An Integrated Approach for Evaluating the Restoration of the Salinity Gradient in Transitional Waters: Monitoring and Numerical Modeling in the Life Lagoon Refresh Case Study

Large lagoons usually show a salinity gradient due to fresh water tributaries with inner areas characterized by lower mean values and higher fluctuation of salinity than seawaterdominated areas. In the Venice Lagoon, this ecotonal environment, characterized in the past by oligo‐mesohaline waters and large intertidal areas vegetated by reedbeds, was greatly reduced by historical human environmental modifications, including the diversion of main rivers outside the Venice Lagoon. The reduction of the fresh water inputs caused a marinization of the lagoon, with an increase in salinity and the loss of the related habitats, biodiversity, and ecosystem services. To counteract this issue, conservation actions, such as the construction of hydraulic infrastructures for the introduction and the regulation of a fresh water flow, can be implemented. The effectiveness of these actions can be preliminarily investigated and then verified through the combined implementation of environmental monitoring and numerical modeling. Through the results of the monitoring activity carried out in Venice Lagoon in the framework of the Life Lagoon Refresh (LIFE16NAT/IT/000663) project, the study of salinity is shown to be a successful and robust combination of different types of monitoring techniques. In particular, the characterization of salinity is obtained by the acquisition of continuous data, field campaigns, and numerical modeling

IMMU-01. TEM-GBM: AN OPEN-LABEL, PHASE I/IIA DOSE-ESCALATION STUDY EVALUATING THE SAFETY AND EFFICACY OF GENETICALLY MODIFIED TIE-2 EXPRESSING MONOCYTES TO DELIVER IFN-A WITHIN GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract Temferon is a macrophage-based treatment relying on ex-vivo transduction of autologous HSPCs to express immune-payloads within the TME. Temferon targets the immune-modulatory molecule IFN-a, to a subset of tumor infiltrating macrophages known as Tie-2 expressing macrophages (TEMs) due to the Tie2 promoter and a post-transcriptional regulation layer represented by miRNA-126 target sequences. As of 31st May 2021, 15-patients received Temferon (D+0) with follow-up of 3 – 693 days. After conditioning neutrophil and platelet engraftment occurred at D+13 and D+13.5, respectively. Temferon-derived differentiated cells, as determined be the number of vector copy per genome, were found within 14 days post treatment and persisted albeit at lower levels up to 18-months. Very low concentrations of IFN-a in the plasma (8.7 pg/ml-D+30) and in the CSF (1.6 pg/ml-D+30) were detected, suggesting tight regulation of transgene expression. Five-deaths occurred at D+322, +340, +402, +478 and +646 due to PD, and one at D+60 due to complications following the conditioning regimen. Eight-patients had progressive disease (range: D-11 to +239) as expected for this tumor type. SAEs include GGT elevation (possibly related to Temferon) and infections, venous thromboembolism, brain abscess, hemiparesis, seizures, anemia and general physical condition deterioration, compatible with ASCT, concomitant medications and PD. Four-patients underwent 2ndsurgery. Recurrent tumors had gene-marked cells and increased expression of ISGs compared to first surgery, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased ISGs than in the progressing lesion, detected in the same patient. Tumor-associated clones expanded in the periphery. TME characterization by scRNA and TCR-sequencing is ongoing. To date, Temferon is well tolerated, with no DLTs identified. The results provide initial evidence of Temferon potential to activate the immune system of GBM patients, as predicted by preclinical studies

Catalytic Multitasking in MetC: one enzyme, multiple reactions

Unlike most modern enzymes, which perform a single reaction, primordial enzymes are believed to have each performed a broad range of reactions. Consequently, most modern enzymes are poor comparative models for inferring the properties of primordial enzymes. The aim of this thesis was to identify and characterise modern enzymes that have evolved to catalyse multiple reactions on multiple substrates, in order to better understand the properties of primordial enzymes. Using bioinformatics and phylogenetics, I have discovered that, in three distinct clades, one enzyme (MetC) has taken over the role of the absent alanine racemase (Alr). Two of the three MetC enzymes have also taken over the role of the absent glutamate racemase (MurI). Even though MetC, MurI and Alr are not homologous. In other organisms, such as Escherichia coli, MetC catalyses the β-elimination of cystathionine in methionine biosynthesis. The E. coli MetC has a small promiscuous alanine racemisation activity, but no detectable glutamate racemisation activity. Two of the clades —the genus Pelagibacter and the family Anaplasmataceae, which includes Wolbachia— were in the same class, the Alphaproteobacteria, but the precise location was under debate in the literature. Therefore, I used phylogenetic methods to determine that they did belong to sister orders and that the AT-richness of their genomes is ancestral, rather than a source of bias. The three enzymes investigated are located in different groups along the MetC tree. Using in vitro activity assays, I found that the enzyme most similar to E. coli MetC, Pelagibacter ubique MetC, could racemise alanine, but could not racemise glutamate. Further away on the tree, Wolbachia MetC has glutamate and alanine racemisation activities that are both stronger than its cystathionine β-elimination activity in terms of turnover numbers (kcat). While, in a basal group, Thermotoga maritima MetC possess the strongest glutamate racemising activity of the enzymes studied. These three enzymes were also able to promiscuously cleave cysteine. The three enzymes had Michaelis constants (KM) for the various substrates that are comparable to those of each dedicated enzyme (MetC, Alr and MurI) in other organisms, while having turnover numbers (kcat) that are much lower than those of each dedicated enzyme. It was conjectured that this balance of kinetic parameters in these enzymes is due to the physiological necessity of operating with low concentrations of each substrate, yet producing a limited amount of product due to the low cellular demand for each metabolite. The crystal structure of T. maritima MetC was solved, showing that it may possess glutamate racemisation activity because an active site tryptophan forms a hydrogen bond with the terminal carboxyl group of the bound glutamate. The structure also revealed a latch-like loop close to the active site entrance. I used directed evolution in an attempt to improve the cystathionine elimination activity of T. maritima MetC. A mutant (S86T/S305C) was identified that possessed a more permissive active site with decreased Michaelis constants for all activities, including those that had not been under selection. This may indicate that wild-type T. maritima MetC has a tight balance of kinetic parameters that needs to be relaxed before it can be evolved into an enzyme with a single activity. Overall, the properties of these enzymes reveal how multiple activities are balanced in a single active site and offer new insights into the likely nature of primordial enzymes

Catalytic Multitasking in MetC: one enzyme, multiple reactions

Unlike most modern enzymes, which perform a single reaction, primordial enzymes are believed to have each performed a broad range of reactions. Consequently, most modern enzymes are poor comparative models for inferring the properties of primordial enzymes. The aim of this thesis was to identify and characterise modern enzymes that have evolved to catalyse multiple reactions on multiple substrates, in order to better understand the properties of primordial enzymes. Using bioinformatics and phylogenetics, I have discovered that, in three distinct clades, one enzyme (MetC) has taken over the role of the absent alanine racemase (Alr). Two of the three MetC enzymes have also taken over the role of the absent glutamate racemase (MurI). Even though MetC, MurI and Alr are not homologous. In other organisms, such as Escherichia coli, MetC catalyses the β-elimination of cystathionine in methionine biosynthesis. The E. coli MetC has a small promiscuous alanine racemisation activity, but no detectable glutamate racemisation activity. Two of the clades —the genus Pelagibacter and the family Anaplasmataceae, which includes Wolbachia— were in the same class, the Alphaproteobacteria, but the precise location was under debate in the literature. Therefore, I used phylogenetic methods to determine that they did belong to sister orders and that the AT-richness of their genomes is ancestral, rather than a source of bias. The three enzymes investigated are located in different groups along the MetC tree. Using in vitro activity assays, I found that the enzyme most similar to E. coli MetC, Pelagibacter ubique MetC, could racemise alanine, but could not racemise glutamate. Further away on the tree, Wolbachia MetC has glutamate and alanine racemisation activities that are both stronger than its cystathionine β-elimination activity in terms of turnover numbers (kcat). While, in a basal group, Thermotoga maritima MetC possess the strongest glutamate racemising activity of the enzymes studied. These three enzymes were also able to promiscuously cleave cysteine. The three enzymes had Michaelis constants (KM) for the various substrates that are comparable to those of each dedicated enzyme (MetC, Alr and MurI) in other organisms, while having turnover numbers (kcat) that are much lower than those of each dedicated enzyme. It was conjectured that this balance of kinetic parameters in these enzymes is due to the physiological necessity of operating with low concentrations of each substrate, yet producing a limited amount of product due to the low cellular demand for each metabolite. The crystal structure of T. maritima MetC was solved, showing that it may possess glutamate racemisation activity because an active site tryptophan forms a hydrogen bond with the terminal carboxyl group of the bound glutamate. The structure also revealed a latch-like loop close to the active site entrance. I used directed evolution in an attempt to improve the cystathionine elimination activity of T. maritima MetC. A mutant (S86T/S305C) was identified that possessed a more permissive active site with decreased Michaelis constants for all activities, including those that had not been under selection. This may indicate that wild-type T. maritima MetC has a tight balance of kinetic parameters that needs to be relaxed before it can be evolved into an enzyme with a single activity. Overall, the properties of these enzymes reveal how multiple activities are balanced in a single active site and offer new insights into the likely nature of primordial enzymes

Development and Validation of a Comprehensive Model to Estimate Early Allograft Failure Among Patients Requiring Early Liver Retransplant

Importance: Expansion of donor acceptance criteria for liver transplant increased the risk for early allograft failure (EAF), and although EAF prediction is pivotal to optimize transplant outcomes, there is no consensus on specific EAF indicators or timing to evaluate EAF. Recently, the Liver Graft Assessment Following Transplantation (L-GrAFT) algorithm, based on aspartate transaminase, bilirubin, platelet, and international normalized ratio kinetics, was developed from a single-center database gathered from 2002 to 2015. Objective: To develop and validate a simplified comprehensive model estimating at day 10 after liver transplant the EAF risk at day 90 (the Early Allograft Failure Simplified Estimation [EASE] score) and, secondarily, to identify early those patients with unsustainable EAF risk who are suitable for retransplant. Design, setting, and participants: This multicenter cohort study was designed to develop a score capturing a continuum from normal graft function to nonfunction after transplant. Both parenchymal and vascular factors, which provide an indication to list for retransplant, were included among the EAF determinants. The L-GrAFT kinetic approach was adopted and modified with fewer data entries and novel variables. The population included 1609 patients in Italy for the derivation set and 538 patients in the UK for the validation set; all were patients who underwent transplant in 2016 and 2017. Main outcomes and measures: Early allograft failure was defined as graft failure (codified by retransplant or death) for any reason within 90 days after transplant. Results: At day 90 after transplant, the incidence of EAF was 110 of 1609 patients (6.8%) in the derivation set and 41 of 538 patients (7.6%) in the external validation set. Median (interquartile range) ages were 57 (51-62) years in the derivation data set and 56 (49-62) years in the validation data set. The EASE score was developed through 17 entries derived from 8 variables, including the Model for End-stage Liver Disease score, blood transfusion, early thrombosis of hepatic vessels, and kinetic parameters of transaminases, platelet count, and bilirubin. Donor parameters (age, donation after cardiac death, and machine perfusion) were not associated with EAF risk. Results were adjusted for transplant center volume. In receiver operating characteristic curve analyses, the EASE score outperformed L-GrAFT, Model for Early Allograft Function, Early Allograft Dysfunction, Eurotransplant Donor Risk Index, donor age 7 Model for End-stage Liver Disease, and Donor Risk Index scores, estimating day 90 EAF in 87% (95% CI, 83%-91%) of cases in both the derivation data set and the internal validation data set. Patients could be stratified in 5 classes, with those in the highest class exhibiting unsustainable EAF risk. Conclusions and relevance: This study found that the developed EASE score reliably estimated EAF risk. Knowledge of contributing factors may help clinicians to mitigate risk factors and guide them through the challenging clinical decision to allocate patients to early liver retransplant. The EASE score may be used in translational research across transplant centers

The roof wing opening system of the UAE pavilion at EXPO 2020

The UAE Pavilion will be a major attraction at Expo 2020 in Dubai. The roof of the building consists of 28 operable wings made of carbon and glass fiber, having masses ranging from 5 to 18 tons and total lengths in the range of 30 to 65 m that have to be actuated by a dedicated mechanism. In this paper we present the turn-key project for the design, manufacturing, installation, test and commissioning of the Roof Wing Opening System, which represents a unique system world-wide for operating the wings. It consists of one Hydraulic Power Unit with approximately 1 MW of installed power, 2 km of piping working at the nominal pressure of 210 bar, 46 hydraulic cylinders with 1.5 tons of mass each and the complete automation and control subsystem that includes 9 separate PLCs, dedicated software, 2.000 sensors and control points, and over 20 km of harness. One major challenge is the control of the wings. Part of them, due to their huge dimensions and masses, are actuated using two or three hydraulic cylinders that have to be properly synchronized during the movement, preventing unwanted displacements in order to avoid stresses on the wing mechanical structure and ultimately permanent damages. Due to the nature of the project, a final validation of the control algorithms can be done only at system level during the commissioning phase. Therefore, particular care has to be devoted to the verification strategy, anticipating the behavior of the system in the early validation stages and following a V-model approach, in order to identify critical situations and reduce the overall risk. After a brief system description, we will explain how the verification has been approached by using system level simulations and dedicated testing activities on specific subsystems. In particular, we will detail the verification of the control algorithms that has been performed on a dedicated Hardware-Inthe- Loop system first, followed then by dedicated tests on a reduced wing mock-up, allowing the study of the system behavior under the most critical conditions. These include the application of external forces with specified profiles. Finally, we will provide the actual status of the system installation, testing and commissioning activities that have been running in Dubai since January 2019

Machine perfusions in liver transplantation: The evidence-based position paper of the italian society of organ and tissue transplantation.

BACKGROUND AND AIMS The use of machine perfusion in liver transplantation is spreading worldwide but its efficacy has not been demonstrated and its proper clinical use far to be implemented. The Italian Society of Organ and Tissue Transplantation (SITO) promoted the development of an evidence-based position paper. METHODS A three-step approach has been adopted to develop this position paper. Firstly, the Society appointed a Chair and a co-Chair who then assembled a Working Group with specific experience of machine perfusion in liver transplantation. The Guideline Development Group framed the clinical questions into a Patient/Intervention/Control/Outcome (PICO) format, extracted and analyzed available literature, ranked the quality of evidence, prepared and graded the recommendations. Recommendations were then discussed by all the members of the SITO and voted by Delphi round by an Internal Review Board and finally evaluated and scored by a panel of external reviewers. RESULTS All available literature was analyzed, and its quality ranked. Eighteen recommendations regarding the use and the efficacy of ex-situ hypothermic and normothermic machine perfusion and sequential normothermic regional perfusion and ex-situ machine perfusion were prepared and graded according to the GRADE method. CONCLUSION A critical and scientific approach is required for the safe implementation of this new technology

Laparoscopic liver resection of hepatocellular carcinoma located in unfavorable segments: a propensity score-matched analysis from the I Go MILS (Italian Group of Minimally Invasive Liver Surgery) Registry

Objective: Laparoscopic liver resection (LLR) for Hepatocellular Carcinoma (HCC) is one of the most important indications for the minimally invasive approach. Our study aims to analyze the experience of the Italian Group of Minimally Invasive Liver Surgery with laparoscopic surgical treatment of HCC, with a focus on tumor location and how it affects morbidity and mortality. Methods: 38 centers participated in this study; 372 cases of LLR for HCC were prospectively enrolled. Patients were divided into two groups according to the HCC nodule location. Group 1 favorable location and group 2 unfavorable location. Perioperative outcomes were compared between the two groups before and after a propensity score match (PS) 1:1. Results: Before PS in group 2 surgical time was longer; conversion rate was higher; postoperative transfusion and comprehensive complication index were also higher. PS was performed with a cohort of 298 patients (from 18 centers), with 66 and 232 patients with HCC in unfavorable and favorable locations, respectively. After PS matching, 62 patients from group 1 and group 2 each were compared. Operative and postoperative course were similar in patients with HCC in favorable and unfavorable LLR locations. Surgical margins were found to be identical before and after PS. Conclusions: These results show that LLR in patients with HCC can be safely performed in all segments because of the extensive experience of all surgeons from multiple centers in performing traditional open liver surgery as well as laparoscopic surgery