Caratterizzazione strutturale e funzionale della topoisomerasi IB umana e dei suoi mutanti

La DNA topoisomerasi IB umana è un enzima monomerico essenziale nel risolvere problemi topologici generati durante procesi chiave nucleari quali la replicazione, trascrizione, combinazione e riparazione del DNA. Latopo IB rilassa DNA superavvolti sia negativi che positivi in assenza di cationi metallici, attraverso la rottura transiente di un singolo filamento di DNA. Durante il ciclo catalitico il residuo Tyr723 viene usato come nucleofilo per rompere il legame fosfodiesterico del backbone del DNA generando un complesso covalente enzima-DNA all'estremità 3' del filamento tagliato. Il gruppo ossidrile 5' del filamento tagliato è libero di ruotare e di agire come nucleofilo attaccando il gruppo fosfato per rompere il legame tra il DNA e l'enzima risaldando il filamento precedentemente tagliato. Secondo studi cristallografici e di proteolisi limitata la topo IB è organizzata in quattro domini: N-terminale, core, linker e C-terminale che contiene il residuo attivo della Tyr723. L'enzima inoltre risulta essere l'unico bersaglio di farmaci chemioterapici quali la camptotecina ed i suoi derivati. Studi di mutanti ipersensibili, come la topoisomerasi I Asp677Gly-Val703Ile, permettono di comprendere i meccanismi alla base del funzionamento dell'enzima e della sua interazione con la droga ed intercorrelazione tra i diversi domini proteici. A tal fine sono stati eseguiti saggi di caratterizzazione biochimica del mutante in vivo ed in vitro analizzando le principali fasi del ciclo. Lo sviluppo sempre maggiore di nuovo farmaci antitumorali ci ha portato a volgere l'attenzione sull'effetto inibitorio del cEPA e a comprenderne il meccanismo. Gli esperimenti mostrano che il cEPA ha un meccanismo differrente dalla CPT inibendo il taglio dell'enzima sul DNA substrato sebbene permetta la formazione del complesso non covalente enzima-DNA. Di particolare interesse è il dominio N-terminale, in quanto è l'unica regione dell'enzima con una struttura 3D sconosciuta, in quanto non è possibile cristallizzarla. Il dominio è stato overespresso in E. coli BL21 come proteina di fusione con la GST. Sono stati esegiuti studi preliminari di struttura mediante tecniche spettroscopiche di fluorescenza, di dicroismo circolare e di NMR monodimensionale. Questi spettri indicano un eqiuilibrio tra regioni strutturate e non strutturate, ma per ricevere ulteriori informazioni strutturali è necessario eseguire spettri NMR bidimensionali producendo il dominio marcato con isotopi N15 e C13.Human DNA topoisomerase IB (topo IN) is an essential monomeric enzyme involved in resolving the torsional stress assiciated with DNA replication, trascription and chromatin condensation. Topo IB catalyzes changes in the supehelical state of the duplex DNA by transiently breaking a single strand allowing the broken strand to move through the break. Phosphodiester bond energy is preserved during catalysis through the formation of a transient phosphotyrosine bond between the active-site tyrosine and the 3' end of the broken strand. According to X-ray crystallography and biochemical analysyses topo IB is organized into four domains: N-terminal domain,core, linker and C-terminal domain which contains the active site Tyr723. The topo IB is also an important target of the antitumor drug camptothecin for chemotherapy of uman cancers. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant displaying a pronounced CPT sensitivity as been investigated to better understad the action mechanism of the enzyme and the interaction of CPT with the cleavable topoI-DNA complex. The results have shown that the CPT hypersensitivity of the mutations its done to a reduction of its religation rate and demonstrated the occurrence of a long range communication between domains localized far away one from the other.The inhibition efficiency on human topo IB wild type of the cEPA has been investigated analyzing the different steps of the enzyme catalytic cycle. The experiments show that cEPA has a mechanism different from CPT, inhibiting the cleavage of the enzyme on the DNA although permitting the non covalent enzyme-DNA interaction. The human topoI N-terminal domain is the only region of the enzyme with an unknown 3D structure since it is not possible to crystallize it. This domain has been overproduced into E.coli BL21,to carry out preliminary spectroscopic analysis consisting of CD, fluorescence and NMR spectra. The spectra indicate an equilibrium between the structured and unstructured regions. We are now producing the isotope labeled protein,growing the bacterial cells in minimum medium containing N15,in the attempt to gain further structural information by high-field 2D NMR spectroscopy

Excellent intra and inter-observer reproducibility of wrist circumference measurements in obese children and adolescents

In a previous study, we found that wrist circumference, in particular its bone component,was associated with insulin resistance in a population of overweight/obese children. Theaim of the present study was to evaluate the intra- and inter-operator variability in wrist cir-cumference measurement in a population of obese children and adolescents. One hundredand two (54 male and 48 female) obese children and adolescents were consecutivelyenrolled. In all subjects wrist circumferences were measured by two different operators twotimes to assess intra- and inter-operator variability. Statistical analysis was performed usingSAS v.9.4 and JMP v.12. Measurements of wrist circumference showed excellent inter-operator reliability with Intra class Correlation Coefficients (ICC) of 0.96 and ICC of 0.97 forthe first and the second measurement, respectively. The intra-operator reliability was, also,very strong with a Concordance Correlation Coefficient (CCC) of 0.98 for both operators.The high reproducibility demonstrated in our results suggests that wrist circumference mea-surement, being safe, non-invasive and repeatable can be easily used in out-patient set-tings to identify youths with increased risk of insulin-resistance. This can avoid testing theentire population of overweight/obese children for insulin resistance parameter

A review of the main genetic factors influencing the course of COVID-19 in Sardinia: the role of human leukocyte antigen-G

Introduction: A large number of risk and protective factors have been identified during the SARS-CoV-2 pandemic which may influence the outcome of COVID-19. Among these, recent studies have explored the role of HLA-G molecules and their immunomodulatory effects in COVID-19, but there are very few reports exploring the genetic basis of these manifestations. The present study aims to investigate how host genetic factors, including HLA-G gene polymorphisms and sHLA-G, can affect SARS-CoV-2 infection. Materials and Methods: We compared the immune-genetic and phenotypic characteristics between COVID-19 patients (n = 381) with varying degrees of severity of the disease and 420 healthy controls from Sardinia (Italy). Results: HLA-G locus analysis showed that the extended haplotype HLA-G*01:01:01:01/UTR-1 was more prevalent in both COVID-19 patients and controls. In particular, this extended haplotype was more common among patients with mild symptoms than those with severe symptoms [22.7% vs 15.7%, OR = 0.634 (95% CI 0.440 – 0.913); P = 0.016]. Furthermore, the most significant HLA-G 3’UTR polymorphism (rs371194629) shows that the HLA-G 3’UTR Del/Del genotype frequency decreases gradually from 27.6% in paucisymptomatic patients to 15.9% in patients with severe symptoms (X2 = 7.095, P = 0.029), reaching the lowest frequency (7.0%) in ICU patients (X2 = 11.257, P = 0.004). However, no significant differences were observed for the soluble HLA-G levels in patients and controls. Finally, we showed that SARS-CoV-2 infection in the Sardinian population is also influenced by other genetic factors such as β-thalassemia trait (rs11549407C>T in the HBB gene), KIR2DS2/HLA-C C1+ group combination and the HLA-B*58:01, C*07:01, DRB1*03:01 haplotype which exert a protective effect [P = 0.005, P = 0.001 and P = 0.026 respectively]. Conversely, the Neanderthal LZTFL1 gene variant (rs35044562A>G) shows a detrimental consequence on the disease course [P = 0.001]. However, by using a logistic regression model, HLA-G 3’UTR Del/Del genotype was independent from the other significant variables [ORM = 0.4 (95% CI 0.2 – 0.7), PM = 6.5 x 10-4]. Conclusion: Our results reveal novel genetic variants which could potentially serve as biomarkers for disease prognosis and treatment, highlighting the importance of considering genetic factors in the management of COVID-19 patients

A sex-informed approach to improve the personalised decision making process in myelodysplastic syndromes: a multicentre, observational cohort study

Background Sex is a major source of diversity among patients and a sex-informed approach is becoming a new paradigm in precision medicine. We aimed to describe sex diversity in myelodysplastic syndromes in terms of disease genotype, phenotype, and clinical outcome. Moreover, we sought to incorporate sex information into the clinical decision-making process as a fundamental component of patient individuality. Methods In this multicentre, observational cohort study, we retrospectively analysed 13 284 patients aged 18 years or older with a diagnosis of myelodysplastic syndrome according to 2016 WHO criteria included in the EuroMDS network (n=2025), International Working Group for Prognosis in MDS (IWG-PM; n=2387), the Spanish Group of Myelodysplastic Syndromes registry (GESMD; n=7687), or the Dusseldorf MDS registry (n=1185). Recruitment periods for these cohorts were between 1990 and 2016. The correlation between sex and genomic features was analysed in the EuroMDS cohort and validated in the IWG-PM cohort. The effect of sex on clinical outcome, with overall survival as the main endpoint, was analysed in the EuroMDS population and validated in the other three cohorts. Finally, novel prognostic models incorporating sex and genomic information were built and validated, and compared to the widely used revised International Prognostic Scoring System (IPSS-R). This study is registered with ClinicalTrials.gov, NCT04889729. Findings The study included 7792 (58middot7%) men and 5492 (41middot3%) women. 10 906 (82middot1%) patients were White, and race was not reported for 2378 (17middot9%) patients. Sex biases were observed at the single-gene level with mutations in seven genes enriched in men (ASXL1, SRSF2, and ZRSR2 p<0middot0001 in both cohorts; DDX41 not available in the EuroMDS cohort vs p=0middot0062 in the IWG-PM cohort; IDH2 p<0middot0001 in EuroMDS vs p=0middot042 in IWG-PM; TET2 p=0middot031 vs p=0middot035; U2AF1 p=0middot033 vs p<0middot0001) and mutations in two genes were enriched in women (DNMT3A p<0middot0001 in EuroMDS vs p=0middot011 in IWG-PM; TP53 p=0middot030 vs p=0middot037). Additionally, sex biases were observed in co-mutational pathways of founding genomic lesions (splicing-related genes, predominantly in men, p<0middot0001 in both the EuroMDS and IWG-PM cohorts), in DNA methylation (predominantly in men, p=0middot046 in EuroMDS vs p<0middot0001 in IWG-PM), and TP53 mutational pathways (predominantly in women, p=0middot0073 in EuroMDS vs p<0middot0001 in IWG-PM). In the retrospective EuroMDS cohort, men had worse median overall survival (81middot3 months, 95% CI 70middot4-95middot0 in men vs 123middot5 months, 104middot5-127middot5 in women; hazard ratio [HR] 1middot40, 95% CI 1middot26-1middot52; p<0middot0001). This result was confirmed in the prospective validation cohorts (median overall survival was 54middot7 months, 95% CI 52middot4-59middot1 in men vs 74middot4 months, 69middot3-81middot2 in women; HR 1middot30, 95% CI 1middot23-1middot35; p<0middot0001 in the GEMSD MDS registry; 40middot0 months, 95% CI 33middot4-43middot7 in men vs 54middot2 months, 38middot6-63middot8 in women; HR 1middot23, 95% CI 1middot08-1middot36; p<0middot0001 in the Dusseldorf MDS registry). We developed new personalised prognostic tools that included sex information (the sex-informed prognostic scoring system and the sex-informed genomic scoring system). Sex maintained independent prognostic power in all prognostic systems; the highest performance was observed in the model that included both sex and genomic information. A five-to-five mapping between the IPSS-R and new score categories resulted in the re-stratification of 871 (43middot0%) of 2025 patients from the EuroMDS cohort and 1003 (42middot0%) of 2387 patients from the IWG-PM cohort by using the sex-informed prognostic scoring system, and of 1134 (56middot0%) patients from the EuroMDS cohort and 1265 (53middot0%) patients from the IWG-PM cohort by using the sex-informed genomic scoring system. We created a web portal that enables outcome predictions based on a sex-informed personalised approach. Interpretation Our results suggest that a sex-informed approach can improve the personalised decision making process in patients with myelodysplastic syndromes and should be considered in the design of clinical trials including low-risk patients. Copyright (c) 2022 Published by Elsevier Ltd. All rights reserved

Potential for Circular Autopoietic Economy in High River Po Valley

This Gigamap aims to describe and summarize a student work carried out during the semester course Open Systems Design at Politecnico di Torino. This is one of the outputs from an analysis of the High River Po Valley area in the Italian Piedmont (Southwest of Turin) and subsequently an in-depth study of the relationships and “flows” with certain “currencies” between some selected economic and public actors. The analysis was conducted through five economic sectors: nature & ecosystem services, tourism, mobility and infrastructure, local craft activities and agri-food. The investigation of the territorial economy was carried out by giving particular attention to the production sectors characteristic of the territory and examine their production lines. The holistic diagnosis has highlighted the existence of several problems related to the linearity of the production processes. Through the five types of system flows (material, CO2 emissions/energy, water, economic and social flows), we designed new opportunities, new activities and new potential companies, thinking circularly and systemically. The Gigamap will be presented to stakeholders in a public hearing and used to illustrate and incubate a more circular economy that is more resilient and more regenerative. Reading the map The reader can find in the first part an abstract with the aim of the Gigamap, the five topics for the investigation and actors’ selection, a timeline about important events and some peculiarities of the territory. In the middle, the territorial map of a suggested improved territorial economy based on circular flows: the 16 actors are localized on the territory with new circular flows “designed in” between them. The boxes describe connections for new circular opportunities, with flows explained under the territorial map—finally, some data about the territory and the three main outputs emerging from the new circular linkages

Caratterizzazione strutturale e funzionale della topoisomerasi IB umana e dei suoi mutanti

La DNA topoisomerasi IB umana è un enzima monomerico essenziale nel risolvere problemi topologici generati durante procesi chiave nucleari quali la replicazione, trascrizione, combinazione e riparazione del DNA. Latopo IB rilassa DNA superavvolti sia negativi che positivi in assenza di cationi metallici, attraverso la rottura transiente di un singolo filamento di DNA. Durante il ciclo catalitico il residuo Tyr723 viene usato come nucleofilo per rompere il legame fosfodiesterico del backbone del DNA generando un complesso covalente enzima-DNA all'estremità 3' del filamento tagliato. Il gruppo ossidrile 5' del filamento tagliato è libero di ruotare e di agire come nucleofilo attaccando il gruppo fosfato per rompere il legame tra il DNA e l'enzima risaldando il filamento precedentemente tagliato. Secondo studi cristallografici e di proteolisi limitata la topo IB è organizzata in quattro domini: N-terminale, core, linker e C-terminale che contiene il residuo attivo della Tyr723. L'enzima inoltre risulta essere l'unico bersaglio di farmaci chemioterapici quali la camptotecina ed i suoi derivati. Studi di mutanti ipersensibili, come la topoisomerasi I Asp677Gly-Val703Ile, permettono di comprendere i meccanismi alla base del funzionamento dell'enzima e della sua interazione con la droga ed intercorrelazione tra i diversi domini proteici. A tal fine sono stati eseguiti saggi di caratterizzazione biochimica del mutante in vivo ed in vitro analizzando le principali fasi del ciclo. Lo sviluppo sempre maggiore di nuovo farmaci antitumorali ci ha portato a volgere l'attenzione sull'effetto inibitorio del cEPA e a comprenderne il meccanismo. Gli esperimenti mostrano che il cEPA ha un meccanismo differrente dalla CPT inibendo il taglio dell'enzima sul DNA substrato sebbene permetta la formazione del complesso non covalente enzima-DNA. Di particolare interesse è il dominio N-terminale, in quanto è l'unica regione dell'enzima con una struttura 3D sconosciuta, in quanto non è possibile cristallizzarla. Il dominio è stato overespresso in E. coli BL21 come proteina di fusione con la GST. Sono stati esegiuti studi preliminari di struttura mediante tecniche spettroscopiche di fluorescenza, di dicroismo circolare e di NMR monodimensionale. Questi spettri indicano un eqiuilibrio tra regioni strutturate e non strutturate, ma per ricevere ulteriori informazioni strutturali è necessario eseguire spettri NMR bidimensionali producendo il dominio marcato con isotopi N15 e C13.Human DNA topoisomerase IB (topo IN) is an essential monomeric enzyme involved in resolving the torsional stress assiciated with DNA replication, trascription and chromatin condensation. Topo IB catalyzes changes in the supehelical state of the duplex DNA by transiently breaking a single strand allowing the broken strand to move through the break. Phosphodiester bond energy is preserved during catalysis through the formation of a transient phosphotyrosine bond between the active-site tyrosine and the 3' end of the broken strand. According to X-ray crystallography and biochemical analysyses topo IB is organized into four domains: N-terminal domain,core, linker and C-terminal domain which contains the active site Tyr723. The topo IB is also an important target of the antitumor drug camptothecin for chemotherapy of uman cancers. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant displaying a pronounced CPT sensitivity as been investigated to better understad the action mechanism of the enzyme and the interaction of CPT with the cleavable topoI-DNA complex. The results have shown that the CPT hypersensitivity of the mutations its done to a reduction of its religation rate and demonstrated the occurrence of a long range communication between domains localized far away one from the other.The inhibition efficiency on human topo IB wild type of the cEPA has been investigated analyzing the different steps of the enzyme catalytic cycle. The experiments show that cEPA has a mechanism different from CPT, inhibiting the cleavage of the enzyme on the DNA although permitting the non covalent enzyme-DNA interaction. The human topoI N-terminal domain is the only region of the enzyme with an unknown 3D structure since it is not possible to crystallize it. This domain has been overproduced into E.coli BL21,to carry out preliminary spectroscopic analysis consisting of CD, fluorescence and NMR spectra. The spectra indicate an equilibrium between the structured and unstructured regions. We are now producing the isotope labeled protein,growing the bacterial cells in minimum medium containing N15,in the attempt to gain further structural information by high-field 2D NMR spectroscopy

Large Retroperitoneal Perivascular Epithelioid Cell Neoplasm (PEComa): A Case Report and a Brief Review

Objective: To describe a case of retroperitoneal perivascular epithelioid cell tumor (PEComa) and to discuss the main features of this rare pathology. Introduction: PEComas represent a rare cluster of neoplasms with uncertain origin; their precursor cells are spindle-shaped and characterized by a myomelanocytic phenotype, so only immunohistochemical staining makes a definitive diagnosis possible. To date, less than three hundred cases are reported in Literature and retroperitoneal site accounts for 7-8% of overall locations. Case Report: Middle-aged female has visited for abdominal pain and urinary complaints; physical findings and imaging demonstrated a huge inhomogeneous mass occupying right abdomen and arising from renal capsule. After multidisciplinary evaluation, patient has been addressed to open surgery and an en-bloc resection of the mass, with right nephrectomy and adrenalectomy. Immunohistochemical staining made a diagnosis of PEComa possible. After an uneventful postoperative stay, the patient entered a follow up protocol, without signs of local recurrence and distant metastases. Conclusion: Retroperitoneal PEComa often presents as a bulky mass with renal and adrenal involvement. Surgical resection should be aimed to obtain a complete removal with negative margins; this makes compartment surgery and en-bloc resection mandatory. Immunostaining is the key methods for a correct diagnosis

Role Played by Paraoxonase-2 Enzyme in Cell Viability, Proliferation and Sensitivity to Chemotherapy of Oral Squamous Cell Carcinoma Cell Lines

Oral squamous cell carcinoma represents the most aggressive and frequent form of head and neck cancer. Due to drug resistance, the 5-year survival rate of patients with advanced disease is less than 50%. In order to identify molecular targets for effective oral cancer treatment, we focused on paraoxonase-2 enzyme. Indeed, based on data previously obtained from preliminary immunohistochemistry and Western blot analyses performed on tissue specimens, the enzyme was found to be upregulated in tumor compared with normal oral mucosa. Therefore, paraoxonase-2 gene silencing was achieved in HSC-3 and HOC621 oral cancer cell lines, and the effect on cell proliferation, viability, apoptosis induction and sensitivity to cisplatin and 5-fluorouracil treatment was evaluated. Fourier Transform InfraRed Microspectroscopy analyzed alterations of cellular macromolecules upon treatment. Enzyme level and cell proliferation were also determined in cisplatin-resistant clones obtained from HOC621 cell line, as well as in parental cells. Reported data showed that paraoxonase-2 knockdown led to a reduction of cell proliferation and viability, as well as to an enhancement of sensitivity to cisplatin, together with the activation of apoptosis pathway. Spectroscopical data demonstrated that, under treatment with cisplatin, oxidative damage exerted on lipids and proteins was markedly more evident in cells down-regulating paraoxonase-2 compared to controls. Interestingly, enzyme expression, as well as cell proliferation were significantly higher in cisplatin-resistant compared with control HOC621 cells. Taken together these results seem to candidate the enzyme as a promising target for molecular treatment of this neoplasm