 2 or fewer bouts of vigorous activity perweek, and the rate was higher in girls (26%) than in boys (17%). Overall, 26% of US children watched 4 or more hours of television per day and 67% watched at least 2 hours per day. Non-Hispanic black children had the highest rates of watching 4 or more hours of television per day (42%). Boys and girls who watch 4 or more hours of television each day had greater body fat (P<.001) and had a greater body mass index (P<.001) than those who watched less than 2 hours per day. CONCLUSIONS Many US children watch a great deal of television and are inadequately vigorously active. Vigorous activity levels are lowest among girls, non-Hispanic blacks, and Mexican Americans. Intervention strategies to promote lifelong physical activity among US children are needed to stem the adverse health consequences of inactivity.

An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. Tuberculosis, a major health problem in developing countries, has reemerged in recent years in many industrialized countries. The increased susceptibility of immunocompromised individuals to tuberculosis, and many experimental studies indicate that T cell-mediated immunity plays an important role in resistance. The lymphokine interferon gamma (IFN-gamma) is thought to be a principal mediator of macrophage activation and resistance to intracellular pathogens. Mice have been developed which fail to produce IFN-gamma (gko), because of a targeted disruption of the gene for IFN-gamma. Upon infection with Mycobacterium tuberculosis, although they develop granulomas, gko mice fail to produce reactive nitrogen intermediates and are unable to restrict the growth of the bacilli. In contrast to control mice, gko mice exhibit heightened tissue necrosis and succumb to a rapid and fatal course of tuberculosis that could be delayed, but not prevented, by treatment with exogenous recombinant IFN-gamma.


Question: In rhesus macaques, daily subcutaneous injections of emtricitabine are ineffective at protecting against rectally transmitted simian-human immunodeficiency virus.

Prevention of Rectal SHIV Transmission in Macaques by Daily or Intermittent Prophylaxis with Emtricitabine and Tenofovir . BACKGROUND In the absence of an effective vaccine, HIV continues to spread globally, emphasizing the need for novel strategies to limit its transmission. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs could prove to be an effective intervention strategy if highly efficacious and cost-effective PrEP modalities are identified. We evaluated daily and intermittent PrEP regimens of increasing antiviral activity in a macaque model that closely resembles human transmission. METHODS AND FINDINGS We used a repeat-exposure macaque model with 14 weekly rectal virus challenges. Three drug treatments were given once daily, each to a different group of six rhesus macaques. Group 1 was treated subcutaneously with a human-equivalent dose of emtricitabine (FTC), group 2 received orally the human-equivalent dosing of both FTC and tenofovir-disoproxil fumarate (TDF), and group 3 received subcutaneously a similar dosing of FTC and a higher dose of tenofovir. A fourth group of six rhesus macaques (group 4) received intermittently a PrEP regimen similar to group 3 only 2 h before and 24 h after each weekly virus challenge. Results were compared to 18 control macaques that did not receive any drug treatment. The risk of infection in macaques treated in groups 1 and 2 was 3.8- and 7.8-fold lower than in untreated macaques (p = 0.02 and p = 0.008, respectively). All six macaques in group 3 were protected. Breakthrough infections had blunted acute viremias; drug resistance was seen in two of six animals. All six animals in group 4 that received intermittent PrEP were protected. CONCLUSIONS This model suggests that single drugs for daily PrEP can be protective but a combination of antiretroviral drugs may be required to increase the level of protection. Short but potent intermittent PrEP can provide protection comparable to that of daily PrEP in this SHIV/macaque model. These findings support PrEP trials for HIV prevention in humans and identify promising PrEP modalities.

RANK signals from CD4+3− inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla. 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.