title: The Evolution of Dominance in Sporophytic Self-Incompatibility Systems. II. Mate Availability and Recombination

text: Sporophytic self-incompatibility (SSI) is a self-pollen recognition system that enforces outcrossing in plants. Recognition in SSI systems is typically controlled by a complex locus (5-locus) with separate genes that determine pollen and stigma specificity. Experimental studies show that S-alleles can be dominant, recessive, or codominant, and that the dominance level of a given S-allele can depend upon whether pollen or stigma specificity is examined. Here and in the companion paper by Llaurens and colleagues, the evolution of dominance in single-locus SSI is explored using numerical models and simulation. Particular attention is directed at factors that can cause S-allele dominance to differ in pollen versus stigma. The effect of recombination between the S-locus and modifier locus is also examined. The models predict that limitation in the number of compatible mates is required for the evolution of S-allele dominance in the stigma but not in the pollen. Tight linkage between the S-locus and modifier promotes the evolution of S-allele dominance hierarchies. Model results are interpreted with respect to published information on the molecular basis of dominance in SSI systems, and reported S-allele dominance relationships in a variety of species. These studies show that dominant S-alleles are more common in the pollen than in the stigma, a pattern that when interpreted in light of model predictions, suggests that mate limitation may be relatively infrequent in natural populations with SSI.


title: Neurological outcomes of animal models of uterine artery ligation and relevance to human intrauterine growth restriction: a systematic review

text:
AIM This review explores the molecular, neurological, and behavioural outcomes in animal models of uterine artery ligation. We analyse the relevance of this type of model to the pathological and functional phenotypes that are consistent with cerebral palsy and its developmental comorbidities in humans.
 METHOD A literature search of the PubMed database was conducted for research using the uterine artery ligation model published between 1990 and 2013. From the studies included, any relevant neuroanatomical and behavioural deficits were then summarized from each document and used for further analysis.
 RESULTS There were 25 papers that met the criteria included for review, and several outcomes were summarized from the results of these papers. Fetuses with growth restriction demonstrated a gradient of reduced body weight with a relative sparing of brain mass. There was a significant reduction in the size of the somatosensory cortex, hippocampus, and corpus callosum. The motor cortex appeared to be spared of identifiable deficits. Apoptotic proteins were upregulated, while those important to neuronal survival, growth, and differentiation were downregulated. Neuronal apoptosis and astrogliosis occurred diffusely throughout the brain regions. White matter injury involved oligodendrocyte precursor maturation arrest, hypomyelination, and an aberrant organization of existing myelin. Animals with growth restriction demonstrated deficits in gait, memory, object recognition, and spatial processing.
 INTERPRETATION This review concludes that neuronal death, white matter injury, motor abnormalities, and cognitive deficits are important outcomes of uterine artery ligation in animal models. Therefore, this is a clinically relevant type of model, as these findings resemble deficits in human cerebral palsy.


title: The fidelity of the ligation step determines how ends are resolved during Nonhomologous end joining

text: Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures; however, it is unclear how the steps employed for resolution are determined. We sought to address this question by analysing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85 to 6%. End-processing by nucleases and polymerases is increased to compensate, although paths with the fewest number of steps to generate a substrate suitable for ligation are favoured. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors.


title: Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5α/β

text: LL5beta has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)-bound microtubule plus ends to the cell cortex. In this study, we show that LL5beta and its homologue LL5alpha (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins alpha3beta1 and alpha6beta4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin alpha3 at the basal cell cortex. Activation of integrin alpha3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin-integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.


title: DNA topoisomerases: structure, function, and mechanism.

text: DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. In addition, these enzymes fine-tune the steady-state level of DNA supercoiling both to facilitate protein interactions with the DNA and to prevent excessive supercoiling that is deleterious. In recent years, the crystal structures of a number of topoisomerase fragments, representing nearly all the known classes of enzymes, have been solved. These structures provide remarkable insights into the mechanisms of these enzymes and complement previous conclusions based on biochemical analyses. Surprisingly, despite little or no sequence homology, both type IA and type IIA topoisomerases from prokaryotes and the type IIA enzymes from eukaryotes share structural folds that appear to reflect functional motifs within critical regions of the enzymes. The type IB enzymes are structurally distinct from all other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. The structural themes common to all topoisomerases include hinged clamps that open and close to bind DNA, the presence of DNA binding cavities for temporary storage of DNA segments, and the coupling of protein conformational changes to DNA rotation or DNA movement. For the type II topoisomerases, the binding and hydrolysis of ATP further modulate conformational changes in the enzymes to effect changes in DNA topology.


title: Pilot testing of dipsticks as point-of-care assays for rapid diagnosis of poor-quality artemisinin drugs in endemic settings

text:
BACKGROUND Good-quality artemisinin drugs are essential for malaria treatment, but increasing prevalence of poor-quality artemisinin drugs in many endemic countries hinders effective management of malaria cases.
 METHODS To develop a point-of-care assay for rapid identification of counterfeit and substandard artemisinin drugs for resource-limited areas, we used specific monoclonal antibodies against artesunate and artemether, and developed prototypes of lateral flow dipstick assays. In this pilot test, we evaluated the feasibility of these dipsticks under different endemic settings and their performance in the hands of untrained personnel.
 RESULTS The results showed that the dipstick tests can be successfully performed by different investigators with the included instruction sheet. None of the artemether and artesunate drugs collected from public pharmacies in different endemic countries failed the test.
 CONCLUSION It is possible that the simple dipstick assays, with future optimization of test conditions and sensitivity, can be used as a qualitative and semi-quantitative assay for rapid screening of counterfeit artemisinin drugs in endemic settings.


title: Increased Microerythrocyte Count in Homozygous α+-Thalassaemia Contributes to Protection against Severe Malarial Anaemia

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


title: Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

text: We have systematically made a set of precisely defined, single‐gene deletions of all nonessential genes in Escherichia coli K‐12. Open‐reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one‐step method for inactivation of chromosomal genes and primers designed to create in‐frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high‐throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection’—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome‐wide testing of mutational effects in a common strain background, E . coli K‐12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist‐nara.ac.jp/).


title: Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses

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


title: Interleukin-6 receptor pathways in coronary heart disease: a collaborative meta-analysis of 82 studies

text:
BACKGROUND Persistent inflammation has been proposed to contribute to various stages in the pathogenesis of cardiovascular disease. Interleukin-6 receptor (IL6R) signalling propagates downstream inflammation cascades. To assess whether this pathway is causally relevant to coronary heart disease, we studied a functional genetic variant known to affect IL6R signalling.
 METHODS In a collaborative meta-analysis, we studied Asp358Ala (rs2228145) in IL6R in relation to a panel of conventional risk factors and inflammation biomarkers in 125,222 participants. We also compared the frequency of Asp358Ala in 51,441 patients with coronary heart disease and in 136,226 controls. To gain insight into possible mechanisms, we assessed Asp358Ala in relation to localised gene expression and to postlipopolysaccharide stimulation of interleukin 6.
 FINDINGS The minor allele frequency of Asp358Ala was 39%. Asp358Ala was not associated with lipid concentrations, blood pressure, adiposity, dysglycaemia, or smoking (p value for association per minor allele ≥0·04 for each). By contrast, for every copy of 358Ala inherited, mean concentration of IL6R increased by 34·3% (95% CI 30·4-38·2) and of interleukin 6 by 14·6% (10·7-18·4), and mean concentration of C-reactive protein was reduced by 7·5% (5·9-9·1) and of fibrinogen by 1·0% (0·7-1·3). For every copy of 358Ala inherited, risk of coronary heart disease was reduced by 3·4% (1·8-5·0). Asp358Ala was not related to IL6R mRNA levels or interleukin-6 production in monocytes.
 INTERPRETATION Large-scale human genetic and biomarker data are consistent with a causal association between IL6R-related pathways and coronary heart disease.
 FUNDING British Heart Foundation; UK Medical Research Council; UK National Institute of Health Research, Cambridge Biomedical Research Centre; BUPA Foundation.


title: Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

text: Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.


title: Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum, and blood cells of hepatocellular carcinoma patients.

text:
PURPOSE AND EXPERIMENTAL DESIGN Using real-time quantitative methylation-specific PCR (RTQ-MSP), we quantified methylated p16INK4a sequences and determined the fractional concentrations of circulating tumor DNA in plasma, serum, and peripheral blood cells collected preoperatively, intraoperatively, and postoperatively from 49 patients with hepatocellular carcinoma (HCC).
 RESULTS RTQ-MSP was sufficiently sensitive to detect down to 10 genome-equivalents of methylated p16INK4a sequences. Quantitative MSP data were expressed in terms of the methylation index, which was the percentage of bisulfite converted unmethylated and methylated p16INK4a sequences that consisted of methylated p16INK4a sequences. Quantities of methylated p16INK4a sequences were detected in peripheral circulation of 80% (23 of 29) of HCC patients. No significant difference was seen in the detectability and concentrations of methylated p16INK4a sequences (range: 10-4046 genome-equivalents/ml) between preoperative plasma and serum samples from HCC patients. Preoperatively, the p16INK4a methylation indices ranged from 0.2 to 100% and from 0.012 to 0.075% in the patients' plasma and buffy coat samples, respectively. After surgical resection, the median p16INK4a methylation indices in plasma and buffy coat concordantly decreased 12- and 15-fold, respectively. These results demonstrated the clinical usefulness and effectiveness of peripheral blood RTQ-MSP for detecting and monitoring HCC after treatment. Furthermore, none of the intraoperative plasma samples and only two of the intraoperative buffy coat samples were p16INK4a methylation positive.
 CONCLUSIONS Quantification of epigenetic changes in peripheral blood by RTQ-MSP is useful for the detection and monitoring of HCC.


title: Dynamic control of excitatory synapse development by a Rac1 GEF/GAP regulatory complex.

text: The small GTPase Rac1 orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. Here, we identify a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases.


title: 4-1BB Costimulation Ameliorates T Cell Exhaustion Induced by Tonic Signaling of Chimeric Antigen Receptors

text: Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials.


title: Ultrafast Evolution and Loss of CRISPRs Following a Host Shift in a Novel Wildlife Pathogen, Mycoplasma gallisepticum

text: Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ∼2% of ancestral poultry strains and a nucleotide substitution rate of 0.8-1.2×10(-5) per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ∼50% of the CRISPR repertoire founding (1994-95) strains and have lost the CRISPR-associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs.


title: Inducing Tumor Immunity through the Selective Engagement of Activating Fcγ Receptors on Dendritic Cells

text: Induction of tumor-specific immunity requires that dendritic cells (DCs) efficiently capture and present tumor antigens to result in the expansion and activation of tumor-specific cytotoxic T cells. The transition from antigen capture to T cell stimulation requires a maturation signal; in its absence tolerance, rather than immunity may develop. While immune complexes (ICs) are able to enhance antigen capture, they can be poor at inducing DC maturation, naive T cell activation and protective immunity. We now demonstrate that interfering with the inhibitory signal delivered by FcγRIIB on DCs converts ICs to potent maturation agents and results in T cell activation. Applying this approach to immunization with DCs pulsed ex-vivo with ICs, we have generated antigen-specific CD8+ T cells in vivo and achieved efficient protective immunity in a murine melanoma model. These data imply that ICs may normally function to maintain tolerance through the binding to inhibitory FcγRs on DCs, but they can be converted to potent immunogenic stimuli by selective engagement of activating FcγRs. This mechanism suggests a novel approach to the development of tumor vaccines.


title: Homocysteine and the risk of ischemic stroke in a triethnic cohort: the NOrthern MAnhattan Study.

text:
BACKGROUND AND PURPOSE The level of total homocysteine (tHcy) that confers a risk of ischemic stroke is unsettled, and no prospective cohort studies have included sufficient elderly minority subjects. We investigated the association between mild to moderate fasting tHcy level and the incidence of ischemic stroke, myocardial infarction, and vascular death in a multiethnic prospective study.
 METHODS A population-based cohort was followed for vascular events (stroke, myocardial infarction, and vascular death). Baseline values of tHcy and methylmalonic acid were measured among 2939 subjects (mean age, 69+/-10; 61% women, 53% Hispanics, 24% blacks, and 20% whites). Cox proportional models were used to calculate hazard ratios (HRs) and 95% CIs in tHcy categories after adjusting for age, race, education, renal insufficiency, B12 deficiency, and other risk factors.
 RESULTS The adjusted HR for a tHcy level > or =15 micromol/L compared with <10 micromol/L was greatest for vascular death (HR=6.04; 95% CI, 3.44 to 10.60), followed by combined vascular events (HR=2.27; 95% CI, 1.51 to 3.43), ischemic stroke (HR=2.01; 95% CI, 1.00 to 4.05), and nonvascular death (HR=2.02; 95% CI, 1.31 to 3.14). Mild to moderate elevations of tHcy of 10 to 15 micromol/L were not significantly predictive of ischemic stroke, but increased the risk of vascular death (2.27; 95% CI, 1.44 to 3.60) and combined vascular events (1.42; 95% CI, 1.06 to 1.88). The effect of tHcy was stronger among whites and Hispanics, but not a significant risk factor for blacks.
 CONCLUSIONS Total Hcy elevations above 15 micromol/L are an independent risk factor for ischemic stroke, whereas mild elevations of tHcy of 10 to 15 micromol/L are less predictive. The vascular effects of tHcy are greatest among whites and Hispanics, and less among blacks.


title: Expression and purification of recombinant methylated HBHA in Mycobacterium smegmatis.

text: The Heparin-Binding Haemagglutinin (HBHA) is a mycobacterial adhesin involved in the dissemination of Mycobacterium tuberculosis from the site of primary infection and a potential candidate for the development of a new vaccine against tuberculosis. Methylation of HBHA is a novel post-translational event that imparts important immunological properties to the protein. Since recombinant HBHA expressed in Escherichia coli is not methylated, we investigated the possibility of producing recombinant methylated HBHA in fast growing mycobacteria for use in immunological and biochemical studies. The complete coding sequence of HBHA was cloned in the plasmid pMV206, under the control of a strong promoter (hsp60) or its own promoter. The constructs generated were electroporated into Mycobacterium smegmatis and the recombinant strains obtained were analyzed for the presence of the HBHA protein using the anti-HBHA monoclonal antibodies D2 and E4. Our results indicate that expression of high amounts of intact protein can be toxic for the mycobacteria, that methylated HBHA can be obtained in M. smegmatis only when using a promoter sequence weaker than hsp60 and that the expression of the complete structural gene is required in order to obtain methylated HBHA. We constructed a recombinant M. smegmatis strain (pMV3-38) that expresses a histidine-tagged methylated HBHA that can be easily purified. The use of fast-growing strains of M. smegmatis to obtain significant amounts of purified HBHA protein within a short timeframe, should be an effective strategy for the evaluation of a new HBHA-based vaccine candidate for tuberculosis.


title: Detection of alternative lengthening of telomeres by telomere quantitative PCR

text: Alternative lengthening of telomeres (ALT) is one of the two known telomere length maintenance mechanisms that are essential for the unlimited proliferation potential of cancer cells. Existing methods for detecting ALT in tumors require substantial amounts of tumor material and are labor intensive, making it difficult to study prevalence and prognostic significance of ALT in large tumor cohorts. Here, we present a novel strategy utilizing telomere quantitative PCR to diagnose ALT. The protocol is more rapid than conventional methods and scrutinizes two distinct characteristics of ALT cells concurrently: long telomeres and the presence of C-circles (partially double-stranded circles of telomeric C-strand DNA). Requiring only 30 ng of genomic DNA, this protocol will facilitate large-scale studies of ALT in tumors and can be readily adopted by clinical laboratories.


title: Noise and interlocking signaling pathways promote distinct transcription factor dynamics in response to different stresses

text: All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to "hedge their bets" in responding to an uncertain future, and to balance growth and survival in an unpredictable environment.


title: Long-term financing needs for HIV control in sub-Saharan Africa in 2015-2050: a modelling study.

text:
OBJECTIVES To estimate the present value of current and future funding needed for HIV treatment and prevention in 9 sub-Saharan African (SSA) countries that account for 70% of HIV burden in Africa under different scenarios of intervention scale-up. To analyse the gaps between current expenditures and funding obligation, and discuss the policy implications of future financing needs.
 DESIGN We used the Goals module from Spectrum, and applied the most up-to-date cost and coverage data to provide a range of estimates for future financing obligations. The four different scale-up scenarios vary by treatment initiation threshold and service coverage level. We compared the model projections to current domestic and international financial sources available in selected SSA countries.
 RESULTS In the 9 SSA countries, the estimated resources required for HIV prevention and treatment in 2015-2050 range from US$98 billion to maintain current coverage levels for treatment and prevention with eligibility for treatment initiation at CD4 count of <500/mm(3) to US$261 billion if treatment were to be extended to all HIV-positive individuals and prevention scaled up. With the addition of new funding obligations for HIV--which arise implicitly through commitment to achieve higher than current treatment coverage levels--overall financial obligations (sum of debt levels and the present value of the stock of future HIV funding obligations) would rise substantially.
 CONCLUSIONS Investing upfront in scale-up of HIV services to achieve high coverage levels will reduce HIV incidence, prevention and future treatment expenditures by realising long-term preventive effects of ART to reduce HIV transmission. Future obligations are too substantial for most SSA countries to be met from domestic sources alone. New sources of funding, in addition to domestic sources, include innovative financing. Debt sustainability for sustained HIV response is an urgent imperative for affected countries and donors.


title: Cell-mediated immunity and antibody responses elicited by attenuated Salmonella enterica Serovar Typhi strains used as live oral vaccines in humans.

text: The development of improved typhoid vaccines is a high global public health priority. However, their development has been hampered by a lack of information regarding the specific determinants of protective immunity to Salmonella enterica serovar Typhi (S. Typhi) infection in humans. Although antibodies to S. Typhi O, H, and Vi appear to be involved in protection against S. Typhi infection, it is unknown whether such antibodies mediate protection, act in conjunction with other adaptive responses, or serve as a surrogate for the presence of other, more dominant protective immune responses (e.g., cell-mediated immunity [CMI]). CMI responses elicited by immunization of subjects with attenuated S. Typhi oral vaccines include lymphoproliferation; production of type 1 cytokines (e.g., interferon- gamma and tumor necrosis factor- alpha ); and classical major histocompatibility complex (MHC) class Ia-restricted and novel, nonclassical MHC class Ib (human leukocyte antigen [HLA]-E)-restricted CD8(+) cytotoxic T cell responses. In sum, human immunity to S. Typhi elicited by immunization is unexpectedly broad and complex. However, the immunologic correlates of protection remain largely undefined.


title: Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation

text: Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.


title: Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis.

text: A method for detecting multidrug-resistant Mycobacterium tuberculosis by using a reduction of resazurin is described. Eighty clinical isolates were evaluated against isoniazid and rifampin; results at 7 days were compared with those of the proportion method. Specificity and sensitivity were excellent. The method is simple, inexpensive, and rapid and might be used with other antituberculosis drugs.


title: The pervasiveness and plasticity of circadian oscillations: the coupled circadian-oscillators framework.

text:
MOTIVATION Circadian oscillations have been observed in animals, plants, fungi and cyanobacteria and play a fundamental role in coordinating the homeostasis and behavior of biological systems. Genetically encoded molecular clocks found in nearly every cell, based on negative transcription/translation feedback loops and involving only a dozen genes, play a central role in maintaining these oscillations. However, high-throughput gene expression experiments reveal that in a typical tissue, a much larger fraction ([Formula: see text]) of all transcripts oscillate with the day-night cycle and the oscillating species vary with tissue type suggesting that perhaps a much larger fraction of all transcripts, and perhaps also other molecular species, may bear the potential for circadian oscillations.
 RESULTS To better quantify the pervasiveness and plasticity of circadian oscillations, we conduct the first large-scale analysis aggregating the results of 18 circadian transcriptomic studies and 10 circadian metabolomic studies conducted in mice using different tissues and under different conditions. We find that over half of protein coding genes in the cell can produce transcripts that are circadian in at least one set of conditions and similarly for measured metabolites. Genetic or environmental perturbations can disrupt existing oscillations by changing their amplitudes and phases, suppressing them or giving rise to novel circadian oscillations. The oscillating species and their oscillations provide a characteristic signature of the physiological state of the corresponding cell/tissue. Molecular networks comprise many oscillator loops that have been sculpted by evolution over two trillion day-night cycles to have intrinsic circadian frequency. These oscillating loops are coupled by shared nodes in a large network of coupled circadian oscillators where the clock genes form a major hub. Cells can program and re-program their circadian repertoire through epigenetic and other mechanisms.    AVAILABILITY AND IMPLEMENTATION High-resolution and tissue/condition specific circadian data and networks available at http://circadiomics.igb.uci.edu.    CONTACT pfbaldi@ics.uci.edu   SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.


title: Gene–environment interactions in 7610 women with breast cancer: prospective evidence from the Million Women Study

text:
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.


title: Thrombocytopenia as an Indicator of Malaria in Adult Population

text: Objectives. To evaluate the predictive value of thrombocytopenia in malaria. Patients and Methods. It was a prospective observational study on all febrile patients with thrombocytopenia presenting to the Medical Unit of Hayat Abad Medical Complex during November 2008 to November 2010. Results. Of the total of 228 patients with fever and thrombocytopenia, 121 patients (53%) proved to be suffering from malaria. Of them 82 patients (68%) had falciparum malaria while 39 patients (32%) had vivax infection. Of these 121 patients, platelet counts ranged between 25,000 and 150,000/dL with a mean value of 101,000/dL (SD ± 47, 500) and a median of 75,000/dL. Of the 107 patients who were not suffering from malaria, the counts ranged between 10,000 and 150,000/dL with a mean value of 58,000/dL (SD ± 54, 000) and median of 50,000/dL. Conclusions. The presence of thrombocytopenia may be a predictor of malaria in adult population.


title: AMPA receptor ligand binding domain mobility revealed by functional cross linking.

text: Glutamate receptors mediate the majority of excitatory synaptic transmission in the CNS. The AMPA-subtype has rapid kinetics, with activation, deactivation and desensitization proceeding on the millisecond timescale or faster. Crystallographic, biochemical, and functional studies suggest that GluR2 Cys mutants which form intermolecular disulfide cross-links between the lower D2 lobes of the ligand binding cores can be trapped in a conformation that represents the desensitized state. We used multi-channel rapid perfusion techniques to examine the state dependence of cross-linking in these mutants. Under reducing conditions, both wild-type GluR2 and the G725C and S729C mutants have normal activation and desensitization kinetics, but the Cys mutants can be efficiently trapped in nonconducting states when oxidized. In contrast the I664C mutant is only partially inactivated under oxidizing conditions. For S729C, disulfide cross-links form rapidly when receptors are desensitized in the presence of glutamate, but receptors also become trapped at rest, in the absence of agonist. We assessed such spontaneous trapping in various conditions, including CNQX, a competitive antagonist; kainate, a weak partial agonist; or when desensitization was blocked by the L483Y mutation that stabilizes the D1 dimer interface. These experiments suggest that trapping in the absence of glutamate is due to two motions: Spontaneous breaking of the D1 dimer interface and hyperextension of the lower lobes of the ligand binding core. These data show that the glutamate binding domains are surprisingly mobile in the absence of ligand, which could influence receptor activity in the brain.


title: Ncf1 (p47phox) Is Essential for Direct Regulatory T Cell Mediated Suppression of CD4+ Effector T Cells

text:
BACKGROUND Multiple mechanisms have been advanced to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated suppression of CD4+ effector T cells (Teffs) but none appear to completely explain suppression. Previous data indicates that Tregs may affect the microenvironment redox state. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs.
 METHODOLOGY/PRINCIPAL FINDINGS Tregs and Teffs were isolated from the spleens of wild type (WT) C57BL/6 mice or Ncf1(p47phox)-deficient C57BL/6 mice which lack NADPH oxidase function. Teffs were labeled with CFSE and co-cultured with unlabeled Tregs at varying Treg:Teff ratios in the presence of anti-CD3/CD28 coated beads for 3 days in suppression assays. Treg-mediated suppression was quantified by flow cytometric analysis of CFSE dilution in Teffs. The presence of the antioxidants n-acetylcysteine (NAC) or 2-mercaptoethanol or inhibitors of NADPH oxidase (diphenyleneiodonium and VAS-2870) resulted in reduced WT Treg-mediated suppression. The observed suppression was in part dependent upon TGFβ as it was partially blocked with neutralizing antibodies. The suppression of Teff proliferation induced by exogenous TGFβ treatment could be overcome with NAC. Ncf1-deficient Teff were slightly but significantly less sensitive than WT Teff to suppression by exogenous TGFβ. Ncf1-deficient Tregs suppressed Ncf1-deficient Teff very poorly compared to wild type controls. There was partial but incomplete reconstitution of suppression in assays with WT Tregs and Ncf1-deficient Teff.
 CONCLUSIONS/SIGNIFICANCE We present evidence that NADPH oxidase derived ROS plays a role in the direct Treg mediated suppression of CD4+ effector T cells in a process that is blocked by thiol-containing antioxidants, NADPH oxidase inhibitors or a lack of Ncf1 expression in Tregs and Teffs. Oxidants may represent a potential new target for therapeutic modulation of Treg function.


title: Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands.

text: Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an in vivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.


title: Expression profiles during dedifferentiation in newt lens regeneration revealed by expressed sequence tags

text:
PURPOSE The adult newt can regenerate lens from pigmented epithelial cells (PECs) of the dorsal iris via dedifferentiation. The purpose of this research is to obtain sequence resources for a newt lens regeneration study and to obtain insights of dedifferentiation at the molecular level.
 METHODS mRNA was purified from iris during dedifferentiation and its cDNA library was constructed. From the cDNA library 10,449 clones were sequenced and analyzed.
 RESULTS From 10,449 reads, 780 contigs and 1,666 singlets were annotated. The presence of several cancer- and apoptosis-related genes during newt dedifferentiation was revealed. Moreover, several candidate genes, which might participate in reprogramming during dedifferentiation, were also found.
 CONCLUSIONS The expression of cancer- and apoptosis-related genes could be hallmarks during dedifferentiation. The expression sequence tag (EST) resource is useful for the future study of newt dedifferentiation, and the sequence information is available in GenBank (accession numbers; FS290155-FS300559).


title: In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells.

text: A rare set of hematopoietic stem cells (HSC) must undergo a massive expansion to produce mature blood cells. The phenotypic isolation of HSC from mice offers the opportunity to determine directly their proliferation kinetics. We analyzed the proliferation and cell cycle kinetics of long-term self-renewing HSC (LT-HSC) in normal adult mice. At any one time, approximately 5% of LT-HSC were in S/G2/M phases of the cell cycle and another 20% were in G1 phase. BrdUrd incorporation was used to determine the rate at which different cohorts of HSC entered the cell cycle over time. About 50% of LT-HSC incorporated BrdUrd by 6 days and >90% incorporated BrdUrd by 30 days. By 6 months, 99% of LT-HSC had incorporated BrdUrd. We calculated that approximately 8% of LT-HSC asynchronously entered the cell cycle per day. Nested reverse transcription-PCR analysis revealed cyclin D2 expression in a high proportion of LT-HSC. Although approximately 75% of LT-HSC are quiescent in G0 at any one time, all HSC are recruited into cycle regularly such that 99% of LT-HSC divide on average every 57 days.


title: The role of the tissue omega-6/omega-3 fatty acid ratio in regulating tumor angiogenesis

text: Angiogenesis is a necessary step in tumor growth and metastasis. It is well established that the metabolites of omega-6 and omega-3 fatty acids, which must be obtained through the diet and cannot be synthesized de novo in mammals, have differential effects on cellular processes. Omega-6 fatty acid (n−6 FA)-derived metabolites promote angiogenesis by increasing growth factor expression whereas omega-3 fatty acids (n−3 FA) have anti-angiogenic and antitumor properties. However, most studies thus far have failed to account for the role of the n−6 FA/n−3 FA ratio in angiogenesis and instead examined the absolute levels of n−6 and n−3 FA. This review highlights the biochemical interactions between n−6 and n−3 FA and focuses on how the n−6/n−3 FA ratio in tissues modulates tumor angiogenesis. We suggest that future work should consider the n−6/n−3 FA ratio to be a key element in experimental design and analysis. Furthermore, we recommend that clinical interventions should aim to both reduce n−6 metabolites and simultaneously increase n−3 FA intake.


title: The Natural Statistics of Audiovisual Speech

text: Humans, like other animals, are exposed to a continuous stream of signals, which are dynamic, multimodal, extended, and time varying in nature. This complex input space must be transduced and sampled by our sensory systems and transmitted to the brain where it can guide the selection of appropriate actions. To simplify this process, it's been suggested that the brain exploits statistical regularities in the stimulus space. Tests of this idea have largely been confined to unimodal signals and natural scenes. One important class of multisensory signals for which a quantitative input space characterization is unavailable is human speech. We do not understand what signals our brain has to actively piece together from an audiovisual speech stream to arrive at a percept versus what is already embedded in the signal structure of the stream itself. In essence, we do not have a clear understanding of the natural statistics of audiovisual speech. In the present study, we identified the following major statistical features of audiovisual speech. First, we observed robust correlations and close temporal correspondence between the area of the mouth opening and the acoustic envelope. Second, we found the strongest correlation between the area of the mouth opening and vocal tract resonances. Third, we observed that both area of the mouth opening and the voice envelope are temporally modulated in the 2-7 Hz frequency range. Finally, we show that the timing of mouth movements relative to the onset of the voice is consistently between 100 and 300 ms. We interpret these data in the context of recent neural theories of speech which suggest that speech communication is a reciprocally coupled, multisensory event, whereby the outputs of the signaler are matched to the neural processes of the receiver.


title: Establishment of human iPSC-based models for the study and targeting of glioma initiating cells

text: Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis.


title: Thyroid Hormone Receptor Repression Linked to Type I Pneumocyte Associated Respiratory Distress Syndrome

text: Although the lung is a defining feature of air-breathing animals, the pathway controlling the formation of type I pneumocytes, the cells that mediate gas exchange, is poorly understood. In contrast, the glucocorticoid receptor and its cognate ligand have long been known to promote type II pneumocyte maturation; prenatal administration of glucocorticoids is commonly used to attenuate the severity of infant respiratory distress syndrome (RDS). Here we show that knock-in mutations of the nuclear co-repressor SMRT (silencing mediator of retinoid and thyroid hormone receptors) in C57BL/6 mice (SMRTmRID) produces a previously unidentified respiratory distress syndrome caused by prematurity of the type I pneumocyte. Though unresponsive to glucocorticoids, treatment with anti-thyroid hormone drugs (propylthiouracil or methimazole) completely rescues SMRT-induced RDS, suggesting an unrecognized and essential role for the thyroid hormone receptor (TR) in lung development. We show that TR and SMRT control type I pneumocyte differentiation through Klf2, which, in turn, seems to directly activate the type I pneumocyte gene program. Conversely, mice without lung Klf2 lack mature type I pneumocytes and die shortly after birth, closely recapitulating the SMRTmRID phenotype. These results identify TR as a second nuclear receptor involved in lung development, specifically type I pneumocyte differentiation, and suggest a possible new type of therapeutic option in the treatment of RDS that is unresponsive to glucocorticoids.


title: Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance.

text: Recent studies have reported that competitive endogenous RNAs (ceRNAs) can act as sponges for a microRNA (miRNA) through their binding sites and that changes in ceRNA abundances from individual genes can modulate the activity of miRNAs. Consideration of this hypothesis would benefit from knowing the quantitative relationship between a miRNA and its endogenous target sites. Here, we altered intracellular target site abundance through expression of an miR-122 target in hepatocytes and livers and analyzed the effects on miR-122 target genes. Target repression was released in a threshold-like manner at high target site abundance (≥1.5 × 10(5) added target sites per cell), and this threshold was insensitive to the effective levels of the miRNA. Furthermore, in response to extreme metabolic liver disease models, global target site abundance of hepatocytes did not change sufficiently to affect miRNA-mediated repression. Thus, modulation of miRNA target abundance is unlikely to cause significant effects on gene expression and metabolism through a ceRNA effect.


title: Immunological mechanisms for desensitization and tolerance in food allergy

text: Food allergy is a major public health concern in westernized countries, estimated to affect 5 % of children and 3–4 % of adults. Allergen-specific immunotherapy for food allergy is currently being actively evaluated, but is still experimental. The optimal protocol, in terms of the route of administration of the food, target maintenance dose, and duration of maintenance therapy, and the optimal patient for these procedures are still being worked out. The mechanisms underlying successful food desensitization are also unclear, in part, because there is no standard immunotherapy protocol. The mechanisms involved, however, may include mast cell and basophil suppression, development of food-specific IgG4 antibodies, reduction in the food-specific IgE/IgG4 ratio, up-regulation and expansion of natural or inducible regulatory T cells, a skewing from a Th2 to a Th1 profile, and the development of anergy and/or deletion in antigen-specific cells. Additional studies are required to elucidate and understand these mechanisms by which desensitization and tolerance are achieved, which may reveal valuable biomarkers for evaluating and following food allergic patients on immunotherapy.


title: Prodromal non-motor symptoms of Parkinson’s disease

text: The motor symptoms of Parkinson’s disease (PD), bradykinesia, muscular rigidity, and tremor depend upon degeneration of the dopaminergic neurons in the substantia nigra pars compacta. Recent neuropathological studies show that the Lewy bodies, the intraneuronal landmark of PD, accumulate in several neuronal cell types in the brain. An ascending gradient of pathological involvement, from the medulla oblongata to neocortical areas has been reported. Thus the original view of PD as a disease characterized by selective damage of the dopaminergic neurons in the mesencephalon should be updated into the concept of a severe multisystemic neurodegenerative disorder. Additionally, the neuropathological alterations outside the substantia nigra are soundly correlated with the non-motor symptoms of PD. As a result of these findings, interest is growing in the identification of prodromal non-motor symptoms of PD. Indeed, data from the literature suggest that autonomic disturbances, olfactory dysfunctions, depression and sleep disorders (in particular REM-sleep behavior disorder) may represent prodromal non-motor symptoms of PD. Several tests are available to detect most of these symptoms. Thus, the identification of prodromal non-motor symptoms may contribute to the precocious diagnosis of PD, and might be useful in the future to test the efficacy of neuroprotective agents.


title: Erythroid Krüppel-Like Factor Directly Activates the Basic Krüppel-Like Factor Gene in Erythroid Cells �

text: The Sp/Krüppel-like factor (Sp/Klf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Krüppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Krüppel-like factor (Eklf/Klf1). We observe that Bklf mRNA is significantly reduced in erythroid tissues from Eklf-null murine embryos. We find that Bklf is driven primarily by two promoters, a ubiquitously active GC-rich upstream promoter, 1a, and an erythroid downstream promoter, 1b. Transcripts from the two promoters encode identical proteins. Interestingly, both the ubiquitous and the erythroid promoter are dependent on Eklf in erythroid cells. Eklf also activates both promoters in transient assays. Experiments utilizing an inducible form of Eklf demonstrate activation of the endogenous Bklf gene in the presence of an inhibitor of protein synthesis. The kinetics of activation are also consistent with Bklf being a direct Eklf target. Chromatin immunoprecipitation assays confirm that Eklf associates with both Bklf promoters. Eklf is typically an activator of transcription, whereas Bklf is noted as a repressor. Our results support the hypothesis that feedback cross-regulation occurs within the Sp/Klf family in vivo.


title: Noncanonical Wnt Signaling Promotes Obesity-Induced Adipose Tissue Inflammation and Metabolic Dysfunction Independent of Adipose Tissue Expansion

text: Adipose tissue dysfunction plays a pivotal role in the development of insulin resistance in obese individuals. Cell culture studies and gain-of-function mouse models suggest that canonical Wnt proteins modulate adipose tissue expansion. However, no genetic evidence supports a role for endogenous Wnt proteins in adipose tissue dysfunction, and the role of noncanonical Wnt signaling remains largely unexplored. Here we provide evidence from human, mouse, and cell culture studies showing that Wnt5a-mediated, noncanonical Wnt signaling contributes to obesity-associated metabolic dysfunction by increasing adipose tissue inflammation. Wnt5a expression is significantly upregulated in human visceral fat compared with subcutaneous fat in obese individuals. In obese mice, Wnt5a ablation ameliorates insulin resistance, in parallel with reductions in adipose tissue inflammation. Conversely, Wnt5a overexpression in myeloid cells augments adipose tissue inflammation and leads to greater impairments in glucose homeostasis. Wnt5a ablation or overexpression did not affect fat mass or adipocyte size. Mechanistically, Wnt5a promotes the expression of proinflammatory cytokines by macrophages in a Jun NH2-terminal kinase-dependent manner, leading to defective insulin signaling in adipocytes. Exogenous interleukin-6 administration restores insulin resistance in obese Wnt5a-deficient mice, suggesting a central role for this cytokine in Wnt5a-mediated metabolic dysfunction. Taken together, these results demonstrate that noncanonical Wnt signaling contributes to obesity-induced insulin resistance independent of adipose tissue expansion.


title: A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease

text: The 5'-exonuclease Rat1 degrades pre-rRNA spacer fragments and processes the 5'-ends of the 5.8S and 25S rRNAs. UV crosslinking revealed multiple Rat1-binding sites across the pre-rRNA, consistent with its known functions. The major 5.8S 5'-end is generated by Rat1 digestion of the internal transcribed spacer 1 (ITS1) spacer from cleavage site A(3). Processing from A(3) requires the 'A(3)-cluster' proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre-rRNA binding. Surprisingly, A(3)-cluster factors were not crosslinked close to site A(3), but bound sites around the 5.8S 3'- and 25S 5'-regions, which are base paired in mature ribosomes, and in the ITS2 spacer that separates these rRNAs. In contrast, Nop4, a protein required for endonucleolytic cleavage in ITS1, binds the pre-rRNA near the 5'-end of 5.8S. ITS2 was reported to undergo structural remodelling. In vivo chemical probing indicates that A(3)-cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A(3) cluster establish long-range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding.


title: Synergistic activity of the histone deacetylase inhibitor suberoylanilide hydroxamic acid and the bisphosphonate zoledronic acid against prostate cancer cells in vitro.

text: Bisphosphonates are widely used agents for the treatment of malignant bone disease. They inhibit osteoclast-mediated bone resorption and can have direct effects on cancer cells. In this study, we investigated whether the anticancer activity of the third-generation bisphosphonate zoledronic acid (ZOL) could be enhanced by combination with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). We found that ZOL and SAHA cooperated to induce cell death in the prostate cancer cell lines LNCaP and PC-3. The effect was synergistic, as evidenced by combination index isobologram analysis. ZOL and SAHA synergized to induce dissipation of the mitochondrial transmembrane potential, to activate caspase-3, and to trigger DNA fragmentation, showing that the combination of ZOL and SAHA resulted in the initiation of apoptosis. Because ZOL acts by inhibiting the mevalonate pathway, thereby preventing protein prenylation, we explored whether the mevalonate pathway was also the target of the cooperative action of ZOL and SAHA. We found that geranylgeraniol, but not farnesol, significantly reduced ZOL/SAHA-induced cell death, indicating that the synergistic action of the agents was due to the inhibition of geranylgeranylation. Consistently, a direct inhibitor of geranylgeranylation, GGTI-298, synergized with SAHA to induce cell death, whereas an inhibitor of farnesylation, FTI-277, had no effect. In addition, SAHA synergized with mevastatin, an inhibitor of the proximal enzyme in the mevalonate pathway. These in vitro findings provide a rationale for an in vivo exploration into the potential of combining SAHA and ZOL, or other inhibitors of the mevalonate pathway, as an effective strategy for anticancer therapy.


title: The Immune Response to Melanoma Is Limited by Thymic Selection of Self-Antigens

text: The expression of melanoma-associated antigens (MAA) being limited to normal melanocytes and melanomas, MAAs are ideal targets for immunotherapy and melanoma vaccines. As MAAs are derived from self, immune responses to these may be limited by thymic tolerance. The extent to which self-tolerance prevents efficient immune responses to MAAs remains unknown. The autoimmune regulator (AIRE) controls the expression of tissue-specific self-antigens in thymic epithelial cells (TECs). The level of antigens expressed in the TECs determines the fate of auto-reactive thymocytes. Deficiency in AIRE leads in both humans (APECED patients) and mice to enlarged autoreactive immune repertoires. Here we show increased IgG levels to melanoma cells in APECED patients correlating with autoimmune skin features. Similarly, the enlarged T cell repertoire in AIRE(-/-) mice enables them to mount anti-MAA and anti-melanoma responses as shown by increased anti-melanoma antibodies, and enhanced CD4(+) and MAA-specific CD8(+) T cell responses after melanoma challenge. We show that thymic expression of gp100 is under the control of AIRE, leading to increased gp100-specific CD8(+) T cell frequencies in AIRE(-/-) mice. TRP-2 (tyrosinase-related protein), on the other hand, is absent from TECs and consequently TRP-2 specific CD8(+) T cells were found in both AIRE(-/-) and AIRE(+/+) mice. This study emphasizes the importance of investigating thymic expression of self-antigens prior to their inclusion in vaccination and immunotherapy strategies.


title: Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4.

text: Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-2(Delta46-110)) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-2(Delta46-110) cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-2(Delta46-110) ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling-3 (SOCS-3) and Kruppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.


title: MicroScope—an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data

text: MicroScope is an integrated platform dedicated to both the methodical updating of microbial genome annotation and to comparative analysis. The resource provides data from completed and ongoing genome projects (automatic and expert annotations), together with data sources from post-genomic experiments (i.e. transcriptomics, mutant collections) allowing users to perfect and improve the understanding of gene functions. MicroScope (http://www.genoscope.cns.fr/agc/microscope) combines tools and graphical interfaces to analyse genomes and to perform the manual curation of gene annotations in a comparative context. Since its first publication in January 2006, the system (previously named MaGe for Magnifying Genomes) has been continuously extended both in terms of data content and analysis tools. The last update of MicroScope was published in 2009 in the Database journal. Today, the resource contains data for >1600 microbial genomes, of which ∼300 are manually curated and maintained by biologists (1200 personal accounts today). Expert annotations are continuously gathered in the MicroScope database (∼50 000 a year), contributing to the improvement of the quality of microbial genomes annotations. Improved data browsing and searching tools have been added, original tools useful in the context of expert annotation have been developed and integrated and the website has been significantly redesigned to be more user-friendly. Furthermore, in the context of the European project Microme (Framework Program 7 Collaborative Project), MicroScope is becoming a resource providing for the curation and analysis of both genomic and metabolic data. An increasing number of projects are related to the study of environmental bacterial (meta)genomes that are able to metabolize a large variety of chemical compounds that may be of high industrial interest.


title: Comparison of Maximal Oxygen Consumption Between Black and White Prepubertal and Pubertal Children

text: The purpose of this investigation was to determine whether maximal oxygen consumption (VO2max) differed between two selected groups of black and white children and whether a difference existed to determine whether it was related to hematologic profiles, body composition, and/or physical activity/inactivity level. Forty-five prepubertal and 42 pubertal, clinically normal black and white children participated. Dual-energy x-ray absorptiometry was used to determine body composition. A computed tomography scan of the abdomen was used to determine visceral adipose tissue and s.c. adipose tissue. Daily physical activity/inactivity was assessed by questionnaire. Black prepubertal and pubertal children had lower VO2max values when compared with white children (28.8 ± 7.8 versus 35.0 ± 6.5 mL · kg−1 · min−1, p < 0.01; 33.7 ± 6.4 versus 40.4 ± 10.2 mL · kg−1 · min−1, p < 0.05; respectively). Black prepubertal and pubertal children had lower Hb concentrations ([Hb]) and hematocrits than white children (prepubertal: 12.1 ± 0.5 versus 12.8 ± 0.9 g/dL, p < 0.001; 35.6 ± 1.4 versus 37.4 ± 2.3%, p < 0.01, respectively; pubertal: 13.0 ± 0.9 versus 13.6 ± 0.7 g/dL, p < 0.05; 37.7 ± 2.5 versus 39.5 ± 2.1%, p < 0.05, respectively). In conclusion, these findings indicate that black prepubertal and pubertal children had lower VO2max when compared with their white peers matched for age, pubertal stage, and body mass index. This difference in VO2max could be attributed at least in part to comparatively lower [Hb] and more sedentary lifestyle in the black children. Further investigations should study Hb flow rate (a function of [Hb] × maximal cardiac output) in black and white children as it relates to VO2max.


title: Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway.

text: CD4(+) helper T (Th) cells play a crucial role in the delicate balance between host defense and autoimmune disease. Two important populations of helper T cells are the proinflammatory, interleukin-17 (IL-17)-producing (Th17) cells and the anti-inflammatory forkhead box P3-positive (FoxP3(+)) T regulatory (Treg) cells. Here we show that all-trans retinoic acid (ATRA) and other agonists of the retinoic acid receptor alpha (RARalpha) inhibit the formation of Th17 cells and promote FoxP3 expression. Conversely, inhibition of retinoic acid signaling constrains transforming growth factor beta (TGF-beta1) induction of FoxP3. The effect of ATRA is mediated independently of IL-2, signal transducer and activator of transcription 5 (Stat5) and Stat3, representing a novel mechanism for the induction of FoxP3 in CD4 T cells. As previous studies have shown that vitamin A derivatives are protective in animal models of autoimmune disease, the current data suggest a previously unrecognized role for RARalpha in the regulation of CD4(+) T-cell differentiation and provide a mechanism for the anti-inflammatory effects of retinoic acid.


title: British Journal of Nutrition (2003), 89, 295–301 q The Authors 2003 DOI: 10.1079/BJN2002776 Plasma homocysteine concentration is decreased by dietary intervention*

text: High plasma total homocysteine (tHcy) concentration is reported to be a risk factor for vascular diseases. We investigated the extent to which serum folate and plasma tHcy respond to a high intake of natural folate from food. Thirty-seven healthy females volunteered t o participate in a crossover dietary intervention. The study included a baseline period and two 5-week diet periods (low- and high-folate diets) with a 3-week washout in between. The low-folate diet contained one serving of both vegetables and fruit/d, while during the high-folate diet the subjects ate at least seven servings of vegetables, berries, and citrus fruit/d. Serum and erythrocyte (RBC) folate, serum vitamin B (12), and plasma tHcy concentrations were measured at the base-line and at the end of each diet period. The mean concentrations of serum and RBC folate were 11.0 (SD 3.0) nmol/l and 412 (SD 120) nmol/l at the end of the low-folate diet and 78 (95 % CI 62, 94) % and 14 (95 % CI 8, 20) % higher in response to the high-folate diet (P< 0.001). The serum concentration of vitamin B12 remained unchanged during the intervention. The mean plasma tHcy concentration was 8.0 pmol/ at the end of the low-folate diet and decreased by 13 (95% CI 9, 18) % in response to the high-folate diet (P<0.001). In conclusion, a diet high in fresh berries, citrus fruit, and vegetables effectively increases serum and RBC folate and decreases plasma homocysteine.


title: Achieving increased resolution and more pixels with Superresolution Optical Fluctuation Imaging (SOFI)

text: Superresolution Optical Fluctuation Imaging (SOFI) as initially demonstrated allows for a resolution enhancement in imaging by a factor of square-root of two. Here, we demonstrate how to increase the resolution of SOFI images by re-weighting the Optical Transfer Function (OTF). Furthermore, we demonstrate how cross-cumulants can be exploited to obtain a fair approximation of the underlying Point-Spread Function. We show a two-fold increase of resolution (over the diffraction limit) of near-infrared quantum dot labeled tubulin-network of 3T3 fibroblasts.


title: Evaluation of reagent strips in detecting asymptomatic bacteriuria in early pregnancy: prospective case series.

text:
OBJECTIVE To evaluate the performance of reagent test strips in screening pregnant women for asymptomatic bacteriuria at their first visit to an antenatal clinic.
 DESIGN Prospective case series.
 SETTING Antenatal clinic of a large inner city maternity hospital.    SUBJECTS All women attending for their first antenatal clinic. Patients taking antibiotics for any reason and those with urinary tract symptoms were excluded.
 INTERVENTION A midstream urine specimen was divided; half was sent for microscopy and formal bacteriological culture and the other half was tested with a commercial reagent strip test for the presence of blood, protein, nitrite, and leucocyte esterase.
 MAIN OUTCOME MEASURES Sensitivity, specificity, and positive and negative predictive values of the reagent strips in diagnosing asymptomatic bacteriuria (defined as 10(5) colony forming units/ml urine).
 RESULTS Sensitivity was low, with a maximum of 33% when all four tests were used in combination. Specificity was high, with typical values of 99% or more. Positive predictive value reached a maximum of 69% and negative predictive value was typically 95% or more.
 CONCLUSION Urine reagent strips are not sufficiently sensitive to be of use in the screening for asymptomatic bacteriuria and therefore many patients would be missed. In view of the potentially serious sequelae of this condition in pregnant women we recommend that formal bacteriological investigation remain the investigation of choice in this group of patients.


title: Wnt5a functions in planar cell polarity regulation in mice.

text: Planar cell polarity (PCP) refers to the polarization of cells within the plane of a cell sheet. A distinctive epithelial PCP in vertebrates is the uniform orientation of stereociliary bundles of the sensory hair cells in the mammalian cochlea. In addition to establishing epithelial PCP, planar polarization is also required for convergent extension (CE); a polarized cellular movement that occurs during neural tube closure and cochlear extension. Studies in Drosophila and vertebrates have revealed a conserved PCP pathway, including Frizzled (Fz) receptors. Here we use the cochlea as a model system to explore the involvement of known ligands of Fz, Wnt morphogens, in PCP regulation. We show that Wnt5a forms a reciprocal expression pattern with a Wnt antagonist, the secreted frizzled-related protein 3 (Sfrp3 or Frzb), along the axis of planar polarization in the cochlear epithelium. We further demonstrate that Wnt5a antagonizes Frzb in regulating cochlear extension and stereociliary bundle orientation in vitro, and that Wnt5a(-/-) animals have a shortened and widened cochlea. Finally, we show that Wnt5a is required for proper subcellular distribution of a PCP protein, Ltap/Vangl2, and that Wnt5a interacts genetically with Ltap/Vangl2 for uniform orientation of stereocilia, cochlear extension, and neural tube closure. Together, these findings demonstrate that Wnt5a functions in PCP regulation in mice.


title: The RNA Polymerase “Switch Region” Is a Target for Inhibitors

text: The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.


title: How reliable are light traps in estimating biting rates of adult Anopheles gambiae s.l. (Diptera: Culicidae) in the presence of treated bed nets?

text: The sampling efficiency of light trap catches relative to human bait catches in estimating biting rates of the mosquito Anopheles gambiae Giles was investigated in two types of community in southern Sierra Leone: (i) where most of the inhabitants slept under treated bed nets; and (ii) where most of the inhabitants slept without bed nets. The number of female A. gambiae mosquitoes caught in these communities by light trap was strongly correlated (r > or = 0.72) with those from corresponding human biting catches performed either on the same or adjacent nights. It was found that the relative sampling efficiency of light traps varied slightly but significantly with mosquito abundance in villages with treated bed nets, but not in those without them. Nevertheless, the relationship between relative sampling efficiency and mosquito abundance did not differ significantly between the two types of village. Overall, there was insufficient evidence to show that the presence of treated nets altered the relative efficiency of light traps and any bias was only slight, and unlikely to be of any practical importance. Hence, it was concluded that light traps can be used as a surrogate for human bait catches in estimating biting rates of A. gambiae mosquitoes in the two communities.


title: Development of a novel mouse glioma model using lentiviral vectors

text: We report the development of a new method to induce glioblastoma multiforme in adult immunocompetent mice by injecting Cre-loxP–controlled lentiviral vectors expressing oncogenes. Cell type- or region-specific expression of activated forms of the oncoproteins Harvey-Ras and AKT in fewer than 60 glial fibrillary acidic protein–positive cells in the hippocampus, subventricular zone or cortex of mice heterozygous for the gene encoding the tumor suppressor Tp53 were tested. Mice developed glioblastoma multiforme when transduced either in the subventricular zone or the hippocampus. However, tumors were rarely detected when the mice were transduced in the cortex. Transplantation of brain tumor cells into naive recipient mouse brain resulted in the formation of glioblastoma multiforme–like tumors, which contained CD133+ cells, formed tumorspheres and could differentiate into neurons and astrocytes. We suggest that the use of Cre-loxP–controlled lentiviral vectors is a novel way to generate a mouse glioblastoma multiforme model in a region- and cell type-specific manner in adult mice.


title: Research Letters

text: We report a case of preclinical variant Creutzfeldt-Jakob disease (vCJD) in a patient who died from a non-neurological disorder 5 years after receiving a blood transfusion from a donor who subsequently developed vCJD. Protease-resistant prion protein (PrP(res)) was detected by western blot, paraffin-embedded tissue blot, and immunohistochemistry in the spleen, but not in the brain. Immunohistochemistry for prion protein was also positive in a cervical lymph node. The patient was a heterozygote at codon 129 of PRNP, suggesting that susceptibility to vCJD infection is not confined to the methionine homozygous PRNP genotype. These findings have major implications for future estimates and surveillance of vCJD in the UK.


title: Swi1Timeless Prevents Repeat Instability at Fission Yeast Telomeres

text: Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1(Timeless), a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Δ mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of Swi1(Timeless) in regulation of telomere stability in cancer cells.


title: Prediction of guide strand of microRNAs from its sequence and secondary structure

text:
BACKGROUND MicroRNAs (miRNAs) are produced by the sequential processing of a long hairpin RNA transcript by Drosha and Dicer, an RNase III enzymes, and form transitory small RNA duplexes. One strand of the duplex, which incorporates into RNA-induced silencing complex (RISC) and silences the gene expression is called guide strand, or miRNA; while the other strand of duplex is degraded and called the passenger strand, or miRNA*. Predicting the guide strand of miRNA is important for better understanding the RNA interference pathways.
 RESULTS This paper describes support vector machine (SVM) models developed for predicting the guide strands of miRNAs. All models were trained and tested on a dataset consisting of 329 miRNA and 329 miRNA* pairs using five fold cross validation technique. Firstly, models were developed using mono-, di-, and tri-nucleotide composition of miRNA strands and achieved the highest accuracies of 0.588, 0.638 and 0.596 respectively. Secondly, models were developed using split nucleotide composition and achieved maximum accuracies of 0.553, 0.641 and 0.602 for mono-, di-, and tri-nucleotide respectively. Thirdly, models were developed using binary pattern and achieved the highest accuracy of 0.708. Furthermore, when integrating the secondary structure features with binary pattern, an accuracy of 0.719 was seen. Finally, hybrid models were developed by combining various features and achieved maximum accuracy of 0.799 with sensitivity 0.781 and specificity 0.818. Moreover, the performance of this model was tested on an independent dataset that achieved an accuracy of 0.80. In addition, we also compared the performance of our method with various siRNA-designing methods on miRNA and siRNA datasets.
 CONCLUSION In this study, first time a method has been developed to predict guide miRNA strands, of miRNA duplex. This study demonstrates that guide and passenger strand of miRNA precursors can be distinguished using their nucleotide sequence and secondary structure. This method will be useful in understanding microRNA processing and can be implemented in RNA silencing technology to improve the biological and clinical research. A web server has been developed based on SVM models described in this study (http://crdd.osdd.net:8081/RISCbinder/).


title: NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome

text: Cornelia de Lange syndrome (CdLS) is a multiple malformation disorder characterized by dysmorphic facial features, mental retardation, growth delay and limb reduction defects. We indentified and characterized a new gene, NIPBL, that is mutated in individuals with CdLS and determined its structure and the structures of mouse, rat and zebrafish homologs. We named its protein product delangin. Vertebrate delangins have substantial homology to orthologs in flies, worms, plants and fungi, including Scc2-type sister chromatid cohesion proteins, and D. melanogaster Nipped-B. We propose that perturbed delangin function may inappropriately activate DLX genes, thereby contributing to the proximodistal limb patterning defects in CdLS. Genome analyses typically identify individual delangin or Nipped-B-like orthologs in diploid animal and plant genomes. The evolution of an ancestral sister chromatid cohesion protein to acquire an additional role in developmental gene regulation suggests that there are parallels between CdLS and Roberts syndrome.


title: Impaired FANCD2 monoubiquitination and hypersensitivity to camptothecin uniquely characterize Fanconi anemia complementation group M.

text: FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM(-/-) cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM(-/-) cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM(-/-) cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex-dependent and -independent manner.


title: Vesicular Glycolysis Provides On-Board Energy for Fast Axonal Transport

text: Fast axonal transport (FAT) requires consistent energy over long distances to fuel the molecular motors that transport vesicles. We demonstrate that glycolysis provides ATP for the FAT of vesicles. Although inhibiting ATP production from mitochondria did not affect vesicles motility, pharmacological or genetic inhibition of the glycolytic enzyme GAPDH reduced transport in cultured neurons and in Drosophila larvae. GAPDH localizes on vesicles via a huntingtin-dependent mechanism and is transported on fast-moving vesicles within axons. Purified motile vesicles showed GAPDH enzymatic activity and produced ATP. Finally, we show that vesicular GAPDH is necessary and sufficient to provide on-board energy for fast vesicular transport. Although detaching GAPDH from vesicles reduced transport, targeting GAPDH to vesicles was sufficient to promote FAT in GAPDH deficient neurons. This specifically localized glycolytic machinery may supply constant energy, independent of mitochondria, for the processive movement of vesicles over long distances in axons.


title: RB loss in resistant EGFR mutant lung adenocarcinomas that transform to small-cell lung cancer

text: Tyrosine kinase inhibitors are effective treatments for non-small-cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of 1 year of continuous treatment. A fundamental histological transformation from NSCLC to small-cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumour samples and cell lines derived from resistant EGFR mutant patients revealed that Retinoblastoma (RB) is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared with resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC.


title: Periostin Deficiency Increases Bone Damage and Impairs Injury Response to Fatigue Loading in Adult Mice

text: Bone damage removal and callus formation in response to fatigue loading are essential to prevent fractures. Periostin (Postn) is a matricellular protein that mediates adaptive response of cortical bone to loading. Whether and how periostin influences damage and the injury response to fatigue remains unknown. We investigated the skeletal response of Postn(-/-) and Postn(+/+) mice after fatigue stimulus by axial compression of their tibia. In Postn(+/+) mice, cracks number and surface (CsNb, CsS) increased 1h after fatigue, with a decrease in strength compared to non-fatigued tibia. At 15 days, CsNb had started to decline, while CtTV and CtBV increased in fatigued vs non-fatigued tibia, reflecting a woven bone response that was present in 75% of the fatigued bones. Cortical porosity and remodelling also prominently increased in the fatigued tibia of Postn(+/+) mice. At 30 days, paralleling a continuous removal of cortical damage, strength of the fatigued tibia was similar to the non-fatigue tibia. In Postn(-/-) mice, cracks were detectable even in the absence of fatigue, while the amount of collagen crosslinks and tissue hardness was decreased compared to Postn(+/+). Fatigue significantly increased CsNb and CsS in Postn(-/-), but was not associated with changes in CtTV and CtBV, as only 16% of the fatigued bones formed some woven bone. Cortical porosity and remodelling did not increase either after fatigue in Postn(-/-), and the level of damage remained high even after 30 days. As a result, strength remained compromised in Postn(-/-) mice. Contrary to Postn(+/+), which osteocytic lacunae showed a change in the degree of anisotropy (DA) after fatigue, Postn(-/-) showed no DA change. Hence periostin appears to influence bone materials properties, damage accumulation and repair, including local modeling/remodeling processes in response to fatigue. These observations suggest that the level of periostin expression could influence the propensity to fatigue fractures.


title: Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling.

text: The yeast histone variant H2AZ (Htz1) is implicated in transcription activation, prevention of the ectopic spread of heterochromatin, and genome integrity. Our genome-wide localization analysis revealed that Htz1 is widely, but nonrandomly, distributed throughout the genome in an SWR1-dependent manner. We found that Htz1 is enriched in intergenic regions compared with coding regions. Its occupancy is inversely proportional to transcription rates and the enrichment of the RNA polymerase II under different growth conditions. However, Htz1 does not seem to directly regulate transcription repression genome-wide; instead, the presence of Htz1 under the inactivated condition is essential for optimal activation of a subset of genes. In addition, Htz1 is not generally responsible for nucleosome positioning, even at those promoters where Htz1 is highly enriched. Finally, using a biochemical approach, we demonstrate that incorporation of Htz1 into nucleosomes inhibits activities of histone modifiers associated with transcription, Dot1, Set2, and NuA4 and reduces the nucleosome mobilization driven by chromatin remodeling complexes. These lines of evidence collectively suggest that Htz1 may serve to mark quiescent promoters for proper activation.


title: Genomic organisation and transcription characterisation of the gene encoding Leishmania (Leishmania) amazonensis arginase and its protein structure prediction.

text: The genomic organisation of the gene encoding Leishmania (Leishmania) amazonensis arginase as well as its flanking regions were characterised. The size of the transcribed RNA was determined, allowing us to map the genomic sites signalling for RNA trans-splicing and putative polyadenylation regions. The general organisation was compared with genes encoding other proteins already described in organisms of the Trypanosomatid family. The complete nucleotide sequence of the arginase open reading frame was obtained and the three-dimensional structure of the enzyme was inferred by a computational analysis of the deduced amino acid sequence, based on the established crystal structure described for Rattus norvergicus arginase. The human liver arginase sequence was analysed in the same way and the comparison of the presumed structure of both the Leishmania and human enzymes identified some differences that may be exploited in chemotherapeutic studies.


title: Endometrial carcinoma risk among women diagnosed with endometrial hyperplasia: the 34-year experience in a large health plan

text: Classifying endometrial hyperplasia (EH) according to the severity of glandular crowding (simple hyperplasia (SH) vs complex hyperplasia (CH)) and nuclear atypia (simple atypical hyperplasia (SAH) vs complex atypical hyperplasia (CAH)) should predict subsequent endometrial carcinoma risk, but data on progression are lacking. Our nested case–control study of EH progression included 138 cases, who were diagnosed with EH and then with carcinoma (1970–2003) at least 1 year (median, 6.5 years) later, and 241 controls, who were individually matched on age, date, and follow-up duration and counter-matched on EH classification. After centralised pathology panel and medical record review, we generated rate ratios (RRs) and 95% confidence intervals (CIs), adjusted for treatment and repeat biopsies. With disordered proliferative endometrium (DPEM) as the referent, AH significantly increased carcinoma risk (RR=14, 95% CI, 5–38). Risk was highest 1–5 years after AH (RR=48, 95% CI, 8–294), but remained elevated 5 or more years after AH (RR=3.5, 95% CI, 1.0–9.6). Progression risks for SH (RR=2.0, 95% CI, 0.9–4.5) and CH (RR=2.8, 95% CI, 1.0–7.9) were substantially lower and only slightly higher than the progression risk for DPEM. The higher progression risks for AH could foster management guidelines based on markedly different progression risks for atypical vs non-atypical EH.