Novel Fibrillar and Non-Fibrillar Collagens Involved in Fibrotic Scar Formation after Myocardial Infarction.

Following myocardial infarction (MI), adverse remodeling depends on the proper formation of fibrotic scars, composed of type I and III collagen. Our objective was to pinpoint the participation of previously unreported collagens in post-infarction cardiac fibrosis. Gene (qRT-PCR) and protein (immunohistochemistry followed by morphometric analysis) expression of fibrillar (types II and XI) and non-fibrillar (types VIII and XII) collagens were determined in RNA-sequencing data from 92 mice undergoing myocardial ischemia; mice submitted to permanent (non-reperfused MI, n = 8) or transient (reperfused MI, n = 8) coronary occlusion; and eight autopsies from chronic MI patients. In the RNA-sequencing analysis of mice undergoing myocardial ischemia, increased transcriptomic expression of collagen types II, VIII, XI, and XII was reported within the first week, a tendency that persisted 21 days afterwards. In reperfused and non-reperfused experimental MI models, their gene expression was heightened 21 days post-MI induction and positively correlated with infarct size. In chronic MI patients, immunohistochemistry analysis demonstrated their presence in fibrotic scars. Functional analysis indicated that these subunits probably confer tensile strength and ensure the cohesion of interstitial components. Our data reveal that novel collagens are present in the infarcted myocardium. These data could lay the groundwork for unraveling post-MI fibrotic scar composition, which could ultimately influence patient survivorship.

Efecto del suero aislado de pacientes con infarto agudo de miocardio con elevación del segmento ST en las células endoteliales / Effect of serum from patients with ST-segment elevation myocardial infarction on endothelial cells

Introducción y objetivos La reperfusión coronaria produce un daño en la microcirculación y, en concreto, las células endoteliales. Este estudio evalúa el efecto del suero aislado tras la revascularización de pacientes con un infarto agudo de miocardio con elevación del segmento ST (IAMCEST) en la viabilidad celular, el grado de permeabilidad endotelial in vitro y la asociación de estos parámetros con una mayor extensión de los índices de resonancia magnética cardiaca (RMC) relacionados con el daño por reperfusión (edema, hemorragia y obstrucción microvascular). Métodos Se incubaron células endoteliales de arteria coronaria humana con suero aislado 24 h tras la revascularización de 43 pacientes con IAMCEST evaluados mediante RMC y 14 sujetos de control. Se testó el efecto del suero de pacientes con IAMCEST en la pérdida de viabilidad celular por activación de la apoptosis y la necrosis, así como en la permeabilidad y la estructura de la monocapa endotelial. Resultados El suero de pacientes con IAMCEST aumentó la apoptosis (p <0,01) y la necrosis (p <0,05) de células endoteliales de arteria coronaria humana y causó un incremento de la permeabilidad de la monocapa endotelial in vitro (p <0,01) debido a mayores espacios intercelulares (p <0,05 frente a los controles). Una mayor necrosis inducida por suero se asoció con más permeabilidad endotelial in vitro (p <0,05) y con una mayor extensión de los principales índices de daño tras reperfusión y mayor tamaño de infarto. Conclusiones El suero tras la reperfusión de pacientes con IAMCEST induce la apoptosis y la necrosis in vitro de las células endoteliales y la permeabilidad endotelial. Cuanto más potente sea el efecto inductor de necrosis, más deletéreas son las consecuencias en cuanto al daño estructural resultante. (AU)

Introduction and objectives Clinical and experimental studies have shown that, in patients with reperfused ST-segment elevation myocardial infarction (STEMI), abnormalities in the endothelial monolayer are initiated during ischemia but rapidly intensify upon restoration of blood perfusion to the ischemic area. We aimed to evaluate the effect of serum isolated after revascularization from STEMI patients on the degree of endothelial permeability in vitro, by promoting endothelial cell apoptosis and necrosis in vitro. We also investigated the association between the percentage of serum-induced endothelial cell apoptosis or necrosis in vitro and the extent of cardiovascular magnetic resonance (CMR)-derived parameters of reperfusion injury (edema, hemorrhage, and microvascular obstruction). Methods Human coronary artery endothelial cells were incubated with serum isolated 24hours after revascularization from 43 STEMI patients who underwent CMR and 14 control participants. We assessed the effect of STEMI serum on activation of apoptosis and necrosis, as well as on the permeability and structure of the endothelial monolayer. Results Serum from STEMI patients increased apoptosis (P <.01) and necrosis (P <.05) in human coronary artery endothelial cells and caused increased permeability of the endothelial monolayer in vitro (P <.01), due to enlarged intercellular spaces (P <.05 vs control in all cases). Higher serum-induced necrosis was associated with greater endothelial permeability in vitro (P <.05) and with more extensive CMR-derived indices of reperfusion injury and infarct size. Conclusions Postreperfusion serum activates necrosis and apoptosis in endothelial cells and increases the degree of endothelial permeability in vitro. The more potent the necrosis-triggering effect of serum, the more deleterious the consequences in terms of the resulting cardiac structure. (AU)

Impact of Epicardial Adipose Tissue on Infarct Size and Left Ventricular Systolic Function in Patients with Anterior ST-Segment Elevation Myocardial Infarction.

We aimed to assess the correlation of cardiovascular magnetic resonance (CMR)-derived epicardial adipose tissue (EAT) with infarct size (IS) and residual systolic function in ST-segment elevation myocardial infarction (STEMI). We enrolled patients discharged for a first anterior reperfused STEMI submitted to undergo CMR. EAT, left ventricular (LV) ejection fraction (LVEF), and IS were quantified at the 1-week (n = 221) and at 6-month CMR (n = 167). At 1-week CMR, mean EAT was 31 ± 13 mL/m2. Patients with high EAT volume (n = 72) showed larger 1-week IS. After adjustment, EAT extent was independently related to 1-week IS. In patients with large IS at 1 week (>30% of LV mass, n = 88), those with high EAT showed more preserved 6-month LVEF. This association persisted after adjustment and in a 1:1 propensity score-matched patient subset. Overall, EAT decreased at 6 months. In patients with large IS, a greater reduction of EAT was associated with more preserved 6-month LVEF. In STEMI, a higher presence of EAT was associated with a larger IS. Nevertheless, in patients with large infarctions, high EAT and greater subsequent EAT reduction were linked to more preserved LVEF in the chronic phase. This dual and paradoxical effect of EAT fuels the need for further research in this field.

Effect of serum from patients with ST-segment elevation myocardial infarction on endothelial cells.

INTRODUCTION AND OBJECTIVES: Clinical and experimental studies have shown that, in patients with reperfused ST-segment elevation myocardial infarction (STEMI), abnormalities in the endothelial monolayer are initiated during ischemia but rapidly intensify upon restoration of blood perfusion to the ischemic area. We aimed to evaluate the effect of serum isolated after revascularization from STEMI patients on the degree of endothelial permeability in vitro, by promoting endothelial cell apoptosis and necrosis in vitro. We also investigated the association between the percentage of serum-induced endothelial cell apoptosis or necrosis in vitro and the extent of cardiovascular magnetic resonance (CMR)-derived parameters of reperfusion injury (edema, hemorrhage, and microvascular obstruction). METHODS: Human coronary artery endothelial cells were incubated with serum isolated 24hours after revascularization from 43 STEMI patients who underwent CMR and 14 control participants. We assessed the effect of STEMI serum on activation of apoptosis and necrosis, as well as on the permeability and structure of the endothelial monolayer. RESULTS: Serum from STEMI patients increased apoptosis (P <.01) and necrosis (P <.05) in human coronary artery endothelial cells and caused increased permeability of the endothelial monolayer in vitro (P <.01), due to enlarged intercellular spaces (P <.05 vs control in all cases). Higher serum-induced necrosis was associated with greater endothelial permeability in vitro (P <.05) and with more extensive CMR-derived indices of reperfusion injury and infarct size. CONCLUSIONS: Postreperfusion serum activates necrosis and apoptosis in endothelial cells and increases the degree of endothelial permeability in vitro. The more potent the necrosis-triggering effect of serum, the more deleterious the consequences in terms of the resulting cardiac structure.

Novel Targets Regulating the Role of Endothelial Cells and Angiogenesis after Infarction: A RNA Sequencing Analysis.

Endothelial cells (ECs) are a key target for cardioprotection due to their role in preserving cardiac microvasculature and homeostasis after myocardial infarction (MI). Our goal is to identify the genes involved in post-MI EC proliferation, EC apoptosis, and angiogenesis regulation via RNA-sequencing transcriptomic datasets. Using eight studies from the Gene Expression Omnibus, RNA-sequencing data from 92 mice submitted to different times of coronary ischemia or sham were chosen. Functional enrichment analysis was performed based on gene ontology biological processes (BPs). Apoptosis-related BPs are activated up to day 3 after ischemia onset, whereas endothelial proliferation occurs from day 3 onwards, including an overrepresentation of up to 37 genes. Endothelial apoptosis post-MI is triggered via both the extrinsic and intrinsic signaling pathways, as reflected by the overrepresentation of 13 and 2 specific genes, respectively. BPs implicated in new vessel formation are upregulated soon after ischemia onset, whilst the mechanisms aiming at angiogenesis repression can be detected at day 3. Overall, 51 pro-angiogenic and 29 anti-angiogenic factors displayed altered transcriptomic expression post-MI. This is the first study using RNA sequencing datasets to evaluate the genes participating in post-MI endothelium physiology and angiogenesis regulation. These novel data could lay the groundwork to advance understanding of the implication of ECs after MI.

Transgene-free, virus-based gene silencing in plants by artificial microRNAs derived from minimal precursors.

Artificial microRNAs (amiRNAs) are highly specific, 21-nucleotide (nt) small RNAs designed to silence target transcripts. In plants, their application as biotechnological tools for functional genomics or crop improvement is limited by the need of transgenically expressing long primary miRNA (pri-miRNA) precursors to produce the amiRNAs in vivo. Here, we analyzed the minimal structural and sequence requirements for producing effective amiRNAs from the widely used, 521-nt long AtMIR390a pri-miRNA from Arabidopsis thaliana. We functionally screened in Nicotiana benthamiana a large collection of constructs transiently expressing amiRNAs against endogenous genes and from artificially shortened MIR390-based precursors and concluded that highly effective and accurately processed amiRNAs can be produced from a chimeric precursor of only 89 nt. This minimal precursor was further validated in A. thaliana transgenic plants expressing amiRNAs against endogenous genes. Remarkably, minimal but not full-length precursors produce authentic amiRNAs and induce widespread gene silencing in N. benthamiana when expressed from an RNA virus, which can be applied into leaves by spraying infectious crude extracts. Our results reveal that the length of amiRNA precursors can be shortened without affecting silencing efficacy, and that viral vectors including minimal amiRNA precursors can be applied in a transgene-free manner to induce whole-plant gene silencing.

Changes in the extracellular matrix at microvascular obstruction area after reperfused myocardial infarction: A morphometric study.

BACKGROUND: Extracellular matrix (ECM) suffers substantial alterations after myocardial infarction (MI), including the invasion of leukocyte subtypes. Despite a complete reopening at epicardial level, hypoperfusion within the infarcted myocardium, known as microvascular obstruction (MVO), occurs and exerts a negative impact on ventricular remodeling. In this study, ECM composition at MVO regions was described using a morphometric analysis. METHODS: MI was induced in female swine (n = 10) by transitory 90-minute coronary occlusion followed by seven days of reperfusion. Prior to euthanasia, intracoronary thioflavin-S was infused. Within the infarcted myocardium, regions displaying MVO (thioflavin-S-) or no MVO (thioflavin-S+) were isolated and stained to morphometrically compare ECM composition. RESULTS: As reflected by cell invasion through ECM, areas with MVO displayed an enlarged presence of neutrophils and lymphocytes, whilst no differences in the amount of macrophages and myofibroblasts were detected compared to infarcted myocardium without MVO. Indeed, those regions with macroscopic MVO showed lower capillary density than areas without MVO. Lastly, a significant reduction in the extension of total collagen, type I, but not type III, collagen, laminin, and fibronectin together with an augmentation of polysaccharides were noted in areas showing MVO compared to those without microvascular injury. CONCLUSIONS: ECM composition in infarcted regions with MVO isolated from female swine displays a higher presence of inflammatory infiltrate and polysaccharides as well as reduced number of microvessels and collagen content compared to those areas without microvascular hypoperfusion. These characteristics might underlie the development of adverse ventricular remodeling in MI patients with extensive MVO.

Multimerization of HIF enhances transcription of target genes containing the hypoxia ancillary sequence.

Transcriptional activity of the hypoxia inducible factor (HIF) relies on the formation of a heterodimer composed of an oxygen-regulated α-subunit and a stably expressed ß-subunit. Heterodimeric HIF activates expression by binding to RCGTG motifs within promoters of hypoxia-activated genes. Some hypoxia targets also possess an adjacent HIF ancillary sequence (HAS) reported to increase transcription but whose function remains obscure. Here, we investigate the contribution of the HAS element to the hypoxia response and its mechanism of action, using the HAS-containing prolyl 4-hydroxylase subunit α1 (P4HA1) as a gene model in NIH/3T3 mouse embryonic fibroblasts and HEK293 human embryonic kidney cells. Our HIF overexpression experiments demonstrate that the HAS motif is essential for full induction by hypoxia and that the presence of the tandem HAS/HIF, as opposed to HIF-only sequences, provides HIF proteins with the capacity to form complexes of stoichiometry beyond the classical heterodimer, likely tetramers, to cooperatively potentiate hypoxia-induced transcription. We also provide evidence of the crucial role played by the Fα helix of the PAS-B domain of the HIF1ß subunit to support the interaction between heterodimers. Functional analysis showed that human genes containing the HAS/HIF motifs are better responders to hypoxia, and their promoters are enriched for specific transcription factor binding sites. Gene ontology enrichment revealed a predominance of HAS/HIF in genes primarily related to tissue formation and development. Our findings add an extra level of regulation of the hypoxia/HIF signaling through multimerization of HIF proteins on regulatory elements containing the HAS/HIF motifs.

Comparación en el uso de Peróxido de Hidrógeno y de Clorhexidina contra SARS-CoV-2 en la cavidad bucal / Comparison in the use of hydrogen peroxide and chlorhexidine against SARS-COV-2 in the oral cavity

La pandemia de COVID-19 originada por el virus SARS-CoV-2 ha impactado en la atención profesional de los pacientes en la práctica odontológica. Se generan bioaerosoles por el odontólogo o por el propio paciente, que aumentan la posibilidad de diseminación del virus. Ante la urgente necesidad de establecer protocolos estrictos y efectivos de control de infecciones, decidimos investigar la efectividad de dos enjuagues bucales en la saliva de pacientes con diagnóstico positivo de SARS-CoV-2.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has impacted on the professional care of patients. In dentistry, the generation of bioaerosols generated by the dentist or by the patient himself increases the possibility of the spread of the virus. Given the urgent need to establish strict and effective infection control protocols, we decided to investigate the effectiveness of two mouthwashes in the saliva of positive patients.

Grief in children's story books. A systematic integrative review.

BACKGROUND: Grieving is an adaptive process in the face of the death of somebody close. Children grieve the loss of a family member or friend and need support from their caregivers and the professionals who care for them during this process. Failure to talk to children about the death of a family member or friend can lead to prolonged grief. Children's story books are one of the resources available for providing this type of support. OBJECTIVE: To provide the nursing professional with information on story books aimed at children from 7 to 11 years of age as a tool to help them understand and cope with grief. DESIGN: A systematic integrative review was conducted. METHODS: A search was performed in the ISBN database of the Ministry of Culture and the University Libraries Network. Data extraction was performed by two coders using a protocol registered in PROSPERO. RESULTS: Fifty-six books met the inclusion criteria. Twenty-five percent of the deceased characters were grandparents and 30.4% died due to illness. The most frequent emotion was sadness, (43.3%) and the most repeated coping strategy was remembering the deceased person, (28.7%). The grieving process was depicted in 32.1% of the selected stories. CONCLUSION: The children's books reviewed support understanding and coping with grief. However, some limitations were detected, and therefore it is advisable to accompany the child while reading these books to discuss aspects that have not been addressed.

Residence in an Area with Environmental Exposure to Heavy Metals and Neurobehavioral Performance in Children 9-11 Years Old: An Explorative Study.

We explored the association between residence in an area polluted with metals and neurobehavioral performance in children aged 9 to 11. A cross-sectional study was conducted with thirty boys and thirty girls aged 9 to 11 from public schools in a heavily industrialized area, matched by age (±4 months) and gender with 15 boys and 15 girls from public schools in cities without relevant industrial activity. Neurobehavioral performance was assessed with the Behavioral Assessment and Research System. Linear regression models were used, adjusting for age, sex, social class and multimedia activities to predict each of the neurobehavioral outcome variables. No differences in neurobehavioral performance were found when all children with residence in areas with environmental exposure to metals were classified as exposed and the children from the other provinces as unexposed. However, when we compared children living <1 km from an industrial area with respect to those living more than 1 km away, significant differences were found. Children living <1 km away had lower scores on Finger Tapping (p = 0.03), Symbol-Digit (p = 0.07) and Continuous Performance (p = 0.02) than those living farther away. Our results support the hypothesis that residing close to an area with industrial activity (<1 km) is associated with deficits in neurobehavioral performance among children aged 9 to 11.

Cleavage of LOXL1 by BMP1 and ADAMTS14 Proteases Suggests a Role for Proteolytic Processing in the Regulation of LOXL1 Function.

Members of the lysyl oxidase (LOX) family catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-links, an essential process for connective tissue maturation. Proteolysis has emerged as an important level of regulation of LOX enzymes with the cleavage of the LOX isoform by metalloproteinases of the BMP1 (bone morphogenetic protein 1) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families as a model example. Lysyl oxidase-like 1 (LOXL1), an isoform associated with pelvic organ prolapse and pseudoexfoliation (PEX) glaucoma, has also been reported to be proteolytically processed by these proteases. However, precise molecular information on these proteolytic events is not available. In this study, using genetic cellular models, along with proteomic analyses, we describe that LOXL1 is processed by BMP1 and ADAMTS14 and identify the processing sites in the LOXL1 protein sequence. Our data show that BMP1 cleaves LOXL1 in a unique location within the pro-peptide region, whereas ADAMTS14 processes LOXL1 in at least three different sites located within the pro-peptide and in the first residues of the catalytic domain. Taken together, these results suggest a complex regulation of LOXL1 function by BMP1- and ADAMTS14-mediated proteolysis where LOXL1 enzymes retaining variable fragments of N-terminal region may display different capabilities.

Sentimientos relacionados con la muerte en estudiantes de enfermería: un estudio observacional de tres cohortes / Feelings about Death among Nursing Students: A Three-Cohort Observational Study

Objetivo principal: Conocer los sentimientos sobre la muerte en estudiantes de grado de enfermería que aún no han iniciado su prácticum en entornos asistenciales reales. Metodología: Estudio observacional de tres cohortes con análisis de pregunta abierta. La Escala de Miedo a la Muerte de Collet-Lester, el Cuestionario de factores para ayudar a morir en paz y un cuestionario con 6 preguntas abiertas fueron completados por 197 estudiantes de segundo curso de Grado de Enfermería. Resultados principales: Los estudiantes reconocen tener miedo de enfrentar la muerte en el prácticum, principalmente temen que les afecte personalmente, la reacción del paciente, el contacto con la muerte y el no saber qué hacer. Conclusión principal: Los estudiantes consideran que la muerte es algo muy presente en su profesión, expresan temer enfrentarse a ella en el prácticum, mayoritariamente reconocen no tener suficientes estrategias de afrontamiento y les gustaría recibir más formación para saber cómo afrontarla.(AU)

Objective: To describe the feelings about death of students enrolled in the Nursing Degree, who had not initiated their practicum in real care settings. Methods: Three-cohort observational study with open question analysis. The Collet-Lester Fear of Death Scale, the Questionnaire on factors to help dying in peace and ad hoc questionnaire with 6 open-ended questions was administered to 197 undergraduate nursing students. Results: The students recognized being afraid to face death in the practicum, mainly they fear that it affects them personally, the patient's reaction, contact with death and not knowing what to do. Conclusions: The students believed that death was something that was very present in their profession, expressed fear in facing it in the practicum, they mostly recognized not having enough overcoming strategies, and they would like to receive training on how to face death.(AU)

The challenge of determining lysyl oxidase activity: Old methods and novel approaches.

The lysyl oxidase (LOX) family of enzymes catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-linkages, an essential process for extracellular matrix (ECM) maturation. Elevated LOX expression levels leading to increased LOX activity is associated with diverse pathologies including fibrosis, cancer, and cardiovascular diseases. Different protocols have been so far established to detect and quantify LOX activity from tissue samples and cultured cells, all of them showing advantages and drawbacks. This review article presents a critical overview of the main features of currently available methods as well as introduces some recent technologies called to revolutionize our approach to LOX catalysis.

Telomere Length but Not Mitochondrial DNA Copy Number Is Altered in Both Young and Old COPD.

Accelerated ageing is implicated in the pathogenesis of respiratory diseases as chronic obstructive pulmonary disease (COPD), but recent evidence indicates that the COPD can have roots early in life. Here we hypothesise that the accelerated ageing markers might have a role in the pathobiology of young COPD. The objective of this study was to compare two hallmarks of ageing, telomere length (TL), and mitochondrial DNA copy number (mtDNA-CN, as a surrogate marker of mitochondrial dysfunction) in young (≤ 50 years) and old (>50 years) smokers, with and without COPD. Both, TL and mtDNA-CN were measured in whole blood DNA by quantitative PCR [qPCR] in: (1) young ever smokers with (n = 81) or without (n = 166) COPD; and (2) old ever smokers with (n = 159) or without (n = 29) COPD. A multivariable linear regression was used to assess the association of TL and mtDNA-CN with lung function. We observed that in the entire study population, TL and mtDNA-CN decreased with age, and the former but not the latter related to FEV1/FVC (%), FEV1 (% ref.), and DLCO (% ref.). The short telomeres were found both in the young and old patients with severe COPD (FEV1 <50% ref.). In addition, we found that TL and mtDNA-CN were significantly correlated, but their relationship was positive in younger while negative in the older patients with COPD, suggesting a mitochondrial dysfunction. We conclude that TL, but not mtDNA-CN, is associated with the lung function impairment. Both young and old patients with severe COPD have evidence of accelerated ageing (shorter TL) but differ in the direction of the correlation between TL and mtDNA-CN in relation to age.

Implementing a Care Pathway for Complex Chronic Patients from a Nursing Perspective: A Qualitative Study.

A care pathway constitutes a complex care strategy for decision-making and the organization of processes in the care of complex chronic patients, avoiding the fragmentation of care. Health professionals play a decisive role in the implementation, development, and evaluation of care pathways. This study sought to explore nurses' opinions on the care pathway for complex chronic patients three years after its implementation. The study participants were thirteen nurses with different roles who were involved in the care pathway. Thematic content analysis of the semi-structured interviews resulted in four major themes: (a) the strengths of the route; (b) the impact of the route on caregivers; (c) the weaknesses of the route; and (d) the future of the route. Overall, the pathway was positively valued for the benefits it provides to patients, the caregiver, and the administration of professional health care. Participants voiced their concerns regarding: communication and coordination difficulties among professionals across the different levels of care, the need for improved teamwork and consensus among professionals at the same center, and human and material resources. The ongoing evaluation and monitoring of facilitators and barriers is necessary throughout the implementation process, to ensure continuity and quality of care in the health system.

GOECP/SEOR radiotherapy guidelines for thymic epithelial tumours.

Thymic epithelial tumours (TET) are rare, heterogeneous neoplasms that range from resectable indolent tumours to aggressive thymic carcinomas with a strong tendency to metastasize. The pathological diagnosis is complex, in part due to the existence of several different classification systems. The evidence base for the management of TETs is scant and mainly based on non-randomised studies and retrospective series. Consequently, the clinical management of TETs tends to be highly heterogenous, which makes it difficult to improve the evidence level. The role of technological advances in the field of radiotherapy and new systemic therapies in the treatment of TETs has received little attention to date. In the present clinical guidelines, developed by the GOECP/SEOR, we review recent developments in the diagnosis and classification of TETs. We also present a consensus-based therapeutic strategy for each disease stage that takes into consideration the best available evidence. These guidelines focus primarily on the role of radiotherapy, including recent advances, in the management of TETs. The main aim of this document is to promote the standardisation of clinical practice and lay the foundations for future studies to clarify the main unresolved questions related to the optimal management of TET.

Techniques to Assess Collagen Synthesis, Deposition, and Cross-Linking In Vitro.

Synthesis, deposition, and cross-linking of collagen are hallmarks of fibroblast to myofibroblast differentiation. Standard methods for determining collagen from tissue samples are not directly applicable to cell culture conditions, where the overall synthesis and deposition of collagen is clearly unfavorable, mainly due to quantity limitations and dilution of required extracellular remodeling factors. In this chapter, we describe the methods we have established to analyze collagen production and deposition into the extracellular matrix by cultured myo/fibroblasts, as well as to determine lysyl oxidase (LOX) activity in cell supernatants as an index of the capacity of the cell to cross-link collagen in vitro.

Liver epigenome changes in patients with hepatopulmonary syndrome: A pilot study.

The hepatopulmonary syndrome (HPS) is defined by the presence of pulmonary gas exchange abnormalities due to intrapulmonary vascular dilatations in patients with chronic liver disease. Changes in DNA methylation reflect the genomic variation. Since liver transplant (LT) reverts HPS we hypothesized that it may be associated with specific liver epigenetic changes. Thus, the aim of this study was to investigate the role of the liver epigenome in patients with HPS. We extracted DNA from paraffin embedded liver tissue samples from 10 patients with HPS and 10 age-, sex- and MELD (Model for End-stage Liver Disease)-matched controls. DNA methylation was determined using the 850K array (Illumina). Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify modules related to defining physiologic characteristics of HPS. Only 12 out of the 20 liver biopsies (7 HPS and 5 controls) had sufficient quality to be analyzed. None of the 802,688 DNA probes analyzed in the case control comparison achieved a significant False Discovery Rate (FDR). WGCNA identified 5 co-methylated gene-modules associated to HPS markers, mainly related to nervous and neuroendocrine system, apoptotic processes, gut bacterial translocation, angiogenesis and vascular remodeling ontologies. To conclude, HPS is associated with nervous/neuroendocrine system and vascular remodeling related liver epigenetic changes.