Gen expression study in Mytilus galloprovincialis after exposure to Prorocentrum lima using quantitative real-time PCR

[Resumen]: Las mareas rojas (Harmful Algal Blooms, HABs) son un problema ecológico y sanitario cada vez más frecuente en las costas gallegas y en todo el mundo. En Galicia las HABs más comunes son las producidas por microalgas productoras de toxinas diarreicas (Diarrhetic Shellfish Poissoning, DSP). El mejillón Mytilus galloprovincialis es uno de los principales vectores de las toxinas DSP. Además, es un organismo centinela de la contaminación, idóneo para el estudio y comprensión de los efectos genotóxicos y citotóxicos que estas toxinas tienen sobre los bivalvos y los ecosistemas. En Galicia, el mejillón es un importante recurso para la economía local a través de su explotación acuícola. Las HABs pueden suponer unas pérdidas económicas cuantiosas para este sector lo que supone un valor añadido para el estudio y comprensión de estos fenómenos y los efectos que causan. En este trabajo se estudian los posibles efectos que pueden tener las toxinas DSP en la expresión de las caspasas 3/7-1, 3/7-3 y 3/7-4 en M. galloprovinicialis, ya que éstas tienen un papel importante en la apoptosis. Para ello, se usaron muestras de branquia de mejillón tomadas bajo dos condiciones experimentales: una control y otra tratamiento (exposición a 100.000 células/L de Prorocentrum lima durante 24 h). De las muestras se extrajo el ARN y éste se utilizó, a través de una qPCR, para detectar las posibles diferencias de expresión. Aunque los resultados finales no muestran una expresión diferencial, estos aportan más información para la comprensión del proceso apoptótico en M. galloprovinicialis y sus mecanismos de defensa ante las toxinas.[Abstract]: Harmful algal blooms are an ecological and health problem, which is becoming more frequent in the Galician coast and all over the world. In Galicia the more frequent ones are those originated by microalgae producers of DSP toxins (Diarrhetic Shellfish Poissoning). The mediterranean mussel Mytilus galloprovincialis is one of the main vectors of DSP intoxication in humans. Also, it is considered a centinel organism of pollution, perfect for the assessment and understanding of the genotoxic and cytotoxic effects that these phenomena inflict on bivalves and ecosistems. In Galicia, mussel farming is a major economic resource for local economy and HABs are able to produce important losses to this activity. This fact makes the study and understanding of HABs and its effects even more crucial. In this study, the effects that DSP toxins can produce in the expression of caspases 3/7-1, 3/7-3 and 3/7-4 in M. galloprovinicialis are investigated, since these proteins are associated with an important role in the apoptosis. For this aim, gill samples taken under two different experimental conditions were used: control and treated (24 h treatment with Prorocentrum lima, 100,000 cells/L). From the samples the RNA was extracted and used, through a qPCR, to assess a possible differential expression. Even though results do not show a differential gene expression, they contribute to the understanding of the apoptotic process in M. galloprovinicialis and its defense mechanisms against toxins.[Resumo]: As mareas vermellas ( Harmful Algal Blooms, HABs) son un problema ecolóxico e sanitario cada vez máis frecuente nas costas galegas e en todo o mundo. En Galicia as HABs máis comúns son as producidas por microalgas produtoras de toxinas diarreicas (Diarrhetic Shellfish Poissoning, DSP). O mexillón Mytilus galloprovincialis é un dos principais vectores de intoxicación con toxinas DSP. Ademais, é un organismo sentinela da contaminación, idóneo para o estudo e 2 comprensión dos efectos xenotóxicos e citotóxicos que estes fenómenos teñen sobre os bivalvos e os ecosistemas. En Galicia, o mexillón é un importante recurso para a economía local a través da súa explotación acuícola. As HABs poden supoñer unhas perdas económicas cuantiosas para este sector o que supón un valor engadido para o estudo e comprensión destes fenómenos e os efectos que causan. Neste traballo estúdanse os posibles efectos que poden ter as toxinas DSP na expresión das caspasas 3/7-1, 3/7-3 e 3/7-4 en M. galloprovinicialis, xa que éstas estan asociadas a un papel importante na apoptose. Para iso empregáronse mostras de branquia de mexillón tomadas baixo dúas condicions experimentais: unha control e outra tratamento (exposición a Prorocentrum lima, 100.000 células/ L durante 24 h). Destas mostras extraeuse o ARN, que sería utilizado, a través dunha qPCR, para detectar as posibles diferenzas de expresión. Aínda que os resultados finais non mostran unha expresión diferencial, estes achegan máis información para a comprensión do proceso apoptótico en M. galloprovinicialis e os seus mecanismos de defensa ante as toxinas.Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2017/201

Circulating miR-181a-5p as a new biomarker for acute cellular rejection in heart transplantation

[Abstract] Background: Acute cellular rejection (ACR) is a major complication in heart transplantation (HTx). Endomyocardial biopsy is the reference method for early detection of ACR, but a new non-invasive approach is needed. Tentative candidates could be circulating microRNAs. This study aimed to discover and validate microRNAs in serum for ACR detection after HTx. Methods: This prospective, observational, single-center study included 121 HTx patients. ACR was graded according to International Society of Heart and Lung Transplantation classification (0R−3R). First, in the discovery phase, microRNA expression profile was carried out in serum samples from patients at pre-rejection, during, and post-rejection time (0RS1 → 2RS2 → 0RS3). Relative expression (2-ΔCq) of 179 microRNAs per sample was analyzed by reverse transcription quantitative polymerase chain reaction. Second, a microRNA with a significant rise and fall pattern during ACR was selected for the next validation phase, where it was analyzed (reverse transcription quantitative polymerase chain reaction) in serum samples from 2 groups of patients: the no-ACR group (0R grade) and the ACR group (≥2R grade). Finally, a sensitivity analysis (receiver operating characteristic curve) was done to assess microRNA accuracy for ACR detection in HTx. Results: A total of 21 ACR episodes (0RS1 → 2RS2 → 0RS3) with their respective serum samples (n = 63) were included in the discovery phase. Among the 179 microRNAs analyzed, only miR-181a-5p met the rise and fall criteria. In the validation phase, miR-181a-5p relative expression (2-ΔCq) in the ACR group (n = 45) was significantly overexpressed (p < 0.0001) vs the no-ACR group (n = 45). miR-181a-5p showed an area under the curve of 0.804 (95% confidence interval: 0.707-0.880); sensitivity and specificity of 78% and 76%, respectively; and a negative predicted value of 98%.Instituto de Salud Carlos III; PI15/0222

Presence of Bacterial DNA in Thrombotic Material of Patients with Myocardial Infarction

[Abstract] Infectious agents have been suggested to be involved in etiopathogenesis of Acute Coronary Syndrome (ACS). However, the relationship between bacterial infection and acute myocardial infarction (AMI) has not yet been completely clarified. The objective of this study is to detect bacterial DNA in thrombotic material of patients with ACS with ST-segment elevation (STEMI) treated with Primary Percutaneous Coronary Intervention (PPCI). We studied 109 consecutive patients with STEMI, who underwent thrombus aspiration and arterial peripheral blood sampling. Testing for bacterial DNA was performed by probe-based real-time Polymerase Chain Reaction (PCR). 12 probes and primers were used for the detection of Aggregatibacter actinomycetemcomitans, Chlamydia pneumoniae, viridans group streptococci, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannarella forsythia, Treponema denticola, Helycobacter pylori, Mycoplasma pneumoniae, Staphylococus aureus, Prevotella intermedia and Streptococcus mutans. Thus, DNA of four species of bacteria was detected in 10 of the 109 patients studied. The most frequent species was viridans group streptococci (6 patients, 5.5%), followed by Staphylococus aureus (2 patients, 1.8%). Moreover, a patient had DNA of Porphyromonas gingivalis (0.9%); and another patient had DNA of Prevotella intermedia (0.9%). Bacterial DNA was not detected in peripheral blood of any of our patients. In conclusion, DNA of four species of endodontic and periodontal bacteria was detected in thrombotic material of 10 STEMI patients. Bacterial DNA was not detected in the peripheral blood of patients with bacterial DNA in their thrombotic material. Bacteria could be latently present in plaques and might play a role in plaque instability and thrombus formation leading to ACS

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field