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Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells.
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Toxinas Bacterianas , Clostridioides difficile , Esporas Bacterianas , Esporas Bacterianas/metabolismo , Esporas Bacterianas/genética , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Animales , Chlorocebus aethiops , Células Vero , Enterotoxinas/metabolismo , Enterotoxinas/genéticaRESUMEN
YabG is a sporulation-specific protease that is conserved among sporulating bacteria. C. difficile YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabG C207A (catalytically inactive), C. difficile yabG A46D, C. difficile yabG G37E, and C. difficile yabG P153L strains germinated in response to TA alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabGA46D showed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.
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The gold standard for coronavirus disease 2019 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes. In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from saliva samples or RNA isolated from nasopharyngeal (NP) swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern. The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed NP rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2. Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.
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In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.
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Proteínas Bacterianas , Clostridioides difficile , N-Acetil Muramoil-L-Alanina Amidasa , Peptidoglicano , Esporas Bacterianas , Esporas Bacterianas/metabolismo , Esporas Bacterianas/genética , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Clostridioides difficile/enzimología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/genética , Peptidoglicano/metabolismo , Regulación Bacteriana de la Expresión Génica , Factor sigma/metabolismo , Factor sigma/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/enzimología , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/genéticaRESUMEN
PURPOSE: Chemotherapy is established as primary treatment in patients with stage IV colorectal cancer and unresectable metastases. Data from nonrandomized clinical trials have fueled persistent uncertainty if primary tumor resection (PTR) before chemotherapy prolongs survival. We investigated the prognostic value of PTR in patients with newly diagnosed stage IV colon cancer who were not amenable to curative treatment. PATIENTS AND METHODS: Patients enrolled in the multicenter, randomized SYNCHRONOUS and CCRe-IV trials were included in the analysis. Patients with colon cancer with synchronous unresectable metastases were randomly assigned at 100 sites in Austria, Germany, and Spain to undergo PTR or up-front chemotherapy (No PTR group). The chemotherapy regimen was left at discretion of the local team. Patients with tumor-related symptoms, inability to tolerate surgery and/or systemic chemotherapy, and history of another cancer were excluded. The primary end point was overall survival (OS), and the analyses were performed with intention-to-treat. RESULTS: A total of 393 patients were randomly assigned to undergo PTR (n = 187) or no PTR (n = 206) between November 2011 and March 2017. Chemotherapy was not administered to 6.4% in the No PTR group and 24.1% in the PTR group. The median follow-up time was 36.7 months (95% CI, 36.6 to 37.3). The median OS was 16.7 months (95% CI, 13.2 to 19.2) in the PTR group and 18.6 months (95% CI, 16.2 to 22.3) in the No PTR group (P = .191). Comparable OS between the study groups was further confirmed on multivariate analysis (hazard ratio, 0.944 [95% CI, 0.738 to 1.209], P = .65) and across all subgroups. Patients with serious adverse events were more common in the No PTR group (10.2% v 18.0%; P = .027). CONCLUSION: Among patients with colon cancer and synchronous unresectable metastases, PTR before systemic chemotherapy was not associated with prolonged OS.
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Neoplasias del Colon , Humanos , Femenino , Masculino , Anciano , Neoplasias del Colon/patología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/mortalidad , Persona de Mediana Edad , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Estadificación de Neoplasias , Metástasis de la Neoplasia , Anciano de 80 o más Años , AdultoRESUMEN
Some members of the Firmicutes phylum, including many members of the human gut microbiota, are able to differentiate a dormant and highly resistant cell type, the endospore (hereinafter spore for simplicity). Spore-formers can colonize virtually any habitat and, because of their resistance to a wide variety of physical and chemical insults, spores can remain viable in the environment for long periods of time. In the anaerobic enteric pathogen Clostridioides difficile the aetiologic agent is the oxygen-resistant spore, while the toxins produced by actively growing cells are the main cause of the disease symptoms. Here, we review the regulatory circuits that govern entry into sporulation. We also cover the role of spores in the infectious cycle of C. difficile in relation to spore structure and function and the main control points along spore morphogenesis.
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Clostridioides difficile , Microbioma Gastrointestinal , Humanos , Morfogénesis , Oxígeno , Examen FísicoRESUMEN
A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance.
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Clostridioides difficile , Péptido Hidrolasas , Humanos , Péptido Hidrolasas/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Clostridioides , Esporas Bacterianas/metabolismo , Factores de Transcripción/metabolismo , Endopeptidasas/metabolismo , Proteínas Bacterianas/metabolismo , Bacillus subtilis/metabolismoRESUMEN
The human pathogenic bacteria Bacillus cereus, Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of σK is not strictly linked to the state of forespore development in B. cereus.
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Bacillus anthracis , Cactaceae , Humanos , Bacillus cereus , Aeronaves , Bacillus subtilis , Colorantes , Microscopía Electrónica de TransmisiónRESUMEN
Oral vaccines are highly demanded by the aquaculture sector, to allow mass delivery of antigens without using the expensive and labor-intensive injectable vaccines. These later require individual handling of fish, provoking stress-related mortalities. One possible strategy to create injection-free vaccine delivery vehicles is the use of bacterial spores, extremely resistant structures with wide biotechnological applications, including as probiotics, display systems, or adjuvants. Bacterial spores, in particular those of Bacillus subtilis, have been shown to behave as mucosal vaccine adjuvants in mice models. However, such technology has not been extensively explored against fish bacterial disease. In this study, we used a laboratory strain of B. subtilis, for which a variety of genetic manipulation tools are available, to display at its spores surface either a Vibrio antigenic protein, OmpK, or the green fluorescence protein, GFP. When previously vaccinated by immersion with the OmpK- carrying spores, zebrafish survival upon a bacterial challenge with V. anguillarum and V. parahaemolyticus, increased up to 50 - 90% depending on the pathogen targeted. Further, we were able to detect anti-GFP-antibodies in the serum of European seabass juveniles fed diets containing the GFP-carrying spores and anti-V. anguillarum antibodies in the serum of European seabass juveniles fed the OmpK-carrying spores containing diet. More important, seabass survival was increased from 60 to 86% when previously orally vaccinated with in-feed OmpK- carrying spores. Our results indicate that B. subtilis spores can effectively be used as antigen-carriers for oral vaccine delivery in fish.
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Lubina , Enfermedades de los Peces , Vibriosis , Ratones , Animales , Vacunas Bacterianas , Pez Cebra , Vibriosis/prevención & control , Vibriosis/veterinaria , Esporas Bacterianas , Vacunación , TecnologíaRESUMEN
INTRODUCCÍON: Los padres se han involucrado cada vez más en el embarazo y el nacimiento de sus hijos, pero aún se requieren intervenciones paternas que permitan reubicar al padre en su rol de corresponsabilidad en la crianza. OBJETIVO: Observar el comportamiento-actitud paterna hacia el/la hijo(a) y la cantidad de oxitocina (OT) secretada en el nacimiento, en padres preparados de un modo especial para el parto. Método: Estudio piloto de 8 meses, parte de una investigación mayor cuali-cuantitativa de dos fases. La fase cualitativa inicial desarrolló una intervención preparatoria de padres para el nacimiento, con énfasis en la vinculación padre-hijo(a). La fase cuantitativa correspondió al piloto de la intervención paterna antenatal. RESULTADOS: Los padres presenciaron activamente el momento del expulsivo y el encuentro madre-hijo(a). Posteriormente, todos optaron por el contacto físico piel-piel con su hijo(a). La OT paterna experimentó un aumento (no significativo) durante el contacto padre-hijo(a) en comparación con la OT basal (momento inmediato al nacimiento). CONCLUSIONES: Padres preparados, sensibilizados y vinculados con su hijo(a) desde el embarazo experimentarían variaciones de la cantidad de OT cuando realizan contacto piel-piel con su hijo(a) en el nacimiento. Se requiere investigación experimental con una muestra mayor de participantes para concluir de manera categórica.
INTRODUCTION: Fathers have been increasingly involved in the pregnancy and birth of their children, but paternal interventions are still required to relocate the father in his role of co-responsibility in parenting. OBJECTIVE: To observe the paternal behavior-attitude towards the child and the amount of oxytocin (OT) secreted at birth in parents prepared (in a special way) for childbirth. METHOD: Pilot study of 8 months, part of a larger qualitative-quantitative research of two phases. The initial qualitative phase developed a male preparatory intervention for the birth, with emphasis on the father-child bonding. The quantitative phase corresponded to the pilot of the antenatal paternal intervention. RESULTS: Fathers actively witnessed the moment of delivery and the mother-child attachment. Subsequently, all of them opted for physical skin-to-skin contact with their child. Paternal OT experienced a (non-significant) increase during father-child contact, compared to baseline OT (immediately after birth). CONCLUSIONS: Males prepared, sensitized and involved with their child since pregnancy would experience variations in the amount of OT when they make father-child skin-to-skin contact at childbirth. Experimental research with a larger sample of participants is required to categorically reach a conclusion.
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Humanos , Masculino , Femenino , Embarazo , Recién Nacido , Adulto , Relaciones Padres-Hijo , Conducta Paterna/fisiología , Oxitocina/fisiología , Tacto , Proyectos Piloto , Parto , Apego a ObjetosRESUMEN
Introducción: en el contexto de la pandemia mundial por COVID-19 se incorporan nuevas metodologías de enseñanza-aprendizaje para contribuir al logro de los objetivos de aprendizaje frente al cierre de las universidades y de los campos clínicos. Esta investigación evalúa la percepción y la experiencia de los estudiantes de tercer y cuarto año de pregrado de enfermería, frente al uso de escenarios de simulación virtual como complemento de la práctica clínica. Materialesy métodos: un total de 68 estudiantes (26%) respondieron la encuesta de percepción sobre su experiencia del uso de la simulación virtual a través del softwarevSim for Nursing™. Se realizó un análisis cuantitativo y cualitativo de los datos. Resultados: un 89,7% de los estudiantes consideraron que la simulación virtual es útil para el aprendizaje y que contribuye principalmente para la comprensión de los contenidos y desarrollo de las habilidades de valoración clínica.Discusión: la experiencia en la simulación virtual es realista y útil tanto para aprender nuevos conocimientos,para reforzar los saberes previos y así también como una buena preparación para la práctica clínica. Se visualiza su incorporación desde el inicio al término de la carrera. Sin embargo, para áreas como salud mental otras metodologías podrían ser más adecuadas para mejorar las habilidades de comunicación.
Introduction: in the context of the global pandemic due to COVID-19, new teaching-learning methodologies were incorporated to contribute to learning objectives in the face of the closure of the university and clinical fields. This research assesses the perception and experience of third and fourth-year undergraduate nursing students regarding using virtual simulation scenarios as a to complement clinical practice. Materials and methods: 68 students (26%) responded to the perception survey about their experienceusing virtual simulation through the vSim for Nursing™ software. A quantitative and qualitative analysis of the data was carried out. Results: 89.7% of the students considered that virtual simulation is rewarding for learning and that it contributes mainly to the understanding of the contents and the development of clinical assessment skills. Discussion: the experience in virtual simulation is realistic and practical both to learning new knowledge, reinforce previous one and thus also as good preparation for clinical practice. Their incorporation is visualized from the beginning to the end of the degree. However, for areas such as mental health, other methodologies might be more suitable to improve communication skills.
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Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the ß-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.
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Proteínas Bacterianas , Esporas Bacterianas , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Estructura Secundaria de Proteína , Esporas Bacterianas/metabolismoRESUMEN
INTRODUCTION: Healthcare associated infections due to carbapenem-resistant Klebsiella pneumoniae (CRKP) are a major concern in Portuguese hospitals. Whole genome sequencing (WGS) can improve infection control, but this practice is not routinely used by hospital clinical laboratories in Portugal. We simulated the investigation of a CRKP outbreak based on WGS, with the aim of determining, in the minimum possible time, genetic relatedness between CRKP clinical and environmental isolates. MATERIAL AND METHODS: Ten CRKP clinical isolates routinely obtained in the hospital laboratory were used. Forty environmental samples - from sinks and sink drains of ward rooms - were collected. Environmental samples were plated on selective media and presumptive CRKP colonies were isolated. Total DNA was extracted from all putative CRKP isolates and sequenced. Clonal relatedness was determined by multi-locus sequence typing and core genome single nucleotide polymorphism analysis; the presence of carbapenemase genes was evaluated. RESULTS: Clinical isolates were characterized in 48 hours: eight strains were confirmed as CRKP, of which six were of ST13 and carried blaKPC-3. Environmental samples results were obtained in six days: eight CRKP were isolated from which five were of ST13 and carried blaKPC-3. Clinical and environmental ST13 isolates were highly related: ten (of 11) isolates differed from each other in < 0.001% of 2 172 367 core nucleotides. DISCUSSION: WGS can be used as a high-resolution effective tool to investigate healthcare associated infections and track routes of dissemination in real-time. CONCLUSION: In Portugal, routine use of WGS to improve infection control could thrive through collaborative initiatives between hospitals and research institutes.
Introdução: As infeções associadas aos cuidados de saúde por Klebsiella pneumoniae resistente aos carbapenemos (CRKP) são uma preocupação nos hospitais portugueses. A sequenciação total do genoma [whole genome sequencing (WGS)] pode ajudar no controlo de infecção, mas esta prática não é comummente utilizada nos laboratórios clínicos hospitalares em Portugal. O objetivo deste estudo foi simular a investigação de um surto causado por CRKP, utilizando WGS. Pretendia-se testar a utilização desta técnica e determinar, no menor tempo possível, relações genéticas entre estirpes. Material e Métodos: Foram analisados dez isolados clínicos de CRKP. Foram obtidas quarenta amostras ambientais que foram inoculadas em meio seletivo para isolamento de colónias sugestivas de CRKP e depois sequenciado o DNA total dos isolados presumptivamente identificados como CRKP A relação clonal entre as estirpes foi determinada por multi-locus sequence typing e análise de single nucleotide polymorphisms no genoma core. Foi determinada a presença de genes de carbapenemases. Resultados: Os isolados clínicos foram caraterizados em 48 horas: oito isolados foram confirmados como CRKP. A maioria pertencia ao ST13 (n = 6) e possuía o gene blaKPC-3. As amostras ambientais foram caraterizadas em seis dias: foram isoladas oito CRKP, das quais cinco eram ST13 e continham o gene blaKPC-3. Os isolados ST13 clínicos e ambientais eram muito semelhantes entre si: dez dos 11 isolados diferiam entre si em menos de 0,001% dos 2 172 367 nucleótidos core analisados. Discussão: A sequenciação total do genoma pode ser usada como uma ferramenta útil para investigar infecções nosocomiais e rastrear cadeias de disseminação em tempo real. Conclusão: Em Portugal, o uso desta técnica em controlo de infecção pode ser implementado através de colaborações entre hospitais e institutos de investigação.
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Infecciones por Klebsiella , Klebsiella pneumoniae , Antibacterianos/uso terapéutico , Carbapenémicos/farmacología , Brotes de Enfermedades , Humanos , Infecciones por Klebsiella/epidemiología , Klebsiella pneumoniae/genética , Laboratorios Clínicos , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Secuenciación Completa del GenomaRESUMEN
Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.
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Prueba de COVID-19/métodos , COVID-19/virología , Colorimetría/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , SARS-CoV-2/aislamiento & purificación , COVID-19/diagnóstico , Humanos , Pandemias , Portugal/epidemiología , ARN Viral/genética , SARS-CoV-2/genética , Saliva/química , Saliva/virología , Sensibilidad y EspecificidadRESUMEN
Palliative care is on the global health agenda, as only approximately 14% of people who require palliative care receive it [...].
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OBJECTIVES: Enhanced Recovery After Surgery (ERAS) programs include multiple perioperative care elements, which when implemented together are designed to improve recovery after surgery with subsequent reduction in hospital length of stay (LOS). The aim of this study is to examine the impact of ERAS protocol compliance on LOS in patients undergoing advanced ovarian cancer surgery within the context of a randomized clinical trial. METHODS: Patients were enrolled in a prospective, consecutive, interventional randomized clinical trial between June 2014 and March 2018. Women with either suspected or confirmed advanced ovarian cancer with International Federation of Gynecology and Obstetrics (FIGO) stages IIB-IVA and recurrent ovarian cancer, who underwent cytoreduction surgery, were randomly assigned to either a conventional management (CM) protocol or an ERAS protocol. Demographic items, preoperative clinical data, and surgical characteristics of patients were recorded, as were LOS and ERAS protocol compliance. Negative binomial regression was used to model the relation between length of stay and ERAS protocol compliance. RESULTS: We included 49 patients in the CM group and 50 patients in the ERAS group. The overall rate of ERAS compliance was 92%. We observed that increasing ERAS protocol compliance was associated with shorter median LOS, and in patients who underwent higher complex surgeries, the length of stay reduction was greater. CONCLUSION: This study identifies a correlation between increasing ERAS protocol compliance and decreasing LOS in ovarian cancer surgery. This finding underlines the necessity to implement as many ERAS protocol elements as possible to achieve optimal clinical outcome improvements.
Asunto(s)
Recuperación Mejorada Después de la Cirugía , Neoplasias Ováricas , Femenino , Adhesión a Directriz , Humanos , Tiempo de Internación , Recurrencia Local de Neoplasia/cirugía , Neoplasias Ováricas/cirugía , Estudios ProspectivosRESUMEN
The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates the interactions of spores with their environment, modulates spore adhesion and germination, and has been implicated in pathogenesis. In Bacillus cereus, the exosporium consists of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, surrounded by a hairy nap composed of glycoproteins. The morphogenetic protein CotE is necessary for the integrity of the B. cereus exosporium, but how CotE directs exosporium assembly remains unknown. Here, we used super-resolution fluorescence microscopy to follow the localization of SNAP-tagged CotE, CotY, and ExsY during B. cereus sporulation and evidenced the interdependencies among these proteins. Complexes of CotE, CotY, and ExsY are present at all sporulation stages, and the three proteins follow similar localization patterns during endospore formation that are reminiscent of the localization pattern of Bacillus subtilis CotE. We show that B. cereus CotE guides the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process, as previously proposed, but occurs through the formation of two caps, as observed during B. subtilis coat morphogenesis, suggesting that a general principle governs the assembly of the spore surface layers of BacillaceaeIMPORTANCE Spores of Bacillaceae are enveloped in an outermost glycoprotein layer. In the B. cereus group, encompassing the Bacillus anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance and separation from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize the formation of the exosporium during the sporulation of B. cereus, and we studied the localization and interdependencies of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaffold before a complete exosporium or coat is detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore, and we present a model for the assembly of the exosporium.
Asunto(s)
Bacillus cereus/crecimiento & desarrollo , Bacillus cereus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Esporas Bacterianas/fisiología , Microscopía Fluorescente/métodos , Esporas Bacterianas/genéticaRESUMEN
Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50-60% of those bacteria have the ability to produce resilient spores that are important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile and contains several genes of unknown function. We report on the characterization of a signature gene, CD25890, which, as we show is involved in the control of sporulation initiation in C. difficile under certain nutritional conditions. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of CD25890 results in increased expression of spo0A per cell and increased sporulation. The effect of CD25890 on spo0A is likely indirect and mediated through repression of the sinRR´ operon. Deletion of the CD25890 gene, however, does not alter the expression of the genes coding for the cytotoxins or the genes involved in biofilm formation. Our results suggest that CD25890 acts to modulate sporulation in response to the nutrients present in the environment.
Asunto(s)
Proteínas Bacterianas/genética , Clostridioides difficile/metabolismo , Infecciones por Clostridium/microbiología , Genes Bacterianos , Esporas Bacterianas/genética , Animales , Regulación Bacteriana de la Expresión Génica , Humanos , MesocricetusRESUMEN
The Health Promoting University (HPU) concept encourages universities to incorporate health into the university context. HPU initiatives exist worldwide, yet information on how universities translate the HPU concept into actions is scarce. This study aimed to identify the factors influencing the implementation of HPU initiatives in Ibero-American universities. Semi-structured interviews were held with seventeen representatives of universities in Ibero-America that had implemented an HPU initiative. All interviewees had been involved in the initiative and had occupied a position of responsibility for at least 1 year before the study. The interviews were carried out remotely, and the data were analyzed using an inductive approach. The main factors influencing the implementation of an HPU initiative were political support by the university authorities, coordination structure, funding, collaboration inside and outside university and participation of the university community. Among them, political support by the university authorities was considered the most important, although some initiatives succeeded without it and managed to obtain support during the implementation process. This study is one of the first to investigate the factors influencing the implementation of the HPU concept. A better understanding of these factors would enable universities to address them to develop the HPU initiative in the best possible conditions.
Asunto(s)
Promoción de la Salud , Universidades , Humanos , Estados UnidosRESUMEN
BACKGROUND: Type-2-diabetes (T2D) has surfaced as a potential risk factor for Parkinson's disease (PD) in some epidemiological studies. Evidence of glucose metabolism alterations in PD from molecular studies remains conflicting. Amylin, the T2D amyloid protein, has been implicated in PD in pathological studies. We aimed to assess peripheral levels of amylin and insulin in PD patients and control subjects (Cs). METHODS: We conducted an observational cross-sectional study of 111 participants: 73 PD and 38 Cs, similar in age, sex and body mass index. All underwent motor (UPDRS-MDS-III), non-motor (NMSS) and cognitive (MDRS) scales as well as determination of four parameters: fasting glycaemia, glycated haemoglobin, fasting plasma insulin (FPI) and fasting plasma amylin (FPA). RESULTS: FPI was significantly lower in PD than Cs (p = 0.034). In participants with age above cohort-median-age, FPA was higher in PD than Cs (p = 0.046). The FPA/FPI ratio (FPAIR) was significantly higher in PD than Cs (p = 0.024). In PD, modest correlation was found between higher insulin-resistance and NMSS scores. CONCLUSIONS: PD patients had lower FPI and increased FPAIR. In older PD subgroup, FPA was increased. The more the insulin resistance, the higher the non-motor scores. These findings provide an additional link between pathophysiology of diabetes and PD. This might be related to a dissociated insulin and amylin secretion in PD, in line with recent evidence of endocrine pancreas role in PD pathogeny.