Helicobacter pylori prophages: screening, detection, induction and potential therapeutic use

Helicobacter pylori is a microaerophilic bacterium that chronically infects the human gastric mucosa. Infections caused by this pathogen are difficult to treat, mainly due to the increased resistance of this species to conventional antibiotics. Therefore, it is important to develop antibiotic alternative or complementary approaches to tackle H. pylori infections. Bacterio(phages) have proven to be efficient antibacterial agents, however it is very difficult to isolate strictly lytic phages infecting H. pylori. Nevertheless, this bacterial species presents prophages in their genomes and although strictly lytic phages have been consensually preferred for phage therapy purposes, temperate prophages holds a great but an exploited potential. In the present work, we developed a new PCR-based screening method to detect the presence of prophages genes in a set of H. pylori Portuguese clinical strains. The genomes of selected strains were then sequenced using a combined Illumina platform and MinION nanopore-based sequencing strategy. Prophages content was then analysed using the PHASTER tool. After sequencing analysis, UV light was used to induce phages, from which one was further characterized in terms of morphology, host range, stability on an in vitro gastric model, genome analysis and efficacy against a H. pylori culture. The complementarity between Illumina and Nanopore results, allowed us to identify a total of 10 intact, 7 questionable and 47 incomplete prophages on the 14 sequenced strains. One predicted intact prophage was induced successfully, and presents a genome length of 31 162 bp with 37.1 % G+C content. Interestingly, this new podovirus infects five H. pylori strains, and in the gastric in vitro model only a small loss of phage titer was observed in the gastric phase, suggesting that this phage could be adapted to the stomach environment. Farther, this phage demonstrated to be capable of maintaining the H. pylori population at low levels for up to 24 h post-infection with MOIs of 0.01, 0.1 and 1. Overall, a new PCR screening method was developed to detect prophages on H. pylori and positive correlations with sequencing results were observed. Moreover, this new isolated phage seems to have therapeutic potential to treat H. pylori gastric infections.This work was supported by the Portuguese Foundation for Science and Technology – Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and Project PTDC/SAU-PUB/29182/2017 [POCI-01-0145-FEDER-029182]. Rute Ferreira is recipient of a FCT PhD grant with the reference SFRH/BD/146496/2019info:eu-repo/semantics/publishedVersio

Screening and in silico characterization of prophages in Helicobacter pylori genomes

Temperate bacterio(phages) play an important role on the evolution of pathogenic bacteria. Nevertheless, information on their role in Helicobacter pylori (an important gastric pathogen bacterium) is scarce. The present study developed a workflow for the identification of prophages in Portuguese H. pylori clinical strains, proposing the use of a new PCR-based screening method. The genome of strains with different PCR profiles were then sequenced. In the fourteen genomes analysed, nine intact prophages were identified by PHASTER. These prophages were annotated by analogy with other identified phages, where seven contained the integrase gene, corroborating the results obtained in the PCR screening, with only one exception. Still, in PCR screening, the holin gene was identified in 75 % of the strains containing intact phages, but BLASTp homologies only recognized this gene in one of the prophages. Fifty-six percent are podovirus, while in 44 % it was not possible to assign any family, according to the VirFam tool. Using the Resistance Gene Identifier of CARD it was identified the Acinetobacter mutant Lpx gene conferring resistance to colistin in two intact prophages. The BLASTp search identified a putative ABC binding cassette transporter in one of the intact prophages. On the bacterial genomes, 71 % have the CRISPR-Cas system classified as evidence level 1 by CRISPRCasFinder, which typically indicate potentially invalid CRISPR arrays. The use of an initial PCR screening method increased the identification of intact prophage-containing strains from 20 % to 57 %. Furthermore, the few virulence factors identified in prophages, and the possible inactivity of CRISPR-Cas in the bacterial genomes, allow the choice of strains for the isolation of phages for future studies. Overall, our results represent a significant contribution to the knowledge of prophages in H. pylori, and provide valuable insights into their potential use in phage therapy.info:eu-repo/semantics/publishedVersio

Comparison of bioinformatics tools to predict the presence of prophages in Helicobacter pylori genomes

Bacterio(phages) are specific viruses for bacteria, being their natural enemies. When the genome of a phage is integrated into the host bacterial genome, it is named prophage. These are a latent form of phages, in which the viral genes can increase the virulence and/or fitness characteristics of the host. This life cycle - lysogenic - does not cause the bacterial cell to rupture. Prophages have already been identified in most pathogenic bacteria, providing them better chances of survival. In the case of Helicobacter pylori, a human gastric pathogen that causes, among others, chronic gastritis, peptic ulcers, and adenocarcinoma, the presence of important prophage genes in their genomes has already been identified. In our work, a total of 109 complete genomes of human isolates of H. pylori and plasmids, deposited in NCBI archives, between November 5, 2015, and February 21, 2020, and 19 complete genomes of Portuguese clinical isolates, were screened, regarding the presence of prophages. For that, two of the most widely used web servers for identifying putative prophages in bacterial genomes were used: Phaster and Prophage Hunter. With the use of Phaster, 78 prophage sequences were identified, 6 of which were intact (7.7 %). Regarding Prophage Hunter, 199 prophages were identified, in a total of 17 active (8.5 %). The differences observed in the number of prophages identified by each tool is probably due to variances in the identification methods that each tool uses, as already reported. However, the intact sequences identified in Phaster were also predicted, in the same strains, in Prophage Hunter. These results suggest a high probability of these strains having inducible sequences of prophages in their genomes. The use of web servers for the rapid identification and annotation of prophage sequences in bacterial genomes and plasmids has been growing, helping to direct laboratory experiments more easily. In this work, we observed some differences in the results between the two tools used, concluding that new prophage prediction tools using Machine-Learning are required to predict more accurately this important viral sequences.info:eu-repo/semantics/publishedVersio

H. pylori phages: from genome release to hope for use as therapy

The increasing antibiotic-resistant Helicobacter pylori infections worldwide and the ineffectiveness of treatments led the World Health Organization to designate clarithromycin-resistant H. pylori as a high-priority bacterium for antibiotic research and development. (Bacterio)phages, viruses that infect bacteria, showing effectiveness in the treatment of pathogenic bacteria, could be a promising alternative strategy in the fight against H. pylori infections. Material and methods In this work, a collection of 74 Portuguese H. pylori-clinical strains was used to screen for the presence of phage genes, using a new PCR-based method. Selected strains were subsequently sequenced and prophage isolation was attempted using UV radiation. Three phages were isolated, one of which was further characterized genetically and biologically. Results PCR-based detection indicated the presence of target phage sequences in 14 strains, and the induction strategies resulted in the release of a new phage. It presents a genome length of 31,162 bp with a G+C content of 37.1 %. This podovirus showed capability to form phage plaques in five strains, was stable under an in vitro gastric digestion model, and was able to maintain a H. pylori population at low levels for up to 24h post-infection. Conclusion The new PCR screening method proved to be very effective in the selection of strains carrying prophages, resulting in the isolation of a new H. pylori phage. This phage presented very promising characteristics in terms of stability and efficacy, being therefore a small step towards the future use of phage therapy in the fight against H. pylori infections.info:eu-repo/semantics/publishedVersio

Universidad y organizaciones sociales en relación a los procesos de organización popular

A partir del hecho ocurrido el pasado 2 de abril de 2013 y a modo de profundización de los procesos de organización territorial que se venían llevando adelante en el barrio de Villa Elvira, específicamente en la Unidad Básica ubicada en 7 y 629, se comenzó a trabajar desde la Facultad de Periodismo y Comunicación Social en articulación con la Juventud Peronista (JP/BA) y otros jóvenes provenientes de distintos espacios institucionales tales como, Facultad de Trabajo Social, Facultad de Arquitectura y la Facultad de Bellas Artes, para facilitar la llegada del Estado Nacional a barrios marginales que no son reconocidos como tales por el Municipio de la Ciudad de La Plata. Además se llevó adelante la generación de una red de relación con las distintas políticas públicas y sociales que se estaban ejecutando en esos lugares, impulsando la participación de jóvenes de escuelas secundarias, Plan de Finalización de Estudios Secundarios y Primarios (FinES) y en articulación con el Instituto Nacional de Economía Social y Solidaria (INAES) para la conformación de cooperativas de trabajo, Administración Nacional de la Seguridad Social (ANSES), Asignación Universal por Hijo (AUH) y programas como Impulsores de tu país para vos, Yo Mamá, Emprendedoras con Vocación Argentina (EVA), Jóvenes con más y mejor trabajo. Así como, también, trabajando en conjunto con espacios de promoción cultural como tecnopolis y turismo social. Éstas son algunas de las herramientas que el Estado Nacional generó a partir del 2003 con el cambio de paradigma y en repuesta a las demandas que la sociedad argentina venía realizando a los gobiernos neoliberales que llevaron adelante las políticas de ajuste a sectores como educación, salud, empleo, seguridad social, vivienda, industria nacional y soberanía energética, entre otros. (Párrafo extraído del texto a modo de resumen)Eje temático: Territorio - Territorio en foco: intervenciones, producción de conocimiento, y acciones colectivas desde Villa Elvira, Partido de La PlataFacultad de Trabajo Socia

Universidad y organizaciones sociales en relación a los procesos de organización popular

A partir del hecho ocurrido el pasado 2 de abril de 2013 y a modo de profundización de los procesos de organización territorial que se venían llevando adelante en el barrio de Villa Elvira, específicamente en la Unidad Básica ubicada en 7 y 629, se comenzó a trabajar desde la Facultad de Periodismo y Comunicación Social en articulación con la Juventud Peronista (JP/BA) y otros jóvenes provenientes de distintos espacios institucionales tales como, Facultad de Trabajo Social, Facultad de Arquitectura y la Facultad de Bellas Artes, para facilitar la llegada del Estado Nacional a barrios marginales que no son reconocidos como tales por el Municipio de la Ciudad de La Plata. Además se llevó adelante la generación de una red de relación con las distintas políticas públicas y sociales que se estaban ejecutando en esos lugares, impulsando la participación de jóvenes de escuelas secundarias, Plan de Finalización de Estudios Secundarios y Primarios (FinES) y en articulación con el Instituto Nacional de Economía Social y Solidaria (INAES) para la conformación de cooperativas de trabajo, Administración Nacional de la Seguridad Social (ANSES), Asignación Universal por Hijo (AUH) y programas como Impulsores de tu país para vos, Yo Mamá, Emprendedoras con Vocación Argentina (EVA), Jóvenes con más y mejor trabajo. Así como, también, trabajando en conjunto con espacios de promoción cultural como tecnopolis y turismo social. Éstas son algunas de las herramientas que el Estado Nacional generó a partir del 2003 con el cambio de paradigma y en repuesta a las demandas que la sociedad argentina venía realizando a los gobiernos neoliberales que llevaron adelante las políticas de ajuste a sectores como educación, salud, empleo, seguridad social, vivienda, industria nacional y soberanía energética, entre otros. (Párrafo extraído del texto a modo de resumen)Eje temático: Territorio - Territorio en foco: intervenciones, producción de conocimiento, y acciones colectivas desde Villa Elvira, Partido de La PlataFacultad de Trabajo Socia

Eligibility criteria for Menopausal Hormone Therapy (MHT): a position statement from a consortium of scientific societies for the use of MHT in women with medical conditions. MHT Eligibility Criteria Group

This project aims to develop eligibility criteria for menopausal hormone therapy (MHT). The tool should be similar to those already established for contraception A consortium of scientific societies coordinated by the Spanish Menopause Society met to formulate recommendations for the use of MHT by women with medical conditions based on the best available evidence. The project was developed in two phases. As a first step, we conducted 14 systematic reviews and 32 metanalyses on the safety of MHT (in nine areas: age, time of menopause onset, treatment duration, women with thrombotic risk, women with a personal history of cardiovascular disease, women with metabolic syndrome, women with gastrointestinal diseases, survivors of breast cancer or of other cancers, and women who smoke) and on the most relevant pharmacological interactions with MHT. These systematic reviews and metanalyses helped inform a structured process in which a panel of experts defined the eligibility criteria according to a specific framework, which facilitated the discussion and development process. To unify the proposal, the following eligibility criteria have been defined in accordance with the WHO international nomenclature for the different alternatives for MHT (category 1, no restriction on the use of MHT; category 2, the benefits outweigh the risks; category 3, the risks generally outweigh the benefits; category 4, MHT should not be used). Quality was classified as high, moderate, low or very low, based on several factors (including risk of bias, inaccuracy, inconsistency, lack of directionality and publication bias). When no direct evidence was identified, but plausibility, clinical experience or indirect evidence were available, "Expert opinion" was categorized. For the first time, a set of eligibility criteria, based on clinical evidence and developed according to the most rigorous methodological tools, has been defined. This will provide health professionals with a powerful decision-making tool that can be used to manage menopausal symptoms

Slaughter weight rather than sex affects carcass cuts and tissue composition of Bisaro pigs

Carcass cuts and tissue composition were assessed in Bisaro pigs (n=64) from two sexes (31 gilts and 33 entire males) reared until three target slaughter body-weights (BW) means: 17 kg, 32 kg, and 79 kg. Dressing percentage and backfat thickness increased whereas carcass shrinkage decreased with increasing BW. Slaughter weight affected most of the carcass cut proportions, except shoulder and thoracic regions. Bone proportion decreased linearly with increasing slaughter BW, while intermuscular and subcutaneous adipose tissue depots increased concomitantly. Slaughter weight increased the subcutaneous adipose tissue proportion but this impaired intramuscular and intermuscular adipose tissues in the loin primal. The sex of the pigs minimally affected the carcass composition, as only the belly weight and the subcutaneous adipose tissue proportions were greater in gilts than in entire males. Light pigs regardless of sex are recommended to balance the trade-offs between carcass cuts and their non-edible compositional outcomes.Work included in the Portuguese PRODER research Project BISOPORC – Pork extensive production of Bísara breed, in two alternative systems: fattening on concentrate vs chesnut, Project PRODER SI I&DT Medida 4.1 “Cooperação para a Inovação”. The authors are grateful to Laboratory of Carcass and Meat Quality of Agriculture School of Polytechnic Institute of Bragança ‘Cantinho do Alfredo’. The authors are members of the MARCARNE network, funded by CYTED (ref. 116RT0503).info:eu-repo/semantics/publishedVersio

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Background Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH,non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2–F3, or F1 with at least oneaccompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpointsfor the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2–F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1–F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2–F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1–F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca