Comparative Analysis Of Residual Factor VIII Expression from Recurrent F8 Nonsense Mutations Indicates that Localization in the B- domain Favours Readthrough- mediated Protein Output

Background: Nonsense mutations, inserting premature termination codons (PTCs), might undergo, with low frequency (<0.01%), spontaneous suppression (readthrough) with production of full-length proteins upon amino acid insertion at the PTC. This process, dictated by nucleotide/protein sequence features, might have implications for hemophilia A (HA) patients. Aims: To investigate residual factor VIII (FVIII) expression through complementary studies in HA patients’ plasma and exploiting a sensitive in-vitro expression platform. Methods: Detection of plasma FVIII levels (ELISA, aPTT), and expression studies (HEK293 cells) with a highly-sensitive naturally-secreted luciferase (Gaussia, GL) fused to FVIII (FVIII-GL). Results: Plasma samples from HA patients affected by six nonsense mutations (p.R446X, p.R814X, p.K1289X, p.W1726X, p.R1985X, p.R2135X) revealed traces of FVIII. Strikingly, the two B-domain variants (p.R814X, p.K1289X) showed the highest FVIII levels, suggesting a position-dependent effect. Expression studies with the FVIII-GL variants showed that those of the B-domain produced the highest luciferase activity levels, thus supporting in vivo findings. Accordingly, the predicted readthrough-deriving amino acid changes (R446W, R814W, K1289Q/Y, W1726Y, R1985W, R2135W) showed a minor impact for those affecting the B-domain. To verify further our hypothesis, the panel of F8 mutations was rationally expanded to be representative of the majority of patients with nonsense mutations (60%), including the most frequent (50% of patients) in the B-domain. Through our sensitive platform we observed that all F8 nonsense variants led to detectable luciferase activity (0.4-6%). Strikingly, when categorized in two groups (B-domain, n=21; other domains, n=26), secreted luciferase activity of B-domain variants was significantly higher (p<0.0001) as compared with variants located in the other FVIII domains. Conclusions: Our findings for the first time indicate that nonsense mutations in the B-domain, known to tolerate missense changes as those potentially arising from readthrough, are favoured in terms of readthrough-mediated protein output, which might have pathophysiological implications for HA patients

Association analysis of 10 candidate genes causing Mendelian calcium nephrolithiasis in the INCIPE study: a South European general population cohort

Background: Idiopathic calcium nephrolithiasis (ICN) is a common condition with a complex phenotype influenced by both environmental and genetic factors. In our study we investigated the association of allelic variants with the history of nephrolithiasis. Methods: We genotyped and selected 10 candidate genes potentially related to ICN from 3046 subjects participating in the INCIPE survey cohort (Initiative on Nephropathy, of relevance to public health, which is Chronic, possibly in its Initial stages, and carries a Potential risk of major clinical End-points), a study enrolling subjects from the general population in the Veneto region in Italy. Results: Overall, 66 224 variants mapping on the 10 candidate genes were studied. A total of 69 and 18 variants in INCIPE-1 and INCIPE-2, respectively, were significantly associated with stone history (SH). Only two variants, rs36106327 (chr20:54 171 755, intron variant) and rs35792925 (chr20:54 173 157, intron variant) of the CYP24A1 gene were observed to be consistently associated with ICN. Neither variant has been previously reported in association with renal stones or other conditions. Carriers of CYP24A1 variants showed a significant increase in the ratio of 1,25 (OH)2 vitamin D to 25 (OH) vitamin D compared with controls (P = .043). Although not associated with ICN in this study, the rs4811494 CYP24A1 variant that was reported to be causative of nephrolithiasis was very prevalent in heterozygosity (20%). Conclusion: Our data suggest a possible role for CYP24A1 variants in the risk of nephrolithiasis. Genetic validation studies in larger sample sets will be necessary to confirm our findings

Prevalence of portal vein thrombosis in non-alcoholic fatty liver disease: a meta-analysis of observational studies

Portal vein thrombosis (PVT) is a common complication of cirrhosis as a result of portal hypertension and modification in the hemostatic balance. Accumulating evidence now suggests that patients with non-alcoholic fatty liver disease (NAFLD), especially those with advanced forms, have an increased risk of PVT. Hence, we performed a meta-analysis of observational studies to estimate the overall prevalence of PVT in patients with NAFLD and its advanced forms compared with patients with advanced liver diseases from other etiologies. We systematically searched PubMed, Scopus and Web of Science databases from the inception date to December 30th 2022, using predefined keywords, to identify observational studies. Meta-analysis was performed using random-effects modeling. We included five observational studies for a total of 225,571 patients. Of these, 26,840 (11.9%) patients had NAFLD, whereas the PVT prevalence was 8.5% (n = 2,280). When compared with patients with advanced liver diseases from other etiologies, patients with NAFLD and its advanced forms had a higher risk of prevalent PVT (OR 1.34, 100% CI 1.07-1.67 p < 0,01). The between-study heterogeneity was substantial (I-2 = 88%). This meta-analysis suggests that compared with patients with advanced liver diseases from other etiologies, patient with NAFLD and its advanced forms had a higher risk of prevalent PVT. Further research is required to understand the complex link between NAFLD/NASH and PVT development

Glomerular Hyperfiltration: A Marker of Fibrosis Severity in Metabolic Associated Steatotic Liver Disease in an Adult Population

Glomerular hyperfiltration (GH) is an increase in the glomerular filtration rate, possibly progressing to chronic kidney disease (CKD). Metabolic-associated steatotic liver disease (MASLD) is linked to an increased risk of CKD, especially if fibrosis is present; however, the association between GH and MASLD has not been explored. To evaluate GH prevalence in MASLD and its possible correlation with liver fibrosis. 772 consecutive patients with ultrasound MASLD (mean age 47.3 ± 8.9 years, 67.1% males) were enrolled. GH was defined as estimated glomerular filtration rate (eGFR) greater than the upper quartile of values in the cohort. Liver stiffness measurement (LSM) by FibroScan ≥ 7.2 kPa suggested liver fibrosis. GH was present in 20% of patients, liver fibrosis in 30%. In total, 53.4% of the cohort was obese, 40.9% hypertensive, 36.3% diabetic and 70.8% dyslipidaemic. GH patients compared to non-GH were significantly younger (38.4 ± 8.3 vs. 49.5 ± 7.7, p < 0.001), with higher prevalence of LSM > 7.2 kPa (35.5% vs. 29%, p < 0.001), without any difference in metabolic comorbidities. In multivariate analysis, age (OR 0.85, CI 95% 0.82–0.87) and significant fibrosis (OR 1.83; CI 95%1.10–3.03) remained independently associated with GH, regardless of the presence of metabolic alterations and nephrotoxic drugs. GH, an early marker of renal damage, is highly prevalent in MASLD and is associated with hepatic fibrosis. GH may be considered an early marker of both liver and renal disease and its recognition could prompt the management of risk factors aimed at preventing the progression of both hepatic and renal disease

SARS-CoV-2 pandemic. Implications in the management of patients with colorectal cancer

The SARS-CoV-2 pandemic has already reached 3,207,248 patients with more than 225,000 deaths all over the world. Colorectal cancer is the third most diagnosed cancer worldwide, and the healthcare system is struggling to manage daily activities for elective cancer surgery. This review integrates clinical, microbiological, architectural and surgical aspects to develop indications on strategies to manage colorectal cancer patients and ensure safety during the pandemic. Telephone or virtual clinics must be encouraged and phone follow-up should be implemented. Indications for surgery must be rigorous, balancing the advantage of early surgical treatment and risks of treatment delay. To decrease the occupancy rate of intensive care unit beds, elective surgical treatment should be delayed until local endemic control, according to stage of disease. Patients with SARS-CoV-2 infection should be treated only after clinical recovery, two consecutive negative oropharyngeal swabs and, if available, a negative stool sample. Before any elective oncologic procedure, a multidisciplinary oncologic team including an anaesthesiologist and an infectious disease specialist must assess every patient to evaluate the risk of infection and its impact on perioperative morbidity, mortality and oncologic prognosis. The hospital should organise to manage all elective oncologic patients in an "infection-free" area or refer them to a non-SARS-CoV-2 hospital

The large area detector onboard the eXTP mission

The Large Area Detector (LAD) is the high-throughput, spectral-timing instrument onboard the eXTP mission, a flagship mission of the Chinese Academy of Sciences and the China National Space Administration, with a large European participation coordinated by Italy and Spain. The eXTP mission is currently performing its phase B study, with a target launch at the end-2027. The eXTP scientific payload includes four instruments (SFA, PFA, LAD and WFM) offering unprecedented simultaneous wide-band X-ray timing and polarimetry sensitivity. The LAD instrument is based on the design originally proposed for the LOFT mission. It envisages a deployed 3.2 m2 effective area in the 2-30 keV energy range, achieved through the technology of the large-area Silicon Drift Detectors - offering a spectral resolution of up to 200 eV FWHM at 6 keV - and of capillary plate collimators - limiting the field of view to about 1 degree. In this paper we will provide an overview of the LAD instrument design, its current status of development and anticipated performance

Integration of the environmental management aspect in the optimization of the design and planning of energy systems

The increasing concerns regarding the environmental pollution derived from anthropogenic activities, such as the use of fossil fuels for power generation, has driven many interested parties to seek different alternatives, e.g. use of renewable energy sources, use of “cleaner” fuels and use of more effective technologies, in order to minimize and control the quantity of emissions that are produced during the life cycle of conventional energy sources. In addition to these alternatives, the use of an integrated procedure in which the environmental aspect will be taken into account during the design and planning of energy systems could provide a basis on which emissions reduction will be dealt with a life cycle approach. The work presented in this paper focuses on the examination of the possibilities of integrating the environmental aspects in the preliminary phase of the conventional design and planning of energy systems in conjunction with other parameters, such as financial cost, availability, capacity, location, etc. The integration of the environmental parameter to the design is carried out within a context where Eco-design concepts are applied. Due to the multi-parameter nature of the design procedure, the tools that are used are Life Cycle Analysis and Multi-criteria Analysis. The proposed optimization model examines and identifies optimum available options of the use of different energy sources and technologies for the production of electricity and/or heat by minimizing both the financial cost and the environmental impacts, with regard to a multiple objective optimization subject to a set of specific constraints. Implementation of the proposed model in the form of a case study for the island of Rhodes in Greece revealed that an optimized solution both cost and environmental-wise, would be an almost balanced participation of renewables and non-renewable energy sources in the energy mix

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Rescue of multiple Haemophilia A-causing mutations by a single ExSpeU1: the importance of the genomic context

Background: Therapies based on RNA splicing modulation are attracting interest for many disorders.. Variants of the spliceosomal U1snRNA have been successfully exploited to rescue defective exons in cellular and mouse models but no attempts have been done on Hemophilia A, the commonest coagulation disorder. Aims: To explore U1snRNA variants targeting intronic sequences downstreaom of the defective exon (exon-specific U1snRNA; ExSpeU1) to correct F8 exon 5 mutations leading to hemophilia A (HA). Methods: Expression of ExSpeU1s and F8 minigenes harboring the 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 mutations in HEK293T cells and evaluation of F8 mRNA splicing (RT-PCR). Results: Expression studies demonstrated that all mutations, both intronic and exonic, occurring within the 5’ splice site (5’ss) induced aberrant transcripts, with the usage of two cryptic intronic 5’ss at positions c.670+64 and c.670+176. Some changes were also associated to trace level of correct transcripts (~10%) and missense changes had no effect on splicing. In co-transfection experiments, we identified an ExSpeU1 (U1sh7), designed to minimize potential off-target effects, able to properly restore splicing. We showed in vitro that the ExSpeU1 is able to strengthen or restore (~80%) proper 5’ss usage for all splicing mutations, including changes at +1 and +2 positions of 5’ss, commonly considered not rescuable. However, deep investigation of rescued transcripts from +1 and +2 variants revealed the usage of adjacent subtle cryptic 5’ss, leading to frameshift. Conclusions: These data further support the therapeutic potential of the ExSpeU1 RNA, where a single therapeutic RNA can rescue multiple mutations. However, they suggest careful inspection of the genomic context and evaluation of transcripts to avoid over-interpretations

Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants

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