The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

Background Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.This work was mainly supported by ERA Biotech (www.erabiotech.com). Additional support was supplied by grant SGR 2009/703 funded by the Generalitat de Catalunya (www10.gencat.net) and grants CDS2007/00036 of Consolider Ingenio program and TRA 2009/0124 of TRACE program funded by Ministerio de Ciencia e Inovación (MICINN, www.micinn.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage

Background: Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display. Methodology/Principal Findings: Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient. Conclusions/Significance: Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Local erythropoietin and endothelial progenitor cells improve regional cardiac function in acute myocardial infarction

<p>Abstract</p> <p>Background</p> <p>Expanded endothelial progenitor cells (eEPC) improve global left ventricular function in experimental myocardial infarction (MI). Erythropoietin beta (EPO) applied together with eEPC may improve regional myocardial function even further by anti-apoptotic and cardioprotective effects. Aim of this study was to evaluate intramyocardial application of eEPCs and EPO as compared to eEPCs or EPO alone in experimental MI.</p> <p>Methods and Results</p> <p>In vitro experiments revealed that EPO dosed-dependently decreased eEPC and leukocyte apoptosis. Moreover, in the presence of EPO mRNA expression in eEPC of proangiogenic and proinflammatory mediators measured by TaqMan PCR was enhanced. Experimental MI was induced by ligation and reperfusion of the left anterior descending coronary artery of nude rats (n = 8-9). After myocardial transplantation of eEPC and EPO CD68+ leukocyte count and vessel density were enhanced in the border zone of the infarct area. Moreover, apoptosis of transplanted CD31 + TUNEL + eEPC was decreased as compared to transplantation of eEPCs alone. Regional wall motion of the left ventricle was measured using Magnetic Resonance Imaging. After injection of eEPC in the presence of EPO regional wall motion significantly improved as compared to injection of eEPCs or EPO alone.</p> <p>Conclusion</p> <p>Intramyocardial transplantation of eEPC in the presence of EPO during experimental MI improves regional wall motion. This was associated with an increased local inflammation, vasculogenesis and survival of the transplanted cells. Local application of EPO in addition to cell therapy may prove beneficial in myocardial remodeling.</p

Deep-Sequencing Analysis of the Mouse Transcriptome Response to Infection with Brucella melitensis Strains of Differing Virulence

Brucella melitensis is an important zoonotic pathogen that causes brucellosis, a disease that affects sheep, cattle and occasionally humans. B. melitensis strain M5-90, a live attenuated vaccine cultured from B. melitensis strain M28, has been used as an effective tool in the control of brucellosis in goats and sheep in China. However, the molecular changes leading to attenuated virulence and pathogenicity in B. melitensis remain poorly understood. In this study we employed the Illumina Genome Analyzer platform to perform genome-wide digital gene expression (DGE) analysis of mouse peritoneal macrophage responses to B. melitensis infection. Many parallel changes in gene expression profiles were observed in M28- and M5-90-infected macrophages, suggesting that they employ similar survival strategies, notably the induction of anti-inflammatory and antiapoptotic factors. Moreover, 1019 differentially expressed macrophage transcripts were identified 4 h after infection with the different B. melitensis strains, and these differential transcripts notably identified genes involved in the lysosome and mitogen-activated protein kinase (MAPK) pathways. Further analysis employed gene ontology (GO) analysis: high-enrichment GOs identified endocytosis, inflammatory, apoptosis, and transport pathways. Path-Net and Signal-Net analysis highlighted the MAPK pathway as the key regulatory pathway. Moreover, the key differentially expressed genes of the significant pathways were apoptosis-related. These findings demonstrate previously unrecognized changes in gene transcription that are associated with B. melitensis infection of macrophages, and the central signaling pathways identified here merit further investigation. Our data provide new insights into the molecular attenuation mechanism of strain M5-90 and will facilitate the generation of new attenuated vaccine strains with enhanced efficacy

Low potency toxins reveal dense interaction networks in metabolism

Background The chemicals of metabolism are constructed of a small set of atoms and bonds. This may be because chemical structures outside the chemical space in which life operates are incompatible with biochemistry, or because mechanisms to make or utilize such excluded structures has not evolved. In this paper I address the extent to which biochemistry is restricted to a small fraction of the chemical space of possible chemicals, a restricted subset that I call Biochemical Space. I explore evidence that this restriction is at least in part due to selection again specific structures, and suggest a mechanism by which this occurs. Results Chemicals that contain structures that our outside Biochemical Space (UnBiological groups) are more likely to be toxic to a wide range of organisms, even though they have no specifically toxic groups and no obvious mechanism of toxicity. This correlation of UnBiological with toxicity is stronger for low potency (millimolar) toxins. I relate this to the observation that most chemicals interact with many biological structures at low millimolar toxicity. I hypothesise that life has to select its components not only to have a specific set of functions but also to avoid interactions with all the other components of life that might degrade their function. Conclusions The chemistry of life has to form a dense, self-consistent network of chemical structures, and cannot easily be arbitrarily extended. The toxicity of arbitrary chemicals is a reflection of the disruption to that network occasioned by trying to insert a chemical into it without also selecting all the other components to tolerate that chemical. This suggests new ways to test for the toxicity of chemicals, and that engineering organisms to make high concentrations of materials such as chemical precursors or fuels may require more substantial engineering than just of the synthetic pathways involved

Identification and characterization of antibacterial compound(s) of cockroaches (Periplaneta americana)

Infectious diseases remain a significant threat to human health, contributing to more than 17 million deaths, annually. With the worsening trends of drug resistance, there is a need for newer and more powerful antimicrobial agents. We hypothesized that animals living in polluted environments are potential source of antimicrobials. Under polluted milieus, organisms such as cockroaches encounter different types of microbes, including superbugs. Such creatures survive the onslaught of superbugs and are able to ward off disease by producing antimicrobial substances. Here, we characterized antibacterial properties in extracts of various body organs of cockroaches (Periplaneta americana) and showed potent antibacterial activity in crude brain extract against methicillin-resistant Staphylococcus aureus and neuropathogenic E. coli K1. The size-exclusion spin columns revealed that the active compound(s) are less than 10 kDa in molecular mass. Using cytotoxicity assays, it was observed that pre-treatment of bacteria with lysates inhibited bacteria-mediated host cell cytotoxicity. Using spectra obtained with LC-MS on Agilent 1290 infinity liquid chromatograph, coupled with an Agilent 6460 triple quadruple mass spectrometer, tissues lysates were analyzed. Among hundreds of compounds, only a few homologous compounds were identified that contained isoquinoline group, chromene derivatives, thiazine groups, imidazoles, pyrrole containing analogs, sulfonamides, furanones, flavanones, and known to possess broad-spectrum antimicrobial properties, and possess anti-inflammatory, anti-tumour, and analgesic properties. Further identification, characterization and functional studies using individual compounds can act as a breakthrough in developing novel therapeutics against various pathogens including superbugs

ER stress arm XBP1s plays a pivotal role in proteasome inhibition-induced bone formation

Background Bone destruction is a hallmark of multiple myeloma (MM). It has been reported that proteasome inhibitors (PIs) can reduce bone resorption and increase bone formation in MM patients, but the underlying mechanisms remain unclear. Methods Mesenchymal stem cells (MSCs) were treated with various doses of PIs, and the effects of bortezomib or carfilzomib on endoplasmic reticulum (ER) stress signaling pathways were analyzed by western blotting and real-time PCR. Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to determine the osteogenic differentiation in vitro. Specific inhibitors targeting different ER stress signaling and a Tet-on inducible overexpressing system were used to validate the roles of key ER stress components in regulating osteogenic differentiation of MSCs. Chromatin immunoprecipitation (ChIP) assay was used to evaluate transcription factor-promoter interaction. MicroCT was applied to measure the microarchitecture of bone in model mice in vivo. Results We found that both PERK-ATF4 and IRE1α-XBP1s ER stress branches are activated during PI-induced osteogenic differentiation. Inhibition of ATF4 or XBP1s signaling can significantly impair PI-induced osteogenic differentiation. Furthermore, we demonstrated that XBP1s can transcriptionally upregulate ATF4 expression and overexpressing XBP1s can induce the expression of ATF4 and other osteogenic differentiation-related genes and therefore drive osteoblast differentiation. MicroCT analysis further demonstrated that inhibition of XBP1s can strikingly abolish bortezomib-induced bone formation in mouse. Conclusions These results demonstrated that XBP1s is a master regulator of PI-induced osteoblast differentiation. Activation of IRE1α-XBP1s ER stress signaling can promote osteogenesis, thus providing a novel strategy for the treatment of myeloma bone disease

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700