A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

Nitrogen and sulphur management: challenges for organic sources in temperate agricultural systems

A current global trend towards intensification or specialization of agricultural enterprises has been accompanied by increasing public awareness of associated environmental consequences. Air and water pollution from losses of nutrients, such as nitrogen (N) and sulphur (S), are a major concern. Governments have initiated extensive regulatory frameworks, including various land use policies, in an attempt to control or reduce the losses. This paper presents an overview of critical input and loss processes affecting N and S for temperate climates, and provides some background to the discussion in subsequent papers evaluating specific farming systems. Management effects on potential gaseous and leaching losses, the lack of synchrony between supply of nutrients and plant demand, and options for optimizing the efficiency of N and S use are reviewed. Integration of inorganic and organic fertilizer inputs and the equitable re-distribution of nutrients from manure are discussed. The paper concludes by highlighting a need for innovative research that is also targeted to practical approaches for reducing N and S losses, and improving the overall synchrony between supply and demand

Multiple Neural Oscillators and Muscle Feedback Are Required for the Intestinal Fed State Motor Program

After a meal, the gastrointestinal tract exhibits a set of behaviours known as the fed state. A major feature of the fed state is a little understood motor pattern known as segmentation, which is essential for digestion and nutrient absorption. Segmentation manifests as rhythmic local constrictions that do not propagate along the intestine. In guinea-pig jejunum in vitro segmentation constrictions occur in short bursts together with other motor patterns in episodes of activity lasting 40–60 s and separated by quiescent episodes lasting 40–200 s. This activity is induced by luminal nutrients and abolished by blocking activity in the enteric nervous system (ENS). We investigated the enteric circuits that regulate segmentation focusing on a central feature of the ENS: a recurrent excitatory network of intrinsic sensory neurons (ISNs) which are characterized by prolonged after-hyperpolarizing potentials (AHPs) following their action potentials. We first examined the effects of depressing AHPs with blockers of the underlying channels (TRAM-34 and clotrimazole) on motor patterns induced in guinea-pig jejunum, in vitro, by luminal decanoic acid. Contractile episode durations increased markedly, but the frequency and number of constrictions within segmenting bursts and quiescent period durations were unaffected. We used these observations to develop a computational model of activity in ISNs, excitatory and inhibitory motor neurons and the muscle. The model predicted that: i) feedback to ISNs from contractions in the circular muscle is required to produce alternating activity and quiescence with the right durations; ii) transmission from ISNs to excitatory motor neurons is via fast excitatory synaptic potentials (EPSPs) and to inhibitory motor neurons via slow EPSPs. We conclude that two rhythm generators regulate segmentation: one drives contractions within segmentation bursts, the other the occurrence of bursts. The latter depends on AHPs in ISNs and feedback to these neurons from contraction of the circular muscle

Living with prostate cancer: randomised controlled trial of a multimodal supportive care intervention for men with prostate cancer

Background: Prostate cancer is the most common male cancer in developed countries and diagnosis and treatment carries with it substantial morbidity and related unmet supportive care needs. These difficulties may be amplified by physical inactivity and obesity. We propose to apply a multimodal intervention approach that targets both unmet supportive care needs and physical activity.Methods/design: A two arm randomised controlled trial will compare usual care to a multimodal supportive care intervention &ldquo;Living with Prostate Cancer&rdquo; that will combine self-management with tele-based group peer support. A series of previously validated and reliable self-report measures will be administered to men at four time points: baseline/recruitment (when men are approximately 3-6 months post-diagnosis) and at 3, 6, and 12 months after recruitment and intervention commencement. Social constraints, social support, self-efficacy, group cohesion and therapeutic alliance will be included as potential moderators/mediators of intervention effect. Primary outcomes are unmet supportive care needs and physical activity levels. Secondary outcomes are domain-specific and healthrelated quality of life (QoL); psychological distress; benefit finding; body mass index and waist circumference. Disease variables (e.g. cancer grade, stage) will be assessed through medical and cancer registry records. An economic evaluation will be conducted alongside the randomised trial.Discussion: This study will address a critical but as yet unanswered research question: to identify a populationbased way to reduce unmet supportive care needs; promote regular physical activity; and improve disease-specific and health-related QoL for prostate cancer survivors. The study will also determine the cost-effectiveness of the intervention.<br /

Common Variants at 10 Genomic Loci Influence Hemoglobin A(1C) Levels via Glycemic and Nonglycemic Pathways

OBJECTIVE Glycated hemoglobin (HbA1c), used to monitor and diagnose diabetes, is influenced by average glycemia over a 2- to 3-month period. Genetic factors affecting expression, turnover, and abnormal glycation of hemoglobin could also be associated with increased levels of HbA1c. We aimed to identify such genetic factors and investigate the extent to which they influence diabetes classification based on HbA1c levels. RESEARCH DESIGN AND METHODS We studied associations with HbA1c in up to 46,368 nondiabetic adults of European descent from 23 genome-wide association studies (GWAS) and 8 cohorts with de novo genotyped single nucleotide polymorphisms (SNPs). We combined studies using inverse-variance meta-analysis and tested mediation by glycemia using conditional analyses. We estimated the global effect of HbA1c loci using a multilocus risk score, and used net reclassification to estimate genetic effects on diabetes screening. RESULTS Ten loci reached genome-wide significant association with HbA1c, including six new loci near FN3K (lead SNP/P value, rs1046896/P = 1.6 × 10−26), HFE (rs1800562/P = 2.6 × 10−20), TMPRSS6 (rs855791/P = 2.7 × 10−14), ANK1 (rs4737009/P = 6.1 × 10−12), SPTA1 (rs2779116/P = 2.8 × 10−9) and ATP11A/TUBGCP3 (rs7998202/P = 5.2 × 10−9), and four known HbA1c loci: HK1 (rs16926246/P = 3.1 × 10−54), MTNR1B (rs1387153/P = 4.0 × 10−11), GCK (rs1799884/P = 1.5 × 10−20) and G6PC2/ABCB11 (rs552976/P = 8.2 × 10−18). We show that associations with HbA1c are partly a function of hyperglycemia associated with 3 of the 10 loci (GCK, G6PC2 and MTNR1B). The seven nonglycemic loci accounted for a 0.19 (% HbA1c) difference between the extreme 10% tails of the risk score, and would reclassify ∼2% of a general white population screened for diabetes with HbA1c. CONCLUSIONS GWAS identified 10 genetic loci reproducibly associated with HbA1c. Six are novel and seven map to loci where rarer variants cause hereditary anemias and iron storage disorders. Common variants at these loci likely influence HbA1c levels via erythrocyte biology, and confer a small but detectable reclassification of diabetes diagnosis by HbA1c

Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule

Characterizing the Role of Cell-Wall β-1,3-Exoglucanase Xog1p in Candida albicans Adhesion by the Human Antimicrobial Peptide LL-37

Candida albicans is the major fungal pathogen of humans. Its adhesion to host-cell surfaces is the first critical step during mucosal infection. Antimicrobial peptides play important roles in the first line of mucosal immunity against C. albicans infection. LL-37 is the only member of the human cathelicidin antimicrobial peptide family and is commonly expressed in various tissues, including epithelium. We previously showed that LL-37 significantly reduced C. albicans adhesion to plastic, oral epidermoid OECM-1 cells, and urinary bladders of female BALB/c mice. The inhibitory effect of LL-37 on cell adhesion occurred via the binding of LL-37 to cell-wall carbohydrates. Here we showed that formation of LL-37–cell-wall protein complexes potentially inhibits C. albicans adhesion to polystyrene. Using phage display and ELISA, we identified 10 peptide sequences that could bind LL-37. A BLAST search revealed that four sequences in the major C. albicans cell-wall β-1,3-exoglucanase, Xog1p, were highly similar to the consensus sequence derived from the 10 biopanned peptides. One Xog1p-derived peptide, Xog1p90–115, and recombinant Xog1p associated with LL-37, thereby reversing the inhibitory effect of LL-37 on C. albicans adhesion. LL-37 reduced Xog1p activity and thus interrupted cell-wall remodeling. Moreover, deletion of XOG1 or another β-1,3-exoglucanase-encoding gene EXG2 showed that only when XOG1 was deleted did cellular exoglucanase activity, cell adhesion and LL-37 binding decrease. Antibodies against Xog1p also decreased cell adhesion. These data reveal that Xog1p, originally identified from LL-37 binding, has a role in C. albicans adhesion to polystyrene and, by inference, attach to host cells via direct or indirect manners. Compounds that target Xog1p might find use as drugs that prevent C. albicans infection. Additionally, LL-37 could potentially be used to screen for other cell-wall components involved in fungal cell adhesion

Computational Identification of Uncharacterized Cruzain Binding Sites

Chagas disease, caused by the unicellular parasite Trypanosoma cruzi, claims 50,000 lives annually and is the leading cause of infectious myocarditis in the world. As current antichagastic therapies like nifurtimox and benznidazole are highly toxic, ineffective at parasite eradication, and subject to increasing resistance, novel therapeutics are urgently needed. Cruzain, the major cysteine protease of Trypanosoma cruzi, is one attractive drug target. In the current work, molecular dynamics simulations and a sequence alignment of a non-redundant, unbiased set of peptidase C1 family members are used to identify uncharacterized cruzain binding sites. The two sites identified may serve as targets for future pharmacological intervention