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R-loops are three-stranded RNA-DNA hybrid structures that play important regulatory roles, but excessive or deregulated R-loops formation can trigger DNA damage and genome instability. Digestion of R-loops is mainly relying on the action of two specialized ribonucleases: RNaseH1 and RNaseH2. RNaseH2 is the main enzyme carrying out the removal of misincorporated rNMPs during DNA replication or repair, through the Ribonucleotide Excision Repair (RER) pathway. We have recently shown that the human RNA helicase DDX3X possessed RNaseH2-like activity, being able to substitute RNaseH2 in reconstituted RER reactions. Here, using synthetic R-loop mimicking substrates, we could show that human DDX3X alone was able to both displace and degrade the ssRNA strand hybridized to DNA. Moreover, DDX3X was found to physically interact with human RNaseH2. Such interaction suppressed the nuclease and helicase activities of DDX3X, but stimulated severalfold the catalytic activity of the trimeric RNaseH2, but not of RNaseH1. Finally, silencing of DDX3X in human cells caused accumulation of RNA-DNA hybrids and phosphorylated RPA foci. These results support a role of DDX3X as a scaffolding protein and auxiliary factor for RNaseH2 during R-loop degradation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hepatocellular carcinoma (HCC) is the most common type of liver solid tumor and the second leading cause of cancer-related deaths worldwide. Although new treatment options have been recently approved, the development of tumor resistance and the poor prognosis for advanced HCC make the current standard of care unsatisfying. In this scenario, the non-receptor tyrosine kinase (TK) c-Src emerged as a promising target for developing new anti-HCC agents. Our group reported a large library of pyrazolo[3,4-d]pyrimidines active as potent c-Src inhibitors. Starting from these data, we applied a molecular hybridization approach to combine the in-house pyrazolo[3,4-d]pyrimidine SI192 with the approved TK inhibitor (TKI) dasatinib, with the aim of identifying a new generation of Src inhibitors. Enzymatic results prompted us to design second-generation compounds with a better binding profile based on a hit optimization protocol comprised of molecular modeling and on-paper rational design. This investigation led to the identification of a few nanomolar Src inhibitors active toward two HCC cell lines (HepG2 and HUH-7) selected according to their high and low c-Src expression, respectively. In particular, 7e showed an IC 50 value of 0.7 nM toward Src and a relevant antiproliferative efficacy on HepG2 cells after 72h (IC 50  = 2.47 μM). Furthermore, 7e exhibited a cytotoxic profile better than dasatinib. The ADME profile suggested that 7e deserves further investigation as a promising TKI in cancer therapies. Finally, 7e's ability to inhibit HepG2 cell proliferation, elicit an irreversible cytotoxic effect, arrest cellular migration, and induce apoptotic-mediated cell death was assessed., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Emmanuele Crespan reports financial support was provided by Italian Association for Cancer Research. Emmanuele Crespan reports financial support was provided by MIUR Projects PRIN 2022. Giovanni Maga reports financial support was provided by Italian Association for Cancer Research. Giovanni Maga reports financial support was provided by MIUR Projects PRIN 2022. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Cancer cells exhibit metabolic plasticity to meet oncogene-driven dependencies while coping with nutrient availability. A better understanding of how systemic metabolism impacts the accumulation of metabolites that reprogram the tumor microenvironment (TME) and drive cancer could facilitate development of precision nutrition approaches. Using the Hi-MYC prostate cancer mouse model, we demonstrated that an obesogenic high-fat diet (HFD) rich in saturated fats accelerates the development of c-MYC-driven invasive prostate cancer through metabolic rewiring. Although c-MYC modulated key metabolic pathways, interaction with an obesogenic HFD was necessary to induce glycolysis and lactate accumulation in tumors. These metabolic changes were associated with augmented infiltration of CD206+ and PD-L1+ tumor-associated macrophages (TAM) and FOXP3+ regulatory T cells, as well as with the activation of transcriptional programs linked to disease progression and therapy resistance. Lactate itself also stimulated neoangiogenesis and prostate cancer cell migration, which were significantly reduced following treatment with the lactate dehydrogenase inhibitor FX11. In patients with prostate cancer, high saturated fat intake and increased body mass index were associated with tumor glycolytic features that promote the infiltration of M2-like TAMs. Finally, upregulation of lactate dehydrogenase, indicative of a lactagenic phenotype, was associated with a shorter time to biochemical recurrence in independent clinical cohorts. This work identifies cooperation between genetic drivers and systemic metabolism to hijack the TME and promote prostate cancer progression through oncometabolite accumulation. This sets the stage for the assessment of lactate as a prognostic biomarker and supports strategies of dietary intervention and direct lactagenesis blockade in treating advanced prostate cancer., Significance: Lactate accumulation driven by high-fat diet and MYC reprograms the tumor microenvironment and promotes prostate cancer progression, supporting the potential of lactate as a biomarker and therapeutic target in prostate cancer. See related commentary by Frigo, p. 1742., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: Family and community nurses (FCNs) play a crucial role in delivering primary care to patients within their homes and communities. A key aspect of their role involves various health interventions, which are influenced by their unique competencies, such as health promotion, advanced clinical knowledge, and strong interpersonal skills. However, it is essential to understand which specific health outcomes these interventions impact to better understand the relationship between FCNs' skills and the health results., Objective: This study aims to outline the steps we will take to develop a set of core outcomes. These outcomes will be particularly sensitive to the health interventions carried out by FCNs, providing a clearer picture of their practice's impact., Methods: A Delphi survey will be used for this research, conducted from January to December 2024. The process will involve 5 steps and input from 3 stakeholder categories. These stakeholders will help identify a preliminary list of outcomes that will form the basis of our core outcome set (COS)., Results: This guideline will be beneficial for a wide range of stakeholders involved in COS development, including COS developers, trialists, systematic reviewers, journal editors, policy makers, and patient groups. As of January 2024, we have successfully completed the first stage of the study, with the stakeholder group approving the reported outcomes and assigning participant lists for each stakeholder group., Conclusions: This study will provide a roadmap for identifying the key health outcomes influenced by the interventions of FCNs. The multistakeholder, multiphase approach will ensure a comprehensive and inclusive process. Ultimately, the findings will enhance our understanding of FCNs' impact on health outcomes, leading to more effective primary care strategies and policies., International Registered Report Identifier (irrid): PRR1-10.2196/51084., (©Sara Russo, Rosario Caruso, Gianluca Conte, Arianna Magon, Ida Vangone, Barbara Bascape', Giulia Maga, Malgorzata Pasek, Cristina Arrigoni. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 29.03.2024.)

Respiratory tract infections involving a variety of microorganisms such as viruses, bacteria, and fungi are a prominent cause of morbidity and mortality globally, exacerbating various pre-existing respiratory and non-respiratory conditions. Moreover, the ability of bacteria and viruses to coexist might impact the development and severity of lung infections, promoting bacterial colonization and subsequent disease exacerbation. Secondary bacterial infections following viral infections represent a complex challenge to be overcome from a therapeutic point of view. We report herein our efforts in the development of new bithiazole derivatives showing broad-spectrum antimicrobial activity against both viruses and bacteria. A series of 4-trifluoromethyl bithiazole analogues was synthesized and screened against selected viruses (hRVA16, EVD68, and ZIKV) and a panel of Gram-positive and Gram-negative bacteria. Among them, two promising broad-spectrum antimicrobial compounds (8a and 8j) have been identified: both compounds showed low micromolar activity against all tested viruses, 8a showed synergistic activity against E. coli and A. baumannii in the presence of a subinhibitory concentration of colistin, while 8j showed a broader spectrum of activity against Gram-positive and Gram-negative bacteria. Activity against antibiotic-resistant clinical isolates is also reported. Given the ever-increasing need to adequately address viral and bacterial infections or co-infections, this study paves the way for the development of new agents with broad antimicrobial properties and synergistic activity with common antivirals and antibacterials., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Introduction: mHealth apps are the most commonly used applications by women for seeking information and support for breastfeeding. The primary goal of this study is to provide a quality assessment of Italian-language mHealth apps for breastfeeding using the Italian version of the Mobile App Rating Scale (I-MARS)., Methods: A systematic search was conducted on the Apple App Store, Google Play Store, and Windows Store. Inclusion criteria were: mHealth apps available in Italian, free of charge, and focused on the theme of breastfeeding. The quality assessment of the eligible mHealth apps was carried out on the latest available version using the I-MARS, by two authors independently., Results: A total of 381 mHealth apps were identified, of which 38 mHealth apps were included and evaluated (n=9 on Google Play Store, n=29 on Apple App Store). The average total score of the objective dimension of I-MARS was found to be adequate, equal to 3,07±0,75, with a Cohen's Kappa score of 65,8%. The subjective dimension of the I-MARS, however, achieved a low average total score of 2,44±1,03, with a Cohen's Kappa score of 60,5%. In this regard, the subjective dimension of the I-MARS shows lower values compared to the subjective evaluation of the users (3,28±1,95)., Discussion: The informational quality dimension of mHealth apps was found to be lacking and/or inadequate in 80% of cases. 53% of the selected mHealth apps presented average quality scores that were adequate. The results of this study thus lay the groundwork for future recommendations for the development and proper use of mHealth apps for the protection, promotion, and support of breastfeeding.

Background: Breastfeeding self-efficacy has been proven to play a predictive role in enhancing breastfeeding initiation and continuation. Breastfeeding self-efficacy measurement tools have facilitated healthcare professionals' early identification and support of women at higher risk of early discontinuation of breastfeeding., Research Aim: The aim of this study was to assess the psychometric properties of breastfeeding self-efficacy measurement tools., Method: A systematic review was carried out in three phases. Phase One comprised a systematic literature review performed in PubMed, SCOPUS, Web of Science, and Cochrane Database of Systematic Reviews from February 2021 to January 2023, including 36 studies for final analysis. Phase Two provided a quality appraisal of the psychometric properties of each of the seven breastfeeding self-efficacy measurement tools, according to COnsensus-based Standards for the selection of health Measurement Instrument checklist (COSMIN) guidelines. Phase Three summarized and graded the overall quality of evidence using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) modified approach., Result: The included articles comprised 9,225 participants and seven breastfeeding self-efficacy measurement tools. The Breastfeeding Self-Efficacy Scale, Breastfeeding Self-Efficacy Scale - Short Form (BSES-SF), and Prenatal Breastfeeding Self-Efficacy Scale were supported by Grade A evidence sustaining their validity and reliability to assess breastfeeding self-efficacy in the continuum of maternity care. The BSES-SF is the most feasible tool in clinical practice and the most utilized internationally, available in 15 languages., Conclusion: This systematic review provided a Grade A recommendation on breastfeeding measurement tools that will be helpful both for clinical and research purposes.Registered in the International Prospective Register of Systematic Reviews (PROSPERO; CRD42021238450)., Competing Interests: Disclosures and Conflicts of InterestAll authors involved declare that they do not have conflicts of interest. There was a student/advisor relationship between Cristina Arrigoni and Giulia Maga. Cristina Arrigoni was the PhD supervisor of Giulia Maga. The collaboration between the Italian and British authors reflects only spontaneous and academic collaborations on topics where authors have a shared research focus.

Src is a non-receptor tyrosine kinase (TK) whose involvement in cancer, including glioblastoma (GBM), has been extensively demonstrated. In this context, we started from our in-house library of pyrazolo[3,4- d ]pyrimidines that are active as Src and/or Bcr-Abl TK inhibitors and performed a lead optimization study to discover a new generation derivative that is suitable for Src kinase targeting. We synthesized a library of 19 compounds, 2a-s . Among these, compound 2a (SI388) was identified as the most potent Src inhibitor. Based on the cell-free results, we investigated the effect of SI388 in 2D and 3D GBM cellular models. Interestingly, SI388 significantly inhibits Src kinase, and therefore affects cell viability, tumorigenicity and enhances cancer cell sensitivity to ionizing radiation.

This study protocol aims to describe the rationale and developmental strategy of the first study in the Italian context which aimed to define a Midwifery Interventions Classification, an evidence-based, standardized taxonomy and classification of midwifery interventions. Midwifery interventions require a specific definition, developed through a consensus-building process by stakeholders to develop the Italian taxonomy of the Midwifery Interventions Classification with the potential for international transferability, implementation, and scaling up. A multi-round Delphi study was designed between June and September 2022, and data collection is planned between February 2023 and February 2024. The developmental phase of the study is based on a literature review to select meaningful midwifery interventions from the international literature, aiming to identify an evidence-based list of midwifery interventions. This phase led to including 16 articles derived from a systematic search performed on PubMed, CINAHL, and Scopus; 164 midwifery interventions were selected from the data extraction performed on the 16 included articles. Healthcare professionals, researchers, and service users will be eligible panelists for the Delphi surveys. The protocol designed a dynamic number of consultation rounds based on the ratings and interim analysis. A nine-point Likert scoring system is designed to evaluate midwifery interventions. Attrition and attrition bias will be evaluated. The results from the study designed in this protocol will inform the development of the Italian taxonomy of the Midwifery Interventions Classification. A shared classification of midwifery interventions will support audit and quality improvement, education, and comparable data collections for research, sustaining public recognition of midwifery interventions to promote optimal maternal and newborn health.

The nucleocapsid protein Np of SARS-CoV-2 is involved in the replication, transcription, and packaging of the viral genome, but it also plays a role in the modulation of the host cell innate immunity and inflammation response. Ectopic expression of Np alone was able to induce significant changes in the proteome of human cells. The cellular RNA helicase DDX1 was among the proteins whose levels were increased by Np expression. DDX1 and its related helicase DDX3X were found to physically interact with Np and to increase 2- to 4-fold its affinity for double-stranded RNA in a helicase-independent manner. Conversely, Np inhibited the RNA helicase activity of both proteins. These functional interactions among Np and DDX1 and DDX3X highlight novel possible roles played by these host RNA helicases in the viral life cycle.

The therapeutic use of tyrosine kinase inhibitors (TKIs) represents one of the successful strategies for the treatment of glioblastoma (GBM). Pyrazolo[3,4- d ]pyrimidines have already been reported as promising small molecules active as c-Src/Abl dual inhibitors. Herein, we present a series of pyrazolo[3,4- d ]pyrimidine derivatives, selected from our in-house library, to identify a promising candidate active against GBM. The inhibitory activity against c-Src and Abl was investigated, and the antiproliferative profile against four GBM cell lines was studied. For the most active compounds endowed with antiproliferative efficacy in the low-micromolar range, the effects toward nontumoral, healthy cell lines (fibroblasts FIBRO 2-93 and keratinocytes HaCaT) was investigated. Lastly, the in silico and in vitro ADME properties of all compounds were also assessed. Among the tested compounds, the promising inhibitory activity against c-Src and Abl ( K i 3.14 µM and 0.44 µM, respectively), the irreversible, apoptotic-mediated death toward U-87, LN18, LN229, and DBTRG GBM cell lines (IC 50 6.8 µM, 10.8 µM, 6.9 µM, and 8.5 µM, respectively), the significant reduction in GBM cell migration, the safe profile toward FIBRO 2-93 and HaCaT healthy cell lines (CC 50 91.7 µM and 126.5 µM, respectively), the high metabolic stability, and the excellent passive permeability across gastrointestinal and blood-brain barriers led us to select compound 5 for further in vivo assays.

Current therapy against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are based on the use of Remdesivir 1 , Molnupiravir 2 , and the recently identified Nirmatrelvir 3 . Unfortunately, these three drugs showed some limitations regarding potency and possible drug-drug interactions. A series of derivatives coming from a decoration approach of the privileged scaffold s-triazines were synthesized and evaluated against SAR-CoV-2. One derivative emerged as the hit of the series for its micromolar antiviral activity and low cytotoxicity. Mode of action and pharmacokinetic in vitro preliminary studies further confirm the role as candidates for a future optimization campaign of the most active derivative identified with this work.

Inhibition of c-Src is considered one of the most studied approaches to cancer treatment, with several heterocyclic compounds approved during the last 15 years as chemotherapeutic agents. Starting from the biological evaluation of an in-house collection of small molecules, indolinone was selected as the most promising scaffold. In this work, several functionalised indolinones were synthesised and their inhibitory potency and cytotoxic activity were assayed. The pharmacological profile of the most active compounds, supported by molecular modelling studies, revealed that the presence of an amino group increased the affinity towards the ATP-binding site of c-Src. At the same time, bulkier derivatizations seemed to improve the interactions within the enzymatic pocket. Overall, these data represent an early stage towards the optimisation of new, easy-to-be functionalised indolinones as potential c-Src inhibitors.

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cystic fibrosis (CF) is a genetic disease caused by loss-of-function mutations in the CFTR gene, which codes for a defective ion channel. This causes an electrolyte imbalance and results in a spiral of negative effects on multiple organs, most notably the accumulation of thick mucus in the lungs, chronic respiratory tract infections and inflammation leading to pulmonary exacerbation and premature death. Progressive decline of lung function is mainly linked to persistent or recurring infections, mostly caused by bacteria, which require treatments with antibiotics and represent one of the major life-limiting factors in subjects with CF. Treatment of such a complex disease require multiple drugs with a consequent therapeutic burden and complications caused by drug-drug interactions and rapid emergence of bacterial drug resistance. We report herein our recent efforts in developing innovative multifunctional antibiotics specifically tailored to CF by a direct action on bacterial topoisomerases and a potential indirect effect on the pulmonary mucociliary clearance mediated by ΔF508-CFTR correction. The obtained results may pave the way for the development of a simplified therapeutic approach with a single agent acting as multifunctional Antibacterial-Corrector., (© 2022 Wiley-VCH GmbH.)

RNA helicases of the DEAD-box family are involved in several metabolic pathways, from transcription and translation to cell proliferation, innate immunity and stress response. Given their multiple roles, it is not surprising that their deregulation or mutation is linked to different pathological conditions, including cancer. However, while in some cases the loss of function of a given DEAD-box helicase promotes tumor transformation, indicating an oncosuppressive role, in other contexts the overexpression of the same enzyme favors cancer progression, thus acting as a typical oncogene. The roles of two well-characterized members of this family, DDX3X and DDX5, as both oncogenes and oncosuppressors have been documented in several cancer types. Understanding the interplay of the different cellular contexts, as defined by the molecular interaction networks of DDX3X and DDX5 in different tumors, with the cancer-specific roles played by these proteins could help to explain their apparently conflicting roles as cancer drivers or suppressors.

Objective: This study aimed to develop and validate a midwifery core outcome set (M-COS) for Italian settings based on a salutogenic framework of maternity care., Design: A multi-phase and multi-method study was performed. In phase one, we conducted a literature review to identify a preliminary set of outcomes sensitive to midwifery care. In phase two, the qualitative and quantitative content validity of the M-COS was tested. Finally, in the third phase, construct validity was explored through a cross-sectional study to assess the psychometric properties of the M-COS through exploratory and confirmative factor analysis. This study was conducted from December 2019 to April 2020 in Italy., Participants: Three main groups of experts/midwives were involved. Group One (n = 10) was involved in the content validity phase, while the other two groups (Group Two and Group Three) were involved in the construct validity phase (n = 300)., Results: The M-COS includes six outcome domains and thirty-one core outcomes perceived as sensitive to midwifery care, namely: mortality and morbidity (n = 6 outcomes), childbirth (n = 3), postnatal period (n = 6), maternal health (n = 11), maternal-infant bonding (n = 3), and maternal self-care (n = 2). All domains showed good evidence of internal consistency., Conclusion: The Italian M-COS is a novel tool that will facilitate the consistent measurement of core outcomes sensitive to midwifery care from the antenatal to the postnatal period in Italian settings. This initial work will be followed by further studies, including validation by service users., Implications for Practice: The use of the M-COS in clinical practice would facilitate evidence-based data collection and thus contribute to promoting high-quality maternity care., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This study aimed to systematically synthesize evidence regarding burnout and post-traumatic stress disorder (PTSD) among nurses engaged in the frontline during the COVID-19 pandemic, highlighting their risk and protective factors. The specific literature on nurses' mental health outcomes still remains not synthesized. A systematic review was performed (PROSPERO: CRD42021227939), searching literature published in 2020 on Pubmed, Scopus, CINAHL, and PsycInfo. We quantitatively pooled means of included studies measuring burnout and PTSD with the same tools. Twenty-five studies were included in this review. Seven (3766 nurses) were included in the meta-analysis for estimating means of depersonalization and emotional exhaustion assessed using the Maslach Burnout Inventory, respectively: 7,40 (95%CI=6,00-8,80) and 22,82 (95%CI=19,24-26,41). Likely, 12 studies were used to estimate two pooled means for PTSD, one for six studies adopting the Impact of Event Scale-Revised (1551 nurses), and six adopting the PTSD Scale for DSM-5 (8547 nurses). The main risk and protective factors of both outcomes were female sex and younger age, work-related variables, and physical and mental factors, such as concerns, skin lesions from wearing personal protective equipment. This systematic review portrayed the situation described in literature during 2020 on nurses' burnout and PTSD during the COVID-19 pandemic. Although the outcomes' levels described in the included studies are diverse, the broad situation appears alarming, and supportive multi-level strategies, considering individual and system-level, should be planned to decrease the described worsening scenario within the clinical settings avoid middle and long-term negative consequences.

Over half a century since the description of the first antiviral drug, "old" re-emerging viruses and "new" emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIβ, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIβ block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIβ inhibition, the role of PI4KIIIβ in SARS-CoV-2 entry/replication is debated., (© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

The worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC 50  = 0.5-5.3 μM) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC 50  = 0.5 μM, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

DDX3X is an ATP-dependent RNA helicase that has recently attracted interest for its involvement in viral replication and oncogenic progression. Starting from hit compounds previously identified by our group, we have designed and synthesized a new series of DDX3X inhibitors that effectively blocked its helicase activity. These new compounds were able to inhibit the proliferation of cell lines from different cancer types, also in DDX3X low-expressing cancer cell lines. According to the absorption, distribution, metabolism, elimination properties, and antitumoral activity, compound BA103 was chosen to be further investigated in glioblastoma models. BA103 determined a significant reduction in the proliferation and migration of U87 and U251 cells, downregulating the oncogenic protein β-catenin. An in vivo evaluation demonstrated that BA103 was able to reach the brain and reduce the tumor growth in xenograft and orthotopic models without evident side effects. This study represents the first demonstration that DDX3X-targeted small molecules are feasible and promising drugs also in glioblastoma.

Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2'-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi-Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

We designed and synthesized 21 new indolylarylsulfones (IASs) as new HIV-1 NNRTIs. Among these, IAS 12 exhibited a remarkable antiviral activity against single and double mutants (K103N EC 50 = <0.7 nM; Y181C EC 50 = <0.7 nM; Y188L EC 50 = 21.3 nM; K103N-Y181C EC 50 = 6.2 nM), resulting equally or more active than previuosly reported IAS 6 and some approved anti-HIV-1 drugs. Docking and molecular dynamics simulations of compound 12 in complex with WT, Y181C, Y188L, K103N and K103N-Y181C RTs clarified a general binding mode that was consistent with biological results. Kinetic experiments disclosed that derivative 12 preferentially binds WT and K103N-Y181C RTs to binary and ternary complexes, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 37,000 people in Italy and has caused widespread socioeconomic disruption. Urgent measures are needed to contain and control the virus, particularly diagnostic kits for detection and surveillance, therapeutics to reduce mortality among the severely affected, and vaccines to protect the remaining population. Here we discuss the potential role of plant molecular farming in the rapid and scalable supply of protein antigens as reagents and vaccine candidates, antibodies for virus detection and passive immunotherapy, other therapeutic proteins, and virus-like particles as novel vaccine platforms. We calculate the amount of infrastructure and production capacity needed to deal with predictable subsequent waves of COVID-19 in Italy by pooling expertise in plant molecular farming, epidemiology and the Italian health system. We calculate the investment required in molecular farming infrastructure that would enable us to capitalize on this technology, and provide a roadmap for the development of diagnostic reagents and biopharmaceuticals using molecular farming in plants to complement production methods based on the cultivation of microbes and mammalian cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Lico, Santi, Baschieri, Noris, Marusic, Donini, Pedrazzini, Maga, Franconi, Di Bonito and Avesani.)

As intracellular parasites, viruses hijack the host cell metabolic machinery for their replication. Among other cellular proteins, the DEAD-box (DDX) RNA helicases have been shown to be hijacked by coronaviruses and to participate in essential DDX-mediated viral replication steps. Human DDX RNA helicases play essential roles in a broad array of biological processes and serve multiple roles at the virus-host interface. The viral proteins responsible for DDX interactions are highly conserved among coronaviruses, suggesting that they might also play conserved functions in the SARS-CoV-2 replication cycle. In this review, we provide an update of the structural and functional data of DDX as possible key factors involved in SARS-CoV-2 hijacking mechanisms. We also attempt to fill the existing gaps in the available structural information through homology modeling. Based on this information, we propose possible paths exploited by the virus to replicate more efficiently by taking advantage of host DDX proteins. As a general rule, sequestration of DDX helicases by SARS-CoV-2 is expected to play a pro-viral role in two ways: by enhancing key steps of the virus life cycle and, at the same time, by suppressing the host innate immune response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Squeglia, Romano, Ruggiero, Maga and Berisio.)

Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5' of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols β and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)