51,253 research outputs found

    Mapping Super-Relaxed States of Myosin Heads in Sarcomeres using Oblique Angle Fluorescent Microscopy

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    We have utilised modern methods of super-resolution fluorescent microscopy to spatially map fluorescently labelled ATP molecules in relaxed rabbit psoas skeletal muscles. For our imaging process, we have labelled ATP molecules with Rhodamine and Z-lines with Alexa488. Data from imaging these fluorophores have been collected using oblique angle fluorescent microscopy and further analysed to map super relaxed states (SRX) of myosin heads on the thick filament. Our experiments have concluded that most SRX of myosin heads were found in the C-zone of the thick filament, while other zones of thick filament had smaller populations of SRX. Further introduction of mavacamten (MAVA) to our imaging system has revealed an increase in SRX in both D and P zones, while the C zone population of SRX had remained constant. Further experiments must be conducted to establish a clear pattern and further proof our findings

    A Microfluidic Device as a Drug Carrier

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    The development of nanomedicine or medical nanotechnology, has brought important new ways to the development of medicines and biotechnology products. As a result of groundbreaking discoveries in the use of nanoscale materials significant commercialization initiatives have been launched and are at the forefront of the rapidly expanding field of nanotechnology by using smart particles. Microfluidic technologies use nano-and micro-scale manufacturing technologies to develop controlled and reproducible liquid microenvironments. Lead compounds with controlled physicochemical properties can be obtained using microfluidics, characterized by high productivity, and evaluated by biomimetic methods. Microfluidics, for example, can not only produce nanoparticles in a well-controlled, reproducible, and high-throughput manner, but it can also continuously create three-dimensional environments to mimic physiological and/or pathological processes. Materials with smart properties can be manipulated to respond in a controllable and reversible way, modifying some of their properties as a result of external stimuli such as mechanical stress or a certain temperature. All in all, microfluidic technology offers a potential platform for the rapid synthesis of various novel drug delivery systems. Therefore, these smart particles are equally necessary as the drug in drug delivery

    Drivers and Direct Impacts of Lean Mass Dynamics on the Stopover Ecology and Migratory Pace of Nearctic-Neotropical Migrant Songbirds in Spring

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    Annual migration in songbirds is one of the most demanding life-history stages. It represents a period of high mortality, yet there is still much unknown about the ecological correlates that influence its successful completion. After long non-stop migratory flights, birds require a stopover period to rest and replenish depleted energy reserves. Birds use fat as the primary fuel to power long-distance flights. However, birds also burn lean tissue, which results in significant reductions in muscle and organ masses. The discovery and quantification of lean mass catabolism represented a paradigm shift in migration ecology because non-fat components were thought to remain homeostatic. Because rebuilding protein is slow, muscle and organ breakdown during migration may dramatically prolong stopover periods and delay overall migration time, which in turn dramatically reduces breeding success. Therefore, the breakdown of lean tissue, the conditions that lead to it, and its consequences are important considerations in understanding the migration strategies of birds. Through this dissertation research, I aim to understand the impact of weather on body condition and how physiological condition impacts subsequent migratory performance. I investigate (1) how weather impacts the lean mass of songbirds after crossing an ecological barrier, and (2) how body condition after crossing an ecological barrier affects stopover duration, refueling rate, and habitat use. My predictions are that higher nightly temperatures or drier conditions experienced during migratory flight will correspond with lower lean body mass on arrival; and that birds with lower lean body mass will require longer stopovers, different habitat, or higher foraging effort to continue migration. I used an integrative approach, combining the field and lab, to better understand how weather experienced during flight can impact the body condition of migratory birds and how this can influence the entire migratory cycle. By using Quantitative Magnetic Resonance (QMR) technology in combination with a novel automated radio-telemetry system, my research provides unprecedented access to detailed physiological and movement data for small migratory songbirds. This research underlines that successfully crossing the Gulf of Mexico may be a key driver of physiological and morphological adaptations. My findings challenge the current paradigm that birds with low lean mass require longer stopover and demonstrates that species under time constraints may shorten stopover even when in poor condition, departing in sub-optimal body condition

    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

    Different brain oxidative and neuroinflammation status in rats during prolonged abstinence depending on their ethanol relapse-like drinking behavior: Effects of ethanol reintroduction

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    Rationale: Accumulating evidence suggests that chronic alcohol consumption is associated with excessive oxidative damage and neuroinflammatory processes and these events have been associated to early alcohol withdrawal. In the present research we wonder if brain oxidative stress and neuroinflammation remains altered during prolonged withdrawal situations and whether these alterations can be correlated with relapse behavior in alcohol consumption. The effects of alcohol reintroduction were also evaluated. Methods: We have used a model based on the alcohol deprivation effect (ADE) within a cohort of wild-type male Wistar rats. Two subpopulations were identified according to the alcohol relapse-like drinking behavior displayed (ADE and NO-ADE subpopulations). Oxidized and reduced glutathione content was determined within the hippocampus and the amygdala using a mass spectrometry method. The levels of mRNA of seven different inflammatory mediators in the prefrontal cortex of rats were quantified. All the analyses were performed in two different conditions: after 21-day alcohol deprivation (prolonged abstinence) and after 24 h of ethanol reintroduction in both subpopulations. Results: ADE and NO-ADE rats showed different endophenotypes. ADE rats always displayed a significant lower alcohol intake rate and ethanol preference than NO-ADE rats. The results also demonstrated the existence of altered brain redox and neuroinflammation status after prolonged abstinence exclusively in ADE rats. Moreover, when ethanol was reintroduced in the ADE subpopulation, altered oxidative stress and neuroinflammatory markers were restored. Conclusions: Present findings provide new mechanisms underlying the neurobiology of relapse behavior and suggest the development of new pharmacological approaches to treat alcohol-induced relapse
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