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Lyme carditis is an extracutaneous manifestation of Lyme disease characterized by episodes of atrioventricular block of varying degrees and additional, less reported cardiomyopathies. The molecular changes associated with the response to Borrelia burgdorferi over the course of infection are poorly understood. Here, we identify broad transcriptomic and proteomic changes in the heart during infection that reveal a profound down-regulation of mitochondrial components. We also describe the long-term functional modulation of macrophages exposed to live bacteria, characterized by an augmented glycolytic output, increased spirochetal binding and internalization, and reduced inflammatory responses. In vitro, glycolysis inhibition reduces the production of tumor necrosis factor (TNF) by memory macrophages, whereas in vivo, it produces the reversion of the memory phenotype, the recovery of tissue mitochondrial components, and decreased inflammation and spirochetal burdens. These results show that B. burgdorferi induces long-term, memory-like responses in macrophages with tissue-wide consequences that are amenable to be manipulated in vivo.
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Borrelia burgdorferi/inmunología , Cardiomiopatías/etiología , Memoria Inmunológica , Enfermedad de Lyme/inmunología , Macrófagos/fisiología , Animales , Cardiomiopatías/inmunología , Cardiomiopatías/microbiología , Cardiomiopatías/patología , Células Cultivadas , Endocarditis Bacteriana/complicaciones , Endocarditis Bacteriana/inmunología , Endocarditis Bacteriana/microbiología , Endocarditis Bacteriana/patología , Femenino , Células HEK293 , Corazón/microbiología , Humanos , Enfermedad de Lyme/patología , Activación de Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/microbiología , Miocitos Cardíacos/patología , Células RAW 264.7RESUMEN
RNA modifications, including N-7-methylguanosine (m7G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m7G modifications is of paramount significance, given recent associations between altered m7G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m7G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m7G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m7G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH4, neutral pH and the addition of 7-methylguanosine monophosphate (m7GMP) during the reducing reaction. Notably, compared to NaBH4-based methods, Bo-Seq enhances the m7G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m7G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m7G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m7G in gene expression and its implications in cancer biology.
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Guanosina/análogos & derivados , Nucleótidos , ARN , Humanos , ARN/química , Nucleótidos/metabolismo , Metilación , Metiltransferasas/genética , Procesamiento Postranscripcional del ARNRESUMEN
Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology.
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Carcinogénesis , Neoplasias de la Próstata , Masculino , Humanos , Carcinogénesis/genética , Transformación Celular Neoplásica , Neoplasias de la Próstata/genética , Transcripción Genética , Procesamiento Postranscripcional del ARN , Metiltransferasas/genéticaRESUMEN
STUDY QUESTION: Is it possible to use free and extracellular vesicle-associated microRNAs (miRNAs) from human endometrial fluid (EF) samples as non-invasive biomarkers for implantative endometrium? SUMMARY ANSWER: The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner. WHAT IS KNOWN ALREADY: miRNAs and extracellular vesicles (EVs) from EF have been described as mediators of the embryo-endometrium crosstalk. Therefore, the analysis of miRNA from this fluid could become a non-invasive technique for recognizing implantative endometrium. This analysis could potentially help improve the implantation rates in ART. STUDY DESIGN, SIZE, DURATION: In this prospective study, we first optimized different protocols for EVs and miRNA analyses using the EF of a setup cohort (n = 72). Then, we examined differentially expressed miRNAs in the EF of women with successful embryo implantation (discovery cohort n = 15/validation cohort n = 30) in comparison with those for whom the implantation had failed (discovery cohort n = 15/validation cohort n = 30). Successful embryo implantation was considered when pregnancy was confirmed by vaginal ultrasound showing a gestational sac 4 weeks after embryo transfer (ET). PARTICIPANTS/MATERIALS, SETTING, METHODS: The EF of the setup cohort was obtained before starting fertility treatment during the natural cycle, 16-21 days after the beginning of menstruation. For the discovery and validation cohorts, the EF was collected from women undergoing frozen ET on Day 5, and the samples were collected immediately before ET. In this study, we compared five different methods; two of them based on direct extraction of RNA and the other three with an EV enrichment step before the RNA extraction. Small RNA sequencing was performed to determine the most efficient method and find a predictive model differentiating between implantative and non-implantative endometrium. The models were confirmed using quantitative PCR in two sets of samples (discovery and validation cohorts) with different implantation outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: The protocols using EV enrichment detected more miRNAs than the methods based on direct RNA extraction. The two most efficient protocols (using polymer-based precipitation (PBP): PBP-M and PBP-N) were used to obtain two predictive models (based on three miRNAs) allowing us to distinguish between an implantative and non-implantative endometrium. The first Model 1 (PBP-M) (discovery: AUC = 0.93; P-value = 0.003; validation: AUC = 0.69; P-value = 0.019) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (discovery: AUC = 0.92; P-value = 0.0002; validation: AUC = 0.78; P-value = 0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Functional analysis of these miRNAs showed strong association with key implantation processes such as in utero embryonic development or transforming growth factor-beta signaling. LARGE SCALE DATA: The FASTQ data are available in the GEO database (access number GSE178917). LIMITATIONS, REASONS FOR CAUTION: One important factor to consider is the inherent variability among the women involved in the trial and among the transferred embryos. The embryos were pre-selected based on morphology, but neither genetic nor molecular studies were conducted, which would have improved the accuracy of our tests. In addition, a limitation in miRNA library construction is the low amount of input RNA. WIDER IMPLICATIONS OF THE FINDINGS: We describe new non-invasive protocols to analyze miRNAs from small volumes of EF. These protocols could be implemented in clinical practice to assess the status of the endometrium before attempting ET. Such evaluation could help to avoid the loss of embryos transferred to a non-implantative endometrium. STUDY FUNDING/COMPETING INTEREST(S): J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). It was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the National Plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not play any role in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. The authors declare no competing interests.
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Endometrio , MicroARNs , Biomarcadores , Femenino , Humanos , MicroARNs/genética , Polímeros , Embarazo , Estudios Prospectivos , Factores de Crecimiento TransformadoresRESUMEN
Tumour progression and therapy tolerance are highly regulated and complex processes largely dependent on the plasticity of cancer cells and their capacity to respond to stress. The higher plasticity of cancer cells highlights the need for identifying targetable molecular pathways that challenge cancer cell survival. Here, we show that N7-guanosine methylation (m7G) of tRNAs, mediated by METTL1, regulates survival to stress conditions in cancer cells. Mechanistically, we find that m7G in tRNAs protects them from stress-induced cleavage and processing into 5' tRNA fragments. Our analyses reveal that the loss of tRNA m7G methylation activates stress response pathways, sensitising cancer cells to stress. Furthermore, we find that the loss of METTL1 reduces tumour growth and increases cytotoxic stress in vivo. Our study uncovers the role of m7G methylation of tRNAs in stress responses and highlights the potential of targeting METTL1 to sensitise cancer cells to chemotherapy.
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Anti-TNF therapy can induce and maintain a remission status during intestinal bowel disease. However, up to 30% of patients do not respond to this therapy by mechanisms that are unknown. Here, we show that the absence of MCJ, a natural inhibitor of the respiratory chain Complex I, induces gut microbiota changes that are critical determinants of the lack of response in a murine model of DSS-induced inflammation. First, we found that MCJ expression is restricted to macrophages in human colonic tissue. Therefore, we demonstrate by transcriptomic analysis of colon macrophages from DSS-induced mice that MCJ-deficiency is linked to the expression of genes belonging to the FcγR signaling pathway and contains an anti-TNF refractory gene signature identified in ulcerative colitis patients. The gut microbial composition changes observed upon DSS treatment in the MCJ-deficient mice revealed the increased presence of specific colitogenic members, including Ruminococcus gnavus and Oscillospira, which could be associated with the non-response to TNF inhibitors. Further, we show that the presence of a microbiota associated resistance to treatment is dominant and transmissible to responsive individuals. Collectively, our findings underscore the critical role played by macrophage mitochondrial function in the gut ecological niche that can substantially affect not only the severity of inflammation but also the ability to successfully respond to current therapies.
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Colitis Ulcerosa , Colitis , Microbioma Gastrointestinal , Microbiota , Humanos , Animales , Ratones , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Inhibidores del Factor de Necrosis Tumoral/efectos adversos , Inhibidores del Factor de Necrosis Tumoral/metabolismo , Colitis/inducido químicamente , Microbioma Gastrointestinal/fisiología , Colon/metabolismo , Inflamación/metabolismo , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum. Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut.
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Inmunidad Innata , Lactobacillaceae/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Adulto , Anciano , Animales , Péptidos Antimicrobianos/inmunología , Femenino , Humanos , Memoria Inmunológica , Interleucina-10/inmunología , Macrófagos/microbiología , Masculino , Ratones , Microbiota , Persona de Mediana Edad , Monocitos/microbiología , Células RAW 264.7 , Saliva/microbiología , SimbiosisRESUMEN
The development of the human brain starts in the first weeks of embryo differentiation. However, there are many relevant neurodevelopmental processes that take place after birth and during lifespan. Such a fine and changing scenario requires the coordinated expression of thousands of genes to achieve the proper specialization and inter-connectivity. In this context, microRNAs (miRNAs), which can modulate mRNA stability and translation, are gaining recognition for their involvement in both brain development and neurodevelopmental disorders. Therefore, cerebrospinal fluid (CSF) miRNAs should be perfectly differentiated in relevant age periods. In this study, we aimed to highlight the biological variability of miRNA expression in the CSF throughout life, which is also crucial for biomarker discovery in CNS pathologies, especially in children, where they are desperately needed. We analyzed the CSF microRNAome of 14 healthy children (aged 0-7.4 years) by smallRNA-Seq and compared it with previously published data in adults (N = 7) and elders (N = 11). miR-423-5p and miR-22-3p were overexpressed in the < 1 and > 3 years groups, respectively. Additionally, we detected 18 miRNAs that reached their highest peak of expression at different time-points during the lifespan and sets of miRNAs that were exclusively expressed in a specific age group. On the contrary, miR-191-5p showed stable expression in CSF from the first year of life. Our results remark the complex differential miRNA expression profile that can be observed through life, which underlines the need for including appropriate age-matched controls when the expression of CSF miRNAs is analyzed in different pathological contexts. Graphical abstract.
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Envejecimiento/líquido cefalorraquídeo , Envejecimiento/genética , MicroARNs/líquido cefalorraquídeo , Niño , Preescolar , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes Esenciales , Humanos , Lactante , Recién Nacido , Masculino , MicroARNs/genéticaRESUMEN
Extracellular vesicles (EVs) mediate cell-to-cell crosstalk whose content can induce changes in acceptor cells and their microenvironment. MLP29 cells are mouse liver progenitor cells that release EVs loaded with signaling cues that could affect cell fate. In the current work, we incubated 3T3-L1 mouse fibroblasts with MLP29-derived EVs, and then analyzed changes by proteomics and transcriptomics. Results showed a general downregulation of protein and transcript expression related to proliferative and metabolic routes dependent on TGF-beta. We also observed an increase in the ERBB2 interacting protein (ERBIN) and Cxcl2, together with an induction of ribosome biogenesis and interferon-related response molecules, suggesting the activation of immune system signaling.
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Dyslexia and attention deficit hyperactivity disorder (ADHD) are two complex neuro-behaviorally disorders that co-occur more often than expected, so that reading disability has been linked to inattention symptoms. We examined 4 SNPs located on genes previously associated to dyslexia (KIAA0319, DCDC2, DYX1C1 and FOXP2) and 3 SNPs within genes related to ADHD (COMT, MAOA and DBH) in a cohort of Spanish children (N = 2078) that met the criteria of having one, both or none of these disorders (dyslexia and ADHD). We used a case-control approach comparing different groups of samples based on each individual diagnosis. In addition, we also performed a quantitative trait analysis with psychometric measures on the general population (N = 3357). The results indicated that the significance values for some markers change depending on the phenotypic groups compared and/or when considering pair-wise marker interactions. Furthermore, our quantitative trait study showed significant genetic associations with specific cognitive processes. These outcomes advocate the importance of establishing rigorous and homogeneous criteria for the diagnosis of cognitive disorders, as well as the relevance of considering cognitive endophenotypes.
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Trastorno por Déficit de Atención con Hiperactividad/epidemiología , Trastorno por Déficit de Atención con Hiperactividad/genética , Dislexia/epidemiología , Dislexia/genética , Estudios de Asociación Genética , Trastorno por Déficit de Atención con Hiperactividad/fisiopatología , Niño , Cognición , Estudios de Cohortes , Comorbilidad , Dislexia/fisiopatología , Femenino , Técnicas de Genotipaje , Humanos , Masculino , Polimorfismo de Nucleótido Simple , España/epidemiologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0206431.].
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Objetivo: explorar los efectos del confinamiento por la pandemia COVID-19 en el estilo de vida de la población general y profesionales sanitarios (PS). Método: estudio descriptivo transversal. Se realizó muestreo no probabilístico por bola de nieve a través de WhatsApp. Se difundió un cuestionario realizado ad hoc previamente pilotado sobre variables sociodemográficas, clínicas y hábitos de vida durante el confinamiento. Para el análisis se utilizaron las pruebas paramétricas t de Student-Fisher, Chi cuadrado de Pearson y odds ratio (OR) con IC95%. Resultados: participaron 184 PS y 460 no sanitarios. La muestra tenía una media de 40,4 (DE: 12,8) años, principalmente eran mujeres (66,9%) con formación universitaria (65,8%). Más PS contaron con una prueba positiva para COVID-19, OR= 17,1 (9,1-32,1) o síntomas compatibles con COVID-19, OR= 2,1 (1,4-3,1). La repercusión psicológica del confinamiento fue superior en PS, OR= 1,5 (1,0-2,1). No se hallaron diferencias significativas en las actividades efectuadas durante la cuarentena exceptuando el teletrabajo (p< 0,001). El 68,0% de la población no sanitaria autorrefirió su grado de preocupación como Muy preocupado y Fuertemente preocupado, similar a los PS (65,8%) (p= 0,218). Los hábitos saludables se mantuvieron durante el confinamiento, a excepción del ejercicio físico que empeora en ambos grupos (66,3% de sanitarios y 66,4% de no sanitarios). El consumo de tabaco y/o alcohol aumentó entre un 20 y un 30% en ambos grupos. Conclusiones: se perciben cambios entre PS y no sanitarios en cuanto a la ocupación durante el confinamiento, el impacto psicológico o la tendencia al contagio. Es imprescindible introducir recursos para el cuidado de la salud mental, especialmente en PS.(AU)
Objective: to explore the effects of lockdown for the COVID-19 pandemic on the lifestyles of the overall population and healthcare professionals (HCPs). Method: a cross-sectional descriptive study. Non-probability snowball sampling was conducted through WhatsApp. After the pilot survey, an ad-hoc questionnaire was distributed on sociodemographic, clinical and lifestyle variables during lockdown. The parametric tests used for analysis were Student-Fishers t and Pearsons Chi-squared test, and odds ratio with CI95%. Results: the study included 184 HCPs and 460 non-HCPs. The median age of the sample was 40.4 (SD: 12.8) years, they were mostly women (66.9%) with university education (65.8%). More HCPs had positive test results for COVID-19, OR= 17.1 (9.1-32.1) or symptoms compatible with COVID-19, OR= 2.1 (1.4-3.1). The psychological impact of lockdown was higher in HCPs, OR= 1.5 (1.0-2.1). There were no significant differences in the activities carried out during quarantine, except for telework (p< 0.001). The degree of concern self-reported by 68.0% of the non-HCP population was Very concerned and Highly concerned, similarly to HCPs (65.8%) (p= 0.218). Healthy lifestyles were maintained during lockdown, except for physical exercise which was worse in both groups (66.3% of HCPs and 66.4% of non-HCPs). Smoking and/or alcohol intake increased between 20 and 30% in both groups. Conclusions: changes were perceived among HCPs and non-HCPs regarding occupation during lockdown, psychological impact or tendency to get infected. It is essential to introduce resources for mental healthcare, particularly for HCPs.(AU)
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Humanos , Masculino , Femenino , Aislamiento Social , Personal de Salud , Población , Pandemias , Infecciones por Coronavirus , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Cuarentena , Estilo de Vida Saludable , Agotamiento Profesional , Agotamiento Psicológico , Estrés Psicológico , Epidemiología Descriptiva , Estudios Transversales , Encuestas y Cuestionarios , Enfermería , Hábitos , Estilo de VidaRESUMEN
Single-cell genome and transcriptome characterizations will probe to be decisive within the stem cells research, especially to describe appropriately the genetic impact of the diverse stem cells populations that are present in each organism. In the present chapter, we describe in detail how to prepare sequencing libraries out of single cells, for whole genome DNA and mRNA sequencing.
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Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de la Célula Individual , Transcriptoma , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de la Célula Individual/métodosRESUMEN
Proteolytic signalling cascades control a wide range of physiological responses. In order to respond rapidly, protease activity must be maintained at a basal level: the component zymogens must be sequentially activated and actively degraded. At the same time, signalling cascades must respond precisely: high target specificity is required. The insects have a wide range of trapping- and tight-binding protease inhibitors, which can regulate the activity of individual proteases. In addition, the interactions between component proteases of a signalling cascade can be modified by serine protease homologues. The suicide-inhibition mechanism of serpin family inhibitors gives rapid turnover of both protease and inhibitor, but target specificity is inherently broad. Similarly, the TEP/macroglobulins have extremely broad target specificity, which suits them for roles as hormone transport proteins and sensors of pathogenic virulence factors. The tight-binding inhibitors, on the other hand, have a lock-and-key mechanism capable of high target specificity. In addition, proteins containing multiple tight-binding inhibitory domains may act as scaffolds for the assembly of signalling complexes. Proteolytic cascades regulated by combinations of different types of inhibitor could combine the rapidity of suicide-inhibitors with the specificity lock-and-key inhibitors. This would allow precise control of physiological responses and may turn out to be a general rule.