Impact of the Mitochondrial Genetic Background in Complex III Deficiency

BACKGROUND: In recent years clinical evidence has emphasized the importance of the mtDNA genetic background that hosts a primary pathogenic mutation in the clinical expression of mitochondrial disorders, but little experimental confirmation has been provided. We have analyzed the pathogenic role of a novel homoplasmic mutation (m.15533 A>G) in the cytochrome b (MT-CYB) gene in a patient presenting with lactic acidosis, seizures, mild mental delay, and behaviour abnormalities. METHODOLOGY: Spectrophotometric analyses of the respiratory chain enzyme activities were performed in different tissues, the whole muscle mitochondrial DNA of the patient was sequenced, and the novel mutation was confirmed by PCR-RFLP. Transmitochondrial cybrids were constructed to confirm the pathogenicity of the mutation, and assembly/stability studies were carried out in fibroblasts and cybrids by means of mitochondrial translation inhibition in combination with blue native gel electrophoresis. PRINCIPAL FINDINGS: Biochemical analyses revealed a decrease in respiratory chain complex III activity in patient's skeletal muscle, and a combined enzyme defect of complexes III and IV in fibroblasts. Mutant transmitochondrial cybrids restored normal enzyme activities and steady-state protein levels, the mutation was mildly conserved along evolution, and the proband's mother and maternal aunt, both clinically unaffected, also harboured the homoplasmic mutation. These data suggested a nuclear genetic origin of the disease. However, by forcing the de novo functioning of the OXPHOS system, a severe delay in the biogenesis of the respiratory chain complexes was observed in the mutants, which demonstrated a direct functional effect of the mitochondrial genetic background. CONCLUSIONS: Our results point to possible pitfalls in the detection of pathogenic mitochondrial mutations, and highlight the role of the genetic mtDNA background in the development of mitochondrial disorders

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Free Form Deformation -based Image Registration Improves Accuracy of Traction Force Microscopy

Traction Force Microscopy (TFM) is a widespread method used to recover cellular tractions from the deformation that they cause in their surrounding substrate. Particle Image Velocimetry (PIV) is commonly used to quantify the substrate’s deformations, due to its simplicity and efficiency. However, PIV relies on a block-matching scheme that easily underestimates the deformations. This is especially relevant in the case of large, locally non-uniform deformations as those usually found in the vicinity of a cell’s adhesions to the substrate. To overcome these limitations, we formulate the calculation of the deformation of the substrate in TFM as a non-rigid image registration process that warps the image of the unstressed material to match the image of the stressed one. In particular, we propose to use a B-spline -based Free Form Deformation (FFD) algorithm that uses a connected deformable mesh to model a wide range of flexible deformations caused by cellular tractions. Our FFD approach is validated in 3D fields using synthetic (simulated) data as well as with experimental data obtained using isolated endothelial cells lying on a deformable, polyacrylamide substrate. Our results show that FFD outperforms PIV providing a deformation field that allows a better recovery of the magnitude and orientation of tractions. Together, these results demonstrate the added value of the FFD algorithm for improving the accuracy of traction recovery.status: publishe

Serum miR-181b-5p predicts ascites onset in patients with compensated cirrhosis.

Background & Aims Treatment with non-selective beta-blockers (NSBBs) reduces the risk of ascites, which is the most common decompensating event in cirrhosis. This study aimed to assess the ability of a serum microRNA (miRNA) signature to predict ascites formation and the hemodynamic response to NSBBs in compensated cirrhosis. Methods Serum levels of miR-452-5p, miR-429, miR-885-5p, miR-181b-5p, and miR-122-5p were analyzed in patients with compensated cirrhosis (N = 105). Hepatic venous pressure gradient (HVPG) was measured at baseline, after intravenous propranolol, and 1 year after randomization to NSBBs (n = 52) or placebo (n = 53) (PREDESCI trial). miRNAs were analyzed at baseline and at 1 year. Results Nineteen patients (18%) developed ascites, of whom 17 developed ascites after 1 year. miR-181b-5p levels at 1 year, but not at baseline, were higher in patients that developed ascites. The AUC of miR-181b-5p at 1 year to predict ascites was 0.7 (95% CI 0.59-0.78). miR-429 levels were lower at baseline in acute HVPG responders to NSBBs (AUC 0.65; 95% CI, 0.53-0.76), but levels at baseline and at 1 year were not associated with the HVPG response to NSBBs at 1 year. Conclusions Serum miR-181b-5p is a promising non-invasive biomarker to identify patients with compensated cirrhosis at risk of ascites development. Lay summary Ascites marks the transition from the compensated to decompensated stage in cirrhosis and indicates a worsening in prognosis. There are currently no easily accessible tools to identify patients with compensated cirrhosis at risk of developing ascites. We evaluated the levels of novel molecules termed microRNAs in the blood of patients with compensated cirrhosis and observed that miR-181b-5p can predict which patients are going to develop ascites

Serum miR-181b-5p predicts ascites onset in patients with compensated cirrhosis

Altres ajuts: Ministerio de Ciencia e Innovación; Fondo Europeo de Desarrollo Regional (FEDER); Gilead Sciences (GLD19/00045).Background & Aims: Treatment with non-selective beta-blockers (NSBBs) reduces the risk of ascites, which is the most common decompensating event in cirrhosis. This study aimed to assess the ability of a serum microRNA (miRNA) signature to predict ascites formation and the hemodynamic response to NSBBs in compensated cirrhosis. Methods: Serum levels of miR-452-5p, miR-429, miR-885-5p, miR-181b-5p, and miR-122-5p were analyzed in patients with compensated cirrhosis (N = 105). Hepatic venous pressure gradient (HVPG) was measured at baseline, after intravenous propranolol, and 1 year after randomization to NSBBs (n = 52) or placebo (n = 53) (PREDESCI trial). miRNAs were analyzed at baseline and at 1 year. Results: Nineteen patients (18%) developed ascites, of whom 17 developed ascites after 1 year. miR-181b-5p levels at 1 year, but not at baseline, were higher in patients that developed ascites. The AUC of miR-181b-5p at 1 year to predict ascites was 0.7 (95% CI 0.59-0.78). miR-429 levels were lower at baseline in acute HVPG responders to NSBBs (AUC 0.65; 95% CI, 0.53-0.76), but levels at baseline and at 1 year were not associated with the HVPG response to NSBBs at 1 year. Conclusions: Serum miR-181b-5p is a promising non-invasive biomarker to identify patients with compensated cirrhosis at risk of ascites development. Lay summary: Ascites marks the transition from the compensated to decompensated stage in cirrhosis and indicates a worsening in prognosis. There are currently no easily accessible tools to identify patients with compensated cirrhosis at risk of developing ascites. We evaluated the levels of novel molecules termed microRNAs in the blood of patients with compensated cirrhosis and observed that miR-181b-5p can predict which patients are going to develop ascites

Metabolomics as a tool to predict the risk of decompensation or liver related death in patients with compensated cirrhosis.

BACKGROUND AIMS Patients with compensated cirrhosis with clinically significant portal hypertension (CSPH: HVPG >10 mmHg) have a high risk of decompensation. HVPG is, however, an invasive procedure not available in all centers. The present study aims to assess whether metabolomics can improve the capacity of clinical models in predicting clinical outcome in these compensated patients. APPROACH RESULTS This is a nested study from the PREDESCI cohort (a RCT of non-selective beta blockers (NSBB) versus placebo in 201 patients with compensated cirrhosis and CSPH) including 167 patients for whom a blood sample was collected. A targeted metabolomic serum analysis, using UHPLC-MS, was performed. Metabolites underwent univariate time-to event cox regression analysis. Top ranked metabolites were selected using LogRank P-value to generate a stepwise cox model. Comparison between models was done using DeLong's test. Eighty-two patients with CSPH were randomized to NSBB and 85 to placebo. Thirty-three patients developed the main endpoint (decompensation/liver-related death). The model including HVPG, Child-Pugh and treatment received (HVPG/Clinical model) had a C-index of 0.748 [CI95% 0.664-0.827]. Addition of two metabolites, Ceramide (d18:1/22:0) and Methionine (HVPG/Clinical/Metabolite model) significantly improved model's performance (C-index of 0.808 [CI95% 0.735-0.882]; P=0.032). The combination of these two metabolites together with Child-Pugh and type of treatment received (Clinical/Metabolite model) had a C-Index of 0.785 [CI95% 0.710-0.860] not significantly different from the HVPG based models including or not metabolites. CONCLUSIONS In patients with compensated cirrhosis and CSPH, metabolomics improves the capacity of clinical models and achieves similar predictive capacity than models including HVPG

BN-PAGE analysis of mitochondrial respiratory chain complexes in control and mutant fibroblasts and cybrids.

<p>Mitochondrial particles were isolated as described in Methods and 40 µg of protein were analyzed on a 5–15% BN-polyacrylamide gel for the separation of multisubunit complexes. Western-blot analysis was performed using antibodies against the indicated OXPHOS subunits. CI, fully-assembled complex I. CIII₂, complex III dimer. CIV, complex IV. CIII₂+IV indicates the presence of the supercomplex containing complexes III and IV. C1 and C2, control fibroblasts. P, patient's fibroblasts. C, control cybrid. Two independent mutant cybrids are indicated as #1 and #2.</p

Assembly kinetics of respiratory chain complexes in control and mutant cybrids.

<p>Two different control cybrids belonging to haplogroup H, and two independent <i>MT-CYB</i> mutant clones were treated for 6 days with doxycycline (an inhibitor of mitochondrial translation), the medium was replaced by doxycycline-free medium and cells were collected at 0, 6, 15, 24, 48, 72, and 96 hours (indicated as t0–t16). SS indicates the steady-state expression levels of the respiratory chain complexes (A) Example of one control and one mutant clone. 40 µg of crude mitochondrial pellets were analyzed by BN-PAGE in combination with complex I and complex IV-IGA assays. (B) Duplicate gels were blotted and incubated with antibodies against the NDUFA9 complex I subunit, complex III core2 protein, complex IV COX5A subunit and complex II SDHA subunit. (C) The signals from the blots were quantified, expressed as percentage of the untreated cells (SS), normalized with the complex II SDHA subunit and plotted. The restoration curves constitute the mean values ± SD obtained from the two controls and the two independent mutant cybrids. Upper left panel, complex I assembly rates. Upper right panel, complex III assembly rates. Lower left panel, complex IV assembly rates. Lower right panel, supercomplex CIII₂+IV assembly kinetics.</p

Residual enzyme activities of mitochondrial respiratory chain complexes in different tissues from the index patient.

<p>Enzyme activities are expressed as</p><p>*cU/U citrate synthase (CS) and</p><p>**nmol.min⁻¹.mg prot⁻¹. CS activity is expressed as mU/mg protein. Abnormal values are indicated in bold. nd, not determined. Complex I, CI; Complex II, CII; Complex III, CIII; Complex IV, CIV.</p