Evolution of the impact of the COVID-19 pandemic on heart transplant recipients: Decreasing risk, improving perspective.

BACKGROUND AND METHODS: Heart transplant recipients (HTr) have a higher probability of suffer from severe coronavirus disease-2019 (COVID-19) in comparison to general population, but their risk has changed over the course of the pandemic in relation to various factors. We conducted a prospective study including all HTr at risk of COVID-19 in a tertiary center between February 2020 and October 2022. The aim was to analyze how the prognosis (incidence of pneumonia and mortality) of COVID-19 in HTr has evolved over time, contextualizing variants, vaccination, and other treatments. RESULTS: Of 308 HTr included, 124 got the infection (39.2%). COVID and non-COVID HTr had similar baseline characteristics. COVID-19 patients with pneumonia had a poorer prognosis than those with less severe presentations, with a higher rate of hospitalization (93.3 vs. 14.1%, p < .001) and death (41.0 vs. 1.2%, p < .001). Multivariate analysis identified age ≥60 years (odds ratio [OR] 3.65, 95% confidence interval [CI] 1.16-11.49, p = .027), and chronic kidney disease ≥3a (OR 4.95, 95% CI 1.39-17.54, p = .014) as predictors of pneumonia. Two-dose vaccination (OR 0.20, CI 95% 0.05-0.72, p = .02) and early remdesivir administration (OR 0.17, CI 0.03-0.90, p = .037) were protective factors. Over the course of the pandemic considering three periods in the follow-up (prevaccination February-December 2020, postvaccination January-December 2021, and post early remdesivir indication January-October 2022), we observed a reduction in pneumonia incidence from 62% to 19% (p < .001); and mortality (from 23% to 4%, p < .001). CONCLUSIONS: The prognosis of COVID-19 in HTr has improved over time, likely due to vaccination and early administration of remdesivir.

A bile-based microRNA signature for differentiating malignant from benign pancreaticobiliary disease.

Differentiating between pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA) is crucial for the appropriate course of treatment, especially with advancements in the role of neoadjuvant chemotherapies for PDAC, compared to CCA. Furthermore, benign pancreaticobiliary diseases can mimic malignant disease, and indeterminate lesions may require repeated investigations to achieve a diagnosis. As bile flows in close proximity to these lesions, we aimed to establish a bile-based microRNA (miRNA) signature to discriminate between malignant and benign pancreaticobiliary diseases. We performed miRNA discovery by global profiling of 800 miRNAs using the NanoString nCounter platform in prospectively collected bile samples from malignant (n = 43) and benign (n = 14) pancreaticobiliary disease. Differentially expressed miRNAs were validated by RT-qPCR and further assessed in an independent validation cohort of bile from malignant (n = 37) and benign (n = 38) pancreaticobiliary disease. MiR-148a-3p was identified as a discriminatory marker that effectively distinguished malignant from benign pancreaticobiliary disease in the discovery cohort (AUC = 0.797 [95% CI 0.68-0.92]), the validation cohort (AUC = 0.772 [95% CI 0.66-0.88]), and in the combined cohorts (AUC = 0.752 [95% CI 0.67-0.84]). We also established a two-miRNA signature (miR-125b-5p and miR-194-5p) that distinguished PDAC from CCA (validation: AUC = 0.815 [95% CI 0.67-0.96]; and combined cohorts: AUC = 0.814 [95% CI 0.70-0.93]). Our research stands as the largest, multicentric, global profiling study of miRNAs in the bile from patients with pancreaticobiliary disease. We demonstrated their potential as clinically useful diagnostic tools for the detection and differentiation of malignant pancreaticobiliary disease. These bile miRNA biomarkers could be developed to complement current approaches for diagnosing pancreaticobiliary cancers.

Healthcare outcomes in patients with HIV infection at a tertiary hospital during the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic has affected the care of patients with other diseases. Difficulty in access to healthcare during these months has been especially relevant for persons with HIV infection (PWH). This study therefore sought to ascertain the clinical outcomes and effectiveness of the measures implemented among PWH in a region with one of the highest incidence rates in Europe. METHODS: Retrospective, observational, pre-post intervention study to compare the outcomes of PWH attended at a high-complexity healthcare hospital from March to October 2020 and during the same months across the period 2016-2019. The intervention consisted of home drug deliveries and preferential use of non face-to-face consultations. The effectiveness of the measures implemented was determined by reference to the number of emergency visits, hospitalisations, mortality rate, and percentage of PWH with viral load >50copies, before and after the two pandemic waves. RESULTS: A total of 2760 PWH were attended from January 2016 to October 2020. During the pandemic, there was a monthly mean of 106.87 telephone consultations and 2075 home deliveries of medical drugs dispensed to ambulatory patients. No statistically significant differences were found between the rate of admission of patients with COVID-HIV co-infection and that of the remaining patients (1172.76 admissions/100,000 population vs. 1424.29, p=0.401) or in mortality (11.54% vs. 12.96%, p=0.939). The percentage of PWH with viral load >50copies was similar before and after the pandemic (1.20% pre-pandemic vs. 0.51% in 2020, p=0.078). CONCLUSION: Our results show that the strategies implemented during the first 8 months of the pandemic prevented any deterioration in the control and follow-up parameters routinely used on PWH. Furthermore, they contribute to the debate about how telemedicine and telepharmacy can fit into future healthcare models.

Serum GDF-15 Levels Accurately Differentiate Patients with Primary Mitochondrial Myopathy, Manifesting with Exercise Intolerance and Fatigue, from Patients with Chronic Fatigue Syndrome.

Primary mitochondrial myopathies (PMM) are a clinically and genetically highly heterogeneous group that, in some cases, may manifest exclusively as fatigue and exercise intolerance, with minimal or no signs on examination. On these occasions, the symptoms can be confused with the much more common chronic fatigue syndrome (CFS). Nonetheless, other possibilities must be excluded for the final diagnosis of CFS, with PMM being one of the primary differential diagnoses. For this reason, many patients with CFS undergo extensive studies, including extensive genetic testing and muscle biopsies, to rule out this possibility. This study evaluated the diagnostic performance of growth differentiation factor-15 (GDF-15) as a potential biomarker to distinguish which patient with chronic fatigue has a mitochondrial disorder. We studied 34 adult patients with symptoms of fatigue and exercise intolerance with a definitive diagnosis of PMM (7), CFS (22), or other non-mitochondrial disorders (5). The results indicate that GDF-15 can accurately discriminate between patients with PMM and CFS (AUC = 0.95) and between PMM and patients with fatigue due to other non-mitochondrial disorders (AUC = 0.94). Therefore, GDF-15 emerges as a promising biomarker to select which patients with fatigue should undergo further studies to exclude mitochondrial disease.

Healthcare outcomes in patients with HIV infection at a tertiary hospital during the COVID-19 pandemic / Resultados en la atención a pacientes con infección por VIH en un hospital terciario durante la pandemia COVID-19

Background: The COVID-19 pandemic has affected the care of patients with other diseases. Difficulty in access to healthcare during these months has been especially relevant for persons with HIV infection (PWH). This study therefore sought to ascertain the clinical outcomes and effectiveness of the measures implemented among PWH in a region with one of the highest incidence rates in Europe. Methods: Retrospective, observational, pre-post intervention study to compare the outcomes of PWH attended at a high-complexity healthcare hospital from March to October 2020 and during the same months across the period 2016–2019. The intervention consisted of home drug deliveries and preferential use of non face-to-face consultations. The effectiveness of the measures implemented was determined by reference to the number of emergency visits, hospitalisations, mortality rate, and percentage of PWH with viral load >50copies, before and after the two pandemic waves. Results: A total of 2760 PWH were attended from January 2016 to October 2020. During the pandemic, there was a monthly mean of 106.87 telephone consultations and 2075 home deliveries of medical drugs dispensed to ambulatory patients. No statistically significant differences were found between the rate of admission of patients with COVID-HIV co-infection and that of the remaining patients (1172.76 admissions/100,000 population vs. 1424.29, p=0.401) or in mortality (11.54% vs. 12.96%, p=0.939). The percentage of PWH with viral load >50copies was similar before and after the pandemic (1.20% pre-pandemic vs. 0.51% in 2020, p=0.078). Conclusion: Our results show that the strategies implemented during the first 8 months of the pandemic prevented any deterioration in the control and follow-up parameters routinely used on PWH. Furthermore, they contribute to the debate about how telemedicine and telepharmacy can fit into future healthcare models.(AU)

Introducción: La pandemia causada por el SARS-CoV-2 ha afectado a la atención de pacientes con otras enfermedades. La dificultad en el acceso a la asistencia sanitaria durante estos meses es especialmente relevante en las personas con infección por VIH (PCV). El objetivo del estudio fue conocer los resultados clínicos y la efectividad de las medidas implementadas en PCV en una de las regiones con mayor incidencia de Europa. Métodos: Estudio observacional retrospectivo, pre-postintervención, comparando los resultados de PCV atendidos en un hospital de alta complejidad entre marzo-octubre de 2020 y el mismo periodo de 2016 a 2019. La intervención consistió en el envío a domicilio de medicamentos y la realización preferente de consultas no presenciales. La efectividad de las medidas implementadas se determinó por el número de visitas a urgencias, hospitalizaciones, mortalidad y porcentaje de PCV con carga viral>50 copias antes y después de 2 olas pandémicas. Resultados: Se atendieron 2.760 PCV entre enero de 2016 y octubre de 2020. Durante la pandemia se realizaron una media mensual de 106,87 consultas telefónicas y 2.075 envíos a domicilio de medicamentos de dispensación ambulatoria. No se encontraron diferencias estadísticamente significativas en la frecuentación de pacientes con coinfección COVID-VIH respecto al resto (1.172,76 ingresos/100.000 habitantes vs. 1.424,29, p=0,401), ni en su mortalidad (11,54 vs. 12,96%, p=0,939). El porcentaje de PCV con carga viral>50 copias fue similar antes y después de la pandemia (1,20% prepandemia vs. 0,51% en 2020, p=0,078). Conclusión: Nuestros resultados revelan que las estrategias implementadas durante los 8 primeros meses de pandemia han evitado el deterioro en parámetros de control y seguimiento empleados habitualmente en PCV. Además, contribuyen a la reflexión sobre el encaje de la telemedicina y telefarmacia en modelos asistenciales futuros.(AU)

Healthcare outcomes in patients with HIV infection at a tertiary hospital during the COVID-19 pandemic.

Background: The COVID-19 pandemic has affected the care of patients with other diseases. Difficulty in access to healthcare during these months has been especially relevant for persons with HIV infection (PWH). This study therefore sought to ascertain the clinical outcomes and effectiveness of the measures implemented among PWH in a region with one of the highest incidence rates in Europe. Methods: Retrospective, observational, pre-post intervention study to compare the outcomes of PWH attended at a high-complexity healthcare hospital from March to October 2020 and during the same months across the period 2016-2019. The intervention consisted of home drug deliveries and preferential use of non face-to-face consultations. The effectiveness of the measures implemented was determined by reference to the number of emergency visits, hospitalisations, mortality rate, and percentage of PWH with viral load >50 copies, before and after the two pandemic waves. Results: A total of 2760 PWH were attended from January 2016 to October 2020. During the pandemic, there was a monthly mean of 106.87 telephone consultations and 2075 home deliveries of medical drugs dispensed to ambulatory patients. No statistically significant differences were found between the rate of admission of patients with COVID-HIV co-infection and that of the remaining patients (1172.76 admissions/100,000 population vs. 1424.29, p = 0.401) or in mortality (11.54% vs. 12.96%, p = 0.939). The percentage of PWH with viral load >50 copies was similar before and after the pandemic (1.20% pre-pandemic vs. 0.51% in 2020, p = 0.078). Conclusion: Our results show that the strategies implemented during the first 8 months of the pandemic prevented any deterioration in the control and follow-up parameters routinely used on PWH. Furthermore, they contribute to the debate about how telemedicine and telepharmacy can fit into future healthcare models.

Introducción: La pandemia causada por el SARS-CoV-2 ha afectado a la atención de pacientes con otras enfermedades. La dificultad en el acceso a la asistencia sanitaria durante estos meses es especialmente relevante en las personas con infección por VIH (PCV). El objetivo del estudio fue conocer los resultados clínicos y la efectividad de las medidas implementadas en PCV en una de las regiones con mayor incidencia de Europa. Métodos: Estudio observacional retrospectivo, pre-postintervención, comparando los resultados de PCV atendidos en un hospital de alta complejidad entre marzo-octubre de 2020 y el mismo periodo de 2016 a 2019. La intervención consistió en el envío a domicilio de medicamentos y la realización preferente de consultas no presenciales. La efectividad de las medidas implementadas se determinó por el número de visitas a urgencias, hospitalizaciones, mortalidad y porcentaje de PCV con carga viral > 50 copias antes y después de 2 olas pandémicas. Resultados: Se atendieron 2.760 PCV entre enero de 2016 y octubre de 2020. Durante la pandemia se realizaron una media mensual de 106,87 consultas telefónicas y 2.075 envíos a domicilio de medicamentos de dispensación ambulatoria. No se encontraron diferencias estadísticamente significativas en la frecuentación de pacientes con coinfección COVID-VIH respecto al resto (1.172,76 ingresos/100.000 habitantes vs. 1.424,29, p = 0,401), ni en su mortalidad (11,54 vs. 12,96%, p = 0,939). El porcentaje de PCV con carga viral > 50 copias fue similar antes y después de la pandemia (1,20% prepandemia vs. 0,51% en 2020, p = 0,078). Conclusión: Nuestros resultados revelan que las estrategias implementadas durante los 8 primeros meses de pandemia han evitado el deterioro en parámetros de control y seguimiento empleados habitualmente en PCV. Además, contribuyen a la reflexión sobre el encaje de la telemedicina y telefarmacia en modelos asistenciales futuros.

Pluripotency factors regulate the onset of Hox cluster activation in the early embryo.

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3' end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs.

Role of Chromatin Replication in Transcriptional Plasticity, Cell Differentiation and Disease.

Chromatin organization is essential to maintain a correct regulation of gene expression and establish cell identity. However, during cell division, the replication of the genetic material produces a global disorganization of chromatin structure. In this paper, we describe the new scientific breakthroughs that have revealed the nature of the post-replicative chromatin and the mechanisms that facilitate its restoration. Moreover, we highlight the implications of these chromatin alterations in gene expression control and their impact on key biological processes, such as cell differentiation, cell reprogramming or human diseases linked to cell proliferation, such as cancer.

Systems level profiling of chemotherapy-induced stress resolution in cancer cells reveals druggable trade-offs.

Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization.

The Implications of ncRNAs in the Development of Human Diseases.

The mammalian genome comprehends a small minority of genes that encode for proteins (barely 2% of the total genome in humans) and an immense majority of genes that are transcribed into RNA but not encoded for proteins (ncRNAs). These non-coding genes are intimately related to the expression regulation of protein-coding genes. The ncRNAs subtypes differ in their size, so there are long non-coding genes (lncRNAs) and other smaller ones, like microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs). Due to their important role in the maintenance of cellular functioning, any deregulation of the expression profiles of these ncRNAs can dissemble in the development of different types of diseases. Among them, we can highlight some of high incidence in the population, such as cancer, neurodegenerative, or cardiovascular disorders. In addition, thanks to the enormous advances in the field of medical genomics, these same ncRNAs are starting to be used as possible drugs, approved by the FDA, as an effective treatment for diseases.

Obtaining EHR-derived datasets for COVID-19 research within a short time: a flexible methodology based on Detailed Clinical Models.

BACKGROUND: COVID-19 ranks as the single largest health incident worldwide in decades. In such a scenario, electronic health records (EHRs) should provide a timely response to healthcare needs and to data uses that go beyond direct medical care and are known as secondary uses, which include biomedical research. However, it is usual for each data analysis initiative to define its own information model in line with its requirements. These specifications share clinical concepts, but differ in format and recording criteria, something that creates data entry redundancy in multiple electronic data capture systems (EDCs) with the consequent investment of effort and time by the organization. OBJECTIVE: This study sought to design and implement a flexible methodology based on detailed clinical models (DCM), which would enable EHRs generated in a tertiary hospital to be effectively reused without loss of meaning and within a short time. MATERIAL AND METHODS: The proposed methodology comprises four stages: (1) specification of an initial set of relevant variables for COVID-19; (2) modeling and formalization of clinical concepts using ISO 13606 standard and SNOMED CT and LOINC terminologies; (3) definition of transformation rules to generate secondary use models from standardized EHRs and development of them using R language; and (4) implementation and validation of the methodology through the generation of the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC-WHO) COVID-19 case report form. This process has been implemented into a 1300-bed tertiary Hospital for a cohort of 4489 patients hospitalized from 25 February 2020 to 10 September 2020. RESULTS: An initial and expandable set of relevant concepts for COVID-19 was identified, modeled and formalized using ISO-13606 standard and SNOMED CT and LOINC terminologies. Similarly, an algorithm was designed and implemented with R and then applied to process EHRs in accordance with standardized concepts, transforming them into secondary use models. Lastly, these resources were applied to obtain a data extract conforming to the ISARIC-WHO COVID-19 case report form, without requiring manual data collection. The methodology allowed obtaining the observation domain of this model with a coverage of over 85% of patients in the majority of concepts. CONCLUSION: This study has furnished a solution to the difficulty of rapidly and efficiently obtaining EHR-derived data for secondary use in COVID-19, capable of adapting to changes in data specifications and applicable to other organizations and other health conditions. The conclusion to be drawn from this initial validation is that this DCM-based methodology allows the effective reuse of EHRs generated in a tertiary Hospital during COVID-19 pandemic, with no additional effort or time for the organization and with a greater data scope than that yielded by conventional manual data collection process in ad-hoc EDCs.

Correlation of Kryptor and Elecsys® immunoassay sFlt-1/PlGF ratio on early diagnosis of preeclampsia and fetal growth restriction: A case-control study.

OBJECTIVES: The measurement of the soluble fms-like tyrosine kinase-1 to placental growth factor (sFlt-1/PlGF) ratio on automated platforms has improved the detection of preeclampsia and fetal growth restriction (PE/FGR). The cut-off points of >38 and ≥85 has been defined for "rule in" and "aid in diagnosis", respectively, using the Elecsys® platform. We aimed to compare the performance of these cut-offs between the Elecsys® and Kryptor platforms at 24-28 weeks. STUDY DESIGN: Observational case-control study of singleton pregnancies at high risk for PE/FGR and sFlt-1/PlGF measurement at 24-28 weeks' gestation: 21 cases (9 early PE/FGR with delivery <32 weeks) were 1:1 matched for body mass index and parity with 21 controls. Correlations of the sFlt-1, PlGF and sFlt-1/PlGF values and diagnostic accuracy of the >38 and ≥85 cutoffs for early and late PE/FGR using Elecsys® and Kryptor assays were evaluated. MAIN OUTCOME MEASURES: PE/FGR cases showed significantly higher median (IQR) sFlt-1/PlGF values at 24-28 weeks vs. controls, using both Elecsys® and Kryptor platforms: 55 (13-254) and 97 (13-530) vs. 4.1 (2.0-6.5) and 3.9 (1.8-7.7), respectively. The sFlt-1/PlGF correlation between both methods was excellent (r2 = 0.95) although lower PlGF and higher sFlt-1/PlGF values were observed with Kryptor. The higher diagnostic accuracy was obtained for early PE/FGR with the ≥85 cutoff (95.2%; 95%CI: 83.8-99.4%) in both platforms. CONCLUSION: sFlt-1/PlGF measurements correlates well between Elecsys® and Kryptor platforms, and the cutoffs of >38 and ≥85 exhibit high diagnostic accuracy for assessing early PE/FGR at 24-28 weeks with both methods.

Updates on the Role of Molecular Alterations and NOTCH Signalling in the Development of Neuroendocrine Neoplasms.

Neuroendocrine neoplasms (NENs) comprise a heterogeneous group of rare malignancies, mainly originating from hormone-secreting cells, which are widespread in human tissues. The identification of mutations in ATRX/DAXX genes in sporadic NENs, as well as the high burden of mutations scattered throughout the multiple endocrine neoplasia type 1 (MEN-1) gene in both sporadic and inherited syndromes, provided new insights into the molecular biology of tumour development. Other molecular mechanisms, such as the NOTCH signalling pathway, have shown to play an important role in the pathogenesis of NENs. NOTCH receptors are expressed on neuroendocrine cells and generally act as tumour suppressor proteins, but in some contexts can function as oncogenes. The biological heterogeneity of NENs suggests that to fully understand the role and the potential therapeutic implications of gene mutations and NOTCH signalling in NENs, a comprehensive analysis of genetic alterations, NOTCH expression patterns and their potential role across all NEN subtypes is required.

RNA sequencing and Prediction Tools for Circular RNAs Analysis.

Circular RNAs (circRNAs) are noncoding and single-stranded RNA transcripts able to form covalently circular-closed structures. They are generated through alternative splicing events and widely expressed from human to viruses. CircRNAs have been appointed as potential regulators of microRNAs (miRNAs), RNA-binding proteins (RPBs), and lineal protein-coding transcripts. Although their mechanism of action remains unclear, the deregulation of circular RNAs has been confirmed in different diseases such as Alzheimer or cancer.The introduction of high-throughput next-generation sequencing (NGS) technology provides millions of short RNA sequences at single-nucleotide level, allowing an accurate and proficient method to measure circular RNAs. Novel protocols based on non-polyadenylated RNAs, rRNA-depleted, and RNA exonuclease-based enrichment approaches (RNase R) have taken even further the possibility of detecting circRNAs.Besides, the identification of circRNAs presence requires the development of specific bioinformatics tools to detect junction-spanning sequences from transcriptome deep-sequencing samples. Thus, recently established bioinformatics' approaches have permitted the discovery of an elevated number of different circRNAs in diverse organisms. In that sense, recent studies have compared different methods and advocate the simultaneous use of more than one prediction tool. For that reason, we want to highlight pipelines such as miARma-Seq that is able to execute various circular RNA identification algorithms in an easy way, without the tedious installation of third-party prerequisites.

Glypican-1 is enriched in circulating-exosomes in pancreatic cancer and correlates with tumor burden.

BACKGROUND: Glypican-1 (GPC1) is expressed in pancreatic ductal adenocarcinoma (PDAC) cells and adjacent stromal fibroblasts. Recently, GPC1 circulating exosomes (crExos) have been shown to be able to detect early stages of PDAC. In this study, we investigated the usefulness of crExos GPC1 as a biomarker for PDAC. METHODS: Plasma was obtained from patients with benign pancreatic disease (n = 16) and PDAC (n = 27) prior to pancreatectomy, and crExos were isolated by ultra-centrifugation. Protein was extracted from surgical specimens (adjacent normal pancreas, n = 13; and PDAC, n = 17). GPC1 levels were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: There was no significant difference in GPC1 levels between normal pancreas and PDAC tissues. This was also true when comparing matched pairs. However, GPC1 levels were enriched in PDAC crExos (n = 11), compared to the source tumors (n = 11; 97 ± 54 vs. 20.9 ± 12.3 pg/mL; P < 0.001). In addition, PDACs with high GPC1 expression tended to have crExos with higher GPC1 levels. Despite these findings, we were unable to distinguish PDAC from benign pancreatic disease using crExos GPC1 levels. Interestingly, we found that in matched pre and post-operative plasma samples there was a significant drop in crExos GPC1 levels after surgical resection for PDAC (n = 11 vs. 11; 97 ± 54 vs. 77.8 ± 32.4 pg/mL; P = 0.0428). Furthermore, we found that patients with high crExos GPC1 levels have significantly larger PDACs (>4 cm; P = 0.012). CONCLUSIONS: High GPC1 crExos may be able to determine PDAC tumor size and disease burden. However, further efforts are needed to elucidate its role as a diagnostic and/or prognostic biomarker using larger cohorts of PDAC patients.

Long-range regulatory interactions at the 4q25 atrial fibrillation risk locus involve PITX2c and ENPEP.

BACKGROUND: Recent genome-wide association studies have uncovered genomic loci that underlie an increased risk for atrial fibrillation, the major cardiac arrhythmia in humans. The most significant locus is located in a gene desert at 4q25, approximately 170 kilobases upstream of PITX2, which codes for a transcription factor involved in embryonic left-right asymmetry and cardiac development. However, how this genomic region functionally and structurally relates to PITX2 and atrial fibrillation is unknown. RESULTS: To characterise its function, we tested genomic fragments from 4q25 for transcriptional activity in a mouse atrial cardiomyocyte cell line and in transgenic mouse embryos, identifying a non-tissue-specific potentiator regulatory element. Chromosome conformation capture revealed that this region physically interacts with the promoter of the cardiac specific isoform of Pitx2. Surprisingly, this regulatory region also interacts with the promoter of the next neighbouring gene, Enpep, which we show to be expressed in regions of the developing mouse heart essential for cardiac electrical activity. CONCLUSIONS: Our data suggest that de-regulation of both PITX2 and ENPEP could contribute to an increased risk of atrial fibrillation in carriers of disease-associated variants, and show the challenges that we face in the functional analysis of genome-wide disease associations.

Integrative analysis of miRNA and mRNA expression profiles in pheochromocytoma and paraganglioma identifies genotype-specific markers and potentially regulated pathways.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. As microRNAs (miRNAs) are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. Through characterization of miRNA profiles in 69 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX, we identified miRNA signatures specific to, as well as common among, the genetic groups of PCCs/PGLs. miRNA expression profiles were validated in an independent series of 30 composed of VHL-, SDHB-, SDHD-, and RET-related formalin-fixed paraffin-embedded PCC/PGL samples using quantitative real-time PCR. Upregulation of miR-210 in VHL- and SDHB-related PCCs/PGLs was verified, while miR-137 and miR-382 were confirmed as generally upregulated in PCCs/PGLs (except in MAX-related tumors). Also, we confirmed overexpression of miR-133b as VHL-specific miRNAs, miR-488 and miR-885-5p as RET-specific miRNAs, and miR-183 and miR-96 as SDHB-specific miRNAs. To determine the potential roles miRNAs play in PCC/PGL pathogenesis, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data that indicated a common enrichment of pathways associated with neuronal and neuroendocrine-like differentiation. We demonstrated that miR-183 and/or miR-96 impede NGF-induced differentiation in PC12 cells. Finally, global proteomic analysis in SDHB and MAX tumors allowed us to determine that miRNA regulation occurs primarily through mRNA degradation in PCCs/PGLs, which partially confirmed our miRNA-mRNA integration results.

Research resource: Transcriptional profiling reveals different pseudohypoxic signatures in SDHB and VHL-related pheochromocytomas.

The six major genes involved in hereditary susceptibility for pheochromocytoma (PCC)/paraganglioma (PGL) (RET, VHL, NF1, SDHB, SDHC, and SDHD) have been recently integrated into the same neuronal apoptotic pathway where mutations in any of these genes lead to cell death. In this model, prolyl hydroxylase 3 (EglN3) abrogation plays a pivotal role, but the molecular mechanisms underlying its inactivation are currently unknown. The aim of the study was to decipher specific alterations associated with the different genetic classes of PCCs/PGLs. With this purpose, 84 genetically characterized tumors were analyzed by means of transcriptional profiling. The analysis revealed a hypoxia-inducible factor (HIF)-related signature common to succinate dehydrogenase (SDH) and von Hippel-Lindau (VHL) tumors, that differentiated them from RET and neurofibromatosis type 1 cases. Both canonical HIF-1α and HIF-2α target genes were overexpressed in the SDH/VHL cluster, suggesting that a global HIF deregulation accounts for this common profile. Nevertheless, when we compared VHL tumors with SDHB cases, which often exhibit a malignant behavior, we found that HIF-1α target genes showed a predominant activation in the VHL PCCs. Expression data from 67 HIF target genes was sufficient to cluster SDHB and VHL tumors into two different groups, demonstrating different pseudo-hypoxic signatures. In addition, VHL-mutated tumors showed an unexpected overexpression of EglN3 mRNA that did not lead to significantly different EglN3 protein levels. These findings pave the way for more specific therapeutic approaches for malignant PCCs/PGLs management based on the patient's genetic alteration.

Tumoral and tissue-specific expression of the major human beta-tubulin isotypes.

The beta-tubulins are microtubule components encoded by a multigene family, which produces slightly different proteins with complex expression patterns. Several widely used anticancer drugs base their activity on beta-tubulin binding, microtubule dynamics alteration, and cell division blockage. The expression of these drug targets in tumoral and normal cells could be of crucial importance for therapy outcome, unfortunately, the complex beta-tubulin expression patterns have been poorly characterized in human. In this study, we developed a quantitative RT-PCR technique that accurately determines the mRNA expression of the eight human beta-tubulin isotypes, encoding class I, IIa, IIb, III, IVa, IVb, V, and VI and applied it to 21 nontumoral tissues and 79 tumor samples belonging to seven cancer types. In the nontumoral tissues, we found that, overall, TUBB (I), TUBB2C (IVb), and TUBB6 (V) were ubiquitous, TUBB1(VI) was hematopoietic cell-specific, and TUBB2A (IIa), TUBB2B (IIb), TUBB3 (III), and TUBB4 (IVa) had high expression in brain; however, the contribution of the different isotypes to the total beta-tubulin content varied for each tissue and had a complex pattern. In tumoral tissues, most isotypes exhibited an altered expression in specific tumor types or related to tumoral characteristics. In general, TUBB3 showed a great increase in expression while TUBB6 expression was largely decreased in most tumors. Thus, normal tissues showed a complex beta-tubulin isotype distribution, which could contribute to the toxicity profile of the microtubule-binding drugs. In addition, the specific isotypes significantly altered in tumors might represent markers for drug response.