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Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

An increase in amyloid-beta (A beta) production is a major pathogenic mechanism associated with Alzheimer's disease (AD)(1,2), but little is known about possible homeostatic control of the amyloidogenic pathway. Here we report that the amyloid precursor protein (APP) intracellular domain (AICD) downregulates Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1 or WASF1) as part of a negative feedback mechanism to limit A beta production. The AICD binds to the Wasf1 promoter, negatively regulates its transcription and downregulates Wasf1 mRNA and protein expression in Neuro 2a (N2a) cells. WAVE1 interacts and colocalizes with APP in the Golgi apparatus. Experimentally reducing WAVE1 in N2a cells decreased the budding of APP-containing vesicles and reduced cell-surface APP, thereby reducing the production of A beta. WAVE1 downregulation was observed in mouse models of AD. Reduction of Wasf1 gene expression dramatically reduced A beta levels and restored memory deficits in a mouse model of AD. A decrease in amounts of WASF1 mRNA was also observed in human AD brains, suggesting clinical relevance of the negative feedback circuit involved in homeostatic regulation of Ab production.

Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin-proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IB degradation assay, Thsp is a slow inhibitor and 4hrs of treatment achieves the same effects as MG-132 at 30min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes invitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.

Studies of linkage disequilibrium (LD) and its variation in the genome are of central importance for understanding evolutionary history, population structure, and selective sweeps. Extreme forms of the latter may result in runs of homozygosity (ROH). In human gene mapping, long ROHs are the basis for homozygosity mapping (HM) with length measured in terms of Mb (10(6) base pairs physical distance). LD varies greatly over the human genome so that long ROHs tend to occur preferentially in regions of high LD and ROHs of the same length in different regions are not strictly comparable. Thus, in human gene mapping, LD appears as a confounder that needs to be taken into account in the interpretation of ROHs. The effect of varying LD can be mitigated by working on a scale of centimorgans (cM, genetic distance) instead of Mb. We demonstrate this effect for HapMap 3 data on chromosome 19 and show examples with different ROH lengths depending on whether physical or genetic lengths are used. These results suggest that HM should preferably be done on genetic rather than physical distances.

Transposable elements (TEs) and repetitive sequences are ubiquitously present in eukaryotic genomes which are in general epigenetically silenced by DNA methylation and/or histone 3 lysine 9 methylation (H3K9me). RNA-directed DNA methylation (RdDM) is the major pathway that initiates de novo DNA methylation in Arabidopsis and sets up a self-reinforcing silencing loop between DNA methylation and H3K9me. However, a key issue is the requirement of a basal level transcript from the target loci to initiate the RNA-based silencing. How the heterochromatic silenced loci are transcribed remains largely unknown. Here, we show that JMJ24, a JmjC domain-containing protein counteracts H3K9me to promote basal level transcription of endogenous silenced loci in Arabidopsis. JMJ24 functionally resembles the fission yeast JmjC protein Epe1. The transcript promoted by JMJ24 is, at least in part, processed to small RNA to initiate the RdDM. Genome-wide transcriptome profiling indicates that transcript levels of TEs are more likely regulated by JMJ24, compared with protein-coding genes. Our data suggest that JMJ24 plays a conserved role in promoting basal level transcription of endogenous silenced loci to reinforce the silencing. We also provide evidence of a physical association between JMJ24 and RNA-dependent RNA polymerase 2 (RDR2), which represents an evolved property of the RNA silencing pathway.

Anemia is a known comorbidity found in chronic obstructive pulmonary disease (COPD) patients. Hypoxemia is common and basically due to ventilation/perfusion (V/Q) mismatch in COPD. Anemia, by decreasing arterial oxygen content, may be a contributing factor for decreased delivery of oxygen to tissues. The objective of this study is to determine if anemia is a factor in qualifying COPD patients for home oxygen therapy. The study was designed as a retrospective, cross-sectional, observational chart review. Patients who were referred for home oxygen therapy evaluation were selected from the computerized patient record system. Demographic data, oxygen saturation at rest and during exercise, pulmonary function test results, hemoglobin level, medications, reason for anemia, comorbid diseases, and smoking status were recorded. The chi(2) tests, independent sample t tests, and logistic regression were used for statistical analysis. Only 356 of total 478 patient referrals had a diagnosis of COPD over a 2-year period. Although 39 of them were excluded, 317 patients were included in the study. The overall rate of anemia was 38% in all COPD patients. Anemia was found significantly more frequent in COPD patients on home oxygen therapy (46%) than those not on home oxygen therapy (18.5%) (P < 0.0001). Mean saturation of peripheral oxygen values were significantly lower in anemic COPD patients both at rest and during exercise (P < 0.0001). Also, in COPD patients, age, Global Initiative for Chronic Obstructive Lung Disease class, smoking status, hemoglobin level, hematocrit, percent of forced expiratory volume in first second, forced expiratory volume in first second/forced vital capacity, residual volume/total lung volume, percent of carbon monoxide diffusion capacity were significantly different between home oxygen therapy and those not on home oxygen therapy (P < 0.05). Multivariate logistic regression showed that anemia remained a strong predictor for long-term oxygen therapy use in COPD patients after adjusting for other significant parameters. Anemic COPD patients are more hypoxic especially during exercise than those who are not anemic. We conclude that anemia is a contributing factor in qualifying COPD patients for home oxygen therapy.

Human dendritic cells (DCs) develop from progressively restricted bone marrow (BM) progenitors: these progenitor cells include granulocyte, monocyte and DC progenitor (GMDP) cells; monocyte and DC progenitor (MDP) cells; and common DC progenitor (CDP) and DC precursor (pre-DC) cells. These four DC progenitors can be defined on the basis of the expression of surface markers such as CD34 and hematopoietin receptors. In this protocol, we describe five multiparametric flow cytometry panels that can be used as a tool (i) to simultaneously detect or phenotype the four DC progenitors, (ii) to isolate DC progenitors to enable in vitro differentiation or (iii) to assess the in vitro differentiation and proliferation of DC progenitors. The entire procedure from isolation of cells to flow cytometry can be completed in 3-7 h. This protocol provides optimized antibody panels, as well as gating strategies, for immunostaining of BM and cord blood specimens to study human DC hematopoiesis in health, disease and vaccine settings.

Background: Institutional review boards (IRBs) distinguish health care quality improvement (QI) and health care quality improvement research (QIR) based primarily on the rigor of the methods used and the purported generalizability of the knowledge gained. Neither of these criteria holds up upon scrutiny. Rather, this apparently false dichotomy may foster under-protection of participants in QI projects and over-protection of participants within QIR. Discussion: Minimal risk projects should entail minimal oversight including waivers for informed consent for both QI and QIR projects. Minimizing the burdens of conducting QIR, while ensuring minimal safeguards for QI projects, is needed to restore this imbalance in oversight. Potentially, such ethical oversight could be provided by the integration of Institutional Review Boards and Clinical Ethical Committees, using a more integrated and streamlined approach such as a two-step process involving a screening review, followed by a review by committee trained in QIR. Standards for such ethical review and training in these standards, coupled with rapid review cycles, could facilitate an appropriate level of oversight within the context of creating and sustaining learning health care systems. Summary: We argue that QI and QIR are not reliably distinguishable. We advocate for approaches that improve protections for QI participants while minimizing over-protection for participants in QIR through reasonable ethical oversight that aligns risk to participants in both QI and QIR with the needs of a learning health care system.

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 angstrom-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA: DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and sigma(A) dissociation. DOI: 10.7554/eLife.08504.001