Structure and function of the neuraminidase produced by Mannheimia haemolytica

The Gram negative bacillus $Mannheimia$ $haemolytica$ is a natural inhabitant of the upper respiratory tract in ruminants and the most common secondary agent of the bovine respiratory disease complex. It is known to produce the extracellular neuraminidase NanH, which has a yet unknown biological role but is suspected to be important for bacterial adhesion to host cells, colonisation, capsule synthesis and biofilm formation. The structure of NanH is not known therefore, the functional domains of NanH, the tertiary structure and the residues involved in catalysis were predicted by sequence homology to the coordinates of other neuraminidases solved by crystallography. The catalytic domain was delimited from residues 23 to 435 and purified. The predicted catalytic residues were substituted in the recombinant NanH for confirmation of their role in hydrolysis of sialic acid. The function of the additional domains is unknown but analysis of NanH sequence and other associated genes found in the chromosome of $M$. $haemolytica$, suggest the presence of an autotransporter domain. The role of NanH in colonisation and infection is not known however, molecular characterisation is presented in this work. These data provide the basic knowledge required for future studies on using Nanh as a therapeutic and prophylactic target

Identification of binding residues between periplasmic adapter protein (PAP) and RND efflux pumps explains PAP-pump promiscuity and roles in antimicrobial resistance

Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or "binding boxes" within the PAP sequence. In the important human pathogen Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a predictive model of pump-contacts that could be exploited in the future for the development of targeted therapeutics and efflux pump inhibitors

Multivalent poultry vaccine development using Protein Glycan Coupling Technology.

BackgroundPoultry is the world's most popular animal-based food and global production has tripled in the past 20 years alone. Low-cost vaccines that can be combined to protect poultry against multiple infections are a current global imperative. Glycoconjugate vaccines, which consist of an immunogenic protein covalently coupled to glycan antigens of the targeted pathogen, have a proven track record in human vaccinology, but have yet to be used for livestock due to prohibitively high manufacturing costs. To overcome this, we use Protein Glycan Coupling Technology (PGCT), which enables the production of glycoconjugates in bacterial cells at considerably reduced costs, to generate a candidate glycan-based live vaccine intended to simultaneously protect against Campylobacter jejuni, avian pathogenic Escherichia coli (APEC) and Clostridium perfringens. Campylobacter is the most common cause of food poisoning, whereas colibacillosis and necrotic enteritis are widespread and devastating infectious diseases in poultry.ResultsWe demonstrate the functional transfer of C. jejuni protein glycosylation (pgl) locus into the genome of APEC χ7122 serotype O78:H9. The integration caused mild attenuation of the χ7122 strain following oral inoculation of chickens without impairing its ability to colonise the respiratory tract. We exploit the χ7122 pgl integrant as bacterial vectors delivering a glycoprotein decorated with the C. jejuni heptasaccharide glycan antigen. To this end we engineered χ7122 pgl to express glycosylated NetB toxoid from C. perfringens and tested its ability to reduce caecal colonisation of chickens by C. jejuni and protect against intra-air sac challenge with the homologous APEC strain.ConclusionsWe generated a candidate glycan-based multivalent live vaccine with the potential to induce protection against key avian and zoonotic pathogens (C. jejuni, APEC, C. perfringens). The live vaccine failed to significantly reduce Campylobacter colonisation under the conditions tested but was protective against homologous APEC challenge. Nevertheless, we present a strategy towards the production of low-cost "live-attenuated multivalent vaccine factories" with the ability to express glycoconjugates in poultry

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Modification of avian pathogenic Escherichia coli χ7122 lipopolysaccharide increases accessibility to glycoconjugate antigens

Abstract Background Worldwide, an estimated 70.7 billion broilers were produced in 2020. With the reduction in use of prophylactic antibiotics as a result of consumer pressure and regulatory oversight alternative approaches, such as vaccination, are required to control bacterial infections. A potential way to produce a multivalent vaccine is via the generation of a glycoconjugate vaccine which consists of an antigenic protein covalently linked to an immunogenic carbohydrate. Protein-glycan coupling technology (PGCT) is an approach to generate glycoconjugates using enzymes that can couple proteins and glycan when produced in bacterial cells. Previous studies have used PGCT to generate a live-attenuated avian pathogenic Escherichia coli (APEC) strain capable of N-glycosylation of target proteins using a chromosomally integrated Campylobacter jejuni pgl locus. However, this proved ineffective against C. jejuni challenge. Results In this study we demonstrate the lack of surface exposure of glycosylated protein in APEC strain χ7122 carrying the pgl locus. Furthermore, we hypothesise that this may be due to the complex cell-surface architecture of E. coli. To this end, we removed the lipopolysaccharide O-antigen of APEC χ7122 pgl+ via deletion of the wecA gene and demonstrate increased surface exposure of glycosylated antigens (NetB and FlpA) in this strain. We hypothesise that increasing the surface expression of the glycosylated protein would increase the chance of host immune cells being exposed to the glycoconjugate, and therefore the generation of an efficacious immune response would be more likely. Conclusions Our results demonstrate an increase in cell surface exposure and therefore accessibility of glycosylated antigens upon removal of lipopolysaccharide antigen from the APEC cell surface. </jats:sec

Evaluation of a FlpA Glycoconjugate Vaccine with Ten <i>N</i>-Heptasaccharide Glycan Moieties to reduce <i>Campylobacter jejuni</i> Colonisation in Chickens

Campylobacter is a major cause of acute gastroenteritis in humans, and infections can be followed by inflammatory neuropathies and other sequelae. Handling or consumption of poultry meat is the primary risk factor for human campylobacteriosis, and C. jejuni remains highly prevalent in retail chicken in many countries. Control of Campylobacter in the avian reservoir is expected to limit the incidence of human disease. Toward this aim, we evaluated a glycoconjugate vaccine comprising the fibronectin-binding adhesin FlpA conjugated to up to ten moieties of the conserved N-linked heptasaccharide glycan of C. jejuni or with FlpA alone. The glycan dose significantly exceeded previous trials using FlpA with two N-glycan moieties. Vaccinated birds were challenged with C. jejuni orally or by exposure to seeder-birds colonised by C. jejuni to mimic natural transmission. No protection against caecal colonisation was observed with FlpA or the FlpA glycoconjugate vaccine. FlpA-specific antibody responses were significantly induced in vaccinated birds at the point of challenge relative to mock-vaccinated birds. A slight but significant antibody response to the N-glycan was detected after vaccination with FlpA-10×GT and challenge. As other laboratories have reported protection against Campylobacter with FlpA and glycoconjugate vaccines in chickens, our data indicate that vaccine-mediated immunity may be sensitive to host- or study-specific variables

Transparencia, combate a la corrupción y Gobierno Abierto: La experiencia de México

La mayoría de las encuestas hacia las elecciones presidenciales en México de 2018 coinciden: el tema que más lastima a los mexicanos del Siglo XXI es la corrupción y la impunidad, junto con la seguridad ciudadana y económica. La promesa de una menor corrupción que acompañó nuestras transiciones, tanto a nivel federal como en las entidades federativas, no se ha cumplido, afectando nuestra confianza en el régimen democrático como el más efectivo para tener mejor calidad de vida. Este libro da cuenta de ello y con la creación del Sistema Nacional Anticorrupción, publicado en el Diario Oficial de la Federación el 25 de mayo de 2015, se abre un espacio para el trabajo colaborativo y de incidencia con las distintas organizaciones que integran el colectivo CIMTRA; para promover a partir de la transparencia las acciones contra la corrupción en los gobiernos. En la medida que las instituciones y los ciudadanos asumamos la corresponsabilidad que tenemos en la construcción de ciudadanía, y respeto a los derechos fundamentales de los ciudadanos se logrará controlar el ejercicio de los poderes públicos y se resignificará la política como el mejor espacio para el mejoramiento de nuestro país