Question: MICAL redox enzymes regulate actin dynamics.

Differential regulation of actin microfilaments by human MICAL proteins.. The Drosophila melanogaster MICAL protein is essential for the neuronal growth cone machinery that functions through plexin- and semaphorin-mediated axonal signaling. Drosophila MICAL is also involved in regulating myofilament organization and synaptic structures, and serves as an actin disassembly factor downstream of plexin-mediated axonal repulsion. In mammalian cells there are three known isoforms, MICAL1, MICAL2 and MICAL3, as well as the MICAL-like proteins MICAL-L1 and MICAL-L2, but little is known of their function, and information comes almost exclusively from neural cells. In this study we show that in non-neural cells human MICALs are required for normal actin organization, and all three MICALs regulate actin stress fibers. Moreover, we provide evidence that the generation of reactive oxygen species by MICAL proteins is crucial for their actin-regulatory function. However, although MICAL1 is auto-inhibited by its C-terminal coiled-coil region, MICAL2 remains constitutively active and affects stress fibers. These data suggest differential but complementary roles for MICAL1 and MICAL2 in actin microfilament regulation.

Continuous glucose monitoring in pregnant women with type 1 diabetes (CONCEPTT): a multicentre international randomised controlled trial. BACKGROUND Pregnant women with type 1 diabetes are a high-risk population who are recommended to strive for optimal glucose control, but neonatal outcomes attributed to maternal hyperglycaemia remain suboptimal. Our aim was to examine the effectiveness of continuous glucose monitoring (CGM) on maternal glucose control and obstetric and neonatal health outcomes. METHODS In this multicentre, open-label, randomised controlled trial, we recruited women aged 18-40 years with type 1 diabetes for a minimum of 12 months who were receiving intensive insulin therapy. Participants were pregnant (≤13 weeks and 6 days' gestation) or planning pregnancy from 31 hospitals in Canada, England, Scotland, Spain, Italy, Ireland, and the USA. We ran two trials in parallel for pregnant participants and for participants planning pregnancy. In both trials, participants were randomly assigned to either CGM in addition to capillary glucose monitoring or capillary glucose monitoring alone. Randomisation was stratified by insulin delivery (pump or injections) and baseline glycated haemoglobin (HbA1c). The primary outcome was change in HbA1c from randomisation to 34 weeks' gestation in pregnant women and to 24 weeks or conception in women planning pregnancy, and was assessed in all randomised participants with baseline assessments. Secondary outcomes included obstetric and neonatal health outcomes, assessed with all available data without imputation. This trial is registered with ClinicalTrials.gov, number NCT01788527. FINDINGS Between March 25, 2013, and March 22, 2016, we randomly assigned 325 women (215 pregnant, 110 planning pregnancy) to capillary glucose monitoring with CGM (108 pregnant and 53 planning pregnancy) or without (107 pregnant and 57 planning pregnancy). We found a small difference in HbA1c in pregnant women using CGM (mean difference -0·19%; 95% CI -0·34 to -0·03; p=0·0207). Pregnant CGM users spent more time in target (68% vs 61%; p=0·0034) and less time hyperglycaemic (27% vs 32%; p=0·0279) than did pregnant control participants, with comparable severe hypoglycaemia episodes (18 CGM and 21 control) and time spent hypoglycaemic (3% vs 4%; p=0·10). Neonatal health outcomes were significantly improved, with lower incidence of large for gestational age (odds ratio 0·51, 95% CI 0·28 to 0·90; p=0·0210), fewer neonatal intensive care admissions lasting more than 24 h (0·48; 0·26 to 0·86; p=0·0157), fewer incidences of neonatal hypoglycaemia (0·45; 0·22 to 0·89; p=0·0250), and 1-day shorter length of hospital stay (p=0·0091). We found no apparent benefit of CGM in women planning pregnancy. Adverse events occurred in 51 (48%) of CGM participants and 43 (40%) of control participants in the pregnancy trial, and in 12 (27%) of CGM participants and 21 (37%) of control participants in the planning pregnancy trial. Serious adverse events occurred in 13 (6%) participants in the pregnancy trial (eight [7%] CGM, five [5%] control) and in three (3%) participants in the planning pregnancy trial (two [4%] CGM and one [2%] control). The most common adverse events were skin reactions occurring in 49 (48%) of 103 CGM participants and eight (8%) of 104 control participants during pregnancy and in 23 (44%) of 52 CGM participants and five (9%) of 57 control participants in the planning pregnancy trial. The most common serious adverse events were gastrointestinal (nausea and vomiting in four participants during pregnancy and three participants planning pregnancy). INTERPRETATION Use of CGM during pregnancy in patients with type 1 diabetes is associated with improved neonatal outcomes, which are likely to be attributed to reduced exposure to maternal hyperglycaemia. CGM should be offered to all pregnant women with type 1 diabetes using intensive insulin therapy. This study is the first to indicate potential for improvements in non-glycaemic health outcomes from CGM use. FUNDING Juvenile Diabetes Research Foundation, Canadian Clinical Trials Network, and National Institute for Health Research.


Question: PrimPol degrades short DNA replication intermediates on the leading strand during DNA replication.

hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity.. Prim-pol is a recently identified DNA primase-polymerase belonging to the archaeao-eukaryotic primase (AEP) superfamily. Here, we characterize a previously unrecognized prim-pol in human cells, which we designate hPrimpol1 (human primase-polymerase 1). hPrimpol1 possesses primase and DNA polymerase activities in vitro, interacts directly with RPA1 and is recruited to sites of DNA damage and stalled replication forks in an RPA1-dependent manner. Cells depleted of hPrimpol1 display increased spontaneous DNA damage and defects in the restart of stalled replication forks. Both RPA1 binding and the primase activity of hPrimpol1 are required for its cellular function during DNA replication. Our results indicate that hPrimpol1 is a novel factor involved in the response to DNA replication stress.

Insulin Signaling and Dietary Restriction Differentially Influence the Decline of Learning and Memory with Age. Of all the age-related declines, memory loss is one of the most devastating. While conditions that increase longevity have been identified, the effects of these longevity-promoting factors on learning and memory are unknown. Here we show that the C. elegans Insulin/IGF-1 receptor mutant daf-2 improves memory performance early in adulthood and maintains learning ability better with age but, surprisingly, demonstrates no extension in long-term memory with age. By contrast, eat-2 mutants, a model of Dietary Restriction (DR), exhibit impaired long-term memory in young adulthood but maintain this level of memory longer with age. We find that crh-1, the C. elegans homolog of the CREB transcription factor, is required for long-term associative memory, but not for learning or short-term memory. The expression of crh-1 declines with age and differs in the longevity mutants, and CREB expression and activity correlate with memory performance. Our results suggest that specific longevity treatments have acute and long-term effects on cognitive functions that decline with age through their regulation of rate-limiting genes required for learning and memory.


Question: Myelin sheaths play a role in action potential propagation.

Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy. We report a newly developed technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser-scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths. Striking color patterns unique to individual myelinated fibers are generated that facilitate their tracing in dense axonal areas. These patterns highlight nodes of Ranvier and Schmidt-Lanterman incisures and can be used to detect various myelin pathologies. Using SCoRe we carried out chronic brain imaging up to 400 μm deep, capturing de novo myelination of mouse cortical axons in vivo. We also established the feasibility of imaging myelinated axons in the human cerebral cortex. SCoRe adds a powerful component to the evolving toolbox for imaging myelination in living animals and potentially in humans.

T cell memory.. T cell memory induced by prior infection or vaccination provides enhanced protection against subsequent microbial infections. The processes involved in generating and maintaining T cell memory are becoming better understood due to recent technological advances in identifying memory T cells and monitoring their behavior and function in vivo. Memory T cells develop in response to a progressive set of cues-starting with signals from antigen-loaded, activated antigen-presenting cells (APCs) and inflammatory mediators induced by the innate immune response, to the poorly defined subsequent signals triggered as the immune response wanes toward homeostasis. The persistence of the resting memory T cells that eventually develop is regulated by cytokines. This chapter discusses recent findings on how memory T cells develop to confer long-term protective immunity.


Question: Lipopolysaccharides have an inflammation independent effect on kidney barrier function.

Modification of kidney barrier function by the urokinase receptor. Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of αvβ3 integrin. Mice lacking uPAR (Plaur−/−) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active β3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate αvβ3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of αvβ3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

TLR-activated B cells suppress T cell-mediated autoimmunity.. TLR sense microbial infections, and control activation of immune responses. Dendritic cells, macrophages, and B lymphocytes express TLR and the TLR-signaling adaptor protein MyD88. The impact of TLR-activated B cells on T cell-mediated inflammation is unknown. In this study, we have used mice carrying B cell-restricted deficiencies in MyD88 or in distinct TLR to examine the impact of TLR-activated B cells on a T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis (EAE). We demonstrate that TLR-signaling in B cells suppresses inflammatory T cell responses (both Th1 and Th17), and stimulates recovery from EAE. Only certain TLR are required on B cells for resolution of EAE, and these are dispensable for disease initiation, indicating that a category of TLR agonists preferentially triggers a suppressive function in B cells and thereby limits autoimmune disease. The TLR agonists controlling the regulatory function of B cells are provided by components of Mycobacterium tuberculosis present in the adjuvant. Thus, MyD88 signaling in B cells antagonizes MyD88 signaling in other cells, which drives differentiation of Th17 cells and is required for induction of EAE. Altogether, our data indicate that B cells link recognition of microbial products via TLR to suppression of a T cell-mediated autoimmune disease.


Question: The innate immune response struggles to remove small numbers of parasites.

Partitioning regulatory mechanisms of within-host malaria dynamics using the effective propagation number.. Immune clearance and resource limitation (via red blood cell depletion) shape the peaks and troughs of malaria parasitemia, which in turn affect disease severity and transmission. Quantitatively partitioning the relative roles of these effects through time is challenging. Using data from rodent malaria, we estimated the effective propagation number, which reflects the relative importance of contrasting within-host control mechanisms through time and is sensitive to the inoculating parasite dose. Our analysis showed that the capacity of innate responses to restrict initial parasite growth saturates with parasite dose and that experimentally enhanced innate immunity can affect parasite density indirectly via resource depletion. Such a statistical approach offers a tool to improve targeting of drugs or vaccines for human therapy by revealing the dynamics and interactions of within-host regulatory mechanisms.

GAPDH Mediates Nitrosylation of Nuclear Proteins. S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.


Question: Genomic sequences involved in alternative splicing responsible for Hutchinson-Gilford progeria syndrome (HGPS) are abundant in the ''progerinonly'' allele of Lmna knock-in models.

Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome. Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease caused by a spontaneous point mutation in lamin A (encoded by LMNA), one of the major architectural elements of the mammalian cell nucleus. The HGPS mutation activates an aberrant cryptic splice site in LMNA pre-mRNA, leading to synthesis of a truncated lamin A protein and concomitant reduction in wild-type lamin A. Fibroblasts from individuals with HGPS have severe morphological abnormalities in nuclear envelope structure. Here we show that the cellular disease phenotype is reversible in cells from individuals with HGPS. Introduction of wild-type lamin A protein does not rescue the cellular disease symptoms. The mutant LMNA mRNA and lamin A protein can be efficiently eliminated by correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site. Upon splicing correction, HGPS fibroblasts assume normal nuclear morphology, the aberrant nuclear distribution and cellular levels of lamina-associated proteins are rescued, defects in heterochromatin-specific histone modifications are corrected and proper expression of several misregulated genes is reestablished. Our results establish proof of principle for the correction of the premature aging phenotype in individuals with HGPS.

SIRT1 deacetylase in SF1 neurons protects against metabolic imbalance.. Chronic feeding on high-calorie diets causes obesity and type 2 diabetes mellitus (T2DM), illnesses that affect hundreds of millions. Thus, understanding the pathways protecting against diet-induced metabolic imbalance is of paramount medical importance. Here, we show that mice lacking SIRT1 in steroidogenic factor 1 (SF1) neurons are hypersensitive to dietary obesity owing to maladaptive energy expenditure. Also, mutant mice have increased susceptibility to developing dietary T2DM due to insulin resistance in skeletal muscle. Mechanistically, these aberrations arise, in part, from impaired metabolic actions of the neuropeptide orexin-A and the hormone leptin. Conversely, mice overexpressing SIRT1 in SF1 neurons are more resistant to diet-induced obesity and insulin resistance due to increased energy expenditure and enhanced skeletal muscle insulin sensitivity. Our results unveil important protective roles of SIRT1 in SF1 neurons against dietary metabolic imbalance.


Question: Sweet taste receptors on the tongue are deactivated by between 1 and 10 mM glucose.

Detection of sweet and umami taste in the absence of taste receptor T1r3.. The tastes of sugars (sweet) and glutamate (umami) are thought to be detected by T1r receptors expressed in taste cells. Molecular genetics and heterologous expression implicate T1r2 plus T1r3 as a sweet-responsive receptor,and T1r1 plus T1r3,as well as a truncated form of the type 4 metabotropic glutamate receptor (taste-mGluR4),as umami-responsive receptors. Here,we show that mice lacking T1r3 showed no preference for artificial sweeteners and had diminished but not abolished behavioral and nerve responses to sugars and umami compounds. These results indicate that T1r3-independent sweet- and umami-responsive receptors and/or pathways exist in taste cells.

The sacroiliac joint in the spondyloarthropathies.. The term spondyloarthropathy (SpA) describes and defines a group of related inflammatory joint disease that share characteristic clinical features and a unique association with the major histocompatibility complex class I molecule HLA-B27. Five subgroups can be differentiated: ankylosing spondylitis, reactive arthritis, psoriatic arthritis, arthritis associated with inflammatory bowel disease, and undifferentiated SpA. The sacroiliac joints are centrally involved in the SpA, most clearly and pathognomonic in ankylosing spondylitis, in which most patients are affected early in the disease. Overcoming some of the diagnostic difficulties of early sacroiliitis, dynamic magnetic resonance imaging was shown to visualize both acute and chronic changes in the sacroiliac joints. The inflammation in the sacroiliac joints in patients with SpA was recently examined in more detail; using immunohistology and in situ hybridrization, T cells, macrophages, and various cytokines were found in infiltrates. Biopsy specimens were obtained under guided computed tomography, and in the same study, intra-articular corticosteroid treatment was successfully undertaken. Further investigation of such biopsy specimens showed the absence of DNA of reactive arthritis-associated bacteria. The pathogenesis of the SpA and the reason for the tropism for the sacroiliac joints is still obscure. The nature of the relation of the genetic background of SpA to initially triggering bacterial infections remains to be established. In chronic disease, autoimmune mechanisms might be more important.


Question: VgrG (Tssl) protein punctures membranes by forming a spike at the tip of the tube apparatus.

PAAR-repeat proteins sharpen and diversify the Type VI secretion system spike. The bacterial type VI secretion system (T6SS) is a large multicomponent, dynamic macromolecular machine that has an important role in the ecology of many Gram-negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules, allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins sharpen the tip of the T6SS spike complex. We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results indicate a new model of the T6SS organelle in which the VgrG-PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event.

Polyphosphate kinase 1, a central node in the stress response network of Mycobacterium tuberculosis, connects the two-component systems MprAB and SenX3-RegX3 and the extracytoplasmic function sigma factor, sigma E.. Polyphosphate (poly P) metabolism regulates the stress response in mycobacteria. Here we describe the regulatory architecture of a signal transduction system involving the two-component system (TCS) SenX3-RegX3, the extracytoplasmic function sigma factor sigma E (SigE) and the poly P-synthesizing enzyme polyphosphate kinase 1 (PPK1). The ppk1 promoter of Mycobacterium tuberculosis is activated under phosphate starvation. This is attenuated upon deletion of an imperfect palindrome likely representing a binding site for the response regulator RegX3, a component of the two-component system SenX3-RegX3 that responds to phosphate starvation. Binding of phosphorylated RegX3 to this site was confirmed by electrophoretic mobility shift assay. The activity of the ppk1 promoter was abrogated upon deletion of a putative SigE binding site. Pull-down of SigE from M. tuberculosis lysates of phosphate-starved cells with a biotinylated DNA harbouring the SigE binding site confirmed the likely binding of SigE to the ppk1 promoter. In vitro transcription corroborated the involvement of SigE in ppk1 transcription. Finally, the overexpression of RseA (anti-SigE) attenuated ppk1 expression under phosphate starvation, supporting the role of SigE in ppk1 transcription. The regulatory elements identified in ppk1 transcription in this study, combined with our earlier observation that PPK1 is itself capable of regulating sigE expression via the MprAB TCS, suggest the presence of multiple positive-feedback loops in this signalling circuit. In combination with the sequestering effect of RseA, we hypothesize that this architecture could be linked to bistability in the system that, in turn, could be a key element of persistence in M. tuberculosis.


Question: The most prevalent adverse events to Semaglutide are cardiovascular.

Effect of Oral Semaglutide Compared With Placebo and Subcutaneous Semaglutide on Glycemic Control in Patients With Type 2 Diabetes: A Randomized Clinical Trial. Importance Glucagon-like peptide-1 (GLP-1) receptor agonists are effective therapies for the treatment of type 2 diabetes and are all currently available as an injection. Objectives To compare the effects of oral semaglutide with placebo (primary) and open-label subcutaneous semaglutide (secondary) on glycemic control in patients with type 2 diabetes. Design, Setting, and Patients Phase 2, randomized, parallel-group, dosage-finding, 26-week trial with 5-week follow-up at 100 sites (hospital clinics, general practices, and clinical research centers) in 14 countries conducted between December 2013 and December 2014. Of 1106 participants assessed, 632 with type 2 diabetes and insufficient glycemic control using diet and exercise alone or a stable dose of metformin were randomized. Randomization was stratified by metformin use. Interventions Once-daily oral semaglutide of 2.5 mg (n = 70), 5 mg (n = 70), 10 mg (n = 70), 20 mg (n = 70), 40-mg 4-week dose escalation (standard escalation; n = 71), 40-mg 8-week dose escalation (slow escalation; n = 70), 40-mg 2-week dose escalation (fast escalation, n = 70), oral placebo (n = 71; double-blind) or once-weekly subcutaneous semaglutide of 1.0 mg (n = 70) for 26 weeks. Main Outcomes and Measures The primary end point was change in hemoglobing A1c (HbA1c) from baseline to week 26. Secondary end points included change from baseline in body weight and adverse events. Results Baseline characteristics were comparable across treatment groups. Of the 632 randomized patients (mean age, 57.1 years [SD, 10.6]; men, 395 (62.7%); diabetes duration, 6.3 years [SD, 5.2]; body weight, 92.3 kg [SD, 16.8]; BMI, 31.7 [SD, 4.3]), 583 (92%) completed the trial. Mean change in HbA1c level from baseline to week 26 decreased with oral semaglutide (dosage-dependent range, −0.7% to −1.9%) and subcutaneous semaglutide (−1.9%) and placebo (−0.3%); oral semaglutide reductions were significant vs placebo (dosage-dependent estimated treatment difference [ETD] range for oral semaglutide vs placebo, –0.4% to –1.6%; P = .01 for 2.5 mg, <.001 for all other dosages). Reductions in body weight were greater with oral semaglutide (dosage-dependent range, −2.1 kg to −6.9 kg) and subcutaneous semaglutide (−6.4 kg) vs placebo (−1.2 kg), and significant for oral semaglutide dosages of 10 mg or more vs placebo (dosage-dependent ETD range, –0.9 to –5.7 kg; P < .001). Adverse events were reported by 63% to 86% (371 of 490 patients) in the oral semaglutide groups, 81% (56 of 69 patients) in the subcutaneous semaglutide group, and 68% (48 of 71 patients) in the placebo group; mild to moderate gastrointestinal events were most common. Conclusions and Relevance Among patients with type 2 diabetes, oral semaglutide resulted in better glycemic control than placebo over 26 weeks. These findings support phase 3 studies to assess longer-term and clinical outcomes, as well as safety. Trial Registration clinicaltrials.gov Identifier: NCT01923181

Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus. BACKGROUND Influenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes. METHODS Alveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively. RESULTS We found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels. CONCLUSIONS Our results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.


Question: Omnivores produce more trimethylamine N-oxide from dietary I-carnitine than vegans.

Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Intestinal microbiota metabolism of choline and phosphatidylcholine produces trimethylamine (TMA), which is further metabolized to a proatherogenic species, trimethylamine-N-oxide (TMAO). We demonstrate here that metabolism by intestinal microbiota of dietary L-carnitine, a trimethylamine abundant in red meat, also produces TMAO and accelerates atherosclerosis in mice. Omnivorous human subjects produced more TMAO than did vegans or vegetarians following ingestion of L-carnitine through a microbiota-dependent mechanism. The presence of specific bacterial taxa in human feces was associated with both plasma TMAO concentration and dietary status. Plasma L-carnitine levels in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for both prevalent cardiovascular disease (CVD) and incident major adverse cardiac events (myocardial infarction, stroke or death), but only among subjects with concurrently high TMAO levels. Chronic dietary L-carnitine supplementation in mice altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increased atherosclerosis, but this did not occur if intestinal microbiota was concurrently suppressed. In mice with an intact intestinal microbiota, dietary supplementation with TMAO or either carnitine or choline reduced in vivo reverse cholesterol transport. Intestinal microbiota may thus contribute to the well-established link between high levels of red meat consumption and CVD risk.

Effect of glucosamine on pain-related disability in patients with chronic low back pain and degenerative lumbar osteoarthritis: a randomized controlled trial.. CONTEXT Chronic low back pain (LBP) with degenerative lumbar osteoarthritis (OA) is widespread in the adult population. Although glucosamine is increasingly used by patients with chronic LBP, little is known about its effect in this setting. OBJECTIVE To investigate the effect of glucosamine in patients with chronic LBP and degenerative lumbar OA. DESIGN, SETTING, AND PARTICIPANTS A double-blind, randomized, placebo-controlled trial conducted at Oslo University Hospital Outpatient Clinic, Oslo, Norway, with 250 patients older than 25 years of age with chronic LBP (>6 months) and degenerative lumbar OA. INTERVENTIONS Daily intake of 1500 mg of oral glucosamine (n = 125) or placebo (n = 125) for 6 months, with assessment of effect after the 6-month intervention period and at 1 year (6 months postintervention). MAIN OUTCOME MEASURES The primary outcome was pain-related disability measured with the Roland Morris Disability Questionnaire (RMDQ). Secondary outcomes were numerical scores from pain-rating scales of patients at rest and during activity, and the quality-of-life EuroQol-5 Dimensions (EQ-5D) instrument. Data collection occurred during the intervention period at baseline, 6 weeks, 3 and 6 months, and again 6 months following the intervention at 1 year. Group differences were analyzed using linear mixed models analysis. RESULTS At baseline, mean RMDQ scores were 9.2 (95% confidence interval [CI], 8.4-10.0) for glucosamine and 9.7 (95% CI, 8.9-10.5) for the placebo group (P = .37). At 6 months, the mean RMDQ score was the same for the glucosamine and placebo groups (5.0; 95% CI, 4.2-5.8). At 1 year, the mean RMDQ scores were 4.8 (95% CI, 3.9-5.6) for glucosamine and 5.5 (95% CI, 4.7-6.4) for the placebo group. No statistically significant difference in change between groups was found when assessed after the 6-month intervention period and at 1 year: RMDQ (P = .72), LBP at rest (P = .91), LBP during activity (P = .97), and quality-of-life EQ-5D (P = .20). Mild adverse events were reported in 40 patients in the glucosamine group and 46 in the placebo group (P = .48). CONCLUSIONS Among patients with chronic LBP and degenerative lumbar OA, 6-month treatment with oral glucosamine compared with placebo did not result in reduced pain-related disability after the 6-month intervention and after 1-year follow-up. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00404079.


Question: Primary cervical cancer screening with HPV detection has lower longitudinal sensitivity than conventional cytology to detect cervical intraepithelial neoplasia grade 2.

Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial.. BACKGROUND Human papillomavirus (HPV) testing is known to be more sensitive, but less specific than cytology for detecting cervical intraepithelial neoplasia (CIN). We assessed the efficacy of cervical-cancer screening policies that are based on HPV testing. METHODS Between March, 2004, and December, 2004, in two separate recruitment phases, women aged 25-60 years were randomly assigned to conventional cytology or to HPV testing in combination with liquid-based cytology (first phase) or alone (second phase). Randomisation was done by computer in two screening centres and by sequential opening of numbered sealed envelopes in the remaining seven centres. During phase one, women who were HPV-positive and aged 35-60 years were referred to colposcopy, whereas women aged 25-34 years were referred to colposcopy only if cytology was also abnormal or HPV testing was persistently positive. During phase two, women in the HPV group were referred for colposcopy if the HPV test was positive. Two rounds of screening occurred in each phase, and all women had cytology testing only at the second round. The primary endpoint was the detection of grade 2 and 3 CIN, and of invasive cervical cancers during the first and second screening rounds. Analysis was done by intention to screen. This trial is registered, number ISRCTN81678807. FINDINGS In total for both phases, 47,001 women were randomly assigned to the cytology group and 47,369 to HPV testing. 33,851 women from the cytology group and 32,998 from the HPV-testing group had a second round of screening. We also retrieved the histological diagnoses from screening done elsewhere. The detection of invasive cervical cancers was similar for the two groups in the first round of screening (nine in the cytology group vs seven in the HPV group, p=0.62); no cases were detected in the HPV group during round two, compared with nine in the cytology group (p=0.004). Overall, in the two rounds of screening, 18 invasive cancers were detected in the cytology group versus seven in the HPV group (p=0.028). Among women aged 35-60 years, at round one the relative detection (HPV vs cytology) was 2.00 (95% CI 1.44-2.77) for CIN2, 2.08 (1.47-2.95) for CIN3, and 2.03 (1.60-2.57) for CIN2 and 3 together. At round two the relative detection was 0.54 (0.23-1.28) for CIN2, 0.48 (0.21-1.11) for CIN3, and 0.51 (0.28-0.93) for CIN2 and 3 together. Among women aged 25-34 years, there was significant heterogeneity between phases in the relative detection of CIN3. At round one the relative detection was 0.93 (0.52-1.64) in phase one and 3.91 (2.02-7.57) in phase two. At round two the relative detection was 1.34 (0.46-3.84) in phase one and 0.20 (0.04-0.93) in phase two. Pooling both phases, the detection ratio of CIN2 for women aged 25-34 years was 4.09 (2.24-7.48) at round one and 0.64 (0.23-1.27) at round two. INTERPRETATION HPV-based screening is more effective than cytology in preventing invasive cervical cancer, by detecting persistent high-grade lesions earlier and providing a longer low-risk period. However, in younger women, HPV screening leads to over-diagnosis of regressive CIN2. FUNDING European Union, Italian Ministry of Health, Regional Health Administrations of Piemonte, Tuscany, Veneto and Emilia-Romagna, and Public Health Agency of Lazio.

G1 arrest and differentiation can occur independently of Rb family function. The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.


Question: Gastric infection with Helicobacter pylori decreases risk of gastric cancer in humans.

Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation.. OBJECTIVE To investigate the association between gastric cancer and prior infection with Helicobacter pylori. DESIGN Case-control comparison of prevalence of IgG antibodies to H pylori in blood samples collected prospectively, before diagnosis of gastric cancer in the cases. Presence of H pylori antibody (greater than 10 micrograms IgG/ml) determined by enzyme linked immunosorbent assay (ELISA). SUBJECTS 29 men with a subsequent diagnosis of gastric cancer and 116 aged matched controls selected from over 22,000 middle aged men participating in two ongoing cohort studies (the British United Provident Association study and the Caerphilly collaborative heart disease study), who had provided blood samples during 1975-1982. RESULTS 20 of the 29 cases (69%) and 54 of the 116 controls (47%) were positive for H pylori specific antibody. The median specific IgG concentration was significantly higher in the cases than controls (90 micrograms/ml v 3.6 micrograms/ml, p less than 0.01). The estimated odds ratio for the risk of gastric cancer in those with a history of infection with H pylori was 2.77 (95% confidence interval 1.04 to 7.97, 2p = 0.039). CONCLUSIONS H pylori infection may be an important cause of gastric cancer; between 35% and 55% of all cases may be associated with such an infection.

Control of oocyte meiotic maturation and fertilization.. Sexual reproduction depends upon meiosis for the generation of haploid gamete nuclei, which unite after fertilization to form the diploid zygote. The oocytes of most animal species arrest during meiotic prophase, and complete meiosis in response to intercellular signaling in a process called meiotic maturation. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase of meiosis I and is accompanied by nuclear envelope breakdown, rearrangement of the cortical cytoskeleton, and meiotic spindle assembly. Thus, the meiotic maturation process is essential for meiosis and prepares the oocyte for fertilization. In C. elegans, the processes of meiotic maturation, ovulation, and fertilization are temporally coupled: sperm utilize the major sperm protein as a hormone to trigger oocyte meiotic maturation, and in turn, the maturing oocyte signals its own ovulation thereby facilitating fertilization. This chapter highlights recent advances in understanding meiotic maturation signaling and gametic interactions required for fertilization.


Question: PCSK9 inhibitors decrease plasma Lp(a) levels.

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

Reverse engineering of TLX oncogenic transcriptional networks identifies RUNX1 as tumor suppressor in T-ALL. The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL.


Question: Bariatric surgery increases rates of colorectal 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.

Loss of lamin B1 results in prolongation of S phase and decondensation of chromosome territories.. Nuclear lamin B1 (LMNB1) constitutes one of the major structural proteins in the lamina mesh. We silenced the expression of LMNB1 by RNA interference in the colon cancer cell line DLD-1 and showed a dramatic redistribution of H3K27me3 from the periphery to a more homogeneous nuclear dispersion. In addition, we observed telomere attrition and an increased frequency of micronuclei and nuclear blebs. By 3D-FISH analyses, we demonstrated that the volume and surface of chromosome territories were significantly larger in LMNB1-depleted cells, suggesting that LMNB1 is required to maintain chromatin condensation in interphase nuclei. These changes led to a prolonged S phase due to activation of Chk1. Finally, silencing of LMNB1 resulted in extensive changes in alternative splicing of multiple genes and in a higher number of enlarged nuclear speckles. Taken together, our results suggest a mechanistic role of the nuclear lamina in the organization of chromosome territories, maintenance of genome integrity and proper gene splicing.


Question: Lack of FGF21 in mice slows the rate of atherosclerotic plaque formation.

Fibroblast Growth Factor 21 Prevents Atherosclerosis by Suppression of Hepatic Sterol Regulatory Element-Binding Protein-2 and Induction of Adiponectin in Mice. BACKGROUND Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. It acts as a key downstream target of both peroxisome proliferator-activated receptor α and γ, the agonists of which have been used for lipid lowering and insulin sensitization, respectively. However, the role of FGF21 in the cardiovascular system remains elusive. METHODS AND RESULTS The roles of FGF21 in atherosclerosis were investigated by evaluating the impact of FGF21 deficiency and replenishment with recombinant FGF21 in apolipoprotein E(-/-) mice. FGF21 deficiency causes a marked exacerbation of atherosclerotic plaque formation and premature death in apolipoprotein E(-/-) mice, which is accompanied by hypoadiponectinemia and severe hypercholesterolemia. Replenishment of FGF21 protects against atherosclerosis in apolipoprotein E(-/-)mice via 2 independent mechanisms, inducing the adipocyte production of adiponectin, which in turn acts on the blood vessels to inhibit neointima formation and macrophage inflammation, and suppressing the hepatic expression of the transcription factor sterol regulatory element-binding protein-2, thereby leading to reduced cholesterol synthesis and attenuation of hypercholesterolemia. Chronic treatment with adiponectin partially reverses atherosclerosis without obvious effects on hypercholesterolemia in FGF21-deficient apolipoprotein E(-/-) mice. By contrast, the cholesterol-lowering effects of FGF21 are abrogated by hepatic expression of sterol regulatory element-binding protein-2. CONCLUSIONS FGF21 protects against atherosclerosis via fine tuning the multiorgan crosstalk among liver, adipose tissue, and blood vessels.

Full-length transcriptome assembly from RNA-Seq data without a reference genome.. Massively parallel sequencing of cDNA has enabled deep and efficient probing of transcriptomes. Current approaches for transcript reconstruction from such data often rely on aligning reads to a reference genome, and are thus unsuitable for samples with a partial or missing reference genome. Here we present the Trinity method for de novo assembly of full-length transcripts and evaluate it on samples from fission yeast, mouse and whitefly, whose reference genome is not yet available. By efficiently constructing and analyzing sets of de Bruijn graphs, Trinity fully reconstructs a large fraction of transcripts, including alternatively spliced isoforms and transcripts from recently duplicated genes. Compared with other de novo transcriptome assemblers, Trinity recovers more full-length transcripts across a broad range of expression levels, with a sensitivity similar to methods that rely on genome alignments. Our approach provides a unified solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome.


Question: Having a main partner worsens HIV outcomes.

Stable partnership and progression to AIDS or death in HIV infected patients receiving highly active antiretroviral therapy: Swiss HIV cohort study.. OBJECTIVES To explore the association between a stable partnership and clinical outcome in HIV infected patients receiving highly active antiretroviral therapy (HAART). DESIGN Prospective cohort study of adults with HIV (Swiss HIV cohort study). SETTING Seven outpatient clinics throughout Switzerland. PARTICIPANTS The 3736 patients in the cohort who started HAART before 2002 (median age 36 years, 29% female, median follow up 3.6 years). MAIN OUTCOME MEASURES Time to AIDS or death (primary endpoint), death alone, increases in CD4 cell count of at least 50 and 100 above baseline, optimal viral suppression (a viral load below 400 copies/ml), and viral rebound. RESULTS During follow up 2985 (80%) participants reported a stable partnership on at least one occasion. When starting HAART, 52% (545/1042) of participants reported a stable partnership; after five years of follow up 46% (190/412) of participants reported a stable partnership. In an analysis stratified by previous antiretroviral therapy and clinical stage when starting HAART (US Centers for Disease Control and Prevention group A, B, or C), the adjusted hazard ratio for progression to AIDS or death was 0.79 (95% confidence interval 0.63 to 0.98) for participants with a stable partnership compared with those without. Adjusted hazards ratios for other endpoints were 0.59 (0.44 to 0.79) for progression to death, 1.15 (1.06 to 1.24) for an increase in CD4 cells of 100 counts/microl or more, and 1.06 (0.98 to 1.14) for optimal viral suppression. CONCLUSIONS A stable partnership is associated with a slower rate of progression to AIDS or death in HIV infected patients receiving HAART.

Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding. The development and maintenance of an epithelium requires finely balanced rates of growth and cell death. However, the mechanical and biochemical mechanisms that ensure proper feedback control of tissue growth, which when deregulated contribute to tumorigenesis, are poorly understood. Here we use the fly notum as a model system to identify a novel process of crowding-induced cell delamination that balances growth to ensure the development of well-ordered cell packing. In crowded regions of the tissue, a proportion of cells undergo a serial loss of cell–cell junctions and a progressive loss of apical area, before being squeezed out by their neighbours. This path of delamination is recapitulated by a simple computational model of epithelial mechanics, in which stochastic cell loss relieves overcrowding as the system tends towards equilibrium. We show that this process of delamination is mechanistically distinct from apoptosis-mediated cell extrusion and precedes the first signs of cell death. Overall, this analysis reveals a simple mechanism that buffers epithelia against variations in growth. Because live-cell delamination constitutes a mechanistic link between epithelial hyperplasia and cell invasion, this is likely to have important implications for our understanding of the early stages of cancer development.


Question: Immune responses in immune cells are geographically segregated.

Inflammatory signaling in human Tuberculosis granulomas is spatially organized. Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased manner. Using laser-capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas have a pro-inflammatory environment that is characterized by the presence of antimicrobial peptides, reactive oxygen species and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum has a comparatively anti-inflammatory signature. These findings are consistent across a set of six human subjects and in rabbits. Although the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. From the protein and lipid snapshots of human and rabbit lesions analyzed here, we hypothesize that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma.

in. Transglutaminase 2 (TGase 2) is a Ca+2-dependent enzyme that catalyzes both intracellular and extracellular cross-linking reactions by transamidation of specific glutamine residues. TGase 2 is known to be involved in the membrane-mediated events required for glucose-stimulated insulin release from the pancreatic beta cells. Here we show that targeted disruption of TGase 2 impairs glucose-stimulated insulin secretion. TGase 2-/- mice show glucose intolerance after intraperitoneal glucose loading. TGase 2-/- mice manifest a tendency to develop hypoglycemia after administration of exogenous insulin as a consequence of enhanced insulin receptor substrate 2 (IRS-2) phosphorylation. We suggest that the increased peripheral sensitivity to insulin partially compensates for the defective secretion in this animal model. TGase 2-/- mouse phenotype resembles that of the maturity-onset diabetes of young (MODY) patients. In the course of screening for human TGase 2 gene in Italian subjects with the clinical features of MODY, we detected a missense mutation (N333S) in the active site of the enzyme. Collectively, these results identify TGase 2 as a potential candidate gene in type 2 diabetes.


Question: FoxO3a activation in neuronal death is inhibited by reactive oxygen species (ROS).

Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase.. The Sir2 deacetylase modulates organismal life-span in various species. However, the molecular mechanisms by which Sir2 increases longevity are largely unknown. We show that in mammalian cells, the Sir2 homolog SIRT1 appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors, a family of proteins that function as sensors of the insulin signaling pathway and as regulators of organismal longevity. SIRT1 and the FOXO transcription factor FOXO3 formed a complex in cells in response to oxidative stress, and SIRT1 deacetylated FOXO3 in vitro and within cells. SIRT1 had a dual effect on FOXO3 function: SIRT1 increased FOXO3's ability to induce cell cycle arrest and resistance to oxidative stress but inhibited FOXO3's ability to induce cell death. Thus, one way in which members of the Sir2 family of proteins may increase organismal longevity is by tipping FOXO-dependent responses away from apoptosis and toward stress resistance.

Development of a syngeneic mouse model for events related to ovarian cancer.. Mouse ovarian surface epithelial cells (MOSEC) were obtained from virgin, mature mice by mild trypsinization and were repeatedly passaged in vitro. Early passage cells (<20 passages) exhibited a cobblestone morphology and contact inhibition of growth. After approximately 20 passages in vitro, cobblestone morphology and contact inhibition of growth was lost. Tumor forming potential was determined by s.c. and i.p. injection of early and late passage cells into athymic and syngeneic C57BL6 mice. Subcutaneous tumors formed in approximately 4 months and were present only at the injection site. Intraperitoneal injection of late passage MOSEC into athymic and syngeneic mice resulted in growth of tumor implants throughout the abdominal cavity, and production of hemorrhagic ascitic fluid. Early passage MOSEC did not form tumors in vivo. Histopathologic analysis of tumors revealed a highly malignant neoplasm containing both carcinomatous and sarcomatous components. Late passage MOSEC expressed cytokeratin and did not produce ovarian steroids in response to gonadotropin stimulation in vitro. Ten clonal lines were established from late passage MOSEC. Each clone formed multiple peritoneal tumors and ascitic fluid after i.p. injection into C57BL6 mice. Three cell lines examined cytogenetically were polyploid with near-tetraploid modal chromosome numbers. Common clonal chromosome gains and losses included +5, +15, +19 and -X, -3, -4. One cell line had a clonal translocation between chromosomes 15 and 18 and another had a small marker chromosome; common structural abnormalities were not observed. These data describe the development of a mouse model for the study of events related to ovarian cancer in humans. The ability of the MOSEC to form extensive tumors within the peritoneal cavity, similar to those seen in women with Stage III and IV cancer, and the ability of the MOSEC to produce tumors in mice with intact immune systems, makes this model unique for investigations of molecular and immune interactions in ovarian cancer development.


Question: C2 works synergistically with A-769662 to activate dephosphorylated AMPK.

Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding.. The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.

Oncogenic pathway signatures in human cancers as a guide to targeted therapies. The development of an oncogenic state is a complex process involving the accumulation of multiple independent mutations that lead to deregulation of cell signalling pathways central to the control of cell growth and cell fate. The ability to define cancer subtypes, recurrence of disease and response to specific therapies using DNA microarray-based gene expression signatures has been demonstrated in multiple studies. Various studies have also demonstrated the potential for using gene expression profiles for the analysis of oncogenic pathways. Here we show that gene expression signatures can be identified that reflect the activation status of several oncogenic pathways. When evaluated in several large collections of human cancers, these gene expression signatures identify patterns of pathway deregulation in tumours and clinically relevant associations with disease outcomes. Combining signature-based predictions across several pathways identifies coordinated patterns of pathway deregulation that distinguish between specific cancers and tumour subtypes. Clustering tumours based on pathway signatures further defines prognosis in respective patient subsets, demonstrating that patterns of oncogenic pathway deregulation underlie the development of the oncogenic phenotype and reflect the biology and outcome of specific cancers. Predictions of pathway deregulation in cancer cell lines are also shown to predict the sensitivity to therapeutic agents that target components of the pathway. Linking pathway deregulation with sensitivity to therapeutics that target components of the pathway provides an opportunity to make use of these oncogenic pathway signatures to guide the use of targeted therapeutics.


Question: Differentiation of progenitor cells to myeloid cells is skewed upon activation of insulin signaling.

Direct sensing of systemic and nutritional signals by hematopoietic progenitors in Drosophila. The Drosophila lymph gland is a haematopoietic organ in which progenitor cells, which are most akin to the common myeloid progenitor in mammals, proliferate and differentiate into three types of mature cell--plasmatocytes, crystal cells and lamellocytes--the functions of which are reminiscent of mammalian myeloid cells. During the first and early second instars of larval development, the lymph gland contains only progenitors, whereas in the third instar, a medial region of the primary lobe of the lymph gland called the medullary zone contains these progenitors, and maturing blood cells are found juxtaposed in a peripheral region designated the cortical zone. A third group of cells referred to as the posterior signalling centre functions as a haematopoietic niche. Similarly to mammalian myeloid cells, Drosophila blood cells respond to multiple stresses including hypoxia, infection and oxidative stress. However, how systemic signals are sensed by myeloid progenitors to regulate cell-fate determination has not been well described. Here, we show that the haematopoietic progenitors of Drosophila are direct targets of systemic (insulin) and nutritional (essential amino acid) signals, and that these systemic signals maintain the progenitors by promoting Wingless (WNT in mammals) signalling. We expect that this study will promote investigation of such possible direct signal sensing mechanisms by mammalian myeloid progenitors.

Methionine restriction decreases visceral fat mass and preserves insulin action in aging male Fischer 344 rats independent of energy restriction.. Reduced dietary methionine intake (0.17% methionine, MR) and calorie restriction (CR) prolong lifespan in male Fischer 344 rats. Although the mechanisms are unclear, both regimens feature lower body weight and reductions in adiposity. Reduced fat deposition in CR is linked to preservation of insulin responsiveness in older animals. These studies examine the relationship between insulin responsiveness and visceral fat in MR and test whether, despite lower food intake observed in MR animals, decreased visceral fat accretion and preservation of insulin sensitivity is not secondary to CR. Accordingly, rats pair fed (pf) control diet (0.86% methinone, CF) to match the food intake of MR for 80 weeks exhibit insulin, glucose, and leptin levels similar to control-fed animals and comparable amounts of visceral fat. Conversely, MR rats show significantly reduced visceral fat compared to CF and PF with concomitant decreases in basal insulin, glucose, and leptin, and increased adiponectin and triiodothyronine. Daily energy expenditure in MR animals significantly exceeds that of both PF and CF. In a separate cohort, insulin responses of older MR animals as measured by oral glucose challenge are similar to young animals. Longitudinal assessments of MR and CF through 112 weeks of age reveal that MR prevents age-associated increases in serum lipids. By 16 weeks, MR animals show a 40% reduction in insulin-like growth factor-1 (IGF-1) that is sustained throughout life; CF IGF-1 levels decline much later, beginning at 112 weeks. Collectively, the results indicate that MR reduces visceral fat and preserves insulin activity in aging rats independent of energy restriction.


Question: Mice lacking Sirt1 in Sf1-expressing neurons have increased susceptibility to diet-induced obesity and insulin resistance.

SIRT1 deacetylase in SF1 neurons protects against metabolic imbalance.. Chronic feeding on high-calorie diets causes obesity and type 2 diabetes mellitus (T2DM), illnesses that affect hundreds of millions. Thus, understanding the pathways protecting against diet-induced metabolic imbalance is of paramount medical importance. Here, we show that mice lacking SIRT1 in steroidogenic factor 1 (SF1) neurons are hypersensitive to dietary obesity owing to maladaptive energy expenditure. Also, mutant mice have increased susceptibility to developing dietary T2DM due to insulin resistance in skeletal muscle. Mechanistically, these aberrations arise, in part, from impaired metabolic actions of the neuropeptide orexin-A and the hormone leptin. Conversely, mice overexpressing SIRT1 in SF1 neurons are more resistant to diet-induced obesity and insulin resistance due to increased energy expenditure and enhanced skeletal muscle insulin sensitivity. Our results unveil important protective roles of SIRT1 in SF1 neurons against dietary metabolic imbalance.

Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing.. Immune sensing of DNA is critical for antiviral immunity but can also trigger autoimmune diseases such as lupus erythematosus (LE). Here we have provided evidence for the involvement of a damage-associated DNA modification in the detection of cytosolic DNA. The oxidized base 8-hydroxyguanosine (8-OHG), a marker of oxidative damage in DNA, potentiated cytosolic immune recognition by decreasing its susceptibility to 3' repair exonuclease 1 (TREX1)-mediated degradation. Oxidizative modifications arose physiologically in pathogen DNA during lysosomal reactive oxygen species (ROS) exposure, as well as in neutrophil extracellular trap (NET) DNA during the oxidative burst. 8-OHG was also abundant in UV-exposed skin lesions of LE patients and colocalized with type I interferon (IFN). Injection of oxidized DNA in the skin of lupus-prone mice induced lesions that closely matched respective lesions in patients. Thus, oxidized DNA represents a prototypic damage-associated molecular pattern (DAMP) with important implications for infection, sterile inflammation, and autoimmunity.


Question: Environmental factors can influence the development of breast cancer.

Gene–environment interactions in 7610 women with breast cancer: prospective evidence from the Million Women Study. BACKGROUND Information is scarce about the combined effects on breast cancer incidence of low-penetrance genetic susceptibility polymorphisms and environmental factors (reproductive, behavioural, and anthropometric risk factors for breast cancer). To test for evidence of gene-environment interactions, we compared genotypic relative risks for breast cancer across the other risk factors in a large UK prospective study. METHODS We tested gene-environment interactions in 7610 women who developed breast cancer and 10 196 controls without the disease, studying the effects of 12 polymorphisms (FGFR2-rs2981582, TNRC9-rs3803662, 2q35-rs13387042, MAP3K1-rs889312, 8q24-rs13281615, 2p-rs4666451, 5p12-rs981782, CASP8-rs1045485, LSP1-rs3817198, 5q-rs30099, TGFB1-rs1982073, and ATM-rs1800054) in relation to prospectively collected information about ten established environmental risk factors (age at menarche, parity, age at first birth, breastfeeding, menopausal status, age at menopause, use of hormone replacement therapy, body-mass index, height, and alcohol consumption). FINDINGS After allowance for multiple testing none of the 120 comparisons yielded significant evidence of a gene-environment interaction. By contrast with previous suggestions, there was little evidence that the genotypic relative risks were affected by use of hormone replacement therapy, either overall or for oestrogen-receptor-positive disease. Only one of the 12 polymorphisms was correlated with any of the ten other risk factors: carriers of the high-risk C allele of MAP3K1-rs889312 were significantly shorter than non-carriers (mean height 162.4 cm [95% CI 162.1-162.7] vs 163.1 cm [162.9-163.2]; p=0.01 after allowance for multiple testing). INTERPRETATION Risks of breast cancer associated with low-penetrance susceptibility polymorphisms do not vary significantly with these ten established environmental risk factors. FUNDING Cancer Research UK and the UK Medical Research Council.

Small molecule screening in human induced pluripotent stem cell-derived terminal cell types.. A need for better clinical outcomes has heightened interest in the use of physiologically relevant human cells in the drug discovery process. Patient-specific human induced pluripotent stem cells may offer a relevant, robust, scalable, and cost-effective model of human disease physiology. Small molecule high throughput screening in human induced pluripotent stem cell-derived cells with the intent of identifying novel therapeutic compounds is starting to influence the drug discovery process; however, the use of these cells presents many high throughput screening development challenges. This technology has the potential to transform the way drug discovery is performed.


Question: Commelina yellow mottle virus (ComYMV) has three typical badnavirus ORFs, capable of encoding proteins 23, 15, and 216 kD.

Properties of Commelina yellow mottle virus's complete DNA sequence, genomic discontinuities and transcript suggest that it is a pararetrovirus.. The non-enveloped bacilliform viruses are the second group of plant viruses known to possess a genome consisting of circular double-stranded DNA. We have characterized the viral transcript and determined the complete sequence of the genome of Commelina mellow mottle virus (CoYMV), a member of this group. Analysis of the viral transcript indicates that the virus encodes a single terminally-redundant genome-length plus 120 nucleotide transcript. A fraction of the transcripts is polyadenylated, although the majority of the transcript is not polyadenylated. Analysis of the genome sequence indicates that the genome is 7489 bp in size and that the transcribed strand contains three open reading frames capable of encoding proteins of 23, 15 and 216 kd. The function of the 25 and 15 kd proteins is unknown. Similarities between the 216 kd polypeptide and the cauliflower mosaic virus coat protein and protease/reverse transcriptase polyprotein suggest that the 216 kd polypeptide is a polyprotein that is proteolytically processed to yield the virion coat protein, a protease, and replicase (reverse transcriptase and ribonuclease H). Each strand of the CoYMV genome is interrupted by site-specific discontinuities. The locations of the 5'-ends of these discontinuities, and the presence and location of a region on the CoYMV transcript capable of annealing with the 3'-end of cytosolic initiator methionine tRNA are consistent with replication by reverse transcription. We have demonstrated that a construct containing 1.3 CoYMV genomes is infective when introduced into Commelina diffusa, the host for CoYMV, using Agrobacterium-mediated infection.

The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP(3), GP(4) and GP(5) envelope glycoproteins, only the M/GP(5) glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP(5). These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines.


Question: ART has no effect on the infectiveness of HIV-positive people.

HIV Treatment as Prevention: Systematic Comparison of Mathematical Models of the Potential Impact of Antiretroviral Therapy on HIV Incidence in South Africa. BACKGROUND Many mathematical models have investigated the impact of expanding access to antiretroviral therapy (ART) on new HIV infections. Comparing results and conclusions across models is challenging because models have addressed slightly different questions and have reported different outcome metrics. This study compares the predictions of several mathematical models simulating the same ART intervention programmes to determine the extent to which models agree about the epidemiological impact of expanded ART. METHODS AND FINDINGS Twelve independent mathematical models evaluated a set of standardised ART intervention scenarios in South Africa and reported a common set of outputs. Intervention scenarios systematically varied the CD4 count threshold for treatment eligibility, access to treatment, and programme retention. For a scenario in which 80% of HIV-infected individuals start treatment on average 1 y after their CD4 count drops below 350 cells/µl and 85% remain on treatment after 3 y, the models projected that HIV incidence would be 35% to 54% lower 8 y after the introduction of ART, compared to a counterfactual scenario in which there is no ART. More variation existed in the estimated long-term (38 y) reductions in incidence. The impact of optimistic interventions including immediate ART initiation varied widely across models, maintaining substantial uncertainty about the theoretical prospect for elimination of HIV from the population using ART alone over the next four decades. The number of person-years of ART per infection averted over 8 y ranged between 5.8 and 18.7. Considering the actual scale-up of ART in South Africa, seven models estimated that current HIV incidence is 17% to 32% lower than it would have been in the absence of ART. Differences between model assumptions about CD4 decline and HIV transmissibility over the course of infection explained only a modest amount of the variation in model results. CONCLUSIONS Mathematical models evaluating the impact of ART vary substantially in structure, complexity, and parameter choices, but all suggest that ART, at high levels of access and with high adherence, has the potential to substantially reduce new HIV infections. There was broad agreement regarding the short-term epidemiologic impact of ambitious treatment scale-up, but more variation in longer term projections and in the efficiency with which treatment can reduce new infections. Differences between model predictions could not be explained by differences in model structure or parameterization that were hypothesized to affect intervention impact.

Effect of hydroxyethyl starch on bleeding after cardiopulmonary bypass: a meta-analysis of randomized trials.. OBJECTIVE The effects of hydroxyethyl starch on bleeding after cardiopulmonary bypass were determined. METHODS A meta-analysis was performed of postoperative blood loss in randomized clinical trials of hydroxyethyl starch versus albumin for fluid management in adult cardiopulmonary bypass surgery. Impacts of hydroxyethyl starch molecular weight and molar substitution were assessed. Randomized trials directly comparing different hydroxyethyl starch solutions were also included. RESULTS Eighteen trials with 970 total patients were included. Compared with albumin, hydroxyethyl starch increased postoperative blood loss by 33.3% of a pooled SD (95% confidence interval, 18.2%-48.3%; P < .001). Risk of reoperation for bleeding was more than doubled by hydroxyethyl starch (relative risk, 2.24; 95% confidence interval, 1.14-4.40; P = .020). Hydroxyethyl starch increased transfusion of red blood cells by 28.4% of a pooled SD (95% confidence interval, 12.2%-44.6%; P < .001), of fresh-frozen plasma by 30.6% (95% confidence interval, 8.0%-53.1%; P = .008), and of platelets by 29.8% (95% confidence interval, 3.4%-56.2%; P = .027). None of these effects differed significantly between hydroxyethyl starch 450/0.7 and 200/0.5. Insufficient data were available for hydroxyethyl starch 130/0.4 versus albumin; however, no significant differences were detected in head-to-head comparisons of hydroxyethyl starch 130/0.4 with 200/0.5. Albumin improved hemodynamics. There were no differences in fluid balance, ventilator time, intensive care unit stay, or mortality. CONCLUSIONS Hydroxyethyl starch increased blood loss, reoperation for bleeding, and blood product transfusion after cardiopulmonary bypass. There was no evidence that these risks could be mitigated by lower molecular weight and substitution.


Question: Induced hepatic stem cells possess the potential to differentiate into hepatocytic cells.

Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.. Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1β and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.

DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.. Increased levels of DNA fragments have frequently been found in the blood plasma of cancer patients. Published data suggest that only a fraction of the DNA in blood plasma is derived from cancer cells. However, it is not known how much of the circulating DNA is from cancer or from noncancer cells. By quantitative methylation-specific PCR of the promoter region of the CDKN2A tumor suppressor gene, we were able to quantify the fraction of plasma DNA derived from tumor cells. In the plasma samples of 30 unselected cancer patients, we detected quantities of tumor DNA from only 3% to as much as 93% of total circulating DNA. We investigated possible origins of nontumor DNA in the plasma and demonstrate here a contribution of T-cell DNA in a few cases only. To investigate the possibility that plasma DNA originates from apoptotic or necrotic cells, we performed studies with apoptotic (staurosporine) and necrotic (staurosporine plus oligomycin) cells in vitro and with mice after induction of apoptotic (anti-CD95) or necrotic (acetaminophen) liver injury. Increasing amounts of DNA were found to be released in the supernatants of cells and in the blood plasma samples of treated animals. A clear discrimination of apoptotic and necrotic plasma DNA was possible by gel electrophoresis. The same characteristic patterns of DNA fragments could be identified in plasma derived from different cancer patients. The data are consistent with the possibility that apoptotic and necrotic cells are a major source for plasma DNA in cancer patients.


Question: The RANK-RANK-Ligand (RANKL) pathway is involved in the development of Aire-expressing medullary thymic epithelial cells (mTECs).

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.

Resistance to gefitinib in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3'-kinase/Akt pathway signaling.. PURPOSE Tyrosine kinase (TK) inhibitors are emerging as a promising new approach to the treatment of HER overexpressing tumors, however optimal use of these agents awaits further definition of the downstream signaling pathways that mediate their effects. We reported previously that both EGFR- and Her2-overexpressing tumors are sensitive to the new EGFR-selective TK inhibitor gefitinib (ZD1839, "Iressa"), and sensitivity to this agent correlated with its ability to down-regulate Akt. However, EGFR-overexpressing MDA-468 cells, which lack PTEN function, are resistant to ZD1839, and ZD1839 is unable to down-regulate Akt activity in these cells. EXPERIMENTAL DESIGN To study the role of PTEN function, we generated MDA468 cells with tet-inducible PTEN expression. RESULTS We show here that the resistance of MDA-468 cells to ZD1839 is attributable to EGFR-independent constitutive Akt activation caused by loss of PTEN function in these cells. Reconstitution of PTEN function through tet-inducible expression restores ZD1839 sensitivity to these cells and reestablishes EGFR-stimulated Akt signaling. Although restoration of PTEN function to tumors is difficult to implement clinically, much of the effects of PTEN loss are attributable to overactive PI3K/Akt pathway signaling, and this overactivity can be modulated by pharmacologic approaches. We show here that pharmacologic down-regulation of constitutive PI3K/Akt pathway signaling using the PI3K inhibitor LY294002 similarly restores EGFR-stimulated Akt signaling and sensitizes MDA-468 cells to ZD1839. CONCLUSIONS Sensitivity to ZD1839 requires intact growth factor receptor-stimulated Akt signaling activity. PTEN loss leads to uncoupling of this signaling pathway and results in ZD1839 resistance, which can be reversed with reintroduction of PTEN or pharmacologic down-regulation of constitutive PI3K/Akt pathway activity. These data have important predictive and therapeutic clinical implications.


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

Prostaglandin E2 promotes intestinal tumor growth via DNA methylation. Although aberrant DNA methylation is considered to be one of the key ways by which tumor-suppressor and DNA-repair genes are silenced during tumor initiation and progression, the mechanisms underlying DNA methylation alterations in cancer remain unclear. Here we show that prostaglandin E(2) (PGE(2)) silences certain tumor-suppressor and DNA-repair genes through DNA methylation to promote tumor growth. These findings uncover a previously unrecognized role for PGE(2) in the promotion of tumor progression.

Tailoring Nutritional Advice for Mexicans Based on Prevalence Profiles of Diet-Related Adaptive Gene Polymorphisms. Diet-related adaptive gene (DRAG) polymorphisms identified in specific populations are associated with chronic disorders in carriers of the adaptive alleles due to changes in dietary and lifestyle patterns in recent times. Mexico's population is comprised of Amerindians (AM) and Mestizos who have variable AM, European (EUR) and African genetic ancestry and an increased risk of nutrition-related chronic diseases. Nutritional advice based on the Mexican genome and the traditional food culture is needed to develop preventive and therapeutic strategies. Therefore, we aimed to provide a prevalence profile of several DRAG polymorphisms in the Mexican population, including Central West (CW) Mexico subpopulations. Geographic heat maps were built using ArcGIS10 (Esri, Redlands, CA, USA) software, based on the published data of the MTHFR C677T (rs1801133), ABCA1 Arg230Cys (rs9282541), APOE T388C (rs429358)/C526T (rs7412), LCT C-13910T (rs4988235) polymorphisms and AMY1 copy number variation (CNV). Also, new data obtained by allelic discrimination-real-time polymerase chain reaction (RT-PCR) assays for the MTHFR, ABCA1, and APOE polymorphisms as well as the AMY1 CNV in the CW Mexico subpopulations with different proportions of AM and EUR ancestry were included. In the CW region, the highest frequency of the MTHFR 677T, ABCA1 230C and APOE ε4 adaptive alleles was observed in the AM groups, followed by Mestizos with intermediate AM ancestry. The LCT-13910T allele frequency was highest in Mestizos-EUR but extremely low in AM, while the AMY1 diploid copy number was 6.82 ± 3.3 copies. Overall, the heat maps showed a heterogeneous distribution of the DRAG polymorphisms, in which the AM groups revealed the highest frequencies of the adaptive alleles followed by Mestizos. Given these genetic differences, genome-based nutritional advice should be tailored in a regionalized and individualized manner according to the available foods and Mexican traditional food culture that may lead to a healthier dietary pattern.


Question: L3MBTL2 binds to H2AK119Ub marked chromatin to form a PRC1 complex.

The polycomb group protein L3mbtl2 assembles an atypical PRC1-family complex that is essential in pluripotent stem cells and early development.. L3mbtl2 has been implicated in transcriptional repression and chromatin compaction but its biological function has not been defined. Here we show that disruption of L3mbtl2 results in embryonic lethality with failure of gastrulation. This correlates with compromised proliferation and abnormal differentiation of L3mbtl2(-/-) embryonic stem (ES) cells. L3mbtl2 regulates genes by recruiting a Polycomb Repressive Complex1 (PRC1)-related complex, resembling the previously described E2F6-complex, and including G9A, Hdac1, and Ring1b. The presence of L3mbtl2 at target genes is associated with H3K9 dimethylation, low histone acetylation, and H2AK119 ubiquitination, but the latter is neither dependent on L3mbtl2 nor sufficient for repression. Genome-wide studies revealed that the L3mbtl2-dependent complex predominantly regulates genes not bound by canonical PRC1 and PRC2. However, some developmental regulators are repressed by the combined activity of all three complexes. Together, we have uncovered a highly selective, essential role for an atypical PRC1-family complex in ES cells and early development.

Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74.. The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding protein (CREB) and that both pathways are modulated by their respective endogenous receptor ligands. By addition of specific pathway modulators against the G protein subunit Galphai, phospholipase C, protein kinase C, calcineurin, p38 MAP kinase, and MEK1, we find that the constitutive and ligand-dependent inductions are mediated by multiple yet similar pathways in both receptors. The NFAT and CREB transcription factors and their upstream activators are known inducers of host and virally encoded genes. We propose that the activity of these virally encoded chemokine receptors coordinates host and potentially viral gene expression similarly. As ORF74 is a known inducer of neoplasia, these findings may have important implications for cytomegalovirus-associated pathogenicity.


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

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

The stimulatory potency of T cell antigens is influenced by the formation of the immunological synapse.. T cell activation is predicated on the interaction between the T cell receptor and peptide-major histocompatibility (pMHC) ligands. The factors that determine the stimulatory potency of a pMHC molecule remain unclear. We describe results showing that a peptide exhibiting many hallmarks of a weak agonist stimulates T cells to proliferate more than the wild-type agonist ligand. An in silico approach suggested that the inability to form the central supramolecular activation cluster (cSMAC) could underlie the increased proliferation. This conclusion was supported by experiments that showed that enhancing cSMAC formation reduced stimulatory capacity of the weak peptide. Our studies highlight the fact that a complex interplay of factors determines the quality of a T cell antigen.


Question: Alteration of origin firing causes changes in termination zones of Okazaki fragments.

Quantitative, genome-wide analysis of eukaryotic replication initiation and termination.. Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.

BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. BCL-2 was isolated from the t(14;18) chromosomal breakpoint in follicular B-cell lymphoma1–3. Bcl-2 has the unique oncogenic role of extending cell survival by inhibiting a variety of apoptotic deaths4–13. An emerging family of Bcl-2 -related proteins share two highly conserved regions14–20 referred to here as Bcl-2 homology 1 and 2 (BH1 and BH2) domains (Fig. 1). This includes Bax which heterodimerizes with Bcl-2 and when overexpressed counteracts Bcl-214. We report here that site-specific mutagenesis of Bcl-2 establishes the two domains as novel dimerization motifs. Substitu-tion of Gly 145 in BHl domain or Trp 188 in BH2 domain completely abrogated Bcl-2's death-repressor activity in inter-leukin-3 deprivation, γ-irradiation and glucocorticoid-induced apoptosis. Mutations that affected Bcl-2's function also disrupted its heterodimerization with Bax, yet still permitted Bcl-2 homo-dimerization. These results establish a functional role for the BH1 and BH2 domains and suggest Bcl-2 exerts its action through heterodimerization with Bax.