300 results on '"Francesco Bernardi"'
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2. Venetoclax is a potent hepsin inhibitor that reduces the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells
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Maria Carmen Rodenas, Julia Peñas-Martínez, Irene Pardo-Sánchez, David Zaragoza-Huesca, Carmen Ortega-Sabater, Jorge Peña-García, Salvador Espín, Guillermo Ricote, Sofía Montenegro, Francisco Ayala-De La Peña, Ginés Luengo-Gil, Andrés Nieto, Francisco García-Molina, Vicente Vicente, Francesco Bernardi, María Luisa Lozano, Victoriano Mulero, Horacio Pérez-Sánchez, Alberto Carmona-Bayonas, and Irene Martínez-Martínez
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hepsin ,colorectal cancer ,metastasis ,thrombosis ,molecular-targeted therapy ,Biology (General) ,QH301-705.5 - Abstract
Introduction: Hepsin is a type II transmembrane serine protease and its expression has been linked to greater tumorigenicity and worse prognosis in different tumors. Recently, our group demonstrated that high hepsin levels from primary tumor were associated with a higher risk of metastasis and thrombosis in localized colorectal cancer patients. This study aims to explore the molecular role of hepsin in colorectal cancer.Methods: Hepsin levels in plasma from resected and metastatic colorectal cancer patients were analyzed by ELISA. The effect of hepsin levels on cell migration, invasion, and proliferation, as well as on the activation of crucial cancer signaling pathways, was performed in vitro using colorectal cancer cells. A thrombin generation assay determined the procoagulant function of hepsin from these cells. A virtual screening of a database containing more than 2000 FDA-approved compounds was performed to screen hepsin inhibitors, and selected compounds were tested in vitro for their ability to suppress hepsin effects in colorectal cancer cells. Xenotransplantation assays were done in zebrafish larvae to study the impact of venetoclax on invasion promoted by hepsin.Results: Our results showed higher plasma hepsin levels in metastatic patients, among which, hepsin was higher in those suffering thrombosis. Hepsin overexpression increased colorectal cancer cell invasion, Erk1/2 and STAT3 phosphorylation, and thrombin generation in plasma. In addition, we identified venetoclax as a potent hepsin inhibitor that reduced the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells. Interestingly, pretreatment with Venetoclax of cells overexpressing hepsin reduced their invasiveness in vivo.Discussion: Our results demonstrate that hepsin overexpression correlates with a more aggressive and prothrombotic tumor phenotype. Likewise, they demonstrate the antitumor role of venetoclax as a hepsin inhibitor, laying the groundwork for molecular-targeted therapy for colorectal cancer.
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- 2023
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3. Whole-Exome Sequencing in a Family with an Unexplained Tendency for Venous Thromboembolism: Multicomponent Prediction of Low-Frequency Variant Deleteriousness and of Individual Protein Interaction
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Barbara Lunghi, Nicole Ziliotto, Dario Balestra, Lucrezia Rossi, Patrizia Della Valle, Pasquale Pignatelli, Mirko Pinotti, Armando D’Angelo, Giovanna Marchetti, and Francesco Bernardi
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venous thromboembolism ,WES analysis ,family studies ,low-frequency variants ,protein interaction pattern ,CRP ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Whole-exome sequencing (WES) in families with an unexplained tendency for venous thromboembolism (VTE) may favor detection of low-frequency variants in genes with known contribution to hemostasis or associated with VTE-related phenotypes. WES analysis in six family members, three of whom affected by documented VTE, filtered for MAF < 0.04 in 192 candidate genes, revealed 22 heterozygous (16 missense and six synonymous) variants in patients. Functional prediction by multi-component bioinformatics tools, implemented by a database/literature search, including ClinVar annotation and QTL analysis, prioritized 12 missense variants, three of which (CRP Leu61Pro, F2 Asn514Lys and NQO1 Arg139Trp) were present in all patients, and the frequent functional variants FGB Arg478Lys and IL1A Ala114Ser. Combinations of prioritized variants in each patient were used to infer functional protein interactions. Different interaction patterns, supported by high-quality evidence, included eight proteins intertwined in the “acute phase” (CRP, F2, SERPINA1 and IL1A) and/or in the “fibrinogen complex” (CRP, F2, PLAT, THBS1, VWF and FGB) significantly enriched terms. In a wide group of candidate genes, this approach highlighted six low-frequency variants (CRP Leu61Pro, F2 Asn514Lys, SERPINA1 Arg63Cys, THBS1 Asp901Glu, VWF Arg1399His and PLAT Arg164Trp), five of which were top ranked for predicted deleteriousness, which in different combinations may contribute to disease susceptibility in members of this family.
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- 2023
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4. OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies
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Claudia Sacchetto, Laura Peretto, Francisco Baralle, Iva Maestri, Francesca Tassi, Francesco Bernardi, Stan F. J. van de Graaf, Franco Pagani, Mirko Pinotti, and Dario Balestra
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OTC deficiency ,Pathogenic mRNA splicing ,Spfash mouse model ,Nucleotide variations ,U1snRNA ,Therapeutics. Pharmacology ,RM1-950 ,Biochemistry ,QD415-436 - Abstract
Abstract Background Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA. Methods Creation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors. Results Comparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects. Moreover, the interplay between the authentic and the adjacent cryptic 5′ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5′ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5′ss, was rescuable by engineered U1snRNA. Conclusions Subtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.
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- 2021
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5. Crosstalk between hemostasis inhibitors and cholesterol biomarkers in multiple sclerosis
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Robert Parambi, Nicole Ziliotto, Francesco Bernardi, Marcello Baroni, Richard W Browne, Dejan Jakimovski, Bianca Weinstock-Guttman, Robert Zivadinov, and Murali Ramanathan
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Cholesterol ,apolipoprotein ,heparin cofactor II ,hemostasis inhibitor ,anticoagulant ,multiple sclerosis ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
The individual roles of cholesterol pathway biomarkers (CPB) and hemostasis inhibitors with neuroimaging outcomes were previously investigated in multiple sclerosis (MS). The purpose of this extension study was to investigate potential crosstalk between plasma CPB [total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and apolipoproteins (Apo) ApoA-I, ApoAII, ApoB, ApoC-II and ApoE] and hemostasis inhibitors [heparin cofactor-II (HCII), protein C (PC), protein S (PS), thrombomodulin, ADAMTS13 and PAI-1] in a cohort of 127 MS patients, and 40 healthy individuals (HI). The associations were assessed with regressions. In MS patients, HCII was positively associated with TC, LDL-C, HDL-C and ApoA-I (p=0.028, 0.027, 0.002 and 0.027, respectively) but negatively associated with ApoCII (p=0.018). PC was positively associated with ApoC-II (p=0.001) and ApoB (p=0.016) whereas PS was associated with TC (p=0.024) and ApoE (p=0.003) in MS. The ApoC-II associations were not observed in HI. The negative association between ApoC-II and HCll was an exception amongst other positive associations between CPB and hemostasis inhibitors in MS. CPB do not modulate the PC associations with neurodegeneration in MS.
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- 2022
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6. Protein S on the surface of plasma lipoproteins: a potential mechanism for protein S delivery to the atherosclerotic plaques?
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Marcello Baroni, Paolo Ferraresi, Nicole Ziliotto, Daria Bortolotti, Pierfilippo Acciarri, Nicola Martinelli, Giovanna Marchetti, Matteo Coen, and Francesco Bernardi
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Protein S ,Atherosclerotic plaques ,Lipoproteins ,Phospholipids ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
The anticoagulant protein S (PS) binds phospholipids with very high affinity, but PS interaction with lipoproteins and lipidrich atherosclerotic plaques remains still poorly defined. We investigated PS in plasma lipoproteins and in atherosclerotic plaques from ten patients undergoing endarterectomy. PS was detected by Western blotting after exposure of the necrotic core to liposomes and was found to maintain its ability to bind phosphatidylserine micelles. The amounts of PS bound to low/very low-density lipoproteins in patient’ plasmas were higher and more variable than those detected in healthy subjects. A direct correlation between bound PS and low-density lipoproteins (LDL), plasma levels was found only in patients (r=0.921, p
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- 2022
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7. Translational readthrough at F8 nonsense variants in the factor VIII B domain contributes to residual expression and lowers inhibitor association
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Maria Francesca Testa, Silvia Lombardi, Francesco Bernardi, Mattia Ferrarese, Donata Belvini, Paolo Radossi, Giancarlo Castaman, Mirko Pinotti, and Alessio Branchini
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
In hemophilia A, F8 nonsense variants, and particularly those affecting the large factor VIII (FVIII) B domain that is dispensable for coagulant activity, display lower association with replacement therapy-related anti-FVIII inhibitory antibodies as retrieved from multiple international databases. Since null genetic conditions favor inhibitor development, we hypothesized that translational readthrough over premature termination codons (PTC) may contribute to immune tolerance by producing full-length proteins through the insertion of amino acid subset(s). To quantitatively evaluate the readthrough output in vitro, we developed a very sensitive luciferase-based system to detect very low full-length FVIII synthesis from a wide panel (n=45; ~60% patients with PTC) of F8 nonsense variants. PTC not associated with inhibitors displayed higher readthrough-driven expression levels than inhibitor-associated PTC, a novel observation. Particularly, higher levels were detected for B-domain variants (n=20) than for variants in other domains (n=25). Studies on plasma from six hemophilia A patients with PTC, integrated by expression of the corresponding nonsense and readthrough-deriving missense variants, consistently revealed higher FVIII levels for B-domain variants. Only one B-domain PTC (Arg814*) was found among the highly represented PTC not sporadically associated with inhibitors, but with the lowest proportion of inhibitor cases (4 out of 57). These original insights into the molecular genetics of hemophilia A, and particularly into genotype-phenotype relationships related with disease treatment, demonstrate that B-domain features favor PTC readthrough output. This provides a potential molecular mechanism contributing to differential PTC-associated inhibitor occurrence, with translational implications for a novel, experimentally based classification of F8 nonsense variants.
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- 2022
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8. Correction: Bernardi et al. OH-Defects in Detrital Quartz Grains from the Julian Basin (NE Italy and Slovenia): A Fourier Transform Infrared Study. Geosciences 2022, 12, 90
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Francesco Bernardi, Henrik Skogby, and Davide Lenaz
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n/a ,Geology ,QE1-996.5 - Abstract
The authors would like to correct the published article [...]
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- 2023
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9. The effect of the chemical chaperone 4-phenylbutyrate on secretion and activity of the p.Q160R missense variant of coagulation factor FVII
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Elisabeth Andersen, Maria Eugenia Chollet, Marcello Baroni, Mirko Pinotti, Francesco Bernardi, Ellen Skarpen, Per Morten Sandset, and Grethe Skretting
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Factor VII deficiency ,Chemical chaperones ,Mutations ,Protein misfolding ,Endoplasmic reticulum ,Trafficking ,Biotechnology ,TP248.13-248.65 ,Biology (General) ,QH301-705.5 ,Biochemistry ,QD415-436 - Abstract
Abstract Background Congenital coagulation factor (F) VII deficiency is a rare bleeding disorder caused by mutations in the F7 gene. The missense factor FVII variant p.Q160R is the disease-causing mutation in all Norwegian FVII deficient patients and results in reduced biological activity and antigen levels of FVII in patient plasma. Previous in vitro studies on this variant demonstrated impaired intracellular trafficking and reduced secretion, possibly due to protein misfolding. The aim of the study was therefore to assess the impact of chemical chaperones on cellular processing and secretion of this variant using a cell model based on overexpression of the recombinant protein. Results Through screening of compounds, we identified 4-phenylbutyrate (4-PBA) to increase the secretion of recombinant (r) FVII-160R by ~ 2.5-fold. Additionally, treatment with 4-PBA resulted in a modest increase in specific biological activity. Intracellular localization studies revealed that upon treatment with 4-PBA, rFVII-160R was secreted through Golgi and Golgi reassembly-stacking protein (GRASP)-structures. Conclusions The present study demonstrates that the chemical chaperone 4-PBA, restores intracellular trafficking and increases the secretion of a missense FVII variant with functional properties in the extrinsic coagulation pathway.
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- 2019
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10. Performance prediction models based on anthropometric, genetic and psychological traits of Croatian sprinters
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Luciana Zaccagni, Barbara Lunghi, Davide Barbieri, Natascia Rinaldo, Sasa Missoni, Tena Šaric, Jelena Šarac, Vesna Babic, Marija Rakovac, Francesco Bernardi, and Emanuela Gualdi-Russo
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sprint performance ,personal best ,anthropometry ,genetic polymorphisms ,competitive anxiety ,Sports medicine ,RC1200-1245 ,Biology (General) ,QH301-705.5 - Abstract
Elite athletes differ from each other in their characteristics according to their discipline. This study aimed to identify performance predictors in elite Croatian sprinters taking into consideration their anthropometric, psychological and genetic characteristics. One hundred and four elite Croatian sprinters (68 males and 36 females) participated in this study. Of them, 38 are currently competing in the 100-metre dash. The others are former sprinters. The participants underwent direct anthropometric assessment. Participants were also tested by means of the Competitive State Anxiety Inventory-2 and for ACE and ACTN3 polymorphisms. Multiple linear regression analysis was applied to identify the best model for performance prediction. Different models were developed for males and females. Anthropometric traits accounted for 44% of the variance in performance for males, 62% for females. Once other traits (psychological for females) were entered into the model, no additional contribution to the variance was observed. The most significant predictors of higher running velocity were bicristal diameter and foot dimensions in males, and leg length and clean one-repetition maximum in females. The findings suggest that performance in sprinters is associated with anthropometric characteristics, with biomechanical implications that may be used to provide a more complete evaluation of sprinters’ performance.
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- 2018
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11. Basophil Blood Cell Count Is Associated With Enhanced Factor II Plasma Coagulant Activity and Increased Risk of Mortality in Patients With Stable Coronary Artery Disease: Not Only Neutrophils as Prognostic Marker in Ischemic Heart Disease
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Francesca Pizzolo, Annalisa Castagna, Oliviero Olivieri, Domenico Girelli, Simonetta Friso, Filippo Stefanoni, Silvia Udali, Veronica Munerotto, Marcello Baroni, Vera Cetera, Giovanni Battista Luciani, Giuseppe Faggian, Francesco Bernardi, and Nicola Martinelli
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basophils ,factor II plasma coagulant activity ,neutrophils ,secondary prevention of coronary artery disease ,white blood cell count ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
Background White blood cell count, which is inexpensive and widely available in clinical practice, has been proposed to provide prognostic information in coronary artery disease (CAD). Elevated levels of white blood cell subtypes may play different roles in atherothrombosis and predict cardiovascular outcomes. Methods and Results The association between white blood cell counts and mortality was evaluated in 823 subjects with angiographically demonstrated and clinically stable CAD in an observational–longitudinal study. The correlation among white blood cell counts and factor II plasma coagulant activity was analyzed in 750 subjects (554 CAD and 196 CAD‐free) not taking anticoagulant drugs. Subjects with overt leukocytosis or leukopenia were excluded. In the longitudinal study after a median follow‐up of 61 months, 160 (19.4%) subjects died, 107 (13.0%) of whom from cardiovascular causes. High levels of neutrophils, monocytes, eosinophils, and basophils were associated with an increased mortality rate. In multiadjusted Cox regression models, only neutrophils and basophils remained predictors of total and cardiovascular mortality. The associations remained significant after adjustment for traditional cardiovascular risk factors and by including D‐dimer and the chemokine CXCL12 in the regression models. Neutrophils and basophils were also significant predictors of factor II plasma coagulant activity variability after adjustment for blood cell counts, age, sex, inflammatory markers, CAD diagnosis, and prothrombin G20210A polymorphism. Factor II plasma coagulant activity was similarly increased in subjects with high neutrophil and basophil counts and in carriers of the prothrombin 20210A allele. Conclusions Both high neutrophil and basophil blood counts may predict mortality in patients with clinically stable CAD and are associated with enhanced factor II plasma coagulant activity, thereby suggesting underlying prothrombotic mechanisms.
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- 2021
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12. Biochemical, molecular and clinical aspects of coagulation factor VII and its role in hemostasis and thrombosis
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Francesco Bernardi and Guglielmo Mariani
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Activated factor VII (FVIIa), the first protease of clotting, expresses its physiological procoagulant potential only after complexing with tissue factor (TF) exposed to blood. Deep knowledge of the FVIIa-TF complex and F7 gene helps to understand the Janus-faced clinical findings associated to low or elevated FVII activity (FVIIc). Congenital FVII deficiency, the most frequent among the recessively inherited bleeding disorders, is caused by heterogeneous mutations in the F7 gene. Complete FVII deficiency causes perinatal lethality. A wide range of bleeding symptoms, from life-threatening intracranial hemorrhage to mild mucosal bleeding, is observed in patients with apparently modest differences in FVIIc levels. Though clinically relevant FVIIc threshold levels are still uncertain, effective management, including prophylaxis, has been devised, substantially improving the quality of life of patients. The exposure of TF in diseased arteries fostered investigation on the role of FVII in cardiovascular disease. FVIIc levels were found to be predictors of cardiovascular death and to be markedly associated to F7 gene variation. These genotype-phenotype relationships are among the most extensively investigated in humans. Genome-wide analyses extended association to numerous loci that, together with F7, explain >50% of FVII level plasma variance. However, the ability of F7 variation to predict thrombosis was not consistently evidenced in the numerous population studies. Main aims of this review are to highlight i) the biological and clinical information that distinguishes FVII deficiency from the other clotting disorders and ii) the impact exerted by genetically predicted FVII level variation on bleeding as well as on the thrombotic states.
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- 2021
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13. Relationships Among Circulating Levels of Hemostasis Inhibitors, Chemokines, Adhesion Molecules, and MRI Characteristics in Multiple Sclerosis
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Nicole Ziliotto, Robert Zivadinov, Dejan Jakimovski, Marcello Baroni, Niels Bergsland, Deepa P. Ramasamy, Bianca Weinstock-Guttman, Murali Ramanathan, Giovanna Marchetti, and Francesco Bernardi
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multiple sclerosis ,neurodegeneration ,cerebral microbleeds ,hemostasis inhibitors ,adhesion molecules ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Background: Several studies suggested cross talk among components of hemostasis, inflammation, and immunity pathways in the pathogenesis, neurodegeneration, and occurrence of cerebral microbleeds (CMBs) in multiple sclerosis (MS).Objectives: This study aimed to evaluate the combined contribution of the hemostasis inhibitor protein C (PC) and chemokine C-C motif ligand 18 (CCL18) levels to brain atrophy in MS and to identify disease-relevant correlations among circulating levels of hemostasis inhibitors, chemokines, and adhesion molecules, particularly in CMB occurrence in MS.Methods: Plasma levels of hemostasis inhibitors (ADAMTS13, PC, and PAI1), CCL18, and soluble adhesion molecules (sNCAM, sICAM1, sVCAM1, and sVAP1) were evaluated by multiplex in 138 MS patients [85 relapsing-remitting (RR-MS) and 53 progressive (P-MS)] and 42 healthy individuals (HI) who underwent 3-T MRI exams. Association of protein levels with MRI outcomes was performed by regression analysis. Correlations among protein levels were assessed by partial correlation and Pearson's correlation.Results: In all patients, regression analysis showed that higher PC levels were associated with lower brain volumes, including the brain parenchyma (p = 0.002), gray matter (p < 0.001), cortex (p = 0.001), deep gray matter (p = 0.001), and thalamus (p = 0.001). These associations were detectable in RR-MS but not in P-MS patients. Higher CCL18 levels were associated with higher T2-lesion volumes in all MS patients (p = 0.03) and in the P-MS (p = 0.003). In the P-MS, higher CCL18 levels were also associated with lower volumes of the gray matter (p = 0.024), cortex (p = 0.043), deep gray matter (p = 0.029), and thalamus (p = 0.022). PC-CCL18 and CCL18-PAI1 levels were positively correlated in both MS and HI, PC–sVAP1 and PAI1–sVCAM1 only in MS, and PC–sICAM1 and PC–sNCAM only in HI. In MS patients with CMBs (n = 12), CCL18–PAI1 and PAI1–sVCAM1 levels were better correlated than those in MS patients without CMBs, and a novel ADAMTS13–sVAP1 level correlation (r = 0.78, p = 0.003) was observed.Conclusions: Differences between clinical phenotype groups in association of PC and CCL18 circulating levels with MRI outcomes might be related to different aspects of neurodegeneration. Disease-related pathway dysregulation is supported by several protein level correlation differences between MS patients and HI. The integrated analysis of plasma proteins and MRI measures provide evidence for new relationships among hemostasis, inflammation, and immunity pathways, relevant for MS and for the occurrence of CMBs.
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- 2020
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14. OH-Defects in Detrital Quartz Grains from the Julian Basin (NE Italy and Slovenia): A Fourier Transform Infrared Study
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Francesco Bernardi, Henrik Skogby, and Davide Lenaz
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detrital quartz ,OH-defects ,Julian Basin ,Italy ,Slovenia ,Geology ,QE1-996.5 - Abstract
In this study, we analyzed up to 80 detrital quartz grains from four lithic greywackes along the stratigraphic column of the Julian Basin, a synorogenic basin in the southeastern Alps between Italy and Slovenia. Fourier transform infrared spectroscopy of detrital quartz was used to investigate the sample set with interest to its OH-defect speciation and content of each associated substitution. According to several recent studies, OH-defects in quartz are correlated to petrogenetic conditions of the source material and can be used as a provenance tool. The aim of this study is to compare results based on this method with previous studies that used other methods, to better constrain the palaeogeographical reconstruction of sedimentary fluxes. Detrital quartz within the samples of the basin shows different patterns of OH-defects and water content, indicating substantial petrogenetic differences between the sediment source rocks. For the oldest analyzed sample (ca. 66 Ma), the distribution of OH-defects suggests a mixed source between igneous and non-igneous rocks, with a predominance of metamorphic material supply. Another sample (56 Ma) reveals a great variability of OH-defects and water content, indicating that the magmatic component dominates over the metamorphic component. The distribution of OH-defects in the samples at the top of the sequence (52–53 Ma) suggests an almost solely metamorphic source. These results are in line with previous studies based on heavy minerals and geochemistry.
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- 2022
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15. Combination of CLEC4M rs868875 G-Carriership and ABO O Genotypes May Predict Faster Decay of FVIII Infused in Hemophilia A Patients
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Barbara Lunghi, Massimo Morfini, Nicola Martinelli, Silvia Linari, Giancarlo Castaman, and Francesco Bernardi
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CLEC4M ,CLEC4M SNPs ,factor VIII ,haemophilia A ,pharmacokinetics ,ABO ,Medicine - Abstract
The C-type lectin CLEC4M binds and internalizes factor VIII (FVIII). Common CLEC4M variants have been associated with FVIII pharmacokinetic (PK) profiles in hemophilia A (HA) patients. The two-compartment PK analysis of plasma-derived (pd-) and full length recombinant FVIII concentrates was conducted in twenty-six patients (FVIII:C ≤ 2 IU/dL). F8, ABO blood-groups, and the CLEC4M rs868875A/G polymorphism were genotyped. CLEC4M genotype groups differed for the elimination rate constant K 1-0 (p < 0.001), half-life (K 1-0 HL), and the Beta rate constant. Patients treated with pd-FVIII also differed in the Alpha phase. In linear regression models, the contribution of the CLEC4M genotypes to FVIII PK parameters remained significant after correction for ABO, age, and VWF antigen levels at PK. Combined CLEC4M rs868875A/G and ABO genotypes displayed significant interaction (K 1-0, p = 0.014). Compared to other combined genotypes, the G-carriers/O genotypes showed half-reduced K 1-0 HL (p = 0.008), and faster FVIII clearance (mean 7.1 ± 2.2 mL/h/kg SE) than in the G-carriers/non-O (mean 2.4 ± 0.3 mL/h/kg SE), (p = 0.038). Comparison in HA patients recruited in several countries suggests that CLEC4M genotypes coherently influence infused FVIII half-life and clearance. Our analysis supports substantially faster FVIII decay associated with the rs868875 G-carrier/ABO O genotypes, which has potential implications for genetically tailored substitutive HA treatment.
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- 2022
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16. Severe bleeding and absent ADP-induced platelet aggregation associated with inherited combined CalDAG-GEFI and P2Y12 deficiencies
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Barbara Lunghi, Anna Lecchi, Rosa Santacroce, Mariangela Scavone, Rita Paniccia, Andrea Artoni, Christian Gachet, Giancarlo Castaman, Maurizio Margaglione, Francesco Bernardi, and Marco Cattaneo
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Published
- 2020
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17. Changes in expression profiles of internal jugular vein wall and plasma protein levels in multiple sclerosis
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Giovanna Marchetti, Nicole Ziliotto, Silvia Meneghetti, Marcello Baroni, Barbara Lunghi, Erica Menegatti, Massimo Pedriali, Fabrizio Salvi, Ilaria Bartolomei, Sofia Straudi, Fabio Manfredini, Rebecca Voltan, Nino Basaglia, Francesco Mascoli, Paolo Zamboni, and Francesco Bernardi
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Gene expression ,Jugular vein wall ,Multiple sclerosis ,Chronic cerebrospinal venous insufficiency ,Venous abnormalities ,Jugular plasma protein levels ,Therapeutics. Pharmacology ,RM1-950 ,Biochemistry ,QD415-436 - Abstract
Abstract Background Multiple sclerosis (MS) is an inflammatory, demyelinating and degenerative disorder of the central nervous system (CNS). Several observations support interactions between vascular and neurodegenerative mechanisms in multiple sclerosis (MS). To investigate the contribution of the extracranial venous compartment, we analysed expression profiles of internal jugular vein (IJV), which drains blood from CNS, and related plasma protein levels. Methods We studied a group of MS patients (n = 19), screened by echo-color Doppler and magnetic resonance venography, who underwent surgical reconstruction of IJV for chronic cerebrospinal venous insufficiency (CCSVI). Microarray-based transcriptome analysis was conducted on specimens of IJV wall from MS patients and from subjects undergoing carotid endarterectomy, as controls. Protein levels were determined by multiplex assay in: i) jugular and peripheral plasma from 17 MS/CCSVI patients; ii) peripheral plasma from 60 progressive MS patients, after repeated sampling and iii) healthy individuals. Results Of the differentially expressed genes (≥ 2 fold-change, multiple testing correction, P
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- 2018
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18. An Altered Splicing Registry Explains the Differential ExSpeU1-Mediated Rescue of Splicing Mutations Causing Haemophilia A
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Dario Balestra, Iva Maestri, Alessio Branchini, Mattia Ferrarese, Francesco Bernardi, and Mirko Pinotti
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RNA splicing ,splicing mutations ,human disease ,ExSpeU1 ,Haemophilia A ,Genetics ,QH426-470 - Abstract
The exon recognition and removal of introns (splicing) from pre-mRNA is a crucial step in the gene expression flow. The process is very complex and therefore susceptible to derangements. Not surprisingly, a significant and still underestimated proportion of disease-causing mutations affects splicing, with those occurring at the 5’ splice site (5’ss) being the most severe ones. This led to the development of a correction approach based on variants of the spliceosomal U1snRNA, which has been proven on splicing mutations in several cellular and mouse models of human disease. Since the alternative splicing mechanisms are strictly related to the sequence context of the exon, we challenged the U1snRNA-mediated strategy in the singular model of the exon 5 of coagulation factor (F)VIII gene (F8) in which the authentic 5’ss is surrounded by various cryptic 5’ss. This scenario is further complicated in the presence of nucleotide changes associated with FVIII deficiency (Haemophilia A), which weaken the authentic 5’ss and create/strengthen cryptic 5’ss. We focused on the splicing mutations (c.602-32A > G, c.602-10T > G, c.602G > A, c.655G > A, c.667G > A, c.669A > G, c.669A > T, c.670G > T, c.670+1G > T, c.670+1G > A, c.670+2T > G, c.670+5G > A, and c.670+6T > C) found in patients with severe to mild Haemophilia A. Minigenes expression studies demonstrated that all mutations occurring within the 5’ss, both intronic or exonic, lead to aberrant transcripts arising from the usage of two cryptic intronic 5’ss at positions c.670+64 and c.670+176. For most of them, the observed proportion of correct transcripts is in accordance with the coagulation phenotype of patients. In co-transfection experiments, we identified a U1snRNA variant targeting an intronic region downstream of the defective exon (Exon Specific U1snRNA, U1sh7) capable to re-direct usage of the proper 5’ss (∼80%) for several mutations. However, deep investigation of rescued transcripts from +1 and +2 variants revealed only the usage of adjacent cryptic 5’ss, leading to frameshifted transcript forms. These data demonstrate that a single ExSpeU1 can efficiently rescue different mutations in the F8 exon 5, and provide the first evidence of the applicability of the U1snRNA-based approach to Haemophilia A.
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- 2019
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19. Combination of Genomic and Transcriptomic Approaches Highlights Vascular and Circadian Clock Components in Multiple Sclerosis
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Chiara Scapoli, Nicole Ziliotto, Barbara Lunghi, Erica Menegatti, Fabrizio Salvi, Paolo Zamboni, Marcello Baroni, Francesco Mascoli, Francesco Bernardi, and Giovanna Marchetti
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multiple sclerosis ,vascular components ,GWAS ,transcriptomics ,WES ,rare variants ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Aiming at exploring vascular components in multiple sclerosis (MS) with brain outflow disturbance, we combined transcriptome analysis in MS internal jugular vein (IJV) wall with WES in MS families with vertical transmission of disease. Main results were the differential expression in IJV wall of 16 MS-GWAS genes and of seven genes (GRIN2A, GRIN2B, IL20RB, IL26, PER3, PITX2, and PPARGC1A) not previously indicated by GWAS but encoding for proteins functionally interacting with MS candidate gene products. Strikingly, 22/23 genes have been previously associated with vascular or neuronal traits/diseases, nine encoded for transcriptional factors/regulators and six (CAMK2G, GRIN2A, GRIN2B, N1RD1, PER3, PPARGC1A) for circadian entrainment/rhythm components. Among the WES low-frequency (MAF ≤ 0.04) SNPs (n = 7) filtered in the 16 genes, the NR1D1 rs17616365 showed significantly different MAF in the Network for Italian Genomes affected cohort than in the 1000 Genome Project Tuscany samples. This pattern was also detected in five nonintronic variants (GRIN2B rs1805482, PER3 rs2640909, PPARGC1A rs2970847, rs8192678, and rs3755863) in genes coding for functional partners. Overall, the study proposes specific markers and low-frequency variants that might help (i) to understand perturbed biological processes in vascular tissues contributing to MS disease, and (ii) to characterize MS susceptibility genes for functional association with disease-pathways.
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- 2021
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20. The Factor VII Variant p.A354V-p.P464Hfs: Clinical versus Intracellular and Biochemical Phenotypes Induced by Chemical Chaperones
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Elisabeth Andersen, Maria Eugenia Chollet, Francesco Bernardi, Alessio Branchini, Marcello Baroni, Guglielmo Mariani, Alberto Dolce, Angelika Batorova, Ellen Skarpen, Christiane Filion Myklebust, Grethe Skretting, and Per Morten Sandset
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factor VII deficiency ,chemical chaperones ,mutations ,protein misfolding ,endoplasmic reticulum ,trafficking ,Technology ,Engineering (General). Civil engineering (General) ,TA1-2040 ,Biology (General) ,QH301-705.5 ,Physics ,QC1-999 ,Chemistry ,QD1-999 - Abstract
(1) Background: Congenital factor (F) VII deficiency is caused by mutations in the F7 gene. Patients with modest differences in FVII levels may display large differences in clinical severity. The variant p.A354V-p.P464Hfs is associated with reduced FVII antigen and activity. The aim of the study was to investigate the clinical manifestation of this variant and the underlying molecular mechanisms. (2) Methods: Analyses were conducted in 37 homozygous patients. The recombinant variant was produced in mammalian cells. (3) Results: We report a large variation in clinical phenotypes, which points out genetic and acquired components beyond F7 mutations as a source of variability. In contrast, patients displayed similarly reduced FVII plasma levels with antigen higher than its activity. Comparative analysis of the recombinant variant and of plasma samples from a subset of patients indicated the presence of an elongated variant with indistinguishable migration. Treatment of cells with the chemical chaperone 4-phenylbutyrate (4-PBA) improved the intracellular trafficking of the variant and increased its secretion to the conditioned medium up to 2-fold. However, the effect of 4-PBA on biological activity was marginal. (4) Conclusions: Chemical chaperones can be used as biochemical tools to study the intracellular fate of a trafficking-defective FVII variant.
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- 2021
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21. Correction of the Exon 2 Duplication in DMD Myoblasts by a Single CRISPR/Cas9 System
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Annalisa Lattanzi, Stephanie Duguez, Arianna Moiani, Araksya Izmiryan, Elena Barbon, Samia Martin, Kamel Mamchaoui, Vincent Mouly, Francesco Bernardi, Fulvio Mavilio, and Matteo Bovolenta
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dystrophin ,CRISPR/Cas9 ,duplication ,lentivirus ,gene editing ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Exonic duplications account for 10%–15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one guide RNA (gRNA) directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications.
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- 2017
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22. Cortical activation following chronic transcranial direct current stimulation in patients with minimally conscious state: a NIRS-based assessment associated to behavioral and plastic response
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Nicola Lamberti, Sofia Straudi, Valentina Bonsangue, Andrea Montis, Susanna Lavezzi, Francesco Bernardi, Giovanna Marchetti, Nicole Ziliotto, Veronica Tisato, Paola Secchiero, Fabio Manfredini, and Nino Basaglia
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Neurovascular diseases ,meeting. ,Diseases of the circulatory (Cardiovascular) system ,RC666-701 - Abstract
Not available
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- 2019
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23. C6orf10 Low-Frequency and Rare Variants in Italian Multiple Sclerosis Patients
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Nicole Ziliotto, Giovanna Marchetti, Chiara Scapoli, Matteo Bovolenta, Silvia Meneghetti, Andrea Benazzo, Barbara Lunghi, Dario Balestra, Lorenza Anna Laino, Nicolò Bozzini, Irene Guidi, Fabrizio Salvi, Sofia Straudi, Donato Gemmati, Erica Menegatti, Paolo Zamboni, and Francesco Bernardi
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multiple sclerosis ,whole exome sequencing ,low-frequency variants ,rare variants ,C6orf10 ,Genetics ,QH426-470 - Abstract
In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value ≤ 5 × 10-6). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) ≤ 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 × 10-7 and p < 1 × 10-20). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3′ region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS.
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- 2019
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24. Coagulation Pathways in Neurological Diseases: Multiple Sclerosis
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Nicole Ziliotto, Francesco Bernardi, Dejan Jakimovski, and Robert Zivadinov
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multiple sclerosis ,coagulation ,extrinsic pathway ,intrinsic pathway ,coagulation inhibitors ,fibrinolytic pathway ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Significant progress has been made in understanding the complex interactions between the coagulation system and inflammation and autoimmunity. Increased blood-brain-barrier (BBB) permeability, a key event in the pathophysiology of multiple sclerosis (MS), leads to the irruption into the central nervous system of blood components that include virtually all coagulation/hemostasis factors. Besides their cytotoxic deposition and role as a possible trigger of the coagulation cascade, hemostasis components cause inflammatory response and immune activation, sustaining neurodegenerative events in MS. Early studies showing the contribution of altered hemostasis in the complex pathophysiology of MS have been strengthened by recent studies using methodologies that permitted deeper investigation. Fibrin(ogen), an abundant protein in plasma, has been identified as a key contributor to neuroinflammation. Perturbed fibrinolysis was found to be a hallmark of progressive MS with abundant cortical fibrin(ogen) deposition. The immune-modulatory function of the intrinsic coagulation pathway still remains to be elucidated in MS. New molecular details in key hemostasis components participating in MS pathophysiology, and particularly involved in inflammatory and immune responses, could favor the development of novel therapeutic targets to ameliorate the evolution of MS. This review article introduces essential information on coagulation factors, inhibitors, and the fibrinolytic pathway, and highlights key aspects of their involvement in the immune system and inflammatory response. It discusses how hemostasis components are (dys)regulated in MS, and summarizes histopathological post-mortem human brain evidence, as well as cerebrospinal fluid, plasma, and serum studies of hemostasis and fibrinolytic pathways in MS. Studies of disease-modifying treatments as potential modifiers of coagulation factor levels, and case reports of autoimmunity affecting hemostasis in MS are also discussed.
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- 2019
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25. Rehabilitation Improves Mitochondrial Energetics in Progressive Multiple Sclerosis: The Significant Role of Robot-Assisted Gait Training and of the Personalized Intensity
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Fabio Manfredini, Sofia Straudi, Nicola Lamberti, Simone Patergnani, Veronica Tisato, Paola Secchiero, Francesco Bernardi, Nicole Ziliotto, Giovanna Marchetti, Nino Basaglia, Massimo Bonora, and Paolo Pinton
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multiple sclerosis ,exercise training ,lactic acid ,pyruvic acid ,Medicine (General) ,R5-920 - Abstract
Abnormal levels of pyruvate and lactate were reported in multiple sclerosis (MS). We studied the response of markers of mitochondrial function to rehabilitation in relation to type, intensity and endurance performance in severely disabled MS patients. Forty-six progressive MS patients were randomized to receive 12 walking sessions of robot-assisted gait training (RAGT, n = 23) or conventional overground therapy (CT, n = 23). Ten healthy subjects were also studied. Blood samples were collected to determine lactate, pyruvate, and glutathione levels and lactate/pyruvate ratio pre–post rehabilitation. In vivo muscle metabolism and endurance walking capacity were assessed by resting muscle oxygen consumption (rmVO2) using near-infrared spectroscopy and by six-minute walking distance (6MWD), respectively. The levels of mitochondrial biomarkers and rmVO2, altered at baseline with respect to healthy subjects, improved after rehabilitation in the whole population. In the two groups, an enhanced response was observed after RAGT compared to CT for lactate (p = 0.012), glutathione (p = 0.08) and rmVO2 (p = 0.07). Metabolic biomarkers and 6MWD improvements were exclusively correlated with a training speed markedly below individual gait speed. In severely disabled MS patients, rehabilitation rebalanced altered serum metabolic and muscle parameters, with RAGT being more effective than CT. A determinable slow training speed was associated with better metabolic and functional recovery. Trial Registration: ClinicalTrials.gov NCT02421731.
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- 2020
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26. Coagulation Factor XII Levels and Intrinsic Thrombin Generation in Multiple Sclerosis
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Nicole Ziliotto, Marcello Baroni, Sofia Straudi, Fabio Manfredini, Rosella Mari, Erica Menegatti, Rebecca Voltan, Paola Secchiero, Paolo Zamboni, Nino Basaglia, Giovanna Marchetti, and Francesco Bernardi
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multiple sclerosis ,coagulation ,factor XII ,intrinsic pathway ,thrombin generation ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
BackgroundFactor XII (FXII) activation initiates the intrinsic (contact) coagulation pathway. It has been recently suggested that FXII could act as an autoimmunity mediator in multiple sclerosis (MS). FXII depositions nearby dentritic cells were detected in the central nervous system of MS patients and increased FXII activity has been reported in plasma of relapsing remitting and secondary progressive MS patients. FXII inhibition has been proposed to treat MS.ObjectiveTo investigate in MS patients multiple FXII-related variables, including the circulating amount of protein, its pro-coagulant function, and their variation over time. To explore kinetic activation features of FXII in thrombin generation (TG).MethodsIn plasma from 74 MS patients and 49 healthy subjects (HS), FXII procoagulant activity (FXII:c) and FXII protein (FXII:Ag) levels were assessed. Their ratio (FXII:ratio) values were derived. Intrinsic TG was evaluated by different triggers.ResultsHigher FXII:Ag levels (p = 0.003) and lower FXII:ratio (p
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- 2018
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27. Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity
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Irving Donadon, John H. McVey, Isabella Garagiola, Alessio Branchini, Mimosa Mortarino, Flora Peyvandi, Francesco Bernardi, and Mirko Pinotti
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Dissection of pleiotropic effects of missense mutations, rarely investigated in inherited diseases, is fundamental to understanding genotype-phenotype relationships. Missense mutations might impair mRNA processing in addition to protein properties. As a model for hemophilia A, we investigated the highly prevalent F8 c.6046c>t/p.R2016W (exon 19) mutation. In expression studies exploiting lentiviral vectors, we demonstrated that the amino acid change impairs both Factor VIII (FVIII) secretion (antigen 11.0±0.4% of wild-type) and activity (6.0±2.9%). Investigations in patients’ ectopic F8 mRNA and with minigenes showed that the corresponding nucleotide change also decreases correct splicing to 70±5%, which is predicted to lower further FVIII activity (4.2±2%), consistently with patients’ levels (a (p.G2013R) reduced exon inclusion to 41±3% and the c.6053a>g (p.E2018G) to 28±2%, similarly to a variant affecting the 5′ splice site (c.6113a>g, p.N2038S, 26±2%), which displayed normal protein features upon recombinant expression. The p.G2013R reduced both antigen (7.0±0.9%) and activity (8.4±0.8%), while the p.E2018G produced a dysfunctional molecule (antigen: 69.0±18.1%; activity: 19.4±2.3%). In conclusion, differentially altered mRNA and protein patterns produce a gradient of residual activity, and clarify genotype-phenotype relationships. Data detail pathogenic mechanisms that, only in combination, account for moderate/severe disease forms, which in turn determine the mutation profile. Taken together we provide a clear example of interplay between mRNA and protein mechanisms of disease that operate in shaping many other inherited disorders.
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- 2018
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28. An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants
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Dario Balestra, Daniela Scalet, Franco Pagani, Malgorzata Ewa Rogalska, Rosella Mari, Francesco Bernardi, and Mirko Pinotti
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Exon-Specific U1snRNA ,hemophilia B ,ExSpeU1 ,mice ,splicing ,Therapeutics. Pharmacology ,RM1-950 - Abstract
In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G) or 3′ (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ≃100 seconds of untreated mice to ≃80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients.
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- 2016
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29. Natural and engineered carboxy-terminal variants: decreased secretion and gain-of-function result in asymptomatic coagulation factor VII deficiency
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Alessio Branchini, Lara Rizzotto, Guglielmo Mariani, Mariasanta Napolitano, Mario Lapecorella, Muriel Giansily-Blaizot, Rosella Mari, Alessandro Canella, Mirko Pinotti, and Francesco Bernardi
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
We report 2 asymptomatic homozygotes for the nonsense p.R462X mutation affecting the carboxy-terminus of coagulation factor VII (FVII, 466 aminoacids). FVII levels of 3–5% and 2.7±0.4% were found in prothrombin time-based and activated factor X (FXa) generation assays with human thromboplastins. Noticeably, FVII antigen levels were barely detectable (0.7±0.2%) which suggested a gain-of-function effect. This effect was more pronounced with bovine thromboplastin (4.8±0.9%) and disappeared with rabbit thromboplastin (0.7±0.2%). This suggests that the mutation influences tissue factor/FVII interactions.Whereas the recombinant rFVII-462X variant confirmed an increase in specific activity (~400%), a panel of nonsense (p.P466X, p.F465X, p.P464X, p.A463X) and missense (p.R462A, p.R462Q, p.R462W) mutations of the FVII carboxy-terminus resulted in reduced secretion but normal specific activity.These data provide evidence for counteracting pleiotropic effects of the p.R462X mutation, which explains the asymptomatic FVII deficiency, and contributes to our understanding of the role of the highly variable carboxy-terminus of coagulation serine proteases.
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- 2012
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30. Chronic sleep deprivation markedly reduces coagulation factor VII expression
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Mirko Pinotti, Cristiano Bertolucci, Elena Frigato, Alessio Branchini, Nicola Cavallari, Kenkichi Baba, Susana Contreras-Alcantara, J. Christopher Ehlen, Francesco Bernardi, Ketema N. Paul, and Gianluca Tosini
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Chronic sleep loss, a common feature of human life in industrialized countries, is associated to cardiovascular disorders. Variations in functional parameters of coagulation might contribute to explain this relationship. By exploiting the mouse model and a specifically designed protocol, we demonstrated that seven days of partial sleep deprivation significantly decreases (−30.5%) the thrombin generation potential in plasma evaluated upon extrinsic (TF/FVIIa pathway) but not intrinsic activation of coagulation. This variation was consistent with a decrease (−49.8%) in the plasma activity levels of factor VII (FVII), the crucial physiologicalal trigger of coagulation, which was even more pronounced at the liver mRNA level (−85.7%). The recovery in normal sleep conditions for three days completely restored thrombin generation and FVII activity in plasma. For the first time, we demonstrate that chronic sleep deprivation on its own reduces, in a reversible manner, the FVII expression levels, thus influencing the TF/FVIIa activation pathway efficiency.
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- 2010
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31. Evaluation of factor V mRNA to define the residual factor V expression levels in severe factor V deficiency
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Barbara Lunghi, Mirko Pinotti, Iva Maestri, Angelika Batorova, and Francesco Bernardi
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
We evaluated FV mRNA in severe factor V deficiency caused by the -12T/A IVS18 mutation, activating a cryptic splice site and leading to premature translation termination. Quantitative evaluation of factor V cDNA from homozygous and heterozygous subjects, and correction for nonsense mediated decay, suggested the presence of 0.1% of normal factor V mRNA.
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- 2008
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32. Combined effect of hemostatic gene polymorphisms and the risk of myocardial infarction in patients with advanced coronary atherosclerosis.
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Nicola Martinelli, Elisabetta Trabetti, Mirko Pinotti, Oliviero Olivieri, Marco Sandri, Simonetta Friso, Francesca Pizzolo, Claudia Bozzini, Pier Paolo Caruso, Ugo Cavallari, Suzanne Cheng, Pier Franco Pignatti, Francesco Bernardi, Roberto Corrocher, and Domenico Girelli
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Medicine ,Science - Abstract
BACKGROUND: Relative little attention has been devoted until now to the combined effects of gene polymorphisms of the hemostatic pathway as risk factors for Myocardial Infarction (MI), the main thrombotic complication of Coronary Artery Disease (CAD). The aim of this study was to evaluate the combined effect of ten common prothrombotic polymorphisms as a determinant of MI. METHODOLOGY/PRINCIPAL FINDINGS: We studied a total of 804 subjects, 489 of whom with angiographically proven severe CAD, with or without MI (n = 307; n = 182; respectively). An additive model considering ten common polymorphisms [Prothrombin 20210G>A, PAI-1 4G/5G, Fibrinogen beta -455G>A, FV Leiden and "R2", FVII -402G>A and -323 del/ins, Platelet ADP Receptor P2Y12 -744T>C, Platelet Glycoproteins Ia (873G>A), and IIIa (1565T>C)] was tested. The prevalence of MI increased linearly with an increasing number of unfavorable alleles (chi(2) for trend = 10.68; P = 0.001). In a multiple logistic regression model, the number of unfavorable alleles remained significantly associated with MI after adjustment for classical risk factors. As compared to subjects with 3-7 alleles, those with few (/=8) alleles had an increased MI risk (OR 2.49, 95%CIs 1.03-6.01). The number of procoagulant alleles correlated directly (r = 0.49, P = 0.006) with endogenous thrombin potential. CONCLUSIONS: The combination of prothrombotic polymorphisms may help to predict MI in patients with advanced CAD.
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- 2008
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33. Modulation of factor VIII pharmacokinetics by genetic components in factor VIII receptors
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Barbara Lunghi, Massimo Morfini, Nicola Martinelli, Alessio Branchini, Silvia Linari, Giancarlo Castaman, and Francesco Bernardi
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Hematology ,General Medicine ,Genetics (clinical) - Abstract
Gene variation in receptors for circulating factor VIII (FVIII) is candidate to explain the large inter-patient variability of infused FVIII pharmacokinetics (PK) in haemophilia A (HA).To compare in an Italian HA cohort (n = 26) the influence on FVIII PK of genetic components in four von Willebrand factor (VWF)/FVIII receptors.Genotypes of low-density lipoprotein receptor (LDLR), asialoglycoprotein receptor minor subunit (ASGR2), family 4 member M (CLEC4M), stabilin2 (STAB2) and ABO blood-group, and VWF:Ag levels were included as independent variables in linear regression analyses of two-compartment model (TCM) - standard half-life (SHL) FVIII PK parameters.In the initial FVIII distribution phase, the STAB2 rs4981022 AA, ASGR2 rs2289645 TT and LDLR rs688 TT genotypes may contribute to increase CWith the limitation of the small number of HA patients, these observations highlight multiple genetic components acting in distinct phases of FVIII PK and contributing to explain FVIII PK variability. This analysis provides candidates for genotype-based, individual tailoring of FVIII substitutive treatment.
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- 2022
34. Combination of Genomic and Transcriptomic Approaches Highlights Vascular and Circadian Clock Components in Multiple Sclerosis
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Chiara Scapoli, Nicole Ziliotto, Barbara Lunghi, Erica Menegatti, Fabrizio Salvi, Paolo Zamboni, Marcello Baroni, Francesco Mascoli, Francesco Bernardi, and Giovanna Marchetti
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vascular components ,QH301-705.5 ,multiple sclerosis ,Polymorphism, Single Nucleotide ,Article ,Catalysis ,Cohort Studies ,Inorganic Chemistry ,transcriptomics ,Gene Frequency ,Circadian Clocks ,Exome Sequencing ,Humans ,GWAS ,Gene Regulatory Networks ,RNA, Messenger ,Physical and Theoretical Chemistry ,Biology (General) ,Molecular Biology ,QD1-999 ,Spectroscopy ,Circadian rhythm ,Gene Expression Profiling ,Circadian entrainment ,Organic Chemistry ,rare variants ,Multiple sclerosis ,Rare variants ,Transcriptomics ,Vascular components ,WES ,Genomics ,General Medicine ,circadian entrainment ,circadian rhythm ,Introns ,Computer Science Applications ,Chemistry ,Gene Expression Regulation ,Italy ,Case-Control Studies ,Blood Vessels ,Transcriptome ,Genome-Wide Association Study - Abstract
Aiming at exploring vascular components in multiple sclerosis (MS) with brain outflow disturbance, we combined transcriptome analysis in MS internal jugular vein (IJV) wall with WES in MS families with vertical transmission of disease. Main results were the differential expression in IJV wall of 16 MS-GWAS genes and of seven genes (GRIN2A, GRIN2B, IL20RB, IL26, PER3, PITX2, and PPARGC1A) not previously indicated by GWAS but encoding for proteins functionally interacting with MS candidate gene products. Strikingly, 22/23 genes have been previously associated with vascular or neuronal traits/diseases, nine encoded for transcriptional factors/regulators and six (CAMK2G, GRIN2A, GRIN2B, N1RD1, PER3, PPARGC1A) for circadian entrainment/rhythm components. Among the WES low-frequency (MAF ≤ 0.04) SNPs (n = 7) filtered in the 16 genes, the NR1D1 rs17616365 showed significantly different MAF in the Network for Italian Genomes affected cohort than in the 1000 Genome Project Tuscany samples. This pattern was also detected in five nonintronic variants (GRIN2B rs1805482, PER3 rs2640909, PPARGC1A rs2970847, rs8192678, and rs3755863) in genes coding for functional partners. Overall, the study proposes specific markers and low-frequency variants that might help (i) to understand perturbed biological processes in vascular tissues contributing to MS disease, and (ii) to characterize MS susceptibility genes for functional association with disease-pathways.
- Published
- 2022
35. Pro-tumor and prothrombotic activities of hepsin in colorectal cancer cells and suppression by venetoclax
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Maria Carmen Rodenas, Julia Peñas-Martínez, Irene Pardo-Sánchez, David Zaragoza-Huesca, Carmen Ortega-Sabater, Jorge Peña-García, Salvador Espín, Guillermo Ricote, Sofía Montenegro, Francisco Ayala de la Peña, Ginés Luengo-Gil, Andrés Nieto, Francisco García-Molina, Vicente Vicente, Francesco Bernardi, Maria Luisa Lozano, Victoriano Mulero, Horacio Pérez-Sánchez, Alberto Carmona-Bayonas, and Irene Martínez-Martínez
- Abstract
Hepsin is a type II transmembrane serine protease whose expression has been linked to greater tumorigenicity and worse prognosis in different tumors such as prostate and gastric cancer. Recently, our group described hepsin expression in the primary biopsy as a potential biomarker of thrombosis and metastasis in localized colorectal cancer patients. Here we explored the role of hepsin in this tumor. Hepsin overexpression increased Caco-2 cell migration and invasion, higher phosphorylation of Erk1/2 and STAT3 and led to more thrombin generation in plasma. Indeed, our study revealed higher plasma levels of hepsin in metastatic colorectal cancer patients, which was associated with a greater tendency toward thrombosis. By virtual screening of a FDA-approved drug library, we identified venetoclax as a potent hepsin inhibitor, reducing the metastatic and prothrombotic phenotype of Caco-2 cells, but not of other colorectal cancer cells without hepsin expression. Interestingly, pre-treating Caco-2 cells overexpressing hepsin with venetoclax reduced its in vivo invasiveness. Taken together, our results demonstrate that elevated hepsin levels correlate with a more aggressive and prothrombotic tumor phenotype. Likewise, they evidence an antitumor role for venetoclax as a hepsin inhibitor. This lays the groundwork for molecular targeted therapy for colorectal cancer.
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- 2022
36. A small-hydro plant model for feasibility analysis of electrification projects in Rural Tanzania.
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Stefano Mandelli, Emanuela Colombo, Andrea Redondi, Francesco Bernardi, Bonaventure B. Saanane, Prosper Mgaya, and Johnstone Malisa
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- 2013
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37. Fusion of engineered albumin with factor IX Padua extends half‐life and improves coagulant activity
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Mattia Ferrarese, Francesco Bernardi, Mirko Pinotti, Alessio Branchini, Jeannette Nilsen, Kristin H Aaen, Torleif Tollefsrud Gjølberg, Silvia Lombardi, Jan Terje Andersen, Lombardi, S, Aaen, K, Nilsen, J, Ferrarese, M, Gjølberg, T, Bernardi, F, Pinotti, M, Andersen, J, and Branchini, A
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Male ,Recombinant Fusion Proteins ,Socio-culturale ,Mice, Transgenic ,Serum Albumin, Human ,Pharmacology ,Hemophilia B ,factor IX Padua ,Factor IX ,03 medical and health sciences ,0302 clinical medicine ,Neonatal Fc receptor ,LS1_5 ,medicine ,LS9_1 ,Animals ,Humans ,Haemophilia B ,Blood Coagulation ,Chemistry ,Platelets, Haemostasis and Thrombosis ,Albumin ,albumin fusion protein ,FcRn receptor ,protein engineering ,Hematology ,Protein engineering ,medicine.disease ,Coagulation Factor IX ,Human serum albumin ,albumin fusion proteins ,human serum albumin ,body regions ,Dissociation constant ,030220 oncology & carcinogenesis ,embryonic structures ,Female ,Specific activity ,Research Paper ,Half-Life ,030215 immunology ,medicine.drug - Abstract
Summary The short half‐life of coagulation factor IX (FIX) for haemophilia B (HB) therapy has been prolonged through fusion with human serum albumin (HSA), which drives the neonatal Fc receptor (FcRn)‐mediated recycling of the chimera. However, patients would greatly benefit from further FIX‐HSA half‐life extension. In the present study, we designed a FIX‐HSA variant through the engineering of both fusion partners. First, we developed a novel cleavable linker combining the two FIX activation sites, which resulted in improved HSA release. Second, insertion of the FIX R338L (Padua) substitution conferred hyperactive features (sevenfold higher specific activity) as for FIX Padua alone. Furthermore, we exploited an engineered HSA (QMP), which conferred enhanced human (h)FcRn binding [dissociation constant (KD) 0·5 nM] over wild‐type FIX‐HSA (KD 164·4 nM). In hFcRn transgenic mice, Padua‐QMP displayed a significantly prolonged half‐life (2·7 days, P
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- 2021
38. Translational readthrough at
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Maria Francesca, Testa, Silvia, Lombardi, Francesco, Bernardi, Mattia, Ferrarese, Donata, Belvini, Paolo, Radossi, Giancarlo, Castaman, Mirko, Pinotti, and Alessio, Branchini
- Abstract
In hemophilia A (HA), F8 nonsense variants, and particularly those affecting the large factor VIII (FVIII) B domain that is dispensable for coagulant activity, display lower association with replacement therapy-related anti-FVIII inhibitory antibodies as retrieved from multiple international databases. Since null genetic conditions favour inhibitor development, we hypothesized that translational readthrough over premature termination codons (PTCs) may contribute to immune tolerance by producing full-length (FL) proteins through the insertion of amino acid subset(s). To quantitatively evaluate in vitro the readthrough output, we developed a very sensitive luciferase-based system to detect very low FL-FVIII synthesis from a wide panel (n=45; ~60% patients with PTCs) of F8 nonsense variants. PTCs not associated with inhibitor displayed higher readthrough-driven expression levels than inhibitor-associated PTCs, a novel observation. Particularly, higher levels were detected for B-domain variants (n=20) than for variants in other domains (n=25). Studies on plasma from six HA patients with PTCs, integrated by expression of the corresponding nonsense and readthrough-deriving missense variants, consistently revealed higher FVIII levels for B-domain variants. Only one B-domain PTC (Arg814*) was found among the highly represented PTCs not sporadically associated with inhibitors, but with the lowest proportion of inhibitor cases (four out of 57). These original findings into HA molecular genetics, and particularly into genotype-phenotype relationships related with disease treatment, demonstrate that B-domain features favour PTC readthrough output. This provides a potential molecular mechanism contributing to differential PTC-associated inhibitor occurrence, with translational implications for a novel, experimentally based classification of F8 nonsense variants.
- Published
- 2022
39. Antiproliferative, antimicrobial and antioxidant properties of Cedrus libani and Pinus pinea wood oils and Juniperus excelsa berry oil
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Antoine M. Saab, Giulio Lupidi, Luca Agostino Vitali, Filippo Maggi, Massimo Bramucci, Armandodoriano Bianco, Luana Quassinti, Dezemona Petrelli, A.G. El Samrani, Francesco Bernardi, Monica Borgatti, Jihad Abboud, Roberto Gambari, M. J. Saab, and Alessandro Venditti
- Subjects
Antioxidant ,010405 organic chemistry ,medicine.medical_treatment ,macromolecular substances ,Plant Science ,Berry ,Biology ,Cedrus libani ,biology.organism_classification ,Antimicrobial ,01 natural sciences ,0104 chemical sciences ,%22">Pinus ,010404 medicinal & biomolecular chemistry ,Botany ,medicine ,Juniperus excelsa ,Ecology, Evolution, Behavior and Systematics - Abstract
In this paper we report the analysis of essential oils from the woods of C. libani and P. pinea and from the berries of J. excelsa. Several differences were observed with respect to previous studie...
- Published
- 2021
40. OH-Defects in Detrital Quartz Grains from the Julian Basin (NE Italy and Slovenia): A Fourier Transform Infrared Study
- Author
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Davide Lenaz, Henrik Skogby, FRANCESCO BERNARDI, Bernardi, Francesco, Skogby, Henrik, and Lenaz, Davide
- Subjects
Italy ,detrital quartz ,Slovenia ,OH-defects ,Julian Basin ,General Earth and Planetary Sciences ,OH-defect - Abstract
In this study, we analyzed up to 80 detrital quartz grains from four lithic greywackes along the stratigraphic column of the Julian Basin, a synorogenic basin in the southeastern Alps between Italy and Slovenia. Fourier transform infrared spectroscopy of detrital quartz was used to investigate the sample set with interest to its OH-defect speciation and content of each associated substitution. According to several recent studies, OH-defects in quartz are correlated to petrogenetic conditions of the source material and can be used as a provenance tool. The aim of this study is to compare results based on this method with previous studies that used other methods, to better constrain the palaeogeographical reconstruction of sedimentary fluxes. Detrital quartz within the samples of the basin shows different patterns of OH-defects and water content, indicating substantial petrogenetic differences between the sediment source rocks. For the oldest analyzed sample (ca. 66 Ma), the distribution of OH-defects suggests a mixed source between igneous and non-igneous rocks, with a predominance of metamorphic material supply. Another sample (56 Ma) reveals a great variability of OH-defects and water content, indicating that the magmatic component dominates over the metamorphic component. The distribution of OH-defects in the samples at the top of the sequence (52–53 Ma) suggests an almost solely metamorphic source. These results are in line with previous studies based on heavy minerals and geochemistry.
- Published
- 2022
41. The p.P1127S pathogenic variant lowers von Willebrand factor levels through higher affinity for the macrophagic scavenger receptor LRP1: Clinical phenotype and pathogenic mechanisms
- Author
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Monica Sacco, Stefano Lancellotti, Alessio Branchini, Maira Tardugno, Maria Francesca Testa, Barbara Lunghi, Francesco Bernardi, Mirko Pinotti, Betti Giusti, Giancarlo Castaman, and Raimondo De Cristofaro
- Subjects
Factor VIII ,asialoglycoprotein receptor 2 ,scavenger receptors ,Settore MED/09 - MEDICINA INTERNA ,Hematology ,von Willebrand factor ,Young Adult ,von Willebrand Diseases ,HEK293 Cells ,Phenotype ,Platelet Glycoprotein GPIb-IX Complex ,protein conformation ,lipoprotein receptor-related protein 1 ,Humans ,Female ,von Willebrand disease ,Low Density Lipoprotein Receptor-Related Protein-1 - Abstract
The index case is a 21-year-old Italian woman with a mild hemorrhagic syndrome and von Willebrand factor antigen (VWF:Ag) = 34.3 U/dl, VWF recombinant glycoprotein Ib (VWF:GpIbR) = 32.8 U/dl, and factor VIII (FVIII) = 55.3 IU/dl.The aim of this study is to characterize from a genetic and biochemical standpoint this low VWF phenotype.Coagulation and biochemical methods were used to study the structural and functional pattern of VWF multimers in the index case's plasma. Recombinant wild-type and p.P1127S VWF variants were produced using human embryonic kidney (HEK)-293 cells. In addition, genetic screening was carried out to detect single nucleotide variants of some scavenger VWF/FVIII receptor genes such as CLEC4M, STAB2, and ASGR2.Genetic investigation revealed that the index case inherited from her mother the heterozygous missense mutation c.3379C T (VWF exon 25), causing the p.P1127S substitution in the VWF D'D3 domain. The index case was also homozygous for the scavenger receptor ASGR2 c.-95 CC-genotype. Desmopressin normalized the VWF level of the patient, although its clearance was faster (tThe p.P1127S variant may cause a low VWF phenotype, stemming from an increased VWF affinity for the scavenger receptor LRP1 and, consequently, an accelerated clearance of VWF.
- Published
- 2022
42. Aptamer-modified FXa generation assays to investigate hypercoagulability in plasma from patients with ischemic heart disease
- Author
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Giovanna Marchetti, Annalisa Castagna, Barry Woodhams, Marcello Baroni, Francesco Bernardi, Oliviero Olivieri, Nicola Martinelli, Barbara Lunghi, Mirko Pinotti, and Filippo Stefanoni
- Subjects
medicine.medical_specialty ,Aptamer ,Socio-culturale ,Coronary Artery Disease ,Factor VIIa ,Disease ,030204 cardiovascular system & hematology ,Aptamers ,Thromboplastin ,Coronary artery disease ,03 medical and health sciences ,Economica ,0302 clinical medicine ,Thrombin ,Tissue factor pathway inhibitor ,Internal medicine ,medicine ,Humans ,Thrombophilia ,business.industry ,Long-term potentiation ,Hematology ,medicine.disease ,Factor VIIa-Antithrombin complex ,Endocrinology ,Quartile ,Diagnostic method ,Hypercoagulopathy ,030220 oncology & carcinogenesis ,Factor Xa ,Ischemic heart ,business ,medicine.drug - Abstract
Background High plasma levels of activated Factor VII-Antithrombin complex (FVIIa-AT) have been associated with an increased risk of cardiovascular mortality in patients with stable coronary artery disease (CAD). Objectives To investigate if FVIIa-AT levels are associated with activated factor X generation (FXaG) in modified assays. Patients/methods Forty CAD patients were characterized for FVIIa-AT levels by ELISA and for FXaG in plasma. Novel fluorogenic FXaG assays, based on aptamers inhibiting thrombin and/or tissue factor pathway inhibitor (TFPI), were set up. Results FXaG correlated with FVIIa-AT levels (RAUC = 0.393, P = 0.012). The combination of thrombin inhibition and FXaG potentiation by using anti-thrombin and anti-TFPI aptamers, respectively, favors the study of time parameters. The progressive decrease in lag time from the lowest to the highest FVIIa-AT quartile was magnified by combining TFPI and thrombin inhibitory aptamers, thus supporting increased FXaG activity in the coagulation initiation phase. By exploring FXaG rates across FVIIa-AT quartiles, the largest relative differences were detectable at the early times (the highest versus the lowest quartile; 5.0-fold, P = 0.005 at 45 s; 3.5-fold, P = 0.001 at 55 s), and progressively decreased over time (2.3-fold, P = 0.002 at 75 s; 1.8-fold, P = 0.008 at 95 s; 1.6-fold, P = 0.022 at 115 s). Association between high FVIIa-AT levels and increased FXaG was independent of F7 −323 A1/A2 polymorphism influencing FVIIa-AT levels. Conclusions High FVIIa-AT plasma levels were associated with increased FXaG. Hypercoagulability features were specifically detectable in the coagulation initiation phase, which may have implications for cardiovascular risk prediction by either FVIIa-AT complex measurement or modified FXaG assays.
- Published
- 2020
43. Translational readthrough ofGLAnonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants
- Author
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Francesco Bernardi, Mattia Ferrarese, Mirko Pinotti, Saverio Marchi, Silvia Lombardi, Paolo Pinton, Alessio Branchini, Lombardi, S, Ferrarese, M, Marchi, S, Pinton, P, Pinotti, M, Bernardi, F, and Branchini, A
- Subjects
Nonsense mutations ,fabry disease ,lysosomal disorders ,translational readthrough ,Nonsense mutation ,Socio-culturale ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Missense mutation ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,lysosomal disorder ,Translational readthrough ,Wild type ,RNA ,Cell Biology ,Molecular biology ,Phenotype ,Amino acid ,Enzyme ,chemistry ,030220 oncology & carcinogenesis - Abstract
Nonsense mutations are relatively frequent in the rare X-linked lysosomal α-galactosidase A (α-Gal) deficiency (Fabry disease; FD), but have been poorly investigated. Here, we evaluated the responsiveness of a wide panel (n = 14) of GLA premature termination codons (PTCs) to the RNA-based approach of drug-induced readthrough through expression of recombinant α-Gal (rGal) nonsense and missense variants. We identified four high-responders to the readthrough-inducing aminoglycoside G418 in terms of full-length protein (C56X/W209X, ≥10% of wild-type rGal) and/or activity (Q119X/W209X/Q321X, ~5-7%), resulting in normal (Q119X/Q321X) or reduced (C56X, 0.27 ± 0.11; W209X, 0.35 ± 0.1) specific activity. To provide mechanistic insights we investigated the predicted amino acid substitutions mediated by readthrough (W209C/R, C56W/R), which resulted in correct lysosomal localization and appreciable protein/activity levels for the W209C/R variants. Differently, the C56W/R variants, albeit appreciably produced and localized into lysosomes, were inactive, thus indicating detrimental effects of substitutions at this position. Noticeably, when co-expressed with the functional W209C or W209R variants, the wild-type rGal displayed a reduced specific activity (0.5 ± 0.2 and 0.6 ± 0.2, respectively) that, considering the dimeric features of the α-Gal enzyme, suggested dominant-negative effects of missense variants through their interaction with the wild-type. Overall, we provide a novel mechanism through which amino acids inserted during readthrough might impact on the functional protein output. Our findings may also have implications for the interpretation of pathological phenotypes in heterozygous FD females, and for other human disorders involving dimeric or oligomeric proteins.
- Published
- 2019
44. Combination of
- Author
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Barbara, Lunghi, Massimo, Morfini, Nicola, Martinelli, Silvia, Linari, Giancarlo, Castaman, and Francesco, Bernardi
- Abstract
The C-type lectin CLEC4M binds and internalizes factor VIII (FVIII). Common
- Published
- 2021
45. F9 missense mutations impairing factor IX activation are associated with pleiotropic plasma phenotypes
- Author
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Alessio Branchini, Massimo Morfini, Barbara Lunghi, Donata Belvini, Paolo Radossi, Loredana Bury, Maria Luisa Serino, Paola Giordano, Dorina Cultrera, Angelo Claudio Molinari, Mariasanta Napolitano, Elisabetta Bigagli, Giancarlo Castaman, Mirko Pinotti, Francesco Bernardi, Paola Agostini, Chiara Biasioli, Teresa Maria Caimi, Filomena Daniele, Alfredo Dragani, Donato Gemmati, Paolo Gresele, Silvia Linari, Gina Rossetti, Cristina Santoro, Rita Santoro, Gianluca Sottilotta, Johanna Svahn, Branchini, Alessio, Morfini, Massimo, Lunghi, Barbara, Belvini, Donata, Radossi, Paolo, Bury, Loredana, Serino, Maria Luisa, Giordano, Paola, Cultrera, Dorina, Molinari, Angelo Claudio, Napolitano, Mariasanta, Bigagli, Elisabetta, Castaman, Giancarlo, Pinotti, Mirko, and Bernardi, Francesco
- Subjects
medicine.medical_specialty ,pharmacogenetics ,Mutation, Missense ,Socio-culturale ,Alpha (ethology) ,aemophilia B ,recombinant proteins ,Hemophilia B ,law.invention ,Factor IX ,Antigen ,law ,Internal medicine ,Genotype ,medicine ,Missense mutation ,Humans ,Haemophilia B ,pharmacokinetic ,Beta (finance) ,Chemistry ,Hematology ,medicine.disease ,Endocrinology ,Phenotype ,factor IX activation ,hemophilia B ,pharmacokinetics ,Recombinant DNA ,Female ,Blood Coagulation Tests ,recombinant protein ,medicine.drug - Abstract
Background Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in hemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels. Objectives To investigate the (1) expression of a complete panel of missense mutations at FIX activation sites and (2) contribution of F9 genotypes on the FIX pharmacokinetics (PK). Methods We checked FIX activity and antigen and activity assays in plasma and after recombinant expression of FIX variants and performed an analysis of infused FIX PK parameters in patients (n = 30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42). Results The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants results in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. Pharmacokinetics analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 h, 95% confidence interval 44.3-114.5) in patients (n = 7) with the R191/R226 substitutions, which in regression analysis were independent predictors (β coefficient 0.699, P = .004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX. Conclusions FIX activity and antigen levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients' phenotypes and substitutive treatment.
- Published
- 2021
46. The Asialoglycoprotein Receptor Minor Subunit Gene Contributes to Pharmacokinetics of Factor VIII Concentrates in Hemophilia A
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Nicola Martinelli, Christian F. Cervellera, Alessio Branchini, Dario Balestra, Sabrina Frusconi, Francesco Bernardi, Massimo Morfini, Giovanna Marchetti, Barbara Lunghi, Silvia Linari, and Giancarlo Castaman
- Subjects
ASGR2 polymorphisms ,Alpha (ethology) ,Asialoglycoprotein Receptor ,Biology ,Hemophilia A ,Hemostatics ,law.invention ,NO ,Pharmacokinetics ,law ,hemic and lymphatic diseases ,White blood cell ,ABO blood group system ,von Willebrand Factor ,medicine ,Humans ,asialoglycoprotein receptor, ASGR2 polymorphisms, factor VIII, hemophilia A, pharmacokinetics ,Beta (finance) ,Factor VIII ,Haplotype ,Hematology ,Molecular biology ,medicine.anatomical_structure ,Recombinant DNA ,Asialoglycoprotein receptor ,5' Untranslated Regions ,pharmacokinetics - Abstract
Background The asialoglycoprotein receptor (ASGPR) binds with high affinity factor VIII (FVIII) through its N-linked oligosaccharides. However, its contribution to the wide inter-individual variation of infused FVIII pharmacokinetics (PK) in hemophilia A (HA) is unknown. Objective To investigate the variability in FVIII PK outcomes in relation to genetic variation in the ASGR2, encoding the ASGPR2 subunit. Methods Thirty-two HA patients with FVIII:C ≤2 IU/dL underwent 66 single-dose FVIII PK studies. PK parameters were evaluated in relation to ASGR2 5′ untranslated region (5′UTR) polymorphisms, which were investigated by recombinant and white blood cell reverse transcription-polymerase chain reaction approaches. Results The 5′UTR polymorphisms determine a frequent and conserved haplotype (HT1) in a regulatory region. The HT1 homozygotes may differ in the amounts of alternatively spliced mRNA transcripts and thus ASGPR2 isoforms. Compared with the other ASGR2 genotypes, the c.-95TT homozygotes (n = 9), showed threefold longer Alpha HL (3.60 hours, 95% confidence interval: 1.44–5.76, p = 0.006), and the c.-95TC heterozygotes (n = 17) showed 25% shorter mean residence time (MRT; 18.5 hours, 15.0–22.0, p = 0.038) and 32% shorter Beta HL (13.5 hours, 10.9–16.0, p = 0.016). These differences were confirmed in patients (n = 27) undergoing PK studies (n = 54) with full-length FVIII only. In different linear regression models, the contribution of the ASGR2 genotypes remained significant after adjustment by ABO genotypes and von Willebrand factor (VWF) antigen levels, and explained 14% (MRT), 15 to 18% (Beta HL), and 22% (Alpha HL) of parameter variability. Conclusion Infused FVIII distribution was modulated by frequent ASGR2 genotypes, independently from and together with ABO and VWF antigen levels, which has potential implications for genetically tailored substitutive treatment in HA.
- Published
- 2021
47. Tailoring the CRISPR system to transactivate coagulation gene promoters in normal and mutated contexts
- Author
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Silvia Pignani, Alessio Branchini, Federico Zappaterra, Francesco Bernardi, Matteo Bovolenta, Antonia Follenzi, Elena Barbon, Mirko Pinotti, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, and École pratique des hautes études (EPHE)
- Subjects
Transcriptional Activation ,0301 basic medicine ,Carcinoma, Hepatocellular ,[SDV]Life Sciences [q-bio] ,TALE-TF ,Biophysics ,Gene Expression ,Socio-culturale ,030204 cardiovascular system & hematology ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,Transactivation ,0302 clinical medicine ,Genes, Reporter ,Structural Biology ,Gene expression ,Biomarkers, Tumor ,Genetics ,medicine ,Humans ,Receptors, Immunologic ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Transcription factor ,Mutation ,Reporter gene ,Binding Sites ,Factor VIII ,CRISPR activation ,Engineered transcription factors ,Promoter mutations ,Chemistry ,Effector ,Endothelial Cells ,Promoter ,Hep G2 Cells ,Factor VII ,Cell biology ,030104 developmental biology ,Hepatocyte Nuclear Factor 4 ,CRISPR-Cas Systems ,RNA, Guide, Kinetoplastida ,Transcription Factors - Abstract
International audience; Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNAF7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNAF8.1, ~8-fold and 3-fold; sgRNAF8.2, ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models.
- Published
- 2019
48. GENETIC DETERMINANTS OF ACTIVATED FACTOR VII-ANTITHROMBIN COMPLEX (FVIIA-AT) PLASMA LEVELS AND MORTALITY IN PATIENTS WITH ANGIOGRAPHICALLY DEMONSTRATED CORONARY ARTERY DISEASE
- Author
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Francesca Pizzolo, Mariangela Veneri, Filippo Sartori, Nicola Osti, Martino Donini, Sara Moruzzi, Filippo Mazzi, Annalisa Castagna, Silvia Udali, Patrizia Pattini, Barbara Lunghi, Marcello Baroni, Francesco Bernardi, Oliviero Olivieri, Simonetta Friso, and Nicola Martinelli
- Subjects
Physiology ,Internal Medicine ,Cardiology and Cardiovascular Medicine - Published
- 2022
49. Performance prediction models based on anthropometric, genetic and psychological traits of Croatian sprinters
- Author
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Francesco Bernardi, Saša Missoni, Davide Barbieri, Emanuela Gualdi-Russo, Tena Šarić, Jelena Šarac, Barbara Lunghi, Natascia Rinaldo, Vesna Babić, Luciana Zaccagni, and Marija Rakovac
- Subjects
Socio-culturale ,Physical Therapy, Sports Therapy and Rehabilitation ,03 medical and health sciences ,0302 clinical medicine ,competitive anxiety ,genetic polymorphisms ,Sprint performance, Personal best, Anthropometry, Genetic polymorphisms, Competitive anxiety ,Physiology (medical) ,Dash ,medicine ,sprint performance ,Orthopedics and Sports Medicine ,Elite athletes ,030212 general & internal medicine ,lcsh:Sports medicine ,lcsh:QH301-705.5 ,Croatian ,Original Paper ,anthropometry ,Leg length ,Sprint performance ,Personal best ,Anthropometry ,Genetic polymorphisms ,Competitive anxiety ,030229 sport sciences ,language.human_language ,lcsh:Biology (General) ,language ,Anxiety ,personal best ,Multiple linear regression analysis ,medicine.symptom ,Psychology ,lcsh:RC1200-1245 ,Demography - Abstract
Elite athletes differ from each other in their characteristics according to their discipline. This study aimed to identify performance predictors in elite Croatian sprinters taking into consideration their anthropometric, psychological and genetic characteristics. One hundred and four elite Croatian sprinters (68 males and 36 females) participated in this study. Of them, 38 are currently competing in the 100-metre dash. The others are former sprinters. The participants underwent direct anthropometric assessment. Participants were also tested by means of the Competitive State Anxiety Inventory-2 and for ACE and ACTN3 polymorphisms. Multiple linear regression analysis was applied to identify the best model for performance prediction. Different models were developed for males and females. Anthropometric traits accounted for 44% of the variance in performance for males, 62% for females. Once other traits (psychological for females) were entered into the model, no additional contribution to the variance was observed. The most significant predictors of higher running velocity were bicristal diameter and foot dimensions in males, and leg length and clean one-repetition maximum in females. The findings suggest that performance in sprinters is associated with anthropometric characteristics, with biomechanical implications that may be used to provide a more complete evaluation of sprinters' performance.
- Published
- 2018
50. Basophil Blood Cell Count Is Associated With Enhanced Factor II Plasma Coagulant Activity and Increased Risk of Mortality in Patients With Stable Coronary Artery Disease: Not Only Neutrophils as Prognostic Marker in Ischemic Heart Disease
- Author
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Filippo Stefanoni, Giuseppe Faggian, Francesca Pizzolo, Veronica Munerotto, Annalisa Castagna, Domenico Girelli, Oliviero Olivieri, Giovanni Battista Luciani, Simonetta Friso, Francesco Bernardi, Silvia Udali, Vera Cetera, Nicola Martinelli, and Marcello Baroni
- Subjects
Male ,Neutrophils ,Myocardial Ischemia ,Disease ,Coronary Artery Disease ,030204 cardiovascular system & hematology ,Basophil ,factor II plasma coagulant activity ,Coronary artery disease ,Blood cell ,Leukocyte Count ,0302 clinical medicine ,Economica ,Risk Factors ,Cardiovascular Disease ,Secondary Prevention ,Coronary Heart Disease ,basophils ,neutrophils ,secondary prevention of coronary artery disease ,white blood cell count ,Original Research ,0303 health sciences ,Middle Aged ,Prognosis ,Basophils ,Survival Rate ,medicine.anatomical_structure ,Italy ,Cardiology ,Female ,Prothrombin ,Cardiology and Cardiovascular Medicine ,medicine.medical_specialty ,Socio-culturale ,Risk Assessment ,03 medical and health sciences ,White blood cell ,Internal medicine ,medicine ,Humans ,In patient ,030304 developmental biology ,Retrospective Studies ,business.industry ,Chronic Ischemic Heart Disease ,medicine.disease ,Increased risk ,business ,Ischemic heart ,Biomarkers ,Follow-Up Studies - Abstract
Background White blood cell count, which is inexpensive and widely available in clinical practice, has been proposed to provide prognostic information in coronary artery disease (CAD). Elevated levels of white blood cell subtypes may play different roles in atherothrombosis and predict cardiovascular outcomes. Methods and Results The association between white blood cell counts and mortality was evaluated in 823 subjects with angiographically demonstrated and clinically stable CAD in an observational–longitudinal study. The correlation among white blood cell counts and factor II plasma coagulant activity was analyzed in 750 subjects (554 CAD and 196 CAD‐free) not taking anticoagulant drugs. Subjects with overt leukocytosis or leukopenia were excluded. In the longitudinal study after a median follow‐up of 61 months, 160 (19.4%) subjects died, 107 (13.0%) of whom from cardiovascular causes. High levels of neutrophils, monocytes, eosinophils, and basophils were associated with an increased mortality rate. In multiadjusted Cox regression models, only neutrophils and basophils remained predictors of total and cardiovascular mortality. The associations remained significant after adjustment for traditional cardiovascular risk factors and by including D‐dimer and the chemokine CXCL12 in the regression models. Neutrophils and basophils were also significant predictors of factor II plasma coagulant activity variability after adjustment for blood cell counts, age, sex, inflammatory markers, CAD diagnosis, and prothrombin G20210A polymorphism. Factor II plasma coagulant activity was similarly increased in subjects with high neutrophil and basophil counts and in carriers of the prothrombin 20210A allele. Conclusions Both high neutrophil and basophil blood counts may predict mortality in patients with clinically stable CAD and are associated with enhanced factor II plasma coagulant activity, thereby suggesting underlying prothrombotic mechanisms.
- Published
- 2021
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