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Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of the COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While all four ORFs caused mitochondrial fragmentation and altered mitochondrial function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes for proteins with critical mitochondrial functions and morphology. Genome-Scale Metabolic Models predicted common and private metabolic flux reprogramming, notably a depressed amino acid metabolism, and an enhanced metabolism of specific lipids distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.

Objetivos: El glicocálix es una estructura que recubre la capa luminal del endotelio vascular cuya degradación se reconoce como una piedra angular en la disfunción endotelial relacionada con el proceso de isquemia-reperfusión. En este sentido, investigamos el papel de los productos de degradación del glicocálix endotelial en pacientes trasplantados de páncreas. Material y métodos: Los niveles de los principales componentes del glicocálix: sindecan-1, ácido hialurónico y heparán sulfato, y el factor de crecimiento endotelial vascular (VEGF) se determinaron en el plasma de 28 pacientes trasplantados mediante ensayos ELISA antes de la cirugía, 10 minutos y 24 horas después de la reperfusión, y al alta del paciente. Resultados: Los pacientes trasplantados presentaron mayor evidencia de daño endotelial 10 minutos después de la reperfusión del injerto versus antes de la cirugía, determinado por los niveles plasmáticos de sindecan-1 (66,54 frente a 155 ng/ml; p<0,0001) y heparán sulfato (4,96 frente a 6,07 ng/ml, p=0,0435). Sin embargo, los niveles de ácido hialurónico se mantuvieron sin cambios significativos durante todo el proceso. Los niveles de VEGF disminuyeron drásticamente en plasma 10 minutos después de la reperfusión del injerto (117 vs 18 pg/ml; p<0,0001). El análisis de regresión mostró una asociación entre los pacientes con niveles elevados de sindecan-1 a los 10 minutos post-reperfusión y el desarrollo de rechazo agudo del páncreas (p=0,011), aparición de complicaciones intestinales (p=0,045), complicaciones quirúrgicas renales (p=0,026) o requerimiento de reintervención urológica (p=0,024). Por otro lado, los niveles de VEGF antes de la cirugía se asociaron negativamente con el desarrollo de infección (p=0,016) y complicaciones quirúrgicas del páncreas como la pancreatitis del injerto (p=0,045) o hemorragia abdominal (p=0,040). Conclusiones: Este estudio proporciona la primera evidencia de daño en el glicocálix endotelial durante el trasplante de páncreas. A

Background: The vascular endothelium is critically involved in many steps of tissue damage originating during ischemia-reperfusion (IR) in human transplantation. An endothelial surface layer, the glycocalyx, forms the primary interface between the blood and the healthy endothelium and its shedding is recognized as a cornerstone in IR-related endothelial dysfunction. We investigated the role of endothelial glycocalyx breakdown products in patients undergoing pancreas transplantation. Methods: Endothelial integrity and remodelling products were measured in plasma from 26 simultaneous reno-pancreas transplants and 2 re-transplants recipients. The levels of syndecan-1, hyaluronan, heparan sulfate and Vascular Endothelial Growth Factor (VEGF) were determined using enzyme-linked immunosorbent assay at different time points: before surgery, 10 minutes and 24 hours after reperfusion, and at patient discharge. Results: Transplant recipients had greater evidence of glycocalyx damage 10 min. after reperfusion of the graft as compared before surgery, measured by plasma levels of syndecan-1 (66.54 vs 155ng/mL; p<0.0001) and heparan sulfate (4.96 vs 6.07 ng/mL, p=0.0435) components. However, plasma hyaluronan levels remained unchanged throughout. The levels of VEGF significantly decreased in plasma 10 min. after graft reperfusion (117 vs 18 pg/mL; p<0.0001). No correlation existed between endothelial markers except for syndecan-1 with VEGF and heparan sulfate at this time point, respectively (r=0.395, p=0.0371; r=0.572, p=0.001). Regression analysis showed that the higher levels of syndecan-1 were more frequent in patients with acute rejection (p=0.011), intestinal complications (p=0.045) and those who developed kidney surgical complications (p=0.026) or needed for a urological reintervention (p=0.024). Graft pancreatitis and pancreatic fistulas were associated with the levels of hyaluronan at 24h post-reperfusion (p=0.04; p=0.04, respectively) and at patient discharge (p=0.012

Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of COVID-19. However, the impacts of individual accessory proteins on host cell metabolic pathways are unknown.Here, SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 were individually transduced into A549 lung carcinoma cells. Furthermore, by combining transcriptomic analysis with functional and metabolic data in accessory protein-specific GSMMs, several alterations were identified that may point to a putative target for investigating novel therapies. In this study, we showed that these accessory proteins induced a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical role in mitochondria function and morphology. On the other hand, while all four ORFs altered mitochondrial dynamics and function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. Genome-Scale Metabolic Models identified both metabolic flux reprogramming features shared across all accessory proteins and specific ones for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. Next, qMTA identified gene knock downs (KDs) that would have the potential to revert the metabolic reprogramming induced by each individual accessory protein, especially in ORF3a and ORF10. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.

Portal hypertension (PH) is a frequent and severe clinical syndrome associated with chronic liver disease. Considering the mechanobiological effects of hydrostatic pressure and shear stress on endothelial cells, we hypothesised that PH might influence the phenotype of liver sinusoidal endothelial cells (LSECs) during disease progression. The aim of this study was to investigate the effects of increased hydrodynamic pressure on LSECs and to identify endothelial-derived biomarkers of PH.

Somatic single-nucleotide variants (SNVs) occur every time a cell divides, appearing even in healthy tissues at low frequencies. These mutations may accumulate as neutral variants during aging, or eventually, promote the development of neoplasia. Here, we present the SP-ddPCR, a droplet digital PCR (ddPCR) based approach that utilizes customized SuperSelective primers aiming at quantifying the proportion of rare SNVs. For that purpose, we selected five potentially pathogenic variants identified by whole-exome sequencing (WES) occurring at low variant allele frequency (VAF) in at-risk colon healthy mucosa of patients diagnosed with colorectal cancer or advanced adenoma. Additionally, two APC SNVs detected in two cancer lesions were added to the study for WES-VAF validation. SuperSelective primers were designed to quantify SNVs at low VAFs both in silico and in clinical samples. In addition to the two APC SNVs in colonic lesions, SP-ddPCR confirmed the presence of three out of five selected SNVs in the normal colonic mucosa with allelic frequencies ≤ 5%. Moreover, SP-ddPCR showed the presence of two potentially pathogenic variants in the distal normal mucosa of patients with colorectal carcinoma. In summary, SP-ddPCR offers a rapid and feasible methodology to validate next-generation sequencing data and accurately quantify rare SNVs, thus providing a potential tool for diagnosis and stratification of at-risk patients based on their mutational profiling.

Introduction : The mechanisms underlying the development of pulmonary hypertension (PH) and its severity in chronic lung diseases (CLD) are unknown. The study aimed to assess the protein expression profile in plasma in patients with chronic obstructive pulmonary disease (COPD) or interstitial lung disease (ILD), according to the presence and severity of PH, and explore similarities with idiopathic pulmonary arterial hypertension (iPAH). Methods: We analysed 115 patients with CLD: 40 non-PH (19 COPD, 21 ILD), 41 with moderate PH (22 COPD, 19 ILD) and 34 with severe PH (21 COPD, 13 ILD); and 38 patients with iPAH. Plasma samples were analysed by liquid chromatography-mass spectrometry. Student t-tests were used to calculate each comparison and false discovery rates computed. Proteins with a fold change >|1.5|, p-value<0.05 and present in >80% cases of the cohort were selected. Results: CLD-PH had up-regulated proteins from the immunoglobulin superfamily (IgSF) compared with non-PH. Compared with CLD patients with moderate PH, those with severe PH had 6 IgSF proteins and lysozyme up-regulated; and keratin-1 down-regulated. Compared with iPAH, patients with CLD- severe PH had up-regulated 6 IgSF proteins, orosomucoid-1 and tenascin-C; and down-regulated IGF-binding protein-3 and tetranectin. Conclusions: Patients with CLD and PH show a distinct proteomic profile, particularly in IgSF expression, which is further amplified in severe PH. Patients with CLD- severe PH also differ from iPAH, specially in IgSF. Further studies are warranted to assess the functionality of IgSF proteins and their clinical implications in CLD-PH.

Background & Aims: Ductular reaction expansion is associated with poor prognosis in patients with advanced liver disease. However, the mechanisms promoting biliary cell proliferation are largely unknown. Here, we identify neutrophils as drivers of biliary cell proliferation and the defective wound-healing response. Methods: The intrahepatic localization of neutrophils was evaluated in patients with chronic liver disease. Neutrophil dynamics were analyzed by intravital microscopy and neutrophil-labeling assays in DDC-treated mice. Neutrophil depletion or inhibition of recruitment was achieved using a Ly6g antibody or a CXCR1/2 inhibitor, respectively. Mice deficient in PAD4 (peptidyl arginine deiminase 4) and ELANE/NE (neutrophil elastase) were used to investigate the mechanisms underlying ductular reaction expansion. Results: In this study we describe a population of ductular reaction-associated neutrophils (DRANs), which are in direct contact with biliary epithelial cells in chronic liver diseases and whose numbers increased in parallel with disease progression. We show that DRANs are immobilized at the site of ductular reaction for a prolonged period of time. In addition, liver neutrophils display a unique phenotypic and transcriptomic profile, showing a decreased phagocytic capacity and increased oxidative burst. Depletion of neutrophils or inhibition of their recruitment reduces DRANs and the expansion of ductular reaction, while mitigating liver fibrosis and angiogenesis. Mechanistically, neutrophils deficient in PAD4 and ELANE abrogate neutrophil-induced biliary cell proliferation, thus indicating the role of neutrophil extracellular traps and elastase release in ductular reaction expansion. Conclusions: Overall, our study reveals the accumulation of DRANs as a hallmark of advanced liver disease and a potential therapeutic target to mitigate ductular reaction and the maladaptive wound-healing response. Impact and implications: Our results indicate that neutrop