Bases moleculares de la interacción esticolisina-membrana sobre la estructura del poro y los efectos de los lípidos.

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 18/06/2021The usefulness of toxicity across the tree of life is far beyond doubt. Most, if not all, organisms produce compounds that can be used for attack and/or defense against external entities. Some of the most specialized of these compounds are toxic proteins, among which pore-forming toxins (PFTs) particularly excel. PFTs are present in all kingdoms of life. Given the wide variety of PFTs, one can expect a multitude of different specificities and mechanisms of action, of which we will certainly take advantage at some point. For that, a thorough characterization of PFTs and their functionality is necessary. In this thesis, we have taken further the characterization of sticholysins, small PFTs produced by the sea anemone Stichodactyla helianthus...La toxicidad es indudablemente útil para los seres vivos. La mayoría, si no todos los organismos, producen compuestos para usarlos contra otros seres vivos, ya sea confines de defensa o de ataque. Algunos de los más especializados de estos compuestos son las proteínas tóxicas, entre las que destacan las toxinas formadoras de poros (ing.,pore-forming toxins, PFT). Estas proteínas pueden encontrarse en todos los reinos dela vida. Dada su amplia diversidad, puede esperarse que tengan una gran variedad de especificidades y mecanismos de acción. Una caracterización detallada de las PFT y su funcionalidad es un requisito fundamental para, en un futuro, poder beneficiarse dela acción de estas toxinas. Por ello, en esta tesis se ha profundizado en el comportamiento de las esticolisinas, pequeñas PFT producidas por la anémona del mar Caribe Stichodactyla helianthus...Nyttan av toxicitet hos olika organismer är långt bortom tvivel. De flesta, om inte alla,organismer producerar föreningar som kan användas för attack och/eller försvar motexterna hot. Några av de mest specialiserade av dessa föreningar är toxiska proteiner,bland vilka por-bildande toxiner (eng., pore-forming toxins, PFT) är särskiltutmärkande. PFT finns i alla livets riken. Med tanke på det stora utbudet av PFT kanman förvänta sig en mängd olika specificiteter och verkningsmekanismer, som vi kankomma att dra nytta i olika tillämpningar. Därför är en grundlig karakterisering avPFT-peptider och deras funktionalitet nödvändig. I denna avhandling har viundersökt och karakteriser olika stickolysiner, vilka är små PFT-peptider produceradeav havsanemonen Stichodactyla helianthus...Fac. de Ciencias QuímicasTRUEunpu

Sistema de gestión de programas de educación ambiental, una aplicación para el desarrollo comunitario

El Sistema para la Gestión de Programas de Educación Ambiental (SGPA), trata de una aplicación WEB que permite gestionar diversos programas de Educación Ambiental con el objeto de fomentar la prevención y cuidado del Medio Ambiente, a los estudiantes del Instituto Tecnológico de Acapulco que realizaran el Servicio Social como requisito para cubrir su programa curricular; y posteriormente con los alumnos de Nivel Medio Superior pertenecientes al mismo sistema tecnológico. Con esta aplicación se pretende que los alumnos que cumplan el requisito para realizar su servicio social, se registren en esta herramienta y propongan algún programa o actividad relacionada con acciones que fomenten a la sociedad el cuidado del Medio Ambiente. Los alumnos registrados con alguno de los programas de educación ambiental, serán evaluados por los responsables de la Oficina de Servicio Social y Sistema de Gestión Ambiental quienes validaran a través de la aplicación, el cumplimiento de los requisitos para la aceptación; facilitando también los trámites, gestión y seguimiento de los procedimientos administrativos que se realizan en la institución. Con estos programas de capacitación, los alumnos adquieren una formación integral, promueven la cultura a través de la educación ambiental y por ende el desarrollo sustentable de las comunidades en la región. Con la gestión de los programas de educación ambiental a través de la aplicación SGPA, se contribuirá con el mejoramiento de calidad de vida de las personas generando impactos de desarrollo sustentable positivos para la región

EVALUACIÓN Y AUTOMATIZACIÓN PARA LA EQUIDAD DE GÉNERO EN EL INSTITUTO TECNOLÓGICO DE ACAPULCO

El presente trabajo de investigación está desarrollado con base en criterios de evaluación que contempla el Modelo de Equidad de Género MEG-2003, y en el cual se pretende conocer y analizar los aspectos de equidad e inequidad de las instituciones educativas que influyen de cierta manera en el desarrollo social y económico. La evaluación realizada en el Instituto Tecnológico de Acapulco considera una serie de aspectos como conocimiento general por parte del personal de la institución, conocimiento e información de normas de equidad por parte del personal directivo, análisis de la plantilla y puestos de los trabajadores, análisis de la matrícula de alumnos de la institución, entre otros. Los aspectos considerados en la evaluación y la información obtenida mediante encuestas y entrevistas son procesados a través de un sistema automatizado, mismo que fue realizado para generar los resultados de forma rápida, precisa y oportuna. El resultado de los diversos criterios se concentra de manera global arrojando una puntuación final, la cual es comparada de manera automática con el parámetro establecido en el sistema, generando un dictamen que indicará si la institución cumple con los requerimientos para ser o no ser certificada. La finalidad es identificar el conocimiento y aplicación del Modelo de Equidad de Género en una institución educativa de nivel superior que evalúe la igualdad de oportunidades entre hombres y mujeres, ya que el conocimiento, la difusión y la aplicación de trabajos relacionados con la equidad contribuyen en gran medida a resolver proyectos más completos y conseguir metas integrales. Considerando un trabajo de investigación que sea evaluado de manera automatizado y que arroje resultados más certeros y oportunos, relacionados con la igualdad de oportunidades para hombres y mujeres, seguramente se contribuirá con la difusión del conocimiento y por consiguiente se tendrán impactos de desarrollo social y económico

Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans 4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH 7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs

Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction

Actinoporins are a group of soluble toxic proteins that bind to membranes containing sphingomyelin (SM) and oligomerize to form pores. Sticholysin II (StnII) is a member of the actinoporin family, produced by Stichodactyla helianthus. Cholesterol (Chol) is known to enhance the activity of StnII. However, the molecular mechanisms behind this activation have remained obscure, although the activation is not Chol specific but rather sterol specific. To further explore how bilayer lipids affect or are affected by StnII, we have used a multiprobe approach (fluorescent analogs of both Chol and SM) in combination with a series of StnII tryptophan (Trp)-mutants, to study StnII/bilayer interactions. First we compared StnII bilayer permeabilization in the presence of Chol or oleoyl-ceramide (OCer). The comparison was done since both Chol and OCer have a 1-hydroxyl which help to orient the molecule in the bilayer (although OCer have additional polar functional groups). Both Chol and OCer also have increased affinity for SM, which StnII may recognize. However, our results show that only Chol was able to activate StnII-induced bilayer permeabilization – OCer failed to active. To further examine possible Chol/StnII interactions, we measured Förster resonance energy transfer (FRET) between Trp in StnII and cholestatrienol (CTL), a fluorescent analog of Chol. We could show higher FRET efficiency between CTL and Trp:s in position 100 and 114 of StnII, when compared to three other Trp positions further away from the bilayer binding region of StnII. Taken together, our results suggest that StnII was able to attract Chol to its vicinity, maybe by showing affinity for Chol. SM interactions are known to be important for StnII binding to bilayers, and Chol is known to facilitate subsequent permeabilization of the bilayers by StnII. Our results help to better understand the role of these important membrane lipids for the bilayer properties of StnII

Sticholysin I–II oligomerization in the absence of membranes

Sticholysins are pore-forming toxins produced by the sea anemone Stichodactyla helianthus. When they encounter a sphingomyelin-containing membrane, these proteins bind to it and oligomerize, a process that ends in pore formation. Mounting evidence indicates that StnII can favour the activity of StnI. Previous results have shown that these two isotoxins can oligomerize together. Furthermore, StnII appeared to potentiate the activity of StnI through the membrane-binding step of the process. Hence, isotoxin interaction should occur prior to membrane encounter. Here, we have used analytical ultracentrifugation to investigate the oligomerization of Stns in solution, both separately and together. Our results indicate that while StnI seems to be more prone to oligomerize in water solution than StnII, a small percentage of StnII in StnI–StnII mixtures promotes oligomerization

Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools

Animal venoms are complex mixtures of highly specialized toxic molecules. Cnidarians and arachnids produce pore-forming proteins (PFPs) directed against the plasma membrane of their target cells. Among PFPs from cnidarians, actinoporins stand out for their small size and molecular simplicity. While native actinoporins require only sphingomyelin for membrane binding, engineered chimeras containing a recognition antibody-derived domain fused to an actinoporin isoform can nonetheless serve as highly specific immunotoxins. Examples of such constructs targeted against malignant cells have been already reported. However, PFPs from arachnid venoms are less well-studied from a structural and functional point of view. Spiders from the Latrodectus genus are professional insect hunters that, as part of their toxic arsenal, produce large PFPs known as latrotoxins. Interestingly, some latrotoxins have been identified as potent and highly-specific insecticides. Given the proteinaceous nature of these toxins, their promising future use as efficient bioinsecticides is discussed throughout this Perspective. Protein engineering and large-scale recombinant production are critical steps for the use of these PFPs as tools to control agriculturally important insect pests. In summary, both families of PFPs, from Cnidaria and Arachnida, appear to be molecules with promising biotechnological applications

Primary vs. Secondary Heart Failure Diagnosis: Differences in Clinical Outcomes, Healthcare Resource Utilization and Cost.

There is scarce information on patients with secondary heart failure diagnosis (sHF). We aimed to compare the characteristics, burden, and outcomes of sHF with those with primary HF diagnosis (pHF). Retrospective, observational study on patients ≥18 years with emergency department (ED) visits during 2018 with pHF and sHF in ED or hospital (ICD-10-CM) diagnostic codes. Baseline characteristics, 30-day and 1-year mortality, readmission and re-ED visit rates, and costs were compared between sHF and pHF. Out of the 797 patients discharged home from the ED, 45.5% had sHF, and these presented lower 1-year hospitalization, re-ED visit rates, and costs. In contrast, out of the 2,286 hospitalized patients, 55% had sHF and 45% pHF. Hospitalized sHF patients had significantly (p < 0.01) greater comorbidity, lower use of recommended HF therapies, longer length of stay (10.8 ± 10.1 vs. 9.7 ± 7.9 days), and higher in-hospital and 1-year mortality (32 vs. 25.8%) with no significant differences in readmission rates and lower 1-year re-ED visit rate. Hospitalized sHF patients had higher total costs (€12,262,422 vs. €9,144,952, p < 0.001), mean cost per patient-year (€9,755 ± 13,395 vs. €8,887 ± 12,059), and average daily cost per patient. Hospitalized sHF patients have a worse initial prognosis, greater use of healthcare resources, and higher costs.This study was an investigator-initiated initiative sponsored and supported by the grant from AstraZeneca Spain. LV receives research funding from the Instituto de Salud Carlos III, Spain (CM20/00104). GM is supported by a pre-doctoral grant by the Spanish Ministry of Education (FPU18/03606).S

Structural and functional characterization of Sticholysin III: A newly discovered actinoporin within the venom of the sea anemone Stichodactyla helianthus.

Actinoporins are a family of pore-forming toxins produced by sea anemones as part of their venomous cocktail. These proteins remain soluble and stably folded in aqueous solution, but when interacting with sphingomyelin-containing lipid membranes, they become integral oligomeric membrane structures that form a pore permeable to cations, which leads to cell death by osmotic shock. Actinoporins appear as multigenic families within the genome of sea anemones: several genes encoding very similar actinoporins are detected within the same species. The Caribbean Sea anemone Stichodactyla helianthus produces three actinoporins (sticholysins I, II and III; StnI, StnII and StnIII) that differ in their toxic potency. For example, StnII is about four-fold more effective than StnI against sheep erythrocytes in causing hemolysis, and both show synergy. However, StnIII, recently discovered in the S. helianthus transcriptome, has not been characterized so far. Here we describe StnIII’s spectroscopic and functional properties and show its potential to interact with the other Stns. StnIII seems to maintain the well-preserved fold of all actinoporins, characterized by a high content of β-sheet, but it is significantly less thermostable. Its functional characterization shows that the critical concentration needed to form active pores is higher than for either StnI or StnII, suggesting differences in behavior when oligomerizing on membrane surfaces. Our results show that StnIII is an interesting and unexpected piece in the puzzle of how this Caribbean Sea anemone species modulates its venomous activity

Pathogenicity and virulence of Listeria monocytogenes: A trip from environmental to medical microbiology

Listeria monocytogenes is a saprophytic gram-positive bacterium, and an opportunistic foodborne pathogen that can produce listeriosis in humans and animals. It has evolved an exceptional ability to adapt to stress conditions encountered in different environments, resulting in a ubiquitous distribution. Because some food preservation methods and disinfection protocols in foodprocessing environments cannot efficiently prevent contaminations, L. monocytogenes constitutes a threat to human health and a challenge to food safety. In the host, Listeria colonizes the gastrointestinal tract, crosses the intestinal barrier, and disseminates through the blood to target organs. In immunocompromised individuals, the elderly, and pregnant women, the pathogen can cross the blood-brain and placental barriers, leading to neurolisteriosis and materno-fetal listeriosis. Molecular and cell biology studies of infection have proven L. monocytogenes to be a versatile pathogen that deploys unique strategies to invade different cell types, survive and move inside the eukaryotic host cell, and spread from cell to cell. Here, we present the multifaceted Listeria life cycle from a comprehensive perspective. We discuss genetic features of pathogenic Listeria species, analyze factors involved in food contamination, and review bacterial strategies to tolerate stresses encountered both during food processing and along the host’s gastrointestinal tract. Then we dissect host–pathogen interactions underlying listerial pathogenesis in mammals from a cell biology and systemic point of view. Finally, we summarize the epidemiology, pathophysiology, and clinical features of listeriosis in humans and animals. This work aims to gather information from different fields crucial for a comprehensive understanding of the pathogenesis of L. monocytogenes