Seroprevalence Survey of Anti-SARS-CoV-2 Antibodies in a Population of Emilia-Romagna Region, Northern Italy

taly was the first Western European country to be severely hit by the COVID-19 pandemic. Variations in seroprevalence rates were reported according to geographical and temporal differences of previous surveys, as well as depending on demographic and occupational factors. In this cross-sectional study, we evaluated the prevalence of anti-SARS-CoV-2 antibodies in a population of the Emilia-Romagna region in Northern Italy after the first wave in the period from 26 September 2020–26 March 2021. We included 5128 subjects who voluntarily underwent serological tests to determine anti-SARS-CoV-2 antibody positivity, including both self-referred individuals (24.2%) and workers adhering to company screening programs (76.8%). Overall, seroprevalence was 11.3%, higher in self-referred (13.8%) than employed-referred (10.5%) individuals. A slightly higher seroprevalence emerged in women compared to men (12.3% and 10.7%), as well as in the extreme age categories (18.6% for 60–69 years, 18.0% for ≥70 years, and 17.1% for <20 years compared to 7.6% for 20–39 years). Healthcare professionals showed the highest prevalence of seropositivity (22.9%), followed by workers in direct contact with customers, such as the communication, finance, and tourism sectors (15.7%). Overall subgroups seroprevalence increased compared to the first wave data but the trends agreed between the first and subsequent waves, except for an increase in the younger age group and in the sector in direct contact with customers. Among the occupational categories, our study confirms that healthcare workers and workers in the sports sector were at high risk of exposure to SARS-CoV-2

Time course of risk factors associated with mortality of 1260 critically ill patients with COVID-19 admitted to 24 Italian intensive care units

Purpose: To evaluate the daily values and trends over time of relevant clinical, ventilatory and laboratory parameters during the intensive care unit (ICU) stay and their association with outcome in critically ill patients with coronavirus disease 19 (COVID-19). Methods: In this retrospective–prospective multicentric study, we enrolled COVID-19 patients admitted to Italian ICUs from February 22 to May 31, 2020. Clinical data were daily recorded. The time course of 18 clinical parameters was evaluated by a polynomial maximum likelihood multilevel linear regression model, while a full joint modeling was fit to study the association with ICU outcome. Results: 1260 consecutive critically ill patients with COVID-19 admitted in 24 ICUs were enrolled. 78% were male with a median age of 63 [55–69] years. At ICU admission, the median ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2) was 122 [89–175] mmHg. 79% of patients underwent invasive mechanical ventilation. The overall mortality was 34%. Both the daily values and trends of respiratory system compliance, PaO2/FiO2, driving pressure, arterial carbon dioxide partial pressure, creatinine, C-reactive protein, ferritin, neutrophil, neutrophil–lymphocyte ratio, and platelets were associated with survival, while for lactate, pH, bilirubin, lymphocyte, and urea only the daily values were associated with survival. The trends of PaO2/FiO2, respiratory system compliance, driving pressure, creatinine, ferritin, and C-reactive protein showed a higher association with survival compared to the daily values. Conclusion: Daily values or trends over time of parameters associated with acute organ dysfunction, acid–base derangement, coagulation impairment, or systemic inflammation were associated with patient survival

Lopinavir/Ritonavir and Darunavir/Cobicistat in Hospitalized COVID-19 Patients: Findings From the Multicenter Italian CORIST Study

Background: Protease inhibitors have been considered as possible therapeutic agents for COVID-19 patients. Objectives: To describe the association between lopinavir/ritonavir (LPV/r) or darunavir/cobicistat (DRV/c) use and in-hospital mortality in COVID-19 patients. Study Design: Multicenter observational study of COVID-19 patients admitted in 33 Italian hospitals. Medications, preexisting conditions, clinical measures, and outcomes were extracted from medical records. Patients were retrospectively divided in three groups, according to use of LPV/r, DRV/c or none of them. Primary outcome in a time-to event analysis was death. We used Cox proportional-hazards models with inverse probability of treatment weighting by multinomial propensity scores. Results: Out of 3,451 patients, 33.3% LPV/r and 13.9% received DRV/c. Patients receiving LPV/r or DRV/c were more likely younger, men, had higher C-reactive protein levels while less likely had hypertension, cardiovascular, pulmonary or kidney disease. After adjustment for propensity scores, LPV/r use was not associated with mortality (HR = 0.94, 95% CI 0.78 to 1.13), whereas treatment with DRV/c was associated with a higher death risk (HR = 1.89, 1.53 to 2.34, E-value = 2.43). This increased risk was more marked in women, in elderly, in patients with higher severity of COVID-19 and in patients receiving other COVID-19 drugs. Conclusions: In a large cohort of Italian patients hospitalized for COVID-19 in a real-life setting, the use of LPV/r treatment did not change death rate, while DRV/c was associated with increased mortality. Within the limits of an observational study, these data do not support the use of LPV/r or DRV/c in COVID-19 patients

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens

Background The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. Results Here, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. Conclusion We conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.Peer reviewe

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens

BackgroundThe Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function.ResultsHere, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory.ConclusionWe conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.</p

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

Claudin-5 Proteins of the Blood-Brain Barrier: Molecular Modeling and Simulations From Structures to Therapeutic Strategies

La barriera emato-encefalica (blood-brain barrier, BBB) è una struttura multicellulare, comprendente periciti, astrociti e cellule endoteliali, che riveste l’ambiente neuronale per mantenere l’omeostasi del Sistema Nervoso Centrale (Central Nervous System, CNS). Le cellule endoteliali formano le pareti dei capillari che si interfacciano con i tessuti neurali e fungono da linea di difesa primaria contro la diffusione passiva incontrollata di soluti dal flusso sanguigno al cervello. Mentre il passaggio di molecole per la via transcellulare è mediato dalla presenza di specifici trasportatori proteici transmembrana, la permeazione attraverso la ristretta regione che separa cellule adiacenti, chiamato lo spazio paracellulare, è limitata da eterogenei complessi di proteine chiamati Giunzioni Strette (Tight Junctions, TJs). I componenti maggioritari delle TJs nella BBB sono le proteine di claudina-5 (Cldn5), che si organizzano in sistemi multimerici per formare filamenti adesi alle membrane basolaterali di cellule contigue, dove prevengono la traslocazione di quasi ogni soluto attraverso la via paracellulare. Nonostante la loro nota rilevanza fisiologica, la precisa disposizione strutturale delle proteine di Cldn5 nei filamenti di TJs è tuttora poco chiara, principalmente a causa mancanza di strutture sperimentali tridimensionali. Ad oggi, due modelli strutturali, denotati come Poro I e Poro II, sono stati introdotti in letteratura come potenziali descrittori dei complessi multimerici di claudine per varie proteine appartenenti a questa famiglia. Entrambi questi modelli propongono una organizzazione tetramerica, nella quale due coppie di molecole appartenenti alle rispettive membrane cellulari interagiscono a livello paracellulare dando forma a una struttura porosa. Sebbene diversi lavori abbiano contribuito a raffinare questi due sistemi grazie ad una preponderante applicazione di metodi computazionali, rimane ancora carente un’esaustiva comparazione delle loro proprietà, così come una valutazione della loro trasferibilità tra diverse claudine omologhe, appartenenti ad altri tessuti. Oltre alla validazione strutturale, fare luce sugli aspetti molecolari della topologia e funzionali dei multimeri di Cldn5 ha implicazioni fondamentali anche in ambito clinico. Infatti, nonostante il ruolo centrale della BBB di garante dell’equilibrio chimico del CNS, la sua presenza rappresenta un notevole ostacolo in caso di patologie neurologiche, impedendo il passaggio di numerosi farmaci e nutrienti diretti al cervello. Un approccio promettente per affrontare questa problematica consiste nell’utilizzo di peptidi affini alla Cldn5. September 2022 – Participation to Ischia Summer Modeling 2022, in Ischia, Italy. The conference designed for theoretically oriented scientists and provided a platform to share ideas and knowledges at the cutting-edge of different fields of molecular sciences, including inter alia, astrochemistry, environmental chemistry, material, and life sciences. 5 Questi oligomeri sarebbero in grado di formare complessi con la proteina, interferendo con la formazione delle TJs, e generando fenestrazioni temporanee attraverso gli spazi paracellulari che potrebbero permettere ai farmaci di penetrare l’endotelio celebrale. In questa tesi, sia l’aspetto di validazione strutturale che di applicazione pratica sono affrontati sfruttando la capacità dei metodi di modeling molecolare di investigare le proprietà strutturali e termodinamiche dei sistemi biologici a livello atomico. In primo luogo, i modelli Poro I e Poro II sono stati assemblati con i monomeri di Cldn5 sia nella forma wild type che con diverse varianti mutate, ed esaminate tramite simulazioni di dinamica molecolare (Molecular Dynamics, MD) nel tempo scala del microsecondo per valutare le proprietà strutturali delle due configurazioni. Inoltre, simulazioni MD accelerate, realizzate con il metodo “umbrella sampling”, sono state effettuate per calcolare i profili di energia libera degli eventi di permeazione ionica attraverso i pori. I risultati di questa analisi suggeriscono che entrambe le configurazioni sono compatibili con la funzione fisiologica della Cldn5 nella BBB, ma solo Poro I è consistente con le evidenze sperimentali descritte per la variante patologica della proteina. Inoltre, in questa fase, la valutazione della trasferibilità di queste due architetture è stata estesa a un altro omologo, il sottotipo claudina-4 (Cldn4), per il quale i calcoli di energia libera hanno indicato una selettività opposta dei due sistemi al passaggio di anioni. Successivamente, nella seconda parte della trattazione, è presentato un protocollo computazionale per progettare e analizzare peptidi dalla lunghezza limitata (fino ad un massimo di 16 residui), solubili nel medium cellulare acquoso e in grado di legare efficientemente le proteine di Cldn5. L’uso di sequenze relativamente corte ha svariati vantaggi, inclusi la facilità di sintesi, una elevata stabilità metabolica, migliore biocompatibilità e un costo contenuto. In aggiunta, permette un più accurato impiego delle tecniche computazionali. La procedura illustrata in questo lavoro è basata su un’estensiva applicazione di metodi di metodi in silico, inclusi il modeling per omologia, simulazioni di docking molecolare e simulazioni MD standard e accelerate, che hanno permesso di identificare un peptide contraddistinto da una significativa affinità per la Cldn5, e indicato come f1-C5C2. Inoltre, questo risultato ha trovato conferma nella caratterizzazione sperimentale del peptide condotta dai collaboratori al Centro di Neuroscienze e Tecnologie Sinaptiche (NSYN) della Fondazione Istituto Italiano di Tecnologia (IIT) di Genova, sottolineando la riuscita integrazione degli approcci in silico e in vitro alla base di questa investigazione. Complessivamente, questa tesi offre una serie di risultati mirati a far progredire la comprensione delle proprietà molecolari dei complessi Cldn nelle TJs, proponendo analisi approfondite per guidare il design razionale di peptidi con un potenziale impatto benefico anche da una prospettiva clinica.The blood-brain barrier (BBB) is a multicellular structure comprising pericytes, astrocytes, and endothelial cells that protects the brain parenchyma and maintains its homeostasis. Endothelial cells form the walls of brain capillary vessels and serve as a primary defense layer against the uncontrolled passive diffusion of solutes from the bloodstream. While the selective transcellular passage of molecules is mediated by the presence of specific transmembrane transporters, the permeation through the narrow region separating adjacent cells, named the paracellular space, is strictly limited by heterogeneous protein complexes named tight junctions (TJs). The major components of TJs in the BBB are claudin-5 (Cldn5) proteins, that arrange into multimeric assemblies to form strands running along the lateral membranes of adjoining cells, where they prevent the translocation of almost each solute across the paracellular route. Despite the well-established physiological relevance of Cldn5 proteins, the precise structural arrangement of these systems within the TJ strands remains elusive, primarily due to the poor availability of experimental structures. To date, two main structural models, denoted as Pore I and Pore II, have been introduced in the literature as building blocks of Cldn paracellular multimeric complexes for various proteins belonging to this family. Interestingly, both these arrangements are based on a tetrameric organization, wherein two pairs of molecules belonging to the respective cell membrane interact at the paracellular level shaping a porous scaffold. Although several studies, mostly computational, contributed to refine and characterize these two models, a comprehensive comparison of their features is still lacking, along with the assessment of their transferability among different Cldn homologs. Beyond the structural validation, shedding light on the molecular determinants of the topological and functional properties of Cldn5 assemblies has paramount implications in clinical settings as well. Indeed, in because of the critical role of the BBB as a guarantor of the chemical environment of the Central Nervous System (CNS), its presence poses a formidable obstacle in case of brain impairments, hindering the direct delivery of drugs. A promising approach to handle this challenge consists in using competitive peptides that target Cldn5 proteins, thus interfering with the formation of TJs and generating temporary fenestrations across the paracellular spaces that could allow the pharmaceuticals to cross the brain endothelium. In this thesis, both the structural validation and the practical application aspects have been addressed by leveraging the powerful capabilities of molecular modeling and simulation methods to investigate biological systems at atomic resolution. Firstly, Pore I and Pore II have been assembled with Cldn5 monomers in both the wild type and various mutated variants and investigated through all-atom molecular dynamics (MD) simulations on the microsecond timescale, to assess the structural properties of the two arrangements. Moreover, biased MD simulations with the umbrella sampling method were performed to compute the free energy profiles of ion permeation across the pore cavities. Results from this analysis suggested that both the pore configurations are compatible with the physiological function of Cldn5 in the BBB, but only Pore I free energy profiles were consistent with the 7 experimental outcomes shown for the pathogenic variant of the protein. To assess the transferability of the two architectures to another Cldn homolog, we chose the anion selective Cldn4, and the free energy calculations revealed again Pore I as the functionally consistent one. Then, in the second part of the dissertation, it is presented a computational workflow to design and characterize peptides of a limited size (up to 16 residues), featuring appropriate solubility and Cldn5 binding properties. The use of relatively short sequences has many advantages, including the ease of synthesis, higher metabolic stability, improved biocompatibility, and limited cost. Moreover, it allows more accurate computational modeling as well. The procedure illustrated in this section is based on a well-rounded application of molecular modeling methods, including homology modeling, molecular docking simulations, and standard and biased MD simulations, that allowed the identification of a peptide, referred to as f1-C5C2, exhibiting a pronounced affinity for Cldn5 proteins. Remarkably, these findings were confirmed by experimental characterization of the peptide carried out by collaborators at the Center for Synaptic Neuroscience and Technology (NSYN) of the Fondazione Istituto Italiano di Tecnologia (IIT) in Genoa, Italy, underscoring the successful integration of the in silico and in vitro approaches at the basis of this investigation. Taken together, the results presented in this thesis aim to advance the understanding of the molecular features of Cldn assemblies within TJ strands, offering valuable insights to drive the rational design of effective peptides with potential beneficial impact from a clinical perspective

Multiscale modelling of claudin-based assemblies: a magnifying glass for novel structures of biological interfaces

Claudins (Cldns) define a family of transmembrane proteins that are the major determinants of the tight junction integrity and tissue selectivity. They promote the formation of either barriers or ion-selective channels at the interface between two facing cells, across the paracellular space. Multiple Cldn subunits form complexes that include cis- (intracellular) interactions along the membrane of a single cell and trans- (intercellular) interactions across adjacent cells. The first description of Cldn assemblies was provided by electron microscopy, while electrophysiology, mutagenesis and cell biology experiments addressed the functional role of different Cldn homologs. However, the investigation of the molecular details of Cldn subunits and complexes are hampered by the lack of experimental native structures, currently limited to Cldn15. The recent implementation of computer-based techniques greatly contributed to the elucidation of Cldn properties. Molecular dynamics simulations and docking calculations were extensively used to refine the first Cldn multimeric model postulated from the crystal structure of Cldn15, and contributed to the introduction of a novel, alternative, arrangement. While both these multimeric assemblies were found to account for the physiological properties of some family members, they gave conflicting results for others. In this review, we illustrate the major findings on Cldn-based systems that were achieved by using state-of-the-art computational methodologies. The information provided by these results could be useful to improve the characterization of the Cldn properties and help the design of new efficient strategies to control the paracellular transport of drugs or other molecules

Computational Assessment of Different Structural Models for Claudin-5 Complexes in Blood-Brain Barrier Tight Junctions

The blood-brain barrier (BBB) strictly regulates the exchange of ions and molecules between the blood and the central nervous system. Tight junctions (TJs) are multimeric structures that control the transport through the paracellular spaces between the adjacent brain endothelial cells of the BBB. Claudin-5 (Cldn5) proteins are essential for TJ formation and assemble into multiprotein complexes via cis-interactions within the same cell membrane and trans-interactions across two contiguous cells. Despite the relevant biological function of Cldn5 proteins and their role as targets of brain drug delivery strategies, the molecular details of their assembly within TJs are still unclear. Two different structural models have been recently introduced, in which Cldn5 dimers belonging to opposite cells join to generate paracellular pores. However, a comparison of these models in terms of ionic transport features is still lacking. In this work, we used molecular dynamics simulations and free energy (FE) calculations to assess the two Cldn5 pore models and investigate the thermodynamic properties of water and physiological ions permeating through them. Despite different FE profiles, both structures present single/multiple FE barriers to ionic permeation, while being permissive to water flux. These results reveal that both models are compatible with the physiological role of Cldn5 TJ strands. By identifying the protein-protein surface at the core of TJ Cldn5 assemblies, our computational investigation provides a basis for the rational design of synthetic peptides and other molecules capable of opening paracellular pores in the BBB