An integrative structural study of the human full-length RAD52 at 2.2 Å resolution.

Human RAD52 (RAD52) is a DNA-binding protein involved in many DNA repair mechanisms and genomic stability maintenance. In the last few years, this protein was discovered to be a promising novel pharmacological target for anticancer strategies. Although the interest in RAD52 has exponentially grown in the previous decade, most information about its structure and mechanism still needs to be elucidated. Here, we report the 2.2 Å resolution cryo-EM reconstruction of the full-length RAD52 (FL-RAD52) protein. This allows us to describe the hydration shell of the N-terminal region of FL-RAD52, which is structured in an undecamer ring. Water molecules coordinate with protein residues to promote stabilization inside and among the protomers and within the inner DNA binding cleft to drive protein-DNA recognition. Additionally, through a multidisciplinary approach involving SEC-SAXS and computational methods, we comprehensively describe the highly flexible and dynamic organization of the C-terminal portion of FL-RAD52. This work discloses unprecedented structural details on the FL-RAD52, which will be critical for characterizing its mechanism of action and inhibitor development, particularly in the context of novel approaches to synthetic lethality and anticancer drug discovery.

Structure-based approaches in synthetic lethality strategies.

Evolution has fostered robust DNA damage response (DDR) mechanisms to combat DNA lesions. However, disruptions in this intricate machinery can render cells overly reliant on the remaining functional but often less accurate DNA repair pathways. This increased dependence on error-prone pathways may result in improper repair and the accumulation of mutations, fostering genomic instability and facilitating the uncontrolled cell proliferation characteristic of cancer initiation and progression. Strategies based on the concept of synthetic lethality (SL) leverage the inherent genomic instability of cancer cells by targeting alternative pathways, thereby inducing selective death of cancer cells. This review emphasizes recent advancements in structural investigations of pivotal SL targets. The significant contribution of structure-based methodologies to SL research underscores their potential impact in characterizing the growing number of SL targets, largely due to advances in next-generation sequencing. Harnessing these approaches is essential for advancing the development of precise and personalized SL therapeutic strategies.

New Horizons of Synthetic Lethality in Cancer: Current Development and Future Perspectives.

In recent years, synthetic lethality has been recognized as a solid paradigm for anticancer therapies. The discovery of a growing number of synthetic lethal targets has led to a significant expansion in the use of synthetic lethality, far beyond poly(ADP-ribose) polymerase inhibitors used to treat BRCA1/2-defective tumors. In particular, molecular targets within DNA damage response have provided a source of inhibitors that have rapidly reached clinical trials. This Perspective focuses on the most recent progress in synthetic lethal targets and their inhibitors, within and beyond the DNA damage response, describing their design and associated therapeutic strategies. We will conclude by discussing the current challenges and new opportunities for this promising field of research, to stimulate discussion in the medicinal chemistry community, allowing the investigation of synthetic lethality to reach its full potential.

A machine learning approach for early identification of patients with severe imported malaria.

BACKGROUND: The aim of this study is to design ad hoc malaria learning (ML) approaches to predict clinical outcome in all patients with imported malaria and, therefore, to identify the best clinical setting. METHODS: This is a single-centre cross-sectional study, patients with confirmed malaria, consecutively hospitalized to the Lazzaro Spallanzani National Institute for Infectious Diseases, Rome, Italy from January 2007 to December 2020, were recruited. Different ML approaches were used to perform the analysis of this dataset: support vector machines, random forests, feature selection approaches and clustering analysis. RESULTS: A total of 259 patients with malaria were enrolled, 89.5% patients were male with a median age of 39 y/o. In 78.3% cases, Plasmodium falciparum was found. The patients were classified as severe malaria in 111 cases. From ML analyses, four parameters, AST, platelet count, total bilirubin and parasitaemia, are associated to a negative outcome. Interestingly, two of them, aminotransferase and platelet are not included in the current list of World Health Organization (WHO) criteria for defining severe malaria. CONCLUSION: In conclusion, the application of ML algorithms as a decision support tool could enable the clinicians to predict the clinical outcome of patients with malaria and consequently to optimize and personalize clinical allocation and treatment.

Collaboration and topic switches in science.

Collaboration is a key driver of science and innovation. Mainly motivated by the need to leverage different capacities and expertise to solve a scientific problem, collaboration is also an excellent source of information about the future behavior of scholars. In particular, it allows us to infer the likelihood that scientists choose future research directions via the intertwined mechanisms of selection and social influence. Here we thoroughly investigate the interplay between collaboration and topic switches. We find that the probability for a scholar to start working on a new topic increases with the number of previous collaborators, with a pattern showing that the effects of individual collaborators are not independent. The higher the productivity and the impact of authors, the more likely their coworkers will start working on new topics. The average number of coauthors per paper is also inversely related to the topic switch probability, suggesting a dilution of this effect as the number of collaborators increases.

An 19F NMR fragment-based approach for the discovery and development of BRCA2-RAD51 inhibitors to pursuit synthetic lethality in combination with PARP inhibition in pancreatic cancer.

The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.

Isolation and Characterization of Monomeric Human RAD51: A Novel Tool for Investigating Homologous Recombination in Cancer.

DNA repair protein RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair DNA double-strand breaks. Due to the structural complexity and dynamics, researchers have not yet clarified the mechanistic details of every step of RAD51 recruitment and DNA repair. RAD51 possesses an intrinsic tendency to form oligomeric structures, which make it challenging to conduct biochemical and biophysical investigations. Here, for the first time, we report on the isolation and characterization of a human monomeric RAD51 recombinant form, obtained through a double mutation, which preserves the protein's integrity and functionality. We investigated different buffers to identify the most suitable condition needed to definitively stabilize the monomer. The monomer of human RAD51 provides the community with a unique biological tool for investigating RAD51-mediated homologous recombination, and paves the way for more reliable structural, mechanistic, and drug discovery studies.

Conditional cash transfers in OECD countries: a realist synthesis.

Conditional Cash Transfers (CCTs) schemes have been adopted mostly in low-income countries as a tool to break the vicious cycle of poverty transmission. Although their use is controversial, behavioral conditionalities have also been widely used in welfare-to-work strategies, minimum income scheme, and labor market "activation" policies in OECD countries. The paper presents the results of a Realist Review to synthesize the evidence of CCTs related to work conditionality, delivered in OECD countries. The evaluation literature of 23 selected CCT programs was analyzed by reconstructing Context-Mechanism-Outcome configurations. The main findings show that CCTs can be an effective counterbalance to work disincentives introduced by welfare measures. The unintended negative impacts, the role of sanctioning, and the causal pathways that may affect the most disadvantaged people and their children are discussed.

Novel Insights into RAD52's Structure, Function, and Druggability for Synthetic Lethality and Innovative Anticancer Therapies.

In recent years, the RAD52 protein has been highlighted as a mediator of many DNA repair mechanisms. While RAD52 was initially considered to be a non-essential auxiliary factor, its inhibition has more recently been demonstrated to be synthetically lethal in cancer cells bearing mutations and inactivation of specific intracellular pathways, such as homologous recombination. RAD52 is now recognized as a novel and critical pharmacological target. In this review, we comprehensively describe the available structural and functional information on RAD52. The review highlights the pathways in which RAD52 is involved and the approaches to RAD52 inhibition. We discuss the multifaceted role of this protein, which has a complex, dynamic, and functional 3D superstructural arrangement. This complexity reinforces the need to further investigate and characterize RAD52 to solve a challenging mechanistic puzzle and pave the way for a robust drug discovery campaign.

The Mechanistic Understanding of RAD51 Defibrillation: A Critical Step in BRCA2-Mediated DNA Repair by Homologous Recombination.

The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a "domino" mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.

Decoding distinctive features of plasma extracellular vesicles in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease's complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell's physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification. METHODS: We analyzed plasma-derived EVs of ALS patients (n = 106) and controls (n = 96), and SOD1G93A and TDP-43Q331K mouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis. RESULTS: Our procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. EVs in the plasma of ALS patients and the two mouse models of ALS had a distinctive size distribution and lower HSP90 levels compared to the controls. In terms of disease progression, the levels of cyclophilin A with the EV size distribution distinguished fast and slow disease progressors, a possibly new means for patient stratification. Immuno-electron microscopy also suggested that phosphorylated TDP-43 is not an intravesicular cargo of plasma-derived EVs. CONCLUSIONS: Our analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

Validation of the Transition Readiness and Appropriateness Measure (TRAM) for the Managing the Link and Strengthening Transition from Child to Adult Mental Healthcare in Europe (MILESTONE) study.

OBJECTIVE: Young people moving from child and adolescent mental health services (CAMHS) to adult mental health services (AMHS) are faced with significant challenges. To improve this state of affairs, there needs to be a recognition of the problem and initiatives and an urgent requirement for appropriate tools for measuring readiness and outcomes at the transfer boundary (16-18 years of age in Europe). The objective of this study was to develop and validate the Transition Readiness and Appropriateness Measure (TRAM) for assessing a young person's readiness for transition, and their outcomes at the transfer boundary. DESIGN: MILESTONE prospective study. SETTING: Eight European Union (EU) countries participating in the EU-funded MILESTONE study. PARTICIPANTS: The first phase (MILESTONE validation study) involved 100 adolescents (pre-transition), young adults (post-transition), parents/carers and both CAMHS and AMHS clinicians. The second phase (MILESTONE cohort study and nested cluster randomised trial) involved over 1000 young people. RESULTS: The development of the TRAM began with a literature review on transitioning and a review of important items regarding transition by a panel of 34 mental health experts. A list of 64 items of potential importance were identified, which together comprised the TRAM. The psychometric properties of the different versions of the TRAM were evaluated and showed that the TRAM had good reliability for all versions and low-to-moderate correlations when compared with other established instruments and a well-defined factor structure. The main results of the cohort study with the nested cluster randomised trial are not reported. CONCLUSION: The TRAM is a reliable instrument for assessing transition readiness and appropriateness. It highlighted the barriers to a successful transition and informed clinicians, identifying areas which clinicians on both sides of the transfer boundary can work on to ease the transition for the young person. TRIAL REGISTRATION NUMBER: ISRCTN83240263 (Registered 23 July 2015), NCT03013595 (Registered 6 January 2017); Pre-results.

The "Obiettivo Antibiotico" Campaign on Prudent Use of Antibiotics in Sicily, Italy: The Pilot Phase.

The issue of antimicrobial resistance (AMR) is a focus of the World Health Organization, which proposes educational interventions targeting the public and healthcare professionals. Here, we present the first attempt at a regionwide multicomponent campaign in Sicily (Italy), called "Obiettivo Antibiotico", which aims to raise the awareness of prudent use of antibiotics in the public and in healthcare professionals. The campaign was designed by an interdisciplinary academic team, and an interactive website was populated with different materials, including key messages, letters, slogans, posters, factsheets, leaflets, and videos. The campaign was launched in November 2018 and, as of 21 December 2018, the website had a total of 1159 unique visitors, of which 190 became champions by pledging to take simple actions to support the fight against AMR. Data from social media showed that the audience was between 18 and 54 years of age, with a high proportion of female participants (64%). Interestingly, the LinkedIn page received more than 1200 followers, and Facebook 685 followers. The number of actions taken (pledges) by the audience was 458, evenly divided between experts (53%) and the general public (47%). Additional efforts are needed to reach more people, thus future efforts should focus on further promotion within the Sicilian region to sustain the engagement with the campaign.

Development of a novel fully-human anti-CD123 antibody to target acute myeloid leukemia.

Monoclonal antibodies are being considered as biopharmaceuticals for the in vivo targeting of acute myeloid leukemia. Here we describe the generation and characterization of a fully-human monoclonal antibody specific to CD123, a surface marker which is overexpressed in a variety of hematological disorders, including acute myeloid leukemia. The cloning and expression of the extracellular portion of CD123 as recombinant Fc fusion allowed the selection and affinity maturation of a human antibody, called H9, which specifically recognized the cognate antigen in biochemical assays and on leukemic cells. The H9 antibody and a previously-described anti-CD123 antibody (CSL362) were reformatted into full immunoglobulin human IgG1 formats, including a variant bearing S293D and I332E mutations to enhance antibody-dependent cell-mediated cytotoxicity (ADCC). The two antibodies recognized different epitopes on the surface of the N-terminal domain of CD123, as revealed by crystallography and SPOT analysis. Both H9 and CSL362 in full immunoglobulin format were able to selectively kill leukemic cells in in vitro ADCC assays, performed both with cell lines and with patient-derived AML blasts. Further, the two antibodies, when reformatted as bispecific BiTE™ reagents by fusion with the anti-CD3 scFv(OKT3) antibody fragment, induced selective killing of AML blasts by patient-derived, autologous T-cells in an in vitro setting, but BiTE(CSL362/OKT3) exhibited a 10-fold higher potency compared to BiTE(H9/OKT3). The availability of two classes of CD123-specific biopharmaceuticals, capable of redirecting the cytolytic activity of NK cells and T cells against AML blasts, may enable novel interventional strategies and combination opportunities for the treatment of AML.

From local development policies to strategic planning-Assessing continuity in institutional coalitions.

In the last two decades, EU policies have had a fundamental role in orienting regional/local development. The objective of this work is set in this context as it intends to analyze the local development programs activated in Sicily in the last three programming periods. The main aim is to explore whether the EU partnership principle influenced cooperation among local actors, assessing the continuity of local institutional coalition in managing different local development programs within the regional development policy system. We focus, in particular, on Strategic Plans (SP) promoted in Sicily in the transition phase between the 2000-2006 and the 2007-2013 periods.

Combining optimization and machine learning techniques for genome-wide prediction of human cell cycle-regulated genes.

MOTIVATION: The identification of cell cycle-regulated genes through the cyclicity of messenger RNAs in genome-wide studies is a difficult task due to the presence of internal and external noise in microarray data. Moreover, the analysis is also complicated by the loss of synchrony occurring in cell cycle experiments, which often results in additional background noise. RESULTS: To overcome these problems, here we propose the LEON (LEarning and OptimizatioN) algorithm, able to characterize the 'cyclicity degree' of a gene expression time profile using a two-step cascade procedure. The first step identifies a potentially cyclic behavior by means of a Support Vector Machine trained with a reliable set of positive and negative examples. The second step selects those genes having peak timing consistency along two cell cycles by means of a non-linear optimization technique using radial basis functions. To prove the effectiveness of our combined approach, we use recently published human fibroblasts cell cycle data and, performing in vivo experiments, we demonstrate that our computational strategy is able not only to confirm well-known cell cycle-regulated genes, but also to predict not yet identified ones. AVAILABILITY AND IMPLEMENTATION: All scripts for implementation can be obtained on request.

Interaction between ticlopidine or warfarin or cardioaspirin with a highly standardized deterpened Ginkgo biloba extract (VR456) in rat and human.

Ginkgo biloba is available in Europe as an over-the-counter drug and it is reported to cause hemorrhage when co-administered with other anti-platelet agents. We set out to study the interactions of ticlopidine with Ginkgo biloba extract or VR456, a new highly standardized deterpened extract from Ginkgo biloba leaves. Male Wistar rats were used to study the effects of ticlopidine (50-100 mg/kg/day), given alone and in combination for 5 days with Ginkgo biloba extract (50 mg/kg/day) or VR456 (50 mg/kg/day), on bleeding time and ex vivo ADP-induced platelet aggregation measurements. In addition, human studies were performed with the compounds under investigation. Combined treatment of ticlopidine and undeterpened Ginkgo biloba extract increased anti-platelet effect and prolonged the bleeding time in the rat. On the contrary, the combination treatment of ticlopidine and VR456 increased anti-platelet effect but did not prolong bleeding time. Moreover, daily administration of 360 mg of VR456 for 14 days to ticlopidine-treated humans did not highlight any unwanted effect and did not alter PT/INR and PTT parameters. Same results have been also obtained in warfarin or in cardioaspirin-treated patients. These data point out the clear role played by the terpenoid, PAF-antagonist fraction of Ginkgo biloba extract in affecting bleeding risk in anticoagulant-treated subjects and suggest VR456 as a possible option treatment in geriatric people subjected to anticoagulant treatment where the use of standard Ginkgo biloba extracts are discouraged.

Schnitzler's syndrome: monoclonal gammopathy associated with chronic urticaria.

Schnitzler's syndrome (SS) is defined by monoclonal gammopathy and chronic urticaria combined with at least two of the following features: fever, arthralgia or arthritis, bone pain, hepato- and/or splenomegaly, palpable lymph nodes, elevated ESR, and leukocytosis. We report a 49-year-old man with monoclonal IgM gammopathy and a 4-year history of recurrent urticarial rash, unexplained fever and arthralgias. The skin biopsy from an acute lesion revealed perivascular lymphocytic infiltrates consisting of CD4+ and CD8+ T lymphocytes. To our knowledge, this is the first report of an immunophenotypic characterization of skin infiltrates in SS. A lower CD4+/CD8+ ratio of circulating T lymphocytes was also detected. SS usually has a benign course, but in 15% of patients a lymphoproliferative disorder develops.

Maillard reaction products inhibit oxidation of human low-density lipoproteins in vitro.

Dietary intake of antioxidants has been associated with a reduced risk of cardiovascular diseases, which is very likely caused by their capability of prevent oxidation of low-density lipoproteins (LDL). During food processing and storage, substances with antioxidative properties are formed by Maillard reactions. In this study, the activity of Maillard products to inhibit copper-induced oxidation of human LDL in vitro was investigated. d-Glucose was heated with an equimolar amount of glycine, l-lysine, or l-arginine, for 1 h under reflux. The increase of the antioxidative activity (AOA) of the Maillard mixtures was highly significant compared to the control mixtures. Additionally, two defined Maillard products with amino reductone structure were tested. 3-Hydroxy-4-(morpholino)-3-buten-2-one (1) and amino hexose reductone (2) showed a significant and dose dependent AOA. Compound 1 was about half as active as ascorbic acid, which served as positive control. Thus, it can be concluded that Maillard products, particularly those with amino reductone structure, have the strong potential to inhibit LDL oxidation.