Fine Structure of Glycosaminoglycans from Fresh and Decellularized Porcine Cardiac Valves and Pericardium

Cardiac valves are dynamic structures, exhibiting a highly specialized architecture consisting of cells and extracellular matrix with a relevant proteoglycan and glycosaminoglycan content, collagen and elastic fibers. Biological valve substitutes are obtained from xenogenic cardiac and pericardial tissues. To overcome the limits of such non viable substitutes, tissue engineering approaches emerged to create cell repopulated decellularized scaffolds. This study was performed to determine the glycosaminoglycans content, distribution, and disaccharides composition in porcine aortic and pulmonary valves and in pericardium before and after a detergent-based decellularization procedure. The fine structural characteristics of galactosaminoglycans chondroitin sulfate and dermatan sulfate were examined by FACE. Furthermore, the mechanical properties of decellularized pericardium and its propensity to be repopulated by in vitro seeded fibroblasts were investigated. Results show that galactosaminoglycans and hyaluronan are differently distributed between pericardium and valves and within heart valves themselves before and after decellularization. The distribution of glycosaminoglycans is also dependent from the vascular district and topographic localization. The decellularization protocol adopted resulted in a relevant but not selective depletion of galactosaminoglycans. As a whole, data suggest that both decellularized porcine heart valves and bovine pericardium represent promising materials bearing the potential for future development of tissue engineered heart valve scaffolds

Fatality rate and predictors of mortality in an Italian cohort of hospitalized COVID-19 patients

Clinical features and natural history of coronavirus disease 2019 (COVID-19) differ widely among different countries and during different phases of the pandemia. Here, we aimed to evaluate the case fatality rate (CFR) and to identify predictors of mortality in a cohort of COVID-19 patients admitted to three hospitals of Northern Italy between March 1 and April 28, 2020. All these patients had a confirmed diagnosis of SARS-CoV-2 infection by molecular methods. During the study period 504/1697 patients died; thus, overall CFR was 29.7%. We looked for predictors of mortality in a subgroup of 486 patients (239 males, 59%; median age 71 years) for whom sufficient clinical data were available at data cut-off. Among the demographic and clinical variables considered, age, a diagnosis of cancer, obesity and current smoking independently predicted mortality. When laboratory data were added to the model in a further subgroup of patients, age, the diagnosis of cancer, and the baseline PaO2/FiO2 ratio were identified as independent predictors of mortality. In conclusion, the CFR of hospitalized patients in Northern Italy during the ascending phase of the COVID-19 pandemic approached 30%. The identification of mortality predictors might contribute to better stratification of individual patient risk

Ingegneria Tissutale delle Valvole Cardiache: valutazione di metodi di decellularizzazione e semine cellulari su Pericardio Bovino e Porcino

BACKGROUND: In the western world the prevalence of valvular heart diseases in general population account for 2%, and is constantly increasing due to ageing phenomena of the population. Current surgical therapies imply the use of mechanical and biological valves for the replacement of the diseased valve. Unfortunately in the long term this substitute valves lead patients to complications or reoperations. By using Tissue Engineering (TE) technologis, a science that combines contributions from engineering and biology, it is being investigated whether it is possible to create a new viable valve substitute that, similarly to healthy human valves, can withstand in vivo mechanical stress and undergo remodelling and growth after implantation, especially in paediatric patients. AIM OF THE STUDY: The purposes of this study are: Understand efficacy of UTRIDOC, TRICOL and TRITDOC decellularisation methods applied to Bovine Pericardium (BP), Calf Pericardium (CP) and Porcine Pericardium (PP), To test recellularisation potential of UTRIDOC, TRICOL and TRITDOC BP, Physical and structural characterisation of CF and PP, before and after TRITDOC treatment. The obtained acellular scaffolds are considered as potential materials to be used for TE heart valve production. MATERIALS AND METHODS: BP has been decellularized using three methods: UTRIDOC, TRICOL and TRITDOC. Istological and immunoistochemical techniques has been used to verify the complete decellularization of tissue and the organization of the Extracellular Matrix (ECM). Bovine fibroblasts (Pericardiocytes) and human endothelial cells (HUVEC) have then been seeded on serosal layer. Many seeding have been done in order to test different cellular densities and culture media. Spectrophotometric Landegren and MTT Tests and Haematoxylin and Eosin (H&E) have been carried on to check the seeding processes. At the same time others samples of BP UTRIDOC have been funzionalized with a syntethic, proadhesive peptide RGD sequence and Pericardiocytes have been seeded. TRITDOC protocol has been also applyed to CP and PP. These Pericardia have been collected and cutted in four anatomical areas: VsxANT, VdxANT, VsxPOST, VdxPOST. Density, thickness and water content HAVE been determined for each area and have been compared with non-tReated Pericardia. To check decellularisation and matrix preservation of these samples the following techniques have been applied: H&E, DAPI, immunohistochemistry and electron transmission microscopy (TEM). Removal of the xenoantigen alpha-Gal has been assessed by means of ELISA test. RESULTS: As been shown the three methods of decellularisation are able to remove cellular component from pericardial tissues and maintain ECM organization. Cellular seeding tests have showed Pericardiocytes adhesion on PB UTRIDOC serosal surface, after 6h and 18h of culture. Moreover, after 7 days Pericardiocyte begin to migrate into ECM of BP UTRIDOC e TRICOL maintaining an active metabolism. When using syntethic, proadhesive peptide RGD, Pericardiocytes better adhere to the BP UTRIDOC. HUVEC cells are able to adhere to the BP UTRIDOC in 1h, 3h, 5h and 24h since seeding, but they does not form a continous layer. However, they form it after 7 days on the BP TRITDOC and they keep espression of vWf and CD31. The whole TRITDOC CP display a significant decrease of water content (p0,05), while density significantly increased (p0,05) and a significant decrease in water content (p0,05). Moreover, for the mentioned above physical features, the VsxANT area in CP and VdxANT area in PP proved to be the most homogeneous specimens, compared to the other areas in the same species. Structural analyses demonstrated complete decellularisation in both CP and PP TRITDOC. Matrix architecture and collagen fibres organization were grossly maintained by the TRITDOC treatment, as exhibited by histology and TEM analysis. The xenoantigen alpha-Gal was completely removed from CP and PP after TRITDOC decellularisation. CONCLUSIONS: the tested decellularisation protocols, UTRIDOC, TRICOL and TRITDOC, produce bovine and porcine pericardial scaffolds that are acellular, non-cytotoxic, and exhibit normal organisation of the ECM. The TRITDOC BP, seems to be suitable for cellular seeding of human endothelial cells HUVEC. TRITDOC decellularisation of CP and PP identified specific pericardial areas more prone for use in TEHV. Further studies should be carried out to assess the mechanical performances of such scaffold, prior and after in-vitro cell seedingPRESUPPOSTI DELLO STUDIO: Nel mondo occidentale la patologia valvolare cardiaca presenta una prevalenza del 2% nella popolazione generale, ed è in costante crescita dato l’invecchiamento della popolazione. La terapia chirurgica attuale utilizza sostituti valvolari meccanici e biologici, che frequentemente portano i pazienti al reintervento o all'insorgenza di complicanze. L'Ingegneria Tissutale (TE), disciplina che unisce il contributo dell'ingegneria e della biologia, potrebbe permettere di creare sostituti valvolari dotati di patrimonio cellulare autologo e vitale in grado di andare incontro a rimodellamento e crescita in accordo con lo stato fisiologico dei pazienti, in particolare quelli pediatrici. SCOPO DELLO STUDIO: Gli scopi di questo studio sono stati: verificare la capacità di decellularizzazione dei metodi UTRIDOC, TRICOL e TRITDOC quando applicati al Pericardio Bovino (PB), al Pericardio Bovino di Vitello (PBV) e al Pericardio Porcino (PP); testare il potenziale di ricellularizzazione del PB UTRIDOC, TRICOL e TRITDOC e caratterizzare strutturalmente e fisicamente il PBV e il PP prima e dopo decellularizzazione TRITDOC che si è rivelato un promettente metodo per l'adesione cellulare endoteliale. Gli scaffolds ottenuti sono stati valutati come biomateriali di base per l'Ingegneria Tissutale delle valvole cardiache (TEHV). MATERIALI E METODI: Il PB è stato decellularizzato utilizzando tre metodi denominati rispettivamente: UTRIDOC, TRICOL E TRITDOC. Verificata la completa decellularizzazione del tessuto e l'organizzazione della Matrice Extracellulare (MEC) tramite tecniche istologiche ed immunoistochimiche, sulla superficie sierosa del tessuto sono stati seminati fibroblasti bovini (Pericardiociti) e cellule endoteliali umane (HUVEC). Le semine sono state eseguite in diverse condizioni sperimentali per testare la densità cellulare più promettente e i terreni di coltura più adatti. I risultati delle semine sono stati indagati attraverso colorazione dei campioni con Ematossilina ed Eosina, Test di Landegren e Test MTT. Il PB UTRIDOC è stato successivamente funzionalizzato con una sequenza peptidica sintetica proadesiva RGD (arginina, glicina, acido aspartico), ed è stata testata l'adesione dei Pericardiociti. Il protocollo TRITDOC è stato poi applicato anche al PBV e al PP. In quest'ultimi esperimenti i pericardi sono stati espiantati dopo sacrificio degli animali e suddivisi in 4 aree anatomiche: VsxANT, VdxANT, VsxPOST, VdxPOST. Per ogni area dei pericardi trattati TRITDOC e non trattati sono stati determinati lo spessore, la densità e il contenuto d’acqua medi. La qualità della decellularizzazione e la preservazione delle matrici sono state valutate tramite istologia, DAPI, immunoistochimica (IHC) e microscopia elettronica a trasmissione (TEM). L'eliminazione dell'antigene xenogenico alpha-Gal dai PBV e dai PP TRITDOC, è stata valutata tramite test ELISA. RISULTATI: Tutti i metodi di decellularizzazione sono in grado di eliminare totalmente la componente cellulare dai tessuti pericardici, inoltre mantengono inalterata l'organizzazione generale della MEC. I test di semina cellulare hanno mostrato che i Pericardiociti aderiscono alla superficie sierosa del PB UTRIDOC dopo 6h e 18h dalla semina e cominciano ad entrare nella MEC dopo 7 giorni nel PB TRICOL e UTRIDOC, mantenendo un metabolismo attivo. In presenza del peptide proadesivo sintetico RGD, i pericardiociti aderiscono meglio al PB UTRIDOC anche utilizzando concentrazioni cellulari di semina minori. Le cellule HUVEC riescono ad aderire alla superficie sierosa del PB UTRIDOC se seminate per 1h, 3h, 5h e 24h, ma non formano un monostrato continuo. Dopo un tempo di 7 giorni dalla semina invece, aderiscono più uniformemente sul PB UTRIDCOC e formano un monostrato sul PB TRITDOC, mantenendo l'espressione dei marcatori endoteliali vWf e CD31. Considerando l’intero PBV, il trattamento TRITDOC causa una riduzione dello spessore (p>0,05), un incremento della densità (p0,05) e il contenuto d’acqua si è ridotto invece in maniera significativa (p0,05). Dopo decellularizzazione, la densità del PP non si è modificata significativamente (p>0,05) rispetto al tessuto nativo. Inoltre, le aree che ricoprono il VsxANT nel PB e il VdxANT nel PP sono risultate le più omogenee per le proprietà fisiche sopra menzionate, sia prima sia dopo decellularizzazione. Le analisi morfo-strutturali eseguite, l’istologia e la valutazione al TEM hanno rilevato una struttura della matrice e un’organizzazione delle fibre collagene sostanzialmente preservata dal trattamento TRITDOC. CONCLUSIONI: I protocolli di decellularizzazione UTRIDOC, TRICOL e TRITDOC producono scaffolds pericardici bovini e porcini acellulari, non citotossici e con una MEC strutturalmente preservata. Il PB TRITDOC sembra lo scaffold più adatto per la semina di cellule umane endoteliali HUVEC. La decellularizzazione del PBV e del PP con il protocollo TRITDOC, ha inoltre permesso di individuare delle aree pericardiche di potenziale interesse per la TEHV. Studi approfonditi sulle prestazioni meccaniche e sul ripopolamento cellulare di queste matrici permetteranno una valutazione più accurata dell’effetto del trattament

Understanding the glycome: an interactive view of glycosylation from glycocompositions to glycoepitopes

Nowadays, due to the advance of experimental techniques in glycomics, large collections of glycan profiles are regularly published. The rapid growth of available glycan data accentuates the lack of innovative tools for visualizing and exploring large amount of information. Scientists resort to using general-purpose spreadsheet applications to create ad hoc data visualization. Thus, results end up being encoded in publication images and text, while valuable curated data is stored in files as supplementary information. To tackle this problem, we have built an interactive pipeline composed with three tools: Glynsight, EpitopeXtractor and Glydin'. Glycan profile data can be imported in Glynsight, which generates a custom interactive glycan profile. Several profiles can be compared and glycan composition is integrated with structural data stored in databases. Glycan structures of interest can then be sent to EpitopeXtractor to perform a glycoepitope extraction. EpitopeXtractor results can be superimposed on the Glydin' glycoepitope network. The network visualization allows fast detection of clusters of glycoepitopes and discovery of potential new targets. Each of these tools is standalone or can be used in conjunction with the others, depending on the data and the specific interest of the user. All the tools composing this pipeline are part of the Glycomics@ExPASy initiative and are available at https://www.expasy.org/glycomics

Understanding the glycome: an interactive view of glycosylation from glycocompositions to glycoepitopes

Nowadays, due to the advance of experimental techniques in glycomics, large collections of glycan profiles are regularly published. The rapid growth of available glycan data accentuates the lack of innovative tools for visualizing and exploring large amount of information. Scientists resort to using general-purpose spreadsheet applications to create ad hoc data visualization. Thus, results end up being encoded in publication images and text, while valuable curated data is stored in files as supplementary information. To tackle this problem, we have built an interactive pipeline composed with three tools: Glynsight, EpitopeXtractor and Glydin'. Glycan profile data can be imported in Glynsight, which generates a custom interactive glycan profile. Several profiles can be compared and glycan composition is integrated with structural data stored in databases. Glycan structures of interest can then be sent to EpitopeXtractor to perform a glycoepitope extraction. EpitopeXtractor results can be superimposed on the Glydin' glycoepitope network. The network visualization allows fast detection of clusters of glycoepitopes and discovery of potential new targets. Each of these tools is standalone or can be used in conjunction with the others, depending on the data and the specific interest of the user. All the tools composing this pipeline are part of the Glycomics@ExPASy initiative and are available at https://www.expasy.org/glycomics

GlyConnect: Glycoproteomics Goes Visual, Interactive, and Analytical

Knowledge of glycoproteins, their site-specific glycosylation patterns, and the glycan structures that they present to their recognition partners in health and disease is gradually being built on using a range of experimental approaches. The data from these analyses are increasingly being standardized and presented in various sources, from supplemental tables in publications to localized servers in investigator laboratories. Bioinformatics tools are now needed to collect these data and enable the user to search, display, and connect glycomics and glycoproteomics to other sources of related proteomics, genomics, and interactomics information. We here introduce GlyConnect (https://glyconnect.expasy.org/), the central platform of the Glycomics@ExPASy portal for glycoinformatics. GlyConnect has been developed to gather, monitor, integrate, and visualize data in a user-friendly way to facilitate the interpretation of collected glycoscience data. GlyConnect is designed to accommodate and integrate multiple data types as they are increasingly produced

Parkin-dependent regulation of the MCU complex component MICU1

The mitochondrial Ca2+ uniporter machinery is a multiprotein complex composed by the Ca2+ selective pore-forming subunit, the mitochondrial uniporter (MCU), and accessory proteins, including MICU1, MICU2 and EMRE. Their concerted action is required to fine-tune the uptake of Ca2+ into the mitochondrial matrix which both sustains cell bioenergetics and regulates the apoptotic response. To adequately fulfil such requirements and avoid impairment in mitochondrial Ca2+ handling, the intracellular turnover of all the MCU components must be tightly regulated. Here we show that the MCU complex regulator MICU1, but not MCU and MICU2, is rapidly and selectively degraded by the Ubiquitin Proteasome System (UPS). Moreover, we show that the multifunctional E3 ubiquitin ligase Parkin (PARK2), whose mutations cause autosomal recessive early-onset Parkinson's disease (PD), is a potential candidate involved in this process since its upregulation strongly decreases the basal level of MICU1. Parkin was found to interact with MICU1 and, interestingly, Parkin Ubl-domain, but not its E3-ubquitin ligase activity, is required for the degradation of MICU1, suggesting that in addition to the well documented role in the control of Parkin basal auto-inhibition, the Ubl-domain might exert important regulatory functions by acting as scaffold for the proteasome-mediated degradation of selected substrates under basal conditions, i.e. to guarantee their turnover. We have found that also MICU2 stability was affected upon Parkin overexpression, probably as a consequence of increased MICU1 degradation. Our findings support a model in which the PD-related E3 ubiquitin ligase Parkin directly participates in the selective regulation of the MCU complex regulator MICU1 and, indirectly, also of the MICU2 gatekeeper, thus indicating that Parkin loss of function could contribute to the impairment of the ability of mitochondria to handle Ca2+ and consequently to the pathogenesis of PD