71,253 research outputs found

    What is the importance of sperm subpopulations?

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    .The study of sperm subpopulations spans three decades. The origin, meaning, and practical significance, however, are less clear. Current technology for assessing sperm morphology (CASA-Morph) and motility (CASA-Mot) has enabled the accurate evaluation of these features, and there are many options for data classification. Subpopulations could occur as a result of the stage of development of each spermatozoon in the subpopulation. Spermatogenesis might contribute to the production of these subpopulations. Insights from evolutionary biology and recent molecular research are indicative of the diversity among male gametes that could occur from unequal sharing of transcripts and other elements through cytoplasmic bridges between spermatids. Sperm cohorts exiting the gonads would contain different RNA and protein contents, affecting the spermatozoon physiology and associations with the surrounding environmental milieu. Subsequently, these differences could affect how spermatozoa interact with the environmental milieu (maturation, mixing with seminal plasma, and interacting with the environmental milieu, or female genital tract and female gamete). The emergence of sperm subpopulations as an outcome of evolution, related to the reproductive strategies of the species, genital tract structures, and copulatory and fertilization processes. This kind of approach in determining the importance of sperm subpopulations in fertilization capacity should have a practical impact for conducting reproductive technologies, inspiring and enabling new ways for the more efficient use of spermatozoa in the medical, animal breeding, and conservation fields. This manuscript is a contribution to the Special Issue in memory of Dr. Duane GarnerS

    Identification of Hindbrain Neural Substrates for Motor Initiation in the hatchling Xenopus laevis Tadpole

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    Animal survival profoundly depends on the ability to detect stimuli in the environment, process them and respond accordingly. In this respect, motor responses to a sensory stimulation evolved into a variety of coordinated movements, which involve the control of brain centres over spinal locomotor circuits. The hatchling Xenopus tadpole, even in its embryonic stage, is able to detect external sensory information and to swim away if the stimulus is considered noxious. To do so, the tadpole relies on well-known ascending sensory pathway, which carries the sensory information to the brain. When the stimulus is strong enough, descending interneurons are activated, leading to the excitation of spinal CPG neurons, which causes the undulatory movement of swimming. However, the activation of descending interneurons that marks the initiation of motor response appears after a long delay from the sensory stimulation. Furthermore, the long-latency response is variable in time, as observed in the slow-summating excitation measured in descending interneurons. These two features, i.e. long-latency and variability, cannot be explained by the firing time and pattern of the ascending sensory pathway of the Xenopus tadpole. Therefore, a novel neuronal population has been proposed to lie in the hindbrain of the tadpole, and being able to 'hold' the sensory information, thus accounting for the long and variable delay of swim initiation. In this work, the role of the hindbrain in the maintenance of the long and variable response to trunk skin stimulation is investigated in the Xenopustadpole at developmental stage 37/38. A multifaceted approach has been used to unravel the neuronal mechanisms underlying the delayed motor response, including behavioural experiments, electrophysiology analysis of fictive swimming, hindbrain extracellular recordings and imaging experiments. Two novel neuronal populations have been identified in the tadpole's hindbrain, which exhibit activation patterns compatible with the role of delaying the excitation of the spinal locomotor circuit. Future work on cellular properties and synaptic connections of these newly discovered populations might shed light on the mechanism of descending control active at embryonic stage. Identifying supraspinal neuronal populations in an embryonic organism could aid in understanding mechanisms of descending motor control in more complex vertebrates

    Uso de las histonas circulantes y sus modificaciones post-traduccionales como biomarcadores en sepsis y shock séptico

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    La sepsis es una afecci√≥n potencialmente mortal causada por una respuesta anormal del hu√©sped a una infecci√≥n, produciendo respuestas fisiol√≥gicas alteradas que da√Īan los propios tejidos del paciente y pueden provocar disfunci√≥n org√°nica e incluso la muerte. Asimismo, algunos pacientes s√©pticos progresan a shock s√©ptico, caracterizado por alteraciones circulatorias, celulares y metab√≥licas sustanciales que aumentan el riesgo de mortalidad. A pesar de que la sepsis se caracteriza por un mal funcionamiento del sistema inmunol√≥gico, lo que a su vez conduce a una respuesta inmune alterada e inmunosupresi√≥n, la alta complejidad de la fisiopatolog√≠a de la sepsis requiere una mayor investigaci√≥n para comprender las respuestas inmunes que ocurren durante la sepsis. Asimismo, las histonas extracelulares circulantes han ganado relevancia como mediadores citot√≥xicos en la sepsis, ya que act√ļan como patrones moleculares asociados a da√Īo, que inducen estr√©s oxidativo y activan el inflamasoma NLRP3. Estos mecanismos median la activaci√≥n de la piroptosis, un mecanismo de muerte celular programada que produce inflamaci√≥n mediante la expresi√≥n de IL-18, IL-1ő≤ and IL-1őĪ. Sin embargo, a pesar de la evidencia de activaci√≥n del inflamasoma en las c√©lulas inmunes durante la sepsis, se desconoce si las histonas extracelulares son capaces de activar los inflamasomas endoteliales y sus consecuencias. En este trabajo destacamos el papel previamente desconocido de las histonas extracelulares, mediando la activaci√≥n del inflamasoma NLRP3 y la piroptosis en las c√©lulas endoteliales, contribuyendo a la disfunci√≥n endotelial y la desregulaci√≥n de la respuesta inmune mediada por el endotelio. Asimismo, tambi√©n demostramos c√≥mo la acetilaci√≥n de histonas disminuye la activaci√≥n de la piroptosis. Adem√°s, demostramos que la piroptosis se produce en pacientes con shock s√©ptico y los niveles de histonas circulantes se correlacionan con la expresi√≥n de citoquinas proinflamatorias y citoquinas piropt√≥ticas, la liberaci√≥n de factores de adhesi√≥n endotelial y la gravedad de la enfermedad. Proponemos la piroptosis mediada por histonas como un nuevo objetivo para desarrollar intervenciones cl√≠nicas. De manera similar, hemos analizado las respuestas inmunorelacionadas que ocurren durante las primeras etapas de la sepsis con el objetivo de proporcionar nuevos datos comparando las cantidades de citoquinas, inmunomoduladores y otros mediadores endoteliales en pacientes cr√≠ticamente enfermos no s√©pticos, s√©pticos y de shock s√©ptico. Nuestro enfoque ayudar√° a caracterizar r√°pidamente las respuestas inmunes alteradas en pacientes s√©pticos y de shock s√©ptico ingresados en la Unidad de Cuidados Intensivos. Finalmente analizamos el papel de la metilaci√≥n del ADN en el control del sistema inmune s√©ptico. Nuestros resultados demostraron el papel central de la metilaci√≥n del ADN modulando la respuesta molecular en los pacientes de shock s√©ptico y contribuyendo a la inmunosupresi√≥n, a trav√©s de la alteraci√≥n de los patrones de metilaci√≥n de los promotores de IL-10 y TREM-2.Sepsis is a life-threatening condition caused by an abnormal host response to an infection that produce altered physiological responses which damages own tissues of the patient and can result in organ dysfunction and in some cases death. Likewise, a subset of septic patients progresses to septic shock, characterized by substantial circulatory, cellular and metabolic abnormalities, which substantially increase the risk of mortality. Sepsis is characterized by a malfunction of the immune system and it can lead to an altered immune response and immunosuppression. Moreover, the high complexity of the pathophysiology of sepsis requires of further investigation to characterize the immune responses in sepsis and septic shock. Likewise, circulating extracellular histones have gained relevance as cytotoxic mediators in sepsis pathophysiology, since they act as damage-associated molecular patterns, which induce oxidative stress and activate NLRP3 inflammasome. Subsequently, inflammasome mediates pyroptosis activation, a programmed cell death mechanism that produces inflammation through the release of IL-18, IL-1ő≤ and IL-1őĪ. However, despite inflammasome activation may occur in immune cells during sepsis, it is unknown if this process also takes place in endothelial cells and particularly whether extracellular histones are capable of activating endothelial inflammasomes and their consequences. In this work we highlight a previously unknown role for extracellular histones, that mediates the activation of NLRP3 inflammasome and pyroptosis in endothelial cells by contributing to endothelial dysfunction and the dysregulation of the immune response mediated by endothelium. Likewise, we demonstrated how histone acetylation decreases pyroptosis activation. Furthermore, we show how pyroptosis occurs in septic shock patients and how circulating histone levels correlate with the expression of pro-inflammatory and pyroptotic cytokines, the release of endothelial adhesion factors and septic shock severity. We propose histone-mediated pyroptosis as a new target to develop clinical interventions. Similarly, we have analyzed the immune-related responses occurring during the early stages of sepsis with the aim of providing new data by comparing the amounts of cytokines, immune modulators and other endothelial mediators in critically-ill non-septic patients, septic and septic shock patients. Our approach will help to rapidly characterize the altered immune responses in septic and septic shock patients admitted in the Intensive Care Unit. Finally, we also analyzed the role of DNA methylation in the control of septic immune system. Our results demonstrated the central role of DNA methylation modulating the molecular response in septic shock patients and contributing to immunosuppression, through the alteration of DNA methylation patterns of IL-10 and TREM2 promoters

    Investigating the potential role for RBMY in cancer

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    Introduction: Head and neck squamous cell carcinoma (HNSCC), is a major healthcare concern with a high male prevalence. We hypothesise that the testis specific mRNA splicing regulator, Y-linked RBMY gene, is aberrantly expressed in HNSCC in part promoting HNSCC through ZFY-short splicing. RBMY has been shown to enhance tumour development in male hepatocellular carcinoma (human tissue specimens and transgenic-mouse models) whilst ZFY-short is predicted to have anti-apoptotic properties and the deletion of RBMY locus on Y-chromosome resulted in lowered ZFY-short expression. Thus, we hypothesize that ZFY-short is generated by RBMY and exerts its anti-apoptotic effects to promote male HNSCC. Methods: Due to the coronavirus lockdown, bench work was restricted to 6 months, therefore, I conducted an extended analysis of RBMY expression in human cancer, including a computational analysis of RBMY gene expression with data from the cBioPortal database. In my bench-work, I attempted to establish GFP- RBMY expressing cell lines and conducted fluorescence microscopy, RT- PCR and qPCR to analyse RBMY expression in HNSCC cell lines and its impact on ZFY-short expression. Results: RBMY is expressed in several cancers, with no driver mutations. RBMY has nuclear localisation and is expressed in 93-UV-147T and UM-SCC-104 cell lines (both HPV16-positive HNSCC cell lines), with increased ZFY-short expression observed in UM- SCC-104. Discussion: Despite RBMY having been shown to be an oncogene in male liver cancer, our analysis of cBioPortal data suggests this activity may be restricted to the small minority of tumours of different cancer types that express RBMY. The paralleled expression of RBMY and ZFY-short in our cell lines indicate an association. UMSCC104 cell line originates from a highly an aggressive and recurrent tumour, RBMY is associated with tumour stemness, thus it is possible that via ZFY-short, RBMY could have promoted the aggressive phenotype in this, and in other HNSCCs

    Elasticidad de membrana plasmática de levaduras mediante estudios de retracción de Nanotubo

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    27 h. figuras; tabls.; ilus. Abstract en espa√Īol e ingl√©s. Contiene Referencia Bibliogr√°fica.Las membranas biol√≥gicas constituyen la estructura celular m√°s com√ļn en la materia viva, pudiendo ser consideradas como un ejemplo de alta tecnolog√≠a natural de microencapsulaci√≥n. Las membranas se comportan como barreras mec√°nicas semipermeables, regulando el tr√°nsito y la se√Īalizaci√≥n de los compartimentos celulares con el exterior. A su vez, son extremadamente din√°micas tanto de manera global como a peque√Īa escala espacial. Las interacciones de los l√≠pidos entre s√≠ y con otras mol√©culas presentes en las membranas (principalmente prote√≠nas) son determinantes para muchas funciones, como la se√Īalizaci√≥n, los procesos de fusi√≥n y fisi√≥n o la adhesi√≥n celular. La forma f√≠sica de una estructura membranosa est√° determinada b√°sicamente por su grado de curvatura intr√≠nseca. De igual manera, esta propiedad evidencia la tendencia que tienen los l√≠pidos a formar fases no lamelares. La disposici√≥n de los l√≠pidos en la bicapa est√° directamente relacionada con su forma intr√≠nseca o geom√©trica, que tambi√©n condiciona el modo en que estas mol√©culas se empaquetan e interaccionan entre s√≠. En este trabajo de tesina se puso foco en la capacidad que tiene la membrana plasm√°tica de cambiar localmente su curvatura. Para realizar estos estudios, deformamos la membrana plasm√°tica localmente, generando tubos de membrana de di√°metro nanom√©trico mediante el empleo de pinzas √≥pticas. El trabajo se realiz√≥ sobre membranas de levaduras Saccharomyces cerevisiae de la cepa BY4741. Estudios previos han demostrado que cuando una bicapa lip√≠dica es sometida a un cambio local de curvatura presenta un comportamiento el√°stico, mientras que las membranas celulares de mam√≠feros son marcadamente viscoel√°sticas. Por otro lado, no se han reportado estudios de deformaciones locales de curvatura en membranas de organismos de otros reinos, que no contienen colesterol sino otros esteroles u hopanoides. Mediante estos experimentos se pretendi√≥ poner a punto la metodolog√≠a a fin de estudiar c√≥mo es la energ√©tica de deformaci√≥n fuera del plano de membrana plasm√°tica de levaduras S. cerevisiae cepa BY4741, la cual contiene un alto porcentaje del esterol ergosterol, en comparaci√≥n con el comportamiento ya conocido de membrana plasm√°tica de c√©lulas de mam√≠fero. En este trabajo se encontr√≥ que la relajaci√≥n de nanotubos de membranas plasm√°ticas de levaduras S. cerevisiae cepa BY4741 ocurre con tiempos caracter√≠sticos en el intervalo 0,02-0,5 s, y si promediamos todos los valores encontrados, obtenemos un valor promedio de 0,2 s ¬Ī 0,1 s. Este valor es similar a lo informado para eritrocitos, as√≠ como para c√©lulas tumorales cerebrales en humanos y c√©lulas de ovario de h√°mster chino. Hasta donde sabemos, no hay estudios de este tipo reportados en microorganismos eucariotas.Fil: Bischof, Andrea Alejandra. Universidad Nacional de C√≥rdoba. Facultad de Ciencias Exactas, F√≠sicas y Naturales; Argentina.Fil: Bischof, Andrea Alejandra. Universidad Nacional de C√≥rdoba. Facultad de Ciencias Qu√≠micas. Departamento de Qu√≠mica Biol√≥gica; Argentina.Fil: Bischof, Andrea Alejandra. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Centro de Investigaciones en Qu√≠mica Biol√≥gica de C√≥rdoba; Argentina

    Prevalence, quantification and antibiotic resistance of Listeria monocytogenes in poultry preparations

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    .A total of 100 samples of fresh poultry preparations were obtained from 10 retail outlets in North-Western Spain. Listeria spp. were found in 73 samples. Isolates were identified through polymerase chain reaction (PCR) as Listeria monocytogenes (56 samples), Listeria innocua (32), Listeria grayi (3), Listeria seeligeri (1) and Listeria spp. (6). In 24 samples, several different Listeria species were found. The loads of L. monocytogenes detected by quantitative polymerase chain reaction (q-PCR) in the 56 positive samples ranged from 0.05) on concentrations of L. monocytogenes. A total of 163 L. monocytogenes isolates were tested (disc diffusion) against 15 antimicrobials of clinical significance. The average number of resistances per isolate was 5.83 ¬Ī 1.64. All strains showed resistance to multiple antimicrobials (between 4 and 11). In all, 80 isolates (49.1%) showed a multi-drug resistant (MDR) phenotype, and two isolates (1.2%) showed an extensively drugresistant (XDR) phenotype. More than 50.0% of isolates showed resistance or reduced susceptibility to oxacillin, cefoxitin, cefotaxime, cefepime, rifampicin, ciprofloxacin, enrofloxacin or nitrofurantoin. This is a cause for concern because these substances are among the antibiotics used to treat human listeriosis, with rifampicin and fluoroquinolones frequently being used. The results from this research work show that poultry preparations are a potential major source of resistant L. monocytogenes strains, since these are present in some samples at high concentrations. This highlights the pressing need to handle poultry preparations correctly, so as to ensure they are sufficiently cooked and to avoid cross-contamination events.S

    Evaluación de la actividad de nanopartículas de plata (AgNPs) como nuevos agentes anti-Trypanosoma cruzi

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    43 h. + Anexo. tabls.; figuras; ilus. Contiene Referencia Bibliogr√°ficaNanopart√≠culas de plata (AgNPs) fueron obtenidas mediante s√≠ntesis natural a partir de un extracto de achicoria (Cichorium intybus) para probar su efecto parasiticida sobre epimastigotes y tripomastigotes de la cepa Y de Trypanosoma cruzi (TcII). La producci√≥n de AgNPs se realiz√≥ mediante reacciones de oxido-reducci√≥n a partir de nitrato de plata (AgNO3). Se caracterizaron el tama√Īo medio y concentraci√≥n de las nanopart√≠culas obtenidas. Se ensay√≥ la citotoxicidad sobre la l√≠nea celular Vero proveniente de mono verde africano (Cercophitecus aethiops) mediante la t√©cnica de captaci√≥n del rojo neutro. La actividad en epimastigotes se evalu√≥ mediante la exposici√≥n durante 96 h a diluciones seriadas de AgNPs a partir del nivel de m√°xima concentraci√≥n no citot√≥xica (MCNC) reportada en el test de citotoxicidad. La actividad oxidativa fue cuantificada con la sonda H2DCFDA usando espectrofluorimetr√≠a. Se realiz√≥ microscop√≠a de fluorescencia sobre los epimastigotes tratados con la IC50 de AgNPs y grupos control a las 96 h. La actividad en tripomastigotes se realiz√≥ con un ensayo en sangre de rat√≥n obtenida a los 15 d√≠as post infecci√≥n. Las AgNPs producidas presentaron una absorbancia m√°xima a una longitud de ‚Čą440nm. El tama√Īo promedio fue de 67 nm y la concentraci√≥n de 23 pM. El ensayo de captaci√≥n del rojo neutro en la linea celular Vero arroj√≥ un valor de MCNC para AgNO3 de 0,0007 mM (700pM) y de 3 pM para las AgNPs sintetizadas. Las AgNPs sobre epimastigotes arrojaron un valor de IC50=1,153 pM para AgNPs a las 96 horas de tratamiento, siendo la MCNC (3 pM) letal para el 100% de los par√°sitos. Las c√©lulas presentaron da√Īo evidente. El control con AgNO3 present√≥ una tasa de inhibici√≥n absoluta. Se observ√≥ actividad oxidativa en c√©lulas expuestas a la IC50. En la forma tripomastigote se logr√≥ una inhibici√≥n m√°xima del ‚Čą60% a la MCNC. La concentraci√≥n efectiva 50 (EC50) fue de 2,58 pM.Fil: Ju√°rez, Marcos Daniel. Universidad Nacional de C√≥rdoba. Facultad de Ciencias Exactas, F√≠sicas y Naturales; Argentina.Fil: Ju√°rez, Marcos Daniel. Universidad Nacional de C√≥rdoba. Facultad de Ciencias M√©dicas. Centro de Investigaci√≥n de la Enfermedad de Chagas y Leishmaniasis; Argentina

    NMR structure of the noncytotoxic őĪ-sarcin mutant őĒ(7-22): The importance of the native conformation of peripheral loops for activity

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    The deletion mutant őĒ(7-22) of őĪ-sarcin, unlike its wild-type protein counterpart, lacks the specific ability to degrade rRNA in intact ribosomes and exhibits an increased unspecific ribonuclease activity and decreased interaction with lipid vesicles. In trying to shed light on these differences, we report here on the three-dimensional structure of the őĒ(7-22) őĪ-sarcin mutant using NMR methods. We also evaluated its dynamic properties on the basis of theoretical models and measured its correlation time (6.2 nsec) by time-resolved fluorescence anisotropy. The global fold characteristic of ribotoxins is preserved in the mutant. The most significant differences with respect to the őĪ-sarcin structure are concentrated in (1) loop 2, (2) loop 3, which adopts a new orientation, and (3) loop 5, which shows multiple conformations and an altered dynamics. The interactions between loop 5 and the N-terminal hairpin are lost in the mutant, producing increased solvent accessibility of the active-site residues. The degree of solvent exposure of the catalytic His 137 is similar to that shown by His 92 in RNase T1. Additionally, the calculated order parameters of residues belonging to loop 5 in the mutant correspond to an internal dynamic behavior more similar to RNase T1 than őĪ-sarcin. On the other hand, changes in the relative orientation of loop 3 move the lysine-rich region 111‚Äď114, crucial for substrate recognition, away from the active site. All of the structural and dynamic data presented here reveal that the mutant is a hybrid of ribotoxins and noncytotoxic ribonucleases, consistent with its biological properties
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