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Study Design: Prospective observational study., Objective: The population of patients with advanced stages of cancer, including metastatic spinal disease, is growing because of better treatment options allowing for longer control of disease. The main goal of treatment for these patients is to improve or maintain their health-related quality of life (HRQOL). A spine oncology-specific outcome measure has been developed by the Spine Oncology Study Group and validated through international studies. We proposed to translate and validate the questionnaire in Italian language., Methods: The cross-cultural adaptation of the questionnaire has been performed according to guidelines previously proposed. After this process, an observational prospective study has been conducted to validate the efficacy of SOSGOQ in Italian language. SOSGOQ has been compared to SF-36 (Short Form Health Survey-36), a generic validated questionnaire to assess HRQOL. Starting from January 2020, SOSGOQ and SF-36 questionnaires were auto-administered to 150 patients affected by spinal metastases who provided written informed consent for study participation., Results: The confirmatory factor analysis on the 4 domains examined showed a good model fit (comparative fit index, .95; RMSEA .07 (90% CI, .05-.09) and SRMR, .05), endorsing construct validity. The analysis of concurrent validity demonstrated strong correlation for physical function, pain and mental health domains with the corresponding domain scores of SF-36. The reliability across item was high with a Cronbach's alpha coefficient of .91., Conclusions: The statistical analysis of the results will allow to accept the Italian version of SOSGOQ as a specific and efficient tool to measure HRQOL in Italian-speaking patients affected by spinal metastases.

Background: SARS-CoV-2 infections have been associated with the onset of thyroid disorders like classic subacute thyroiditis (SAT) or atypical SAT upon severe COVID disease (COV-A-SAT). Little is known about thyroid anti-viral immune responses., Objectives: To define the role of T-cells in COV-A-SAT., Methods: T-cells from COV-A-SAT patients were analyzed by multi-dimensional flow cytometry, UMAP and DiffusionMap dimensionality reduction and FlowSOM clustering. T-cells from COVID-naïve healthy donors, patients with autoimmune thyroiditis (ATD) and with SAT following COVID vaccination were analyzed as controls. T-cells were analyzed four and eight months post-infection in peripheral blood and in thyroid specimen obtained by ultrasound-guided fine needle aspiration. SARS-COV2-specific T-cells were identified by cytokine production induced by SARS-COV2-derived peptides and with COVID peptide-loaded HLA multimers after HLA haplotyping., Results: COV-A-SAT was associated with HLA-DRB1*13 and HLA-B*57. COV-A-SAT patients contained activated Th1- and cytotoxic CD4+ and CD8+ effector cells four months post-infection, which acquired a quiescent memory phenotype after eight months. Anti-SARS-CoV-2-specific T-cell responses were readily detectable in peripheral blood four months post-infection, but were reduced after eight months. CD4+ and CD8+ tissue-resident memory cells (TRM) were present in the thyroid, and circulating CXCR3+T-cells identified as their putative precursors. SARS-CoV-2-specific T-cells were enriched in the thyroid, and acquired a TRM phenotype eight months post-infection., Conclusions: The association of COV-A-SAT with specific HLA haplotypes suggests a genetic predisposition and a key role for T-cells. COV-A-SAT is characterized by a prolonged systemic anti-viral effector T-cell response and the late generation of COVID-specific TRM in the thyroid target tissue., Competing Interests: Declaration of Competing Interest We declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment., Competing Interests: The authors declare no conflict of interest.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CF transmembrane conductance regulator (CFTR) is associated to misfolding and defective gating of the mutant channel. One of the most promising CF drug targets is the ubiquitin ligase RNF5, which promotes F508del-CFTR degradation. Recently, the first ever reported inhibitor of RNF5 was discovered, i.e., the 1,2,4-thiadiazol-5-ylidene inh-2 . Here, we designed and synthesized a series of new analogues to explore the structure-activity relationships (SAR) of this class of compounds. SAR efforts ultimately led to compound 16 , which showed a greater F508del-CFTR corrector activity than inh-2 , good tolerability, and no toxic side effects. Analogue 16 increased the basal level of autophagy similar to what has been described with RNF5 silencing. Furthermore, co-treatment with 16 significantly improved the F508del-CFTR rescue induced by the triple combination elexacaftor/tezacaftor/ivacaftor in CFBE41o - cells. These findings validate the 1,2,4-thiadiazolylidene scaffold for the discovery of novel RNF5 inhibitors and provide evidence to pursue this unprecedented strategy for the treatment of CF.

One-fourth to one-third of women with endometriosis receiving first-line hormonal treatment lacks an adequate response in terms of resolution of painful symptoms. This phenomenon has been ascribed to "progesterone resistance", an entity that was theorized to explain the gap between the ubiquity of retrograde menstruation and the 10% prevalence of endometriosis among women of reproductive age.Nevertheless, the hypothesis of progesterone resistance is not free of controversies. As our understanding of endometriosis is increasing, authors are starting to set aside the traditionally accepted tunnel vision of endometriosis as a strictly pelvic disease, opening to a more comprehensive perspective of the condition. The question is: are patients not responding to first-line treatment because they have an altered signaling pathway for such treatment, or have we been overlooking a series of other pain contributors which may not be resolved by hormonal therapy?Finding an answer to this question is evermore impelling, for two reasons mainly. Firstly, because not recognizing the presence of further pain contributors adds a delay in treatment to the already existing delay in diagnosis of endometriosis. This may lead to chronicity of the untreated pain contributors as well as causing adverse consequences on quality of life and psychological health. Secondly, misinterpreting the consequences of untreated pain contributors as a non-response to standard first-line treatment may imply the adoption of second-line medical therapies or of surgery, which may entail non-negligible side effects and may not be free of physical, psychological and socioeconomic repercussions.The current narrative review aims at providing an overview of all the possible pain contributors in endometriosis, ranging from those strictly organic to those with a greater neuro-psychological component. Including these aspects in a broader psychobiological approach may provide useful suggestions for treating those patients who report persistent pain symptoms despite receiving first-line hormonal medical treatment., (© 2023. The Author(s).)

Colorectal cancer (CRC) is a leading cause of cancer-associated death. In the tumor site, the interplay between effector immune cells and cancer cells determines the balance between tumor elimination or outgrowth. We discovered that the protein TMEM123 is over-expressed in tumour-infiltrating CD4 and CD8 T lymphocytes and it contributes to their effector phenotype. The presence of infiltrating TMEM123+ CD8+ T cells is associated with better overall and metastasis-free survival. TMEM123 localizes in the protrusions of infiltrating T cells, it contributes to lymphocyte migration and cytoskeleton organization. TMEM123 silencing modulates the underlying signaling pathways dependent on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, which are required for synaptic force exertion. Using tumoroid-lymphocyte co-culture assays, we found that lymphocytes form clusters through TMEM123, anchoring to cancer cells and contributing to their killing. We propose an active role for TMEM123 in the anti-cancer activity of T cells within tumour microenvironment., Competing Interests: Author Renata Grifantini is currently employed by CheckmAb Srl. The remaining 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 © 2023 Pesce, Cordiglieri, Bombaci, Eppenberger-Castori, Oliveto, Manara, Crosti, Ercan, Coto, Gobbini, Campagnoli, Donnarumma, Martinelli, Bevilacqua, De Camilli, Gruarin, Sarnicola, Cassinotti, Baldari, Viale, Biffo, Abrignani, Terracciano and Grifantini.)

Background: Cystic fibrosis is caused by mutations impairing expression, trafficking, stability and/or activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The G1244E mutation causes a severe gating defect that it is not completely rescued by ivacaftor but requires the use of a second compound (a co-potentiator). Recently, it has been proposed that the corrector elexacaftor may act also as a co-potentiator., Methods: By using molecular, biochemical and functional analyses we performed an in-depth characterization of the G1244E-CFTR mutant in heterologous and native cell models., Results: Our studies demonstrate that processing and function of the mutant protein, as well as its pharmacological sensitivity, are markedly dependent on cell background. In heterologous expression systems, elexacaftor mainly acted on G1244E-CFTR as a co-potentiator, thus ameliorating the gating defect. On the contrary, in the native nasal epithelial cell model, elexacaftor did not act as a co-potentiator, but it increased mature CFTR expression possibly by improving mutant's defective stability at the plasma membrane., Conclusions: Our study highlights the importance of the cell background in the evaluation of CFTR modulator effects. Further, our results draw attention to the need for the development of novel potentiators having different mechanisms with respect to ivacaftor to improve channel activity for mutants with severe gating defect., Competing Interests: Conflicts of Interest The authors declare no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

S737F is a Cystic Fibrosis (CF) transmembrane conductance regulator (CFTR) missense variant. The aim of our study was to describe the clinical features of a cohort of individuals carrying this variant. In parallel, by exploiting ex vivo functional and molecular analyses on nasal epithelia derived from a subset of S737F carriers, we evaluated its functional impact on CFTR protein as well as its responsiveness to CFTR modulators. We retrospectively collected clinical data of all individuals bearing at least one S737F CFTR variant and followed at the CF Centre of Tuscany region (Italy). Nasal brushing was performed in cooperating individuals. At study end clinical data were available for 10 subjects (mean age: 14 years; range 1-44 years; 3 adult individuals). Five asymptomatic subjects had CF, 2 were CRMS/CFSPID and 3 had an inconclusive diagnosis. Ex vivo analysis on nasal epithelia demonstrated different levels of CF activity. In particular, epithelia derived from asymptomatic CF subjects and from one of the subjects with inconclusive diagnosis showed reduced CFTR activity that could be rescued by treatment with CFTR modulators. On the contrary, in the epithelia derived from the other two individuals with an inconclusive diagnosis, the CFTR-mediated current was similar to that observed in epithelia derived from healthy donors. In vitro functional and biochemical analysis on S737F-CFTR expressed in immortalized bronchial cells highlighted a modest impairment of the channel activity, that was improved by treatment with ivacaftor alone or in combination with tezacaftor/elexacaftor. Our study provide evidence towards the evaluation of CFTR function on ex vivo nasal epithelial cell models as a new assay to help clinicians to classify individuals, in presence of discordance between clinical picture, sweat test and genetic profile.

Atomic layer deposition (ALD)-grown zirconia films underwent irradiation by 100 keV protons at fluences ranging from 1⋅10 12 p + / c m 2 through 5⋅10 14 p + / c m 2 . The induced structural damage was modeled using the stopping and range of ions in matter (SRIM) and compared with the change of the optical properties characterized by ellipsometry, spectrophotometry, and x-ray reflectometry. Proton-induced contamination of the optical surface due to deposition of a carbon-rich layer was determined. Correct estimation of the substrate damage was shown to be critical for reliable evaluation of the optical constants of the irradiated films. The ellipsometric angle Δ is shown to be sensitive to both the presence of the buried damaged zone in the irradiated substrate and the contamination layer on the surface of the samples. The complex chemistry in carbon-doped zirconia accommodating over-stoichiometric oxygen is discussed, along with the impact of the film composition change on the refractive index of the irradiated films.

Many priority pollutants are concentrated in the environment due to human activity. Most are highly toxic to various organisms, including endocrine disruptors EDCs, aromatic polycyclic hydrocarbons PAHs, pesticides. While the effects of single and binary exposure have been widely explored, several pollutants can be simultaneously present at the same time in the environment, in in more or less polluted matrices. Effective pollution control requires the presence and sources of contamination to be identified. Previously we used Drosophila melanogaster to investigate metal pollution. Here, we re-used Drosophila to identify the biomarkers of pollution, and to determine if they can be used for specific types of pollution. Single and combined exposure of Bis(2-ethylhexyl) phthalate (DEHP), bisphenol A, nonylphenol, benzo(a)pyrene, and glyphosate was investigated. The impact of these pollutants on post-embryonic development and the expression pattern of 38 molecular targets were examined using qPCR. During single exposure, different profiles were observed at the molecular level. In complex mixtures, the expression profile resembled that of bisphenol A. In contrast, relatively specific gene expression profiles were obtained for the effects of each pollutant separately. While direct pollutant-gene profiling remains difficult in mixtures, molecular biology analyses enhance pollution monitoring, and should be incorporated in toxicological studies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: David Siaussat reports financial support was provided by Sorbonne University., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood., Methods: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach., Findings: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation., Interpretation: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection., Funding: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola., Competing Interests: Declaration of interests The authors report no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

After a rich history in medicine, randomized control trials (RCTs), both simple and complex, are in increasing use in other areas, such as web-based A/B testing and planning and design of decisions. A main objective of RCTs is to be able to measure parameters, and contrasts in particular, while guarding against biases from hidden confounders. After careful definitions of classical entities such as contrasts, an algebraic method based on circuits is introduced which gives a wide choice of randomization schemes., Competing Interests: Conflict of interestThe authors declare no conflicts of interest associated with this manuscript., (© The Institute of Statistical Mathematics, Tokyo 2022. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

The aim of this work is to investigate the possibility of engineering desired molecular sp 2 structures in graphene oxide, via controlled oxidation of graphite powder, in order to achieve tunable chemical and microstructural properties useful for optoelectronics or sensing applications. Specifically, GO powder is obtained by a modified Hummers method, by using different concentrations of potassium permanganate (KMnO 4 ) in order to change the number of oxygen functionalities in the graphitic structure. Then, a successive alkaline treatment is performed by increasing the KOH concentration. The alkaline treatment induces a noticeable variation of the GO microstructural and chemical properties, which is accompanied by a strong enhancement of photoluminecence. PL and PLE measurements reveal that the configuration of electronic energy states changes as a function of the KMnO 4 and KOH concentration, by introducing further electronic n levels available for n→π* transitions. In particular, the number of sp 2 small domains embedded among oxygen-sp 3 domains, increases under the KOH treatment, due to the addition of OH groups. Most of these sp 2 domains are lifted-off from GO and thrown away in the surnatant giving it high blue photoluminescence excited at λ exc ∼ 319 nm. The employ of combined spectroscopy techniques allows a deep investigation of the microstructural and chemical changes induced by chemical treatments, opening the way to the fine tuning of GO functional properties.

En bloc resection consists in the surgical removal of a vertebral tumor in a single piece with a sufficient margin, to improve survival and reduce recurrence rate. This procedure is technically demanding and correlates with a high complication rate. The purpose of this study is to investigate the risk factors for complications in en bloc resection and evaluate if benefits overcome the risks in term of overall survival. We retrospectively analyzed prospectively collected data of patients treated with en bloc resection between 1980 and 2021. Complications were classified according to SAVES-V2. Overall Survival was estimated using Kaplan-Meier method. A total of 149 patients out of 298 (50%) suffered from at least one complication. Moreover, 220 adverse events were collected (67 intraoperative, 82 early post-operative, 71 late post-operative), 54% of these were classified as grade 3 (in a severity scale from 1 to 6). Ten years overall survival was 67% (95% CI 59-74). The occurrence of relapses was associated to an increased risk of mortality with OR 3.4 (95% CI 2.1-5.5), while complications did not affect the overall survival. Despite a high complication rate, en bloc resection allows for a better control of disease and should be performed in selected patients by specialized surgeons.

Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.

The use of three-dimensional (3D)-printed custom-made implants is spreading in the orthopedics field for the reconstruction of bone losses or for joint replacement, thanks to their unparalleled versatility. In particular, this novel technology opens new perspectives to formulate custom-made fixation strategies for the upper cervical region, sacrum and pelvis, where reconstruction is challenging. We report and analyze the literature concerning upper cervical reconstruction with 3D-printed personalized implants after tumor surgery, and discuss two cases of patients where this technology was used to reconstruct the anterior column after extracapsular debulking of C2 recurrent chordoma at our institution.

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.)

COVID-19 has proven to be particularly serious and life-threatening for patients presenting with pre-existing pathologies. Patients affected by rheumatic musculoskeletal disease (RMD) are likely to have impaired immune responses against SARS-CoV-2 infection due to their compromised immune system and the prolonged use of disease-modifying anti-rheumatic drugs (DMARDs), which include conventional synthetic (cs) DMARDs or biologic and targeted synthetic (b/ts) DMARDs. To provide an integrated analysis of the immune response following SARS-CoV-2 infection in RMD patients treated with different classes of DMARDs we carried out an immunological analysis of the antibody responses toward SARS-CoV-2 nucleocapsid and RBD proteins and an extensive immunophenotypic analysis of the major immune cell populations. We showed that RMD individuals under most DMARD treatments mount a sustained antibody response to the virus, with neutralizing activity. In addition, they displayed a sizable percentage of effector T and B lymphocytes. Among b-DMARDs, we found that anti-TNFα treatments are more favorable drugs to elicit humoral and cellular immune responses as compared to CTLA4-Ig and anti-IL6R inhibitors. This study provides a whole picture of the humoral and cellular immune responses in RMD patients by reassuring the use of DMARD treatments during COVID-19. The study points to TNF-α inhibitors as those DMARDs permitting elicitation of functional antibodies to SARS-CoV-2 and adaptive effector populations available to counteract possible re-infections., Competing Interests: Author EM is employed by Dia.Pro (Diagnostic Bioprobes srl, Milan, Italy). The remaining 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 © 2022 Favalli, Favalli, Gobbini, Zagato, Bombaci, Maioli, Pesce, Donnici, Gruarin, Biggioggero, Curti, Manganaro, Marchisio, Bevilacqua, Martinovic, Fabbris, Sarnicola, Crosti, Marongiu, Granucci, Notarbartolo, Bandera, Gori, De Francesco, Abrignani, Caporali and Grifantini.)

Developing strategies against the SARS-CoV-2 is currently a main research subject. SARS-CoV-2 infects host cells by binding to human ACE2 receptors. Both, virus and ACE2, are highly glycosylated, and exploiting glycans of the SARS-CoV-2 envelope as binding sites for ACE2 represents a virus strategy for attacking the human host. We report here that a family of mannose-binding synthetic carbohydrate-binding agents (CBAs) inhibits SARS-CoV-2 infection, showing broad neutralizing activity vs. several variants of the spike protein. Preliminary tests indicated that the investigated CBAs interact with the spike protein rather than with ACE2. For a lead compound (IDS060), which has been selected among others for its lack of cytotoxicity, evidence of binding to the RBD of the spike protein has been found by NMR experiments, while competitive binding assays in the presence of IDS060 showed inhibition of binding of RBD to hACE2, although neutralizing activity was also observed with variants showing reduced or depleted binding., Competing Interests: The authors declare no competing interests, (© 2022 The Authors.)

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.

Objectives: Given the high occurrence of asymptomatic subsets, the true prevalence of SARS-CoV-2 infection in rheumatic patients is still underestimated. This study aims to evaluate the seroprevalence of SARS-CoV-2 antibodies in rheumatic musculoskeletal diseases (RMD) patients receiving immunomodulatory drugs., Methods: All consecutive patients with rheumatoid arthritis or spondyloarthritis receiving disease-modifying antirheumatic drugs (DMARDs) evaluated between 4th May and 16th June 2020 were included. All participants were tested for anti-SARS-CoV-2 antibodies (IgG, IgM, IgA) by ELISA and were questioned about previous COVID-19 symptoms and clinical course. Results were compared with healthy population from the same region and with a control group of healthy subjects diagnosed with confirmed COVID-19., Results: The study population includes 358 patients. The overall prevalence of anti-SARS-CoV-2 antibodies (18.4%) was higher than prevalence rate based on swab-positivity (1.12%) or clinically suspected cases (10.6%), but consistent with seroprevalence observed in the healthy population. Among seropositive patients 58% were asymptomatic. Mean anti-SARS-CoV-2 titer was comparable with the control group. No differences in seroprevalence were observed according to age, sex, rheumatic disease and treatment with conventional, biologic or targeted synthetic DMARDs, whereas glucocorticoids and comorbidities resulted in higher seroprevalence rate., Conclusions: The results of this study are reassuring about the low impact of RMDs and immunomodulatory therapies on the risk and clinical course of COVID-19 and on humoral immune response to SARS-CoV-2 infection., Competing Interests: EM is employed by Dia.Pro, Diagnostic Bioprobes Srl. The remaining 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 © 2022 Favalli, Gobbini, Bombaci, Maioli, Biggioggero, Pesce, Favalli, Martinovic, Fabbris, Marchisio, Bandiera, Gori, Abrignani, Grifantini and Caporali.)

Cystic fibrosis (CF) is a genetic disease affecting the lungs and pancreas and causing progressive damage. CF is caused by mutations abolishing the function of CFTR, a protein whose role is chloride's mobilization in the epithelial cells of various organs. Recently a therapy focused on small molecules has been chosen as a main approach to contrast CF, designing and synthesizing compounds acting as misfolding (correctors) or defective channel gating (potentiators). Multi-drug therapies have been tested with different combinations of the two series of compounds. Previously, we designed and characterized two series of correctors, namely, hybrids, which were conceived including the aminoarylthiazole (AAT) core, merged with the benzodioxole carboxamide moiety featured by VX-809. In this paper, we herein proceeded with molecular modeling studies guiding the design of a new third series of hybrids, featuring structural variations at the thiazole moiety and modifications on position 4. These derivatives were tested in different assays including a YFP functional assay on models F508del-CFTR CFBE41o-cells, alone and in combination with VX-445, and by using electrophysiological techniques on human primary bronchial epithelia to demonstrate their F508del-CFTR corrector ability. This study is aimed (i) at identifying three molecules ( 9b, 9g, and 9j ), useful as novel CFTR correctors with a good efficacy in rescuing the defect of F508del-CFTR; and (ii) at providing useful information to complete the structure-activity study within all the three series of hybrids as possible CFTR correctors, supporting the development of pharmacophore modelling studies, taking into account all the three series of hybrids. Finally, in silico evaluation of the hybrids pharmacokinetic (PK) properties contributed to highlight hybrid developability as drug-like correctors.

Background: Antibodies against cationic platelet chemokine, platelet factor 4 (PF4/CXCL4), have been described in heparin-induced thrombocytopenia (HIT), but also in patients positive for antiphospholipid antibodies (aPL) even in the absence of heparin treatment and HIT-related clinical manifestations. Anti-PF4 antibodies have been recently described also in subjects who developed thrombosis with thrombocytopenia syndrome (TTS) in association with adenoviral vector-based, but not with mRNA-based, COVID-19 vaccines., Objective: To investigate whether COVID-19 vaccination affects the production of anti-PF4 antibodies in aPL-positive patients and in control groups., Methods: Anti-PF4 immunoglobulins were detected in patients' and controls' serum samples by ELISA and their ability to activate normal platelets was assessed by the platelet aggregation test., Results: Anti-PF4 were found in 9 of 126 aPL-positive patients, 4 of 50 patients with COVID-19, 9 of 49 with other infections, and 1 of 50 aPL-negative patients with systemic lupus erythematosus. Clinical manifestations of TTS were not observed in any aPL patient positive for anti-PF4, whose serum failed to cause platelet aggregation. The administration of COVID-19 vaccines did not affect the production of anti-PF4 immunoglobulins or their ability to cause platelet aggregation in 44 aPL-positive patients tested before and after vaccination., Conclusions: Heparin treatment-independent anti-PF4 antibodies can be found in aPL-positive patients and asymptomatic carriers, but their presence, titre as well as in vitro effect on platelet activation are not affected by COVID-19 vaccination., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by beta-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has rapidly spread across the globe starting from February 2020. It is well established that during viral infection, extracellular vesicles become delivery/presenting vectors of viral material. However, studies regarding extracellular vesicle function in COVID-19 pathology are still scanty. Here, we performed a comparative study on exosomes recovered from the plasma of either MILD or SEVERE COVID-19 patients. We show that although both types of vesicles efficiently display SARS-CoV-2 spike-derived peptides and carry immunomodulatory molecules, only those of MILD patients are capable of efficiently regulating antigen-specific CD4 + T-cell responses. Accordingly, by mass spectrometry, we show that the proteome of exosomes of MILD patients correlates with a proper functioning of the immune system, while that of SEVERE patients is associated with increased and chronic inflammation. Overall, we show that exosomes recovered from the plasma of COVID-19 patients possess SARS-CoV-2-derived protein material, have an active role in enhancing the immune response, and possess a cargo that reflects the pathological state of patients in the acute phase of the disease., 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 © 2022 Pesce, Manfrini, Cordiglieri, Santi, Bandera, Gobbini, Gruarin, Favalli, Bombaci, Cuomo, Collino, Cricrì, Ungaro, Lombardi, Mangioni, Muscatello, Aliberti, Blasi, Gori, Abrignani, De Francesco, Biffo and Grifantini.)

Cystic fibrosis (CF) is caused by different mutations related to the cystic fibrosis transmembrane regulator protein (CFTR), with F508del being the most common. Pioneering the development of CFTR modulators, thanks to the development of effective correctors or potentiators, more recent studies deeply encouraged the administration of triple combination therapeutics. However, combinations of molecules interacting with other proteins involved in functionality of the CFTR channel recently arose as a promising approach to address a large rescue of F508del-CFTR. In this context, the design of compounds properly targeting the molecular chaperone Hsp70, such as the allosteric inhibitor MKT-077, proved to be effective for the development of indirect CFTR modulators, endowed with ability to amplify the accumulation of the rescued protein. Herein we performed structure-based studies of a number of allosteric HSP70 inhibitors, considering the recent X-ray crystallographic structure of the human enzyme. This allowed us to point out the main interaction supporting the binding mode of MKT-077, as well as of the related analogues. In particular, cation-π and π-π stacking with the conserve residue Tyr175 deeply stabilized inhibitor binding at the HSP70 cavity. Molecular docking studies had been followed by QSAR analysis and then by virtual screening of aminoaryl thiazoles ( I - IIIa ) as putative HSP70 inhibitors. Their effectiveness as CFTR modulators has been verified by biological assays, in combination with VX-809, whose positive results confirmed the reliability of the whole applied computational method. Along with this, the "in-silico" prediction of absorption, distribution, metabolism, and excretion (ADME) properties highlighted, once more, that AATs may represent a chemical class to be further investigated for the rational design of novel combination of compounds for CF treatment.

Purpose: In the present report it is described the design, the manufacturing and the successful surgical implant of one of the first 3D custom titanium vertebra realized with Additive Manufacturing technique and its use for the spinal reconstruction after en-bloc resection for primary osteogenic sarcoma., Methods: Clinical case presentation and the design of the 3D custom titanium vertebra was reported. It was also described the complex procedures adopted to evaluate the retrieved device from the histological point of view, as a tumor relapse hit the patient, one year after the reconstruction procedure., Results: The histological evaluation confirmed that the resection technique exerts an important role in promoting bone formation: vertebral body osteotomies favored the reconstruction procedure and maximized the contact area between host bone/vertebral prosthesis thus favoring the bone tissue penetration and device colonization., Conclusion: The sharing of these results is very important as they represent the starting point for improving the knowledge starting from the evidence obtained in a challenging clinical condition and with post-operative treatments that could be never reproduced in preclinical model., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Heavy metals, like many other chemical elements, are naturally present in the environment; however, the concentrations of these metals in various environmental matrices have increased through their intensive use in many human activities (such as industry, mining and agriculture). Among the heavy metals, cadmium (Cd) and mercury (Hg) induce a wide variety of defects in animals. While the effects of these heavy metals have been widely documented, a single exposure paradigm is typically used. Few studies have focused on evaluating combined exposure to these metals. However, in the environment, animals are confronted with a plethora of substances simultaneously; thus, the presence and origin of such substances must be determined to reduce the sources of contamination. Using the model of the fruit fly Drosophila melanogaster, for which many tools are readily available, we investigated how different concentrations of Cd and Hg in single and combined exposures impact post-embryonic development. In parallel, we evaluated the extended expression pattern of 38 molecular targets used as potential biomarkers of exposure through qPCR. Our results showed that both metals caused developmental delays and mortality in dose-dependent responses. Both metals were able to deregulate genes involved in hormonal control, general stress, and oxidative stress. Importantly, we confirmed synergistic interactions between Cd and Hg. Our results indicate the importance of assessing several biomarkers and their kinetics in mixtures. Drosophila represents a useful model for monitoring the toxicity of substances in polluted environments., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5 + T-BET + memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8 + T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8 + T lymphocytes, while CD4 + T cells were less expanded and skewed toward T CM and T H 2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8 + GZMB + effector cells were clonally expanded both during the infection and post-infection, while CD8 + GZMK + lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8 + GZMB + and GZMK + subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8 + T cell population with memory precursor-like features., (Copyright © 2021, American Association for the Advancement of Science.)

Computational drug repositioning is of growing interest to academia and industry, for its ability to rapidly screen a huge number of candidates in silico (exploiting comprehensive drug datasets) together with reduced development cost and time. The potential of drug repositioning has not been fully evaluated yet for cystic fibrosis (CF), a disease mainly caused by deletion of Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. F508del-CFTR is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. CF is still a fatal disease. Nowadays, it is treatable by some CFTR-rescuing drugs, but new-generation drugs with stronger therapeutic benefits and fewer side effects are still awaited. In this manuscript we report about the results of a pilot computational drug repositioning screening in search of F508del-CFTR-targeted drugs performed on AIFA library by means of a dedicated computational pipeline and surface plasmon resonance binding assay to experimentally validate the computational findings., 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.)

Background: Cystic fibrosis (CF) is the autosomal recessive disorder most common in Caucasian populations. It is caused by mutations in the cystic fibrosis transmembrane regulator protein (CFTR). CFTR is predominantly expressed at the apical plasma membranes of the epithelial cells lining several organs, and functions as a cAMP-regulated chloride/bicarbonate channel. To address the underlying causes of cystic fibrosis, two biomolecular activities are required, namely correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel., Objective: In our previous data, we demonstrated that some aminoarylthiazoles (AATs) have peculiar activity acting as correctors and as potentiator-like molecules. Curiously, a compound called 1 has been shown to be markedly active as a potentiator. Now, we have further modified its scaffold at different portions, for the identification of molecules with improved potency and effectiveness on mutant CFTR., Methods: Starting from this active compound, we synthesized a small library trying to improve the activity as potentiators. To extrapolate the contribution of a particular structural portion to bioactivity, we selectively modified one portion at a time., Results: Our study has provided a structure-activity relationship (SAR) on AATs and led to the identification of some compounds, with a particular ability to act as CFTR potentiators., Conclusion: Two compounds 2 and 13 appear to be promising molecules and could be used for the future development of potentiators of the chloride transport defect in cystic fibrosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In genetic diseases, the most prevalent mechanism of pathogenicity is an altered expression of dosage-sensitive genes. Drugs that restore physiological levels of these genes should be effective in treating the associated conditions. We developed a screening strategy, based on a bicistronic dual-reporter vector, for identifying compounds that modulate protein levels, and used it in a pharmacological screening approach. To provide a proof-of-principle, we chose autosomal dominant leukodystrophy (ADLD), an ultra-rare adult-onset neurodegenerative disorder caused by lamin B1 (LMNB1) overexpression. We used a stable Chinese hamster ovary (CHO) cell line that simultaneously expresses an AcGFP reporter fused to LMNB1 and a Ds-Red normalizer. Using high-content imaging analysis, we screened a library of 717 biologically active compounds and approved drugs, and identified alvespimycin, an HSP90 inhibitor, as a positive hit. We confirmed that alvespimycin can reduce LMNB1 levels by 30%-80% in five different cell lines (fibroblasts, NIH3T3, CHO, COS-7, and rat primary glial cells). In ADLD fibroblasts, alvespimycin reduced cytoplasmic LMNB1 by about 50%. We propose this approach for effectively identifying potential drugs for treating genetic diseases associated with deletions/duplications and paving the way toward Phase II clinical trials., (© 2020 Wiley Periodicals LLC.)

Cystic fibrosis (CF) is the autosomal recessive disorder most recurrent in Caucasian populations. It is caused by different mutations in the cystic fibrosis transmembrane regulator protein (CFTR) gene, with F508del being the most common. During the last years, small-molecule therapy chosen to contrast CF relied on compounds that correct CFTR misfolding and ER retention (correctors such as VX-809), or defective channel gating (potentiators such as VX-770). Combination therapy with the two series of drugs has been applied, leading to the approval of several multi-drugs such as Orkambi. Despite this, this treatment proved to be only partially effective making the search for novel modulators an urgent need to contrast CF. Recently, we reported compound 2a as reference compound of a series of aminoarylthiazole-VX-809 hybrid derivatives exhibiting promising F508del-CFTR corrector ability. Herein, we report exploring the docking mode of the prototype VX-809 and of 2a in order to derive useful guidelines for the rational design of novel optimized analogues. To demonstrate experimentally their effective F508del-CFTR-binding and rescuing potential, the most promising derivatives had been synthesized and evaluated in biological assays including YFP functional assay on F508del-CFTR CFBE41o-cells, trans epithelial electrical resistance (TEER) and surface plasmon resonance (SPR). This multidisciplinary strategy led to the discovery of a second series of hybrids including 7j and 7m endowed with higher potency than the prototype., 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.)

Cystic fibrosis is a genetic disease caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator gene, encoding for CFTR protein. The most frequent mutation is the deletion of phenylalanine at position 508 (F508del), which leads to distinct defects in channel gating and cellular processing. In last years, several thiazole containing small molecules, endowed with dual F508del-CFTR modulator activity, proved to be able to target these defects. In search of new chemical entities able to restore CFTR function, we designed and synthesized a small series of sixteen thiazole derivatives. The designed compounds were studied as correctors and potentiators of F508del-CFTR. Although none of the molecules showed significant corrector activity, compounds 10 and 11 exhibited potentiator effects, thus allowing to determine some basic structural features which enable to obtain F508del-CFTR potentiator activity. In silico ADME studies showed that these derivatives obey Lipinski's rule of five and are expected to be orally bioavailable. Therefore, these molecules may represent a good starting point for the design of analogues endowed with improved CFTR potentiator activity and a good pharmacokinetic profile., 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 Ltd. All rights reserved.)

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1 H -pyrido[4,3- b ]indole core as a novel chemotype of potentiators. In-depth structure-activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

In recent years, a number of drugs have been approved for the treatment of cystic fibrosis (CF). Among them, newly released Trikafta, a combination of 3 drugs (VX-661/VX-445/VX-770), holds great promise to radically improve the quality of life for a large portion of patients with CF carrying 1 copy of F508del, the most frequent CF transmembrane conductance regulator (CFTR) mutation. Currently available disease-modifying CF drugs work by rescuing the function of the mutated CFTR anion channel. Recent research has shown that membrane lipids, and the cell lipidome in general, play a significant role in the mechanism of CFTR-defective trafficking and, on the other hand, its rescue. In this paper, by using untargeted lipidomics on CFBE41o- cells, we identified distinctive changes in the bronchial epithelial cell lipidome associated with treatment with Trikafta and other CF drugs. Particularly interesting was the reduction of levels of ceramide, a known molecular player in the induction of apoptosis, which appeared to be associated with a decrease in the susceptibility of cells to undergo apoptosis. This evidence could account for additional beneficial roles of the triple combination of drugs on CF phenotypes.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed at the apical plasma membrane (PM) of different epithelial cells. The most common mutation responsible for the onset of cystic fibrosis (CF), F508del, inhibits the biosynthesis and transport of the protein at PM, and also presents gating and stability defects of the membrane anion channel upon its rescue by the use of correctors and potentiators. This prompted a multiple drug strategy for F508delCFTR aimed simultaneously at its rescue, functional potentiation and PM stabilization. Since ganglioside GM1 is involved in the functional stabilization of transmembrane proteins, we investigated its role as an adjuvant to increase the effectiveness of CFTR modulators. According to our results, we found that GM1 resides in the same PM microenvironment as CFTR. In CF cells, the expression of the mutated channel is accompanied by a decrease in the PM GM1 content. Interestingly, by the exogenous administration of GM1, it becomes a component of the PM, reducing the destabilizing effect of the potentiator VX-770 on rescued CFTR protein expression/function and improving its stabilization. This evidence could represent a starting point for developing innovative therapeutic strategies based on the co-administration of GM1, correctors and potentiators, with the aim of improving F508del CFTR function.

F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC 50 of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.

Rationale: Posterior sternoclavicular joint dislocations (PSCJDs) are particularly rare injuries, accounting for 3% to 5% of sternoclavicular joint dislocations. With very few cases reported in the literature, these injuries are often misdiagnosed and imaging is not always clear, thus making physicians often unaware of them. The present case report aims to investigate a rare case involving a clavicular Salter-Harris II fracture with associated posterior displacement of the diaphysis, a term coined a "pseudodislocation.", Patient Concerns: We present a case of a 14-year-old adolescent who sustained a traumatic injury to the shoulder while falling during a soccer match. His main concern was about recovery time and the return to daily life activities., Diagnoses: Multiple imaging studies imaging (X-rays, computed tomography, magnetic resonance imaging) revealed a Salter-Harris II fracture of the right clavicle with posterior displacement of the diaphysis., Interventions: The patient underwent primary surgery to reduce the fracture, using an articular locking compression plate, and secondary surgery to remove the hardware., Outcomes: Following the removal of the hardware at 60 days after the initial surgery and a number of cycles of physiotherapy the patient reported a pain-free range of motion with slight limitation at extremes. Full return to recreational and everyday life activities were achieved at 3 months from the initial surgery., Lessons: The PSCJDs are challenging injuries, as they are surrounded by delicate structures inside the mediastinum. Attention must be taken while diagnosing and treating these injuries as the risk of complications and iatrogenic injuries is high. To the author's knowledge, this case is one of the first of its kind described in the literature where we have a Salter-Harrys type II fracture associated with a posterior pseudodislocation of the lateral clavicle. Given the positive results of the case, we recommend the above-mentioned treatment protocol in PSCJD with associated Salter-Harris II fractures in adolescent patients.

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster model of eIF6 upregulation, leading to a boost in general translation and the shut-down of the ecdysone biosynthetic pathway. Indeed, translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed Programmed Cell Death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background and Aims: Primary biliary cholangitis is an autoimmune biliary disease characterized by injury of bile ducts, eventually leading to cirrhosis and death. In most cases, anti-mitochondrial antibodies and persistently elevated serum alkaline phosphatase are the basis for the serological diagnosis. Anti-nuclear antibodies are also useful and may indicate a more aggressive diseases course. In patients in which anti-mitochondrial antibodies are not detected, an accurate diagnosis requires liver histology. This study aims at identifying specific biomarkers for the serological diagnosis of primary biliary cholangitis., Methods: Sera from patients affected by primary biliary cholangitis, primary sclerosing cholangitis, hepatitis C virus (with and without cryoglobulinemia), hepatocarcinoma and healthy donors were tested on a protein array representing 1658 human proteins. The most reactive autoantigens were confirmed by DELFIA analysis on expanded cohorts of the same mentioned serum classes, and on autoimmune hepatitis sera, using anti-PDC-E2 as reference biomarker., Results: Two autoantigens, SPATA31A3 and GARP, showed high reactivity with primary biliary cholangitis sera, containing or not anti-mitochondrial antibodies. Their combination with PDC-E2 allowed to discriminate primary biliary cholangitis from all tested control classes with high sensitivity and specificity. We found that GARP expression is upregulated upon exposure to biliary salts in human cholangiocytes, an event involving EGFR and insulin pathways. GARP expression was also detected in biliary duct cells of PBC patients., Conclusions: This study highlighted SPATA31A3 and GARP as new biomarkers for primary biliary cholangitis and unravelled molecular stimuli underlying GARP expression in human cholangiocytes., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Magnetic resonance imaging allows acquiring functional and structural connectivity data from which high-density whole-brain networks can be derived to carry out connectome-wide analyses in normal and clinical populations. Graph theory has been widely applied to investigate the modular structure of brain connections by using centrality measures to identify the "hub" of human connectomes, and community detection methods to delineate subnetworks associated with diverse cognitive and sensorimotor functions. These analyses typically rely on a preprocessing step (pruning) to reduce computational complexity and remove the weakest edges that are most likely affected by experimental noise. However, weak links may contain relevant information about brain connectivity, therefore, the identification of the optimal trade-off between retained and discarded edges is a subject of active research. We introduce a pruning algorithm to identify edges that carry the highest information content. The algorithm selects both strong edges (i.e. edges belonging to shortest paths) and weak edges that are topologically relevant in weakly connected subnetworks. The newly developed "strong-weak" pruning (SWP) algorithm was validated on simulated networks that mimic the structure of human brain networks. It was then applied for the analysis of a real dataset of subjects affected by amyotrophic lateral sclerosis (ALS), both at the early (ALS2) and late (ALS3) stage of the disease, and of healthy control subjects. SWP preprocessing allowed identifying statistically significant differences in the path length of networks between patients and healthy subjects. ALS patients showed a decrease of connectivity between frontal cortex to temporal cortex and parietal cortex and between temporal and occipital cortex. Moreover, degree of centrality measures revealed significantly different hub and centrality scores between patient subgroups. These findings suggest a widespread alteration of network topology in ALS associated with disease progression.

Neutrophils migrate to sites of infection where they phagocytose, degranulate, and/or, in the presence of appropriate stimuli, release decondensed chromatin strands (called neutrophil extracellular traps, NETs) for trapping and possibly killing microorganisms. NET formation is characterized by marked morphological cell changes, in particular within the nucleus. Lytic NET formation can be observed in neutrophils undergoing cell death, which is referred to as NETosis. Dysregulation of NET production and/or degradation can exert pathogenic effects, contributing to the pathogenesis of various diseases, including cystic fibrosis, autoimmune diseases and inflammatory conditions. By employing a phenotypic assay based on high-content imaging and analysis, we screened a library of biologically active compounds and identified vanilloids as a novel class of chemical compounds able to hinder NETosis induction and NET release. Vanilloids also markedly decrease cytosolic ROS production. The identification of novel vanilloid NET inhibitors, able to stop excessive or aberrant NET production might offer new therapeutic options for those disorders displaying NET overproduction.

Purpose To develop and test an artificial intelligence (AI) system, based on deep convolutional neural networks (CNNs), for automated real-time triaging of adult chest radiographs on the basis of the urgency of imaging appearances. Materials and Methods An AI system was developed by using 470 388 fully anonymized institutional adult chest radiographs acquired from 2007 to 2017. The free-text radiology reports were preprocessed by using an in-house natural language processing (NLP) system modeling radiologic language. The NLP system analyzed the free-text report to prioritize each radiograph as critical, urgent, nonurgent, or normal. An AI system for computer vision using an ensemble of two deep CNNs was then trained by using labeled radiographs to predict the clinical priority from radiologic appearances only. The system's performance in radiograph prioritization was tested in a simulation by using an independent set of 15 887 radiographs. Prediction performance was assessed with the area under the receiver operating characteristic curve; sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were also determined. Nonparametric testing of the improvement in time to final report was determined at a nominal significance level of 5%. Results Normal chest radiographs were detected by our AI system with a sensitivity of 71%, specificity of 95%, PPV of 73%, and NPV of 94%. The average reporting delay was reduced from 11.2 to 2.7 days for critical imaging findings (P < .001) and from 7.6 to 4.1 days for urgent imaging findings (P < .001) in the simulation compared with historical data. Conclusion Automated real-time triaging of adult chest radiographs with use of an artificial intelligence system is feasible, with clinically acceptable performance. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Auffermann in this issue.

Machine learning approaches hold great potential for the automated detection of lung nodules on chest radiographs, but training algorithms requires very large amounts of manually annotated radiographs, which are difficult to obtain. The increasing availability of PACS (Picture Archiving and Communication System), is laying the technological foundations needed to make available large volumes of clinical data and images from hospital archives. Binary labels indicating whether a radiograph contains a pulmonary lesion can be extracted at scale, using natural language processing algorithms. In this study, we propose two novel neural networks for the detection of chest radiographs containing pulmonary lesions. Both architectures make use of a large number of weakly-labelled images combined with a smaller number of manually annotated x-rays. The annotated lesions are used during training to deliver a type of visual attention feedback informing the networks about their lesion localisation performance. The first architecture extracts saliency maps from high-level convolutional layers and compares the inferred position of a lesion against the true position when this information is available; a localisation error is then back-propagated along with the softmax classification error. The second approach consists of a recurrent attention model that learns to observe a short sequence of smaller image portions through reinforcement learning; the reward function penalises the exploration of areas, within an image, that are unlikely to contain nodules. Using a repository of over 430,000 historical chest radiographs, we present and discuss the proposed methods over related architectures that use either weakly-labelled or annotated images only., (Copyright © 2019 Elsevier B.V. All rights reserved.)