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Saturated N -heterocycles constitute a vital scaffold for pharmaceutical chemistry but are challenging to access synthetically, particularly asymmetrically. Here, we demonstrate how imine reductases can achieve annulation through tandem inter- and intramolecular reductive amination processes. Imine reductases were used in combination with further enzymes to access unsubstituted, α-substituted, and α,α'-disubstituted N -heterocycles from simple starting materials in one pot and under benign conditions. This work shows the remarkable flexibility of these enzymes to have broad activity against numerous substrates derived from singlular starting materials., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Enterobacter cloacae complex isolates have been reported as an important nosocomial multidrug resistance pathogen. In the present study, we investigated antimicrobial susceptibility and the colistin-resistance rates, their genetic determinants and clonality among clinical E. cloacae complex isolates from different Brazilian states. For this, an initial screening was carried out on 94 clinical isolates of E. clocacae complex received between 2016 and 2018 by LAPIH-FIOCRUZ, using EMB plates containing 4 μg/mL of colistin, followed MIC determination, resulting in the selection of 26 colistin-resistant isolates from the complex. The presence of carbapenemases encoding genes (bla KPC , bla NDM and bla OXA-48 ), plasmidial genes for resistance to polymyxins (mcr1-9) and mutations in chromosomal genes (pmrA, pmrB, phoP and phoQ) described as associated with resistance to polymyxin were screened by PCR and DNA sequencing. Finally, the hsp60 gene was sequenced to identify species of the E. cloacae complex and genetic diversity was evaluated by PFGE and MLST. The results have shown that among 94 E. cloacae complex isolates, 19 (20.2%) were colistin-resistant. The resistant strains exhibited MIC ranging from 4 to 128 µg / mL and E. hormaechei subsp. steigerwaltii was the prevalent species in the complex (31,6%), followed by E. cloacae subsp. cloacae (26,3%). The antimicrobials with the highest susceptibility rate were gentamicin (21%) and tigecycline (26%). Carbapenemases encoding genes (bla KPC n = 5, bla NDM n = 1) were detected in 6 isolates and mcr-9 in one. Among the modifications found in PmrA, PmrB, PhoP e PhoQ (two-component regulatory system), only the S175I substitution in PmrB found in E. cloacae subsp cloacae isolates were considered deleterious (according to the prediction of PROVEAN). By PFGE, 13 profiles were found among E. cloacae complex isolates, with EcD the most frequent. Furthermore, by MLST 10 ST's, and 1 new ST, were identified in E. cloacae. In conclusion, no prevalence of clones or association among carbapenemase production and polymyxin resistance was found between the E. cloacae. Thereby, the results suggest that the increased polymyxin-resistance is related to the selective pressure exerted by the indiscriminate use in hospitals. Lastly, this study highlights the urgent need to elucidate the mechanism involved in the resistance to polymyxin in the E. cloacae complex and the development of measures to control and prevent infections caused by these multiresistant bacteria., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Deciphering the initial steps of SARS-CoV-2 infection, that influence COVID-19 outcomes, is challenging because animal models do not always reproduce human biological processes and in vitro systems do not recapitulate the histoarchitecture and cellular composition of respiratory tissues. To address this, we developed an innovative ex vivo model of whole human lung infection with SARS-CoV-2, leveraging a lung transplantation technique. Through single-cell RNA-seq, we identified that alveolar and monocyte-derived macrophages (AMs and MoMacs) were initial targets of the virus. Exposure of isolated lung AMs, MoMacs, classical monocytes and non-classical monocytes (ncMos) to SARS-CoV-2 variants revealed that while all subsets responded, MoMacs produced higher levels of inflammatory cytokines than AMs, and ncMos contributed the least. A Wuhan lineage appeared to be more potent than a D614G virus, in a dose-dependent manner. Amidst the ambiguity in the literature regarding the initial SARS-CoV-2 cell target, our study reveals that AMs and MoMacs are dominant primary entry points for the virus, and suggests that their responses may conduct subsequent injury, depending on their abundance, the viral strain and dose. Interfering on virus interaction with lung macrophages should be considered in prophylactic strategies., (© 2024. The Author(s).)

Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, some of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) binds PA and the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to interfere with the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin β RanBP5 and subsequent steps in FluPol assembly. Using FluPol mutants selected after passaging in VHH16-expressing cells, we identified the VHH16 binding site at the interface formed by PA residues with the N-terminus of PB1, overlapping or close to binding sites of two host proteins, ANP32A and RNA-polymerase II RPB1 subunit which are critical for virus replication and transcription, respectively. These data suggest that the VHH16 neutralization is likely due to several activities, altering the import of the PA-PB1 dimer into the nucleus as well as inhibiting specifically virus transcription and replication. Thus, the VHH16 binding site represents a new Achilles' heel for FluPol and as such, a potential target for antiviral development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bessonne et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV., (© 2024. The Author(s).)

Introduction: The cases of dermatophytosis are increasing and they are associated with a higher number of therapeutic failures leading the doctor to prescribe combinations of antifungals as therapy. The objective was to evaluate the interaction of terbinafine and ciclopirox, the most commonly antifungals used in the clinic, in dermatophyte isolates., Methodology: The minimum inhibitory concentrations (MIC) of ciclopirox and terbinafine were determined by the broth microdilution method according CLSI and the checkerboard assay was used to evaluate the interaction between the antifungal agents., Results: For terbinafine the mic50 was 0.125 ug/mL and mic90 was 0.250 ug/mL. For ciclopirox the values were 2.0 ug/mL for mic50 and 4.0 ug/mL for mic90. No synergistic interaction was observed for the dermatophyte isolates tested., Conclusion: These results suggest that the use of terbinafine in combination with ciclopirox, which is widely used in the clinic, may not be a good choice for the treatment of onychomycosis., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 SFMM. Published by Elsevier Masson SAS. All rights reserved.)

ABSTRACT The brain aging process triggers cognitive function impairment, such as memory loss and compromised quality of life. Cognitive impairment is based on bioenergetic status, with reduced glucose uptake and metabolism in aged brains. Anaplerotic substrates are reported to promote mitochondrial ATP generation, having been tested in clinical trials for the treatment of neurological disorders and metabolic diseases. Objectives and Methods: To assess whether the improvement in oxidative capacity ameliorates cognitive function in adults (12 weeks), and aged (22-month-old) C57/6BJ mice, they received (1) a ketogenic diet, (2) a ketogenic diet supplemented with the anaplerotic substance, triheptanoin, or (3) a control diet for 12 weeks. Spontaneous alternation and time spent in a previously closed arm in the Y-maze test and time interacting with an unknown object in the novel object recognition test (NORT) were used to evaluate working memory. Acetylcholinesterase (AChE) activity in the prefrontal lobe, brain left hemisphere, and cerebellum was also evaluated. Glucose transporter 3 (GLUT3) expression in the prefrontal lobe was analyzed by western blotting. Results: The ketogenic diet (KD) reduced spontaneous alternation in aged mice, leading to lower AChE activity in the aged prefrontal lobe and cerebellum, and in the parieto-temporal-occipital lobe of adult mice. Furthermore, KD decreased GLUT3 protein expression in the frontal lobe of the adults. Discussion: Supplementation of KD with triheptanoin prevented memory impairment and showed similar values of AChE activity and GLUT3 expression compared to the controls. Our data suggest that triheptanoin has a potential role in the bioenergetic capacity of the brain, improving cognitive function.

Although some studies have shown neuroimaging and neuropsychological alterations in post-COVID-19 patients, fewer combined neuroimaging and neuropsychology evaluations of individuals who presented a mild acute infection. Here we investigated cognitive dysfunction and brain changes in a group of mildly infected individuals. We conducted a cross-sectional study of 97 consecutive subjects (median age of 41 years) without current or history of psychiatric symptoms (including anxiety and depression) after a mild infection, with a median of 79 days (and mean of 97 days) after diagnosis of COVID-19. We performed semi-structured interviews, neurological examinations, 3T-MRI scans, and neuropsychological assessments. For MRI analyses, we included a group of non-infected 77 controls. The MRI study included white matter (WM) investigation with diffusion tensor images (DTI) and functional connectivity with resting-state functional MRI (RS-fMRI). The patients reported memory loss (36%), fatigue (31%) and headache (29%). The quantitative analyses confirmed symptoms of fatigue (83% of participants), excessive somnolence (35%), impaired phonemic verbal fluency (21%), impaired verbal categorical fluency (13%) and impaired logical memory immediate recall (16%). The WM analyses with DTI revealed higher axial diffusivity values in post-infected patients compared to controls. Compared to controls, there were no significant differences in the functional connectivity of the posterior cingulum cortex. There were no significant correlations between neuropsychological scores and neuroimaging features (including DTI and RS-fMRI). Our results suggest persistent cognitive impairment and subtle white matter abnormalities in individuals mildly infected without anxiety or depression symptoms. The longitudinal analyses will clarify whether these alterations are temporary or permanent., (© 2024. The Author(s).)

Background: Autoantibodies against surface neuronal antigens have been associated with specific neurological presentations including autoimmune encephalitis (AE), with variable association with neoplasia and infections., Methods: We described the phenotype and environmental associations of patients with neurological syndromes associated with antibodies against neuronal surface antigens who were referred to a tertiary center in the South of Brazil. All patients were tested for neuronal autoantibodies using cell-based assays. Clinical, radiological, and laboratory findings were retrospectively reviewed., Results: We identified 16 patients, 15 had subacute, and one had a progressive disease course. Among patients with subacute onset, 11 (73 %) were N-Methyl-d-Aspartate receptor (NMDAr-IgG)+, 3 (20 %) were Leucine-rich Glioma-Inactivated-1 (LGI1-IgG)+, and 1 (6 %) was positive for Glycine receptor-IgG. The patient with a progressive disease course had antibodies against IgLON5. Most patients had disease onset in spring and summer suggesting environmental factors for the development of AE. Also, we observed a different pattern of brain lesions when NMDAr-IgG encephalitis followed herpes encephalitis and a previously unreported association with Rosai-Dorfman-Destombe disease. All patients with encephalopathy met criteria for possible AE and all proven NMDAr-IgG+ met criteria for NMDAr-IgG encephalitis. However, only one LGI1-IgG+ patient fulfilled clinical criteria for limbic encephalitis. All but one received high-dose intravenous methylprednisolone, 11 also had intravenous human immunoglobulin, and 4 plasma exchange. Furthermore, all patients received second-line immunotherapy. Importantly, most patients improved with immunotherapy, even when initiated later in the disease course., Conclusion: We identified seasonal variability associated with neuronal surface antibodies suggesting environmental triggers. Also, we described the coexistence of NMDAr-IgG encephalitis with histiocytosis. In our series, most patients received second-line immunotherapy. We observed neurologic improvement after treatment even in cases of delayed diagnosis. Increasing the recognition and availability of tests and treatments for these conditions is of paramount importance in low- and middle-income countries., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Bruna Klein da Costa reports a relationship with Merck KGaA that includes: travel reimbursement. Bruna Klein da Costa reports a relationship with Roche that includes: travel reimbursement. Bruna Klein da Costa reports a relationship with Foundation for Research Support of Rio Grande do Sul State that includes: funding grants. Bruna Klein da Costa reports a relationship with Teva that includes: funding grants. Gisele Hansel reports a relationship with Coordination of Higher Education Personnel Improvement that includes: funding grants. Lucas Porcello Schilling reports a relationship with Biogen that includes: consulting or advisory. Lucas Porcello Schilling reports a relationship with Roche that includes: consulting or advisory. Lucas Porcello Schilling reports a relationship with Ache Pharmaceutical Laboratories SA that includes: speaking and lecture fees. Lucas Porcello Schilling reports a relationship with Apsen that includes: speaking and lecture fees. Lucas Porcello Schilling reports a relationship with Biogen that includes: speaking and lecture fees. Giordani Rodrigues dos Passos reports a relationship with Biogen that includes: speaking and lecture fees and travel reimbursement. Giordani Rodrigues dos Passos reports a relationship with Merck KGaA that includes: travel reimbursement. Giordani Rodrigues dos Passos reports a relationship with Roche that includes: travel reimbursement. Giordani Rodrigues dos Passos reports a relationship with Sanofi Genzyme that includes: travel reimbursement. Giordani Rodrigues dos Passos reports a relationship with Alnylam Pharmaceuticals Inc that includes: speaking and lecture fees. Giordani Rodrigues dos Passos reports a relationship with Novartis that includes: speaking and lecture fees. William Alves Martins reports a relationship with Ache Pharmaceutical Laboratories SA that includes: speaking and lecture fees. Jefferson Becker reports a relationship with Roche that includes: consulting or advisory and speaking and lecture fees. Jefferson Becker reports a relationship with Biogen that includes: consulting or advisory, funding grants, and speaking and lecture fees. Jefferson Becker reports a relationship with Merck KGaA that includes: consulting or advisory and speaking and lecture fees. Jefferson Becker reports a relationship with Novartis that includes: consulting or advisory, funding grants, and speaking and lecture fees. Jefferson Becker reports a relationship with Sanofi that includes: speaking and lecture fees. Jefferson Becker reports a relationship with Horizon Therapeutics plc that includes: consulting or advisory and speaking and lecture fees. Jefferson Becker reports a relationship with EMS that includes: speaking and lecture fees. Jefferson Becker reports a relationship with Janssen Pharmaceuticals Inc that includes: consulting or advisory. Raphael Machado Castilhos reports a relationship with Alzheimer's Association that includes: funding grants. Raphael Machado Castilhos reports a relationship with Foundation for Research Support of Rio Grande do Sul State that includes: funding grants. Andre Palmini reports a relationship with Biogen that includes: speaking and lecture fees. Andre Palmini reports a relationship with Libbs Pharmaceutical that includes: speaking and lecture fees. Andre Palmini reports a relationship with Prati-Donaduzzi Generic Medicines that includes: speaking and lecture fees. Andre Palmini reports a relationship with UCB Inc that includes: speaking and lecture fees. Andre Palmini reports a relationship with Novartis that includes: speaking and lecture fees. Andre Palmini reports a relationship with Abbott that includes: speaking and lecture fees. Andre Palmini reports a relationship with Zodiac pharmaceuticals that includes: speaking and lecture fees. Andre Palmini reports a relationship with Torrent Do Brasil Ltda that includes: speaking and lecture fees. Douglas Kazutoshi Sato reports a relationship with Roche that includes: consulting or advisory and speaking and lecture fees. Douglas Kazutoshi Sato reports a relationship with Biogen that includes: funding grants and speaking and lecture fees. Douglas Kazutoshi Sato reports a relationship with Merck KGaA that includes: consulting or advisory, funding grants, and speaking and lecture fees. Douglas Kazutoshi Sato reports a relationship with Horizon Therapeutics plc that includes: consulting or advisory and speaking and lecture fees. Douglas Kazutoshi Sato reports a relationship with Alexion that includes: consulting or advisory. Douglas Kazutoshi Sato reports a relationship with Foundation for Research Support of Rio Grande do Sul State that includes: funding grants. Douglas Kazutoshi Sato reports a relationship with National Council for Scientific and Technological Development that includes: funding grants. Douglas Kazutoshi Sato reports a relationship with Teva Pharmaceutical Industries Ltd that includes: funding grants. BKC received medical education support from Biogen and Bayer and compensation from work as subinvestigator in a clinical trial sponsored by Sanofi. Received the Du Pré Grant from the Multiple Sclerosis International Federation. GRP has received scholarships from ECTRIMS, Novartis, and the World Federation of Neurology, compensation for work as subinvestigator in trials sponsored by Roche, travel grants from Biogen, Merck, Roche, and Sanofi-Genzyme, fees for editorial content from Merck Serono, Neurodiem, and Roche., (Copyright © 2023. Published by Elsevier B.V.)

Neuroblastoma is the most common extracranial solid tumor in infants, arising from developmentally stalled neural crest-derived cells. Driving tumor differentiation is a promising therapeutic approach for this devastating disease. Here, we show that the CDK4/6 inhibitor palbociclib not only inhibits proliferation but induces extensive neuronal differentiation of adrenergic neuroblastoma cells. Palbociclib-mediated differentiation is manifested by extensive phenotypic and transcriptional changes accompanied by the establishment of an epigenetic program driving expression of mature neuronal features. In vivo palbociclib significantly inhibits tumor growth in mouse neuroblastoma models. Furthermore, dual treatment with retinoic acid resets the oncogenic adrenergic core regulatory circuit of neuroblastoma cells, further suppresses proliferation, and can enhance differentiation, altering gene expression in ways that significantly correlate with improved patient survival. We therefore identify palbociclib as a therapeutic approach to dramatically enhance neuroblastoma differentiation efficacy that could be used in combination with retinoic acid to improve patient outcomes., Competing Interests: Declaration of interests We have filed an application seeking patent protection on the use of the combination CDK4/6 inhibitors and retinoic acid in neuroblastoma., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Dermatophytosis is one of the most common fungal infections worldwide. The distribution of dermatophytes varies across continents, but the genera Trichophyton and Microsporum have emerged as the main isolated agents in humans and animals., Objectives: To validate Drosophila melanogaster flies as a fast and feasible model to study dermatophytic infections., Methods: Wild-type (WT) and Toll-deficient D. melanogaster flies were infected by Trichophyton rubrum, T. mentagrophytes, Microsporum canis and Nannizzia gypsea by pricking with a needle previously dipped in inoculum concentrations ranging from 10 3 to 10 8 colony-forming units/mL. Establishment of infection was confirmed by survival curves, histopathological analysis and fungal burden. Thereafter, flies were treated with terbinafine, itraconazole and clioquinol., Results: WT flies were predominantly resistant to the infection, whereas Toll-deficient flies succumbed to the four dermatophyte genera tested. The antifungal drugs protected flies from the infection, except for N. gypsea whose survival curves did not differ from the untreated group., Conclusions: This pilot study confirms that D. melanogaster is a suitable model to study the virulence and antifungal drug efficacy in dermatophyte species., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

In response to the increasing demand for lung transplantation, ex vivo lung perfusion (EVLP) has extended the number of suitable donor lungs by rehabilitating marginal organs. However despite an expanding use in clinical practice, the responses of the different lung cell types to EVLP are not known. In order to advance our mechanistic understanding and establish a refine tool for improvement of EVLP, we conducted a pioneer study involving single cell RNA-seq on human lungs declined for transplantation. Functional enrichment analyses were performed upon integration of data sets generated at 4 h (clinical duration) and 10 h (prolonged duration) from two human lungs processed to EVLP. Pathways related to inflammation were predicted activated in epithelial and blood endothelial cells, in monocyte-derived macrophages and temporally at 4 h in alveolar macrophages. Pathways related to cytoskeleton signaling/organization were predicted reduced in most cell types mainly at 10 h. We identified a division of labor between cell types for the selected expression of cytokine and chemokine genes that varied according to time. Immune cells including CD4 + and CD8 + T cells, NK cells, mast cells and conventional dendritic cells displayed gene expression patterns indicating blunted activation, already at 4 h in several instances and further more at 10 h. Therefore despite inducing inflammatory responses, EVLP appears to dampen the activation of major lung immune cell types, what may be beneficial to the outcome of transplantation. Our results also support that therapeutics approaches aiming at reducing inflammation upon EVLP should target both the alveolar and vascular compartments., 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 © 2023 Gouin, Vu Manh, Jouneau, Bevilacqua, De Wolf, Glorion, Hannouche, Urien, Estephan, Roux, Magnan, Le Guen, Da Costa, Chevalier, Descamps, Schwartz-Cornil, Dalod and Sage.)

Importance: Titanium-nitride-oxide (TiNO)-coated stents show faster strut coverage compared with drug-eluting stents without excessive intimal-hyperplasia observed in bare metal stents. It is important to study long-term clinical outcomes after treatment of patients with an acute coronary syndrome (ACS) by TiNO-coated stents, which are neither drug-eluting stents nor bare metal stents., Objective: To compare the rate of main composite outcome of cardiac death, myocardial infarction (MI), or ischemia-driven target lesion revascularization at 5 years in patients with ACS randomized to receive either a TiNO-coated stent or a third-generation everolimus-eluting stent (EES)., Design, Setting, and Participants: This multicenter, randomized, controlled, open-label trial was conducted in 12 clinical sites in 5 European countries and enrolled patients from January 2014 to August 2016. Patients presenting with ACS (ST-segment elevation MI, non-ST-segment elevation MI, and unstable angina) with at least 1 de novo lesion were randomized to receive either a TiNO-coated stent or an EES. The present report analyzes the long-term follow-up for the main composite outcome and its individual components. Analysis took place between November 2022 to March 2023., Main Outcome: The primary end point was a composite of cardiac death, MI, or target lesion revascularization at 12-month follow-up., Results: A total of 1491 patients with ACS were randomly assigned to receive either TiNO-coated stents (989 [66.3%]) or EES (502 [33.7%]). The mean (SD) age was 62.7 (10.8) years, and 363 (24.3%) were female. At 5 years, the main composite outcome events occurred in 111 patients (11.2%) in the TiNO group vs 60 patients (12%) in the EES group (hazard ratio [HR], 0.94; 95% CI, 0.69-1.28; P = .69). The rate of cardiac death was 0.9% (9 of 989) vs 3.0% (15 of 502) (HR, 0.30; 95% CI, 0.13-0.69; P = .005), the rate of MI was 4.6% (45 of 989) vs 7.0% (35 of 502) (HR, 0.64; 95% CI, 0.41-0.99; P = .049), the rate of stent thrombosis was 1.2% (12 of 989) vs 2.8% (14 of 502) (HR, 0.43; 95% CI, 0.20-0.93; P = .034), and the rate of target lesion revascularization was 7.4% (73 of 989) vs 6.4% (32 of 502) (HR, 1.16; 95% CI, 0.77-1.76; P = .47) in the TiNO-coated stent arm and in the EES arm, respectively., Conclusion and Relevance: In this study, patients with ACS had a main composite outcome that was not different 5 years after TiNO-coated stent or EES., Trial Registration: ClinicalTrials.gov Identifier: NCT02049229.

Purpose: To investigate the independent and joint associations of cardiorespiratory fitness and body mass index (BMI) with five dimensions of Health-Related Quality of Life (HRQoL) in a cross-sectional sample of Brazilian adolescents., Methods: 619 Brazilian schoolchildren answered a survey, BMI categories (healthy weight and overweight/obesity) were assessed by their weight and height, and they participated in a 20-m shuttle run test. HRQoL was measured using the KIDSCREEN-27 across five dimensions: Physical Well-Being, Psychological Well-Being, Autonomy and Parent Relation, Peers and Social Support, and School Environment. Sex, age, maternal education, physical activity level, and habitual sedentary behaviour were assessed and used as adjusting variables. Cardiorespiratory fitness was categorized in tertiles and independent and joint associations were tested using mixed-effects linear regressions., Results: Higher levels of cardiorespiratory fitness were favourably associated with the physical well-being, psychological well-being, and peer and social support dimensions of HRQoL. Adolescents with overweight/obesity presented higher scores on peer and social support dimensions when compared to healthy-weight adolescents. Independent of the adolescents' BMI categories, better cardiorespiratory fitness was positively associated with physical and psychological well-being when compared with the category of overweight/obesity and low cardiorespiratory fitness. In addition, adolescents with overweight/obesity combined with intermediate cardiorespiratory fitness or high cardiorespiratory fitness had higher scores on the peer and social support dimension., Conclusion: Cardiorespiratory fitness is a strong correlate of HRQoL across most of the dimensions measured, while BMI was a correlate of one dimension of HRQoL. Future studies should evaluate these relationships prospectively and experimentally., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Purpose: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway., Experimental Design: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX)., Results: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth., Conclusions: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma., (©2023 The Authors; Published by the American Association for Cancer Research.)

Age-related macular degeneration (AMD) is the leading cause of severe vision loss and blindness in elderly people worldwide. The damage to the retinal pigment epithelium (RPE) triggered by oxidative stress plays a central role in the onset and progression of AMD and results from the excessive accumulation of reactive oxygen species (ROS) produced mainly by mitochondria. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecular chaperone that contributes to the maintenance of mitochondrial integrity by decreasing the production and accumulation of ROS. The present study aimed to evaluate the presence and the role of TRAP1 in the RPE. Here, we report that TRAP1 is expressed in human adult retinal pigment epithelial cells and is located mainly in the mitochondria. Exposure of RPE cells to hydrogen peroxide decreases the levels of TRAP1. Furthermore, TRAP1 silencing increases intracellular ROS production and decreases mitochondrial respiratory capacity without affecting cell proliferation. Together, these findings offer novel insights into TRAP1 functions in RPE cells, opening possibilities to develop new treatment options for AMD.

The complex and dynamic three-dimensional organization of chromatin within the nucleus makes understanding the control of gene expression challenging, but also opens up possible ways to epigenetically modulate gene expression. Because plants are sessile, they evolved sophisticated ways to rapidly modulate gene expression in response to environmental stress, that are thought to be coordinated by changes in chromatin conformation to mediate specific cellular and physiological responses. However, to what extent and how stress induces dynamic changes in chromatin reorganization remains poorly understood. Here, we comprehensively investigated genome-wide chromatin changes associated with transcriptional reprogramming response to heat stress in tomato. Our data show that heat stress induces rapid changes in chromatin architecture, leading to the transient formation of promoter-enhancer contacts, likely driving the expression of heat-stress responsive genes. Furthermore, we demonstrate that chromatin spatial reorganization requires HSFA1a, a transcription factor (TF) essential for heat stress tolerance in tomato. In light of our findings, we propose that TFs play a key role in controlling dynamic transcriptional responses through 3D reconfiguration of promoter-enhancer contacts., (© 2023. The Author(s).)

Mutations in the Crumbs homolog 1 ( CRB1 ) gene lead to severe inherited retinal dystrophies (IRDs), accounting for nearly 80,000 cases worldwide. To date, there is no therapeutic option for patients suffering from CRB1 -IRDs. Therefore, it is of great interest to evaluate gene editing strategies capable of correcting CRB1 mutations. A retrospective chart review was conducted on ten patients demonstrating one or two of the top ten most prevalent CRB1 mutations and receiving care at Columbia University Irving Medical Center, New York, NY, USA. Patient phenotypes were consistent with previously published data for individual CRB1 mutations. To identify the optimal gene editing strategy for these ten mutations, base and prime editing designs were evaluated. For base editing, we adopted the use of a near-PAMless Cas9 (SpRY Cas9), whereas for prime editing, we evaluated the canonical NGG and NGA prime editors. We demonstrate that for the correction of c.2843G>A, p.(Cys948Tyr), the most prevalent CRB1 mutation, base editing has the potential to generate harmful bystanders. Prime editing, however, avoids these bystanders, highlighting its future potential to halt CRB1 -mediated disease progression. Additional studies investigating prime editing for CRB1 -IRDs are needed, as well as a thorough analysis of prime editing's application, efficiency, and safety in the retina.

Most influenza viruses express the PB1-F2 protein which is regarded as a virulence factor. However, PB1-F2 behaves differently in avian and mammalian hosts, suggesting that this protein may be involved in the species barrier crossings regularly observed in influenza viruses. To better understand the functions associated with this viral protein, we decided to compare the BioID2-derived proximity interactome of a human PB1-F2 from an H3N2 virus with that of an avian PB1-F2 from an H7N1 strain. The results obtained reveal that the two proteins share only a few interactors and thus common functions. The human virus protein is mainly involved in signaling by Rho GTPases while the avian virus protein is mainly involved in ribonucleoprotein complex biogenesis. PB1-F2 H3N2 interactors include several members of the 14-3-3 protein family, a family of regulatory proteins involved in many signaling pathways. We then validated the interaction with 14-3-3 proteins and were able to show that the association of H3N2-PB1-F2 with YWHAH increased the activity of the antiviral sensor MDA5, while H7N1-PB1-F2 had no effect. Collectively, these results show that PB1-F2 can associate with a large range of protein complexes and exert a wide variety of functions. Furthermore, PB1-F2 interactome differs according to the avian or human origin of the protein.

Importance: Each approach for primary total hip arthroplasty (THA) has a long learning curve, so a surgeon's choice to change their preferred approach needs to be guided by clear justifications. However, current evidence does not suggest that any of the THA approaches are more beneficial than others, and the choice of approach is mainly based on the knowledge and experience of the surgeon and individual patient characteristics., Objective: To assess the efficacy and safety associated with different surgical approaches for THA., Data Sources: A comprehensive search of PubMed, EMBASE, and Cochrane databases from inception to March 26, 2022; reference lists of eligible trials; and related reviews., Study Selection: Randomized clinical trials (RCTs) comparing different surgical approaches, including the 2-incision approach, direct anterior approach (DAA), direct lateral approach (DLA), minimally invasive direct lateral approach (MIS-DLA), minimally invasive anterolateral approach (MIS-ALA), posterior approach (PA), minimally invasive posterior approach (MIS-PA), and supercapsular percutaneously assisted total hip arthroplasty (SuperPath), for primary THA., Data Extraction and Synthesis: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses, 2 reviewers independently extracted data on study participants, interventions, and outcomes as well as assessed the risk of bias using the Cochrane risk of bias tool and the certainty of evidence using the Grading of Recommendations, Assessment, Development, and Evaluation framework. A frequentist framework was used to inform a series of random-effects network meta-analyses., Main Outcomes and Measures: The outcomes were hip score (range, 0-100, with higher scores indicating better overall hip condition), pain score (range, 0-100, with higher scores indicating more pain), hospitalization time, operation time, quality of life score, blood loss, cup abduction angle, and cup anteversion angle., Results: Of 2130 retrieved studies, 63 RCTs including 4859 participants (median [IQR] age, 64.0 [60.3-66.5] years; median [IQR] percentage male, 46.74% [38.64%-54.74%]) were eligible for analysis. Eight surgical approaches were evaluated. For hip score, DAA (mean difference [MD], 4.04; 95% CI, 1.92 to 6.16; moderate certainty), MIS-ALA (MD, 3.00; 95% CI, 0.43 to 5.59; moderate certainty), MIS-DLA (MD, 3.37; 95% CI, 1.05 to 5.68; moderate certainty), MIS-PA (MD, 4.46; 95% CI, 1.60 to 7.31; moderate certainty), PA (MD, 4.37; 95% CI, 1.87 to 6.88; high certainty), and SuperPath (MD, 5.00; 95% CI, 0.58 to 9.42; high certainty) were associated with greater improvement in hip score compared with DLA. DLA was associated with lower decrease in pain score than SuperPath (MD, 1.16; 95% CI, 0.13 to 2.20; high certainty) and MIS-DLA (MD, 0.90; 95% CI, 0.04 to 1.76; moderate certainty). PA was associated with shorter operation times compared with 2-incision (MD, -23.85 minutes; 95% CI, -36.60 to -11.10 minutes; high certainty), DAA (MD, -13.94 minutes; 95% CI, -18.79 to -9.08 minutes; moderate certainty), DLA (MD, -10.50 minutes; 95% CI, -16.07 to -4.94 minutes; high certainty), MIS-ALA (MD, -6.76 minutes; 95% CI, -12.86 to -0.65 minutes; moderate certainty), and SuperPath (MD, -13.91 minutes; 95% CI, -21.87 to -5.95 minutes; moderate certainty). The incidence of 6 types of complications did not differ significantly between the approaches., Conclusions and Relevance: In this study, moderate to high certainty evidence indicated that compared with PA, all surgical approaches except DLA were associated with similar improvements of hip score but longer operation time. DLA was associated with smaller improvement of hip score. The safety of the different approaches did not show significant differences. These findings will help health professionals and patients with better clinical decision-making and also provide references for policy makers.

The loss of smell (anosmia) related to SARS-CoV-2 infection is one of the most common symptoms of COVID-19. Olfaction starts in the olfactory epithelium mainly composed of olfactory sensory neurons surrounded by supporting cells called sustentacular cells. It is now clear that the loss of smell is related to the massive infection by SARS-CoV-2 of the sustentacular cells in the olfactory epithelium leading to its desquamation. However, the molecular mechanism behind the destabilization of the olfactory epithelium is less clear. Using golden Syrian hamsters infected with an early circulating SARS-CoV-2 strain harboring the D614G mutation in the spike protein; we show here that rather than being related to a first wave of apoptosis as proposed in previous studies, the innate immune cells play a major role in the destruction of the olfactory epithelium. We observed that while apoptosis remains at a low level in the damaged area of the infected epithelium, the latter is invaded by Iba1 + cells, neutrophils and macrophages. By depleting the neutrophil population or blocking the activity of neutrophil elastase-like proteinases, we could reduce the damage induced by the SARS-CoV-2 infection. Surprisingly, the impairment of neutrophil activity led to a decrease in SARS-CoV-2 infection levels in the olfactory epithelium. Our results indicate a counterproductive role of neutrophils leading to the release of infected cells in the lumen of the nasal cavity and thereby enhanced spreading of the virus in the early phase of the SARS-CoV-2 infection., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The ongoing COVID-19 pandemic continues to pose a need for new and efficient therapeutic strategies. We explored antisense therapy using oligonucleotides targeting the severe acute respiratory syndrome coronavirus (SARS-CoV-2) genome. We predicted in silico four antisense oligonucleotides (ASO gapmers with 100% PTO linkages and LNA modifications at their 5' and 3'ends) targeting viral regions ORF1a, ORF1b, N and the 5'UTR of the SARS-CoV-2 genome. Efficiency of ASOs was tested by transfection in human ACE2-expressing HEK-293T cells and monkey VeroE6/TMPRSS2 cells infected with SARS-CoV-2. The ORF1b-targeting ASO was the most efficient, with a 71% reduction in the number of viral genome copies. N- and 5'UTR-targeting ASOs also significantly reduced viral replication by 55 and 63%, respectively, compared to non-related control ASO (ASO-C). Viral titration revealed a significant decrease in SARS-CoV-2 multiplication both in culture media and in cells. These results show that anti-ORF1b ASO can specifically reduce SARS-CoV-2 genome replication in vitro in two different cell infection models. The present study presents proof-of concept of antisense oligonucleotide technology as a promising therapeutic strategy for COVID-19., Competing Interests: VAB was not employed by Andes Biotechnologies SpA but worked on ASO applications in oncology in collaboration with the company at the Centro de Centro Científico y Tecnológico de Excelencia Ciencia and Vida. 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 Dhorne-Pollet, Fitzpatrick, Da Costa, Bourgon, Eléouët, Meunier, Burzio, Delmas and Barrey.)

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment., Competing Interests: The authors have declared that no competing interests exist.

Abstract: The objective of this study was to determine the association between biases related to attrition, missing data, and the use of intention to treat and changes in effect size estimates in physical therapy randomized trials. A meta-epidemiological study was conducted. A random sample of randomized controlled trials included in meta-analyses in the physical therapy discipline were identified. Data extraction including assessments of the use of intention to treat principle, attrition-related bias, and missing data was conducted independently by two reviewers. To determine the association between these methodological issues and effect sizes, a two-level analysis was conducted using a meta-meta-analytic approach. Three hundred ninety-three trials included in 43 meta-analyses, analyzing 44,622 patients contributed to this study. Trials that did not use the intention-to-treat principle (effect size = -0.13, 95% confidence interval = -0.26 to 0.01) or that were assessed as having inappropriate control of incomplete outcome data tended to underestimate the treatment effect when compared with trials with adequate use of intention to treat and control of incomplete outcome data (effect size = -0.18, 95% confidence interval = -0.29 to -0.08).Researchers and clinicians should pay attention to these methodological issues because they could provide inaccurate effect estimates. Authors and editors should make sure that intention-to-treat and missing data are properly reported in trial reports., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Dipyrone is an analgesic and antipyretic drug commonly used in many countries. Although generally not recommended during pregnancy, it is known that many women use dipyrone during the gestational period. In this study, we investigated the endocrine and reproductive effects of dipyrone in female and male offspring rats exposed in utero from gestational days 10-21. Pregnant rats were treated with dipyrone at 25, 75, and 225 mg/kg/day via oral gavage. Developmental landmarks-anogenital index (AGI), number of nipples, vaginal opening, first estrus, and preputial separation-were evaluated in the offspring. Reproductive parameters, including estrous cycle regularity, daily sperm production, weight and histopathology of reproductive organs, steroid hormone levels, and gene expression of selected markers of reproductive function were assessed at adulthood. At the highest dose, dipyrone induced a significant increase in postimplantation losses/fetal death and delayed parturition in dams. Offspring exposed in utero to the highest dose also exhibited significant changes in some early life markers of endocrine disruption, in particular increased AGI in females, indicating a proandrogenic effect, and increased rate of retained nipples in males, indicating an antiandrogenic response. No changes were observed in markers of puberty onset or reproductive parameters at adulthood. These results suggest that exposure to therapeutically relevant doses of dipyrone may induce mild endocrine disruptive effects that can be detected in late pregnancy and early life. Such effects may be relevant considering dipyrone use by pregnant women and the possibility of coexposures with other endocrine disruptors., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The effects of UV light on the skin have been extensively investigated. However, systematic information about how the exposure to ultraviolet-A (UVA) light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass-spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional assays, we mapped the subcellular reorganization of the proteome of human keratinocytes in response to UVA light. Our workflow quantified and assigned subcellular localization for over 1,600 proteins, of which about 200 were found to redistribute upon UVA exposure. Reorganization of the proteome affected modulators of signaling pathways, cellular metabolism, and DNA damage response. Strikingly, mitochondria were identified as one of the main targets of UVA-induced stress. Further investigation demonstrated that UVA induces mitochondrial fragmentation, up-regulates redox-responsive proteins, and attenuates respiratory rates. These observations emphasize the role of this radiation as a potent metabolic stressor in the skin., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Animal models are essential to understanding COVID-19 pathophysiology and for preclinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap, and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, or bloodstream, resulted in a rapid decrease of SARS-CoV-2 RNA in wild-type larvae. However, when the virus was inoculated in the swim bladder, viral RNA stabilized after 24 h. By immunohistochemistry, epithelial cells containing SARS-CoV-2 nucleoprotein were observed in the swim bladder wall. Our data suggest an abortive infection of the swim bladder. In some animals, several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. A mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro . In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung., 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 Laghi, Rezelj, Boucontet, Frétaud, Da Costa, Boudinot, Salinas, Lutfalla, Vignuzzi and Levraud.)

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N -hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N -hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3'-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5'-end of nascent host mRNA.

Objective: To determine the effectiveness of TENS at relieving pain and improving physical function as compared to placebo TENS, and to determine its safety, in patients with knee osteoarthritis., Methods: Multi-centre, parallel, 1:1 randomized, double-blind, placebo-controlled clinical trial conducted in six outpatient clinics in Switzerland. We included 220 participants with knee osteoarthritis recruited between October 15, 2012, and October 15, 2014. Patients were randomized to 3 weeks of treatment with TENS (n = 108) or placebo TENS (n = 112). Our pre-specified primary endpoint was knee pain at the end of 3-weeks treatment assessed with the WOMAC pain subscale. Secondary outcome measures included WOMAC physical function subscale and safety outcomes., Results: There was no difference between TENS and placebo TENS in WOMAC pain at the end of treatment (mean difference -0.06; 95%CI -0.41 to 0.29; P = 0.74), nor throughout the trial duration (P = 0.98). Subgroup analyses did not indicate an interaction between patient/treatment characteristics and treatment effect on WOMAC pain at the end of treatment (P-interaction ≥0.22). The occurrence of adverse events was similar across groups, with 10.4% and 10.6% of patients reporting events in the TENS and placebo TENS groups, respectively (P = 0.95). No relevant differences were observed in secondary outcomes., Conclusions: TENS does not improve knee osteoarthritis pain when compared to placebo TENS. Therapists should consider other potentially more effective treatment modalities to decrease knee osteoarthritis pain and facilitate strengthening and aerobic exercise. Our findings are conclusive and further trials comparing TENS and placebo TENS in this patient population are not necessary., (Copyright © 2021 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). The remyelination process requires the activation, migration and differentiation of oligodendrocyte progenitor cells (OPC) in demyelinated areas. The metabolic dysfunction in chronic demyelinating lesions impairs the activation of OPCs, the myelin debris clearance by microglia decreases with age, along with diminished secretion of factors promoting OPC differentiation. Conventional magnetic resonance imaging (MRI) sequences have limited ability to differentiate unmyelinated and remyelinated lesions. Advanced MRI sequences based on magnetization transfer ratio (MTR), myelin water fraction (MWF) and diffusion tensor imaging (DTI) have been used to evaluate remyelination in clinical trials. More recently, the q-space myelin map (qMM) has been used on experimental and exploratory clinical studies. The improvement of myelin-specific MRI sequences with high reliability and standardization among centers will allow a more accurate evaluation of new therapies to improve remyelination. These new remyelination promoting treatments alone or in combination with current options may reduce the risk of long-term disability in MS., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Influenza A viruses cause important diseases in both human and animal. The PB1-F2 protein is a virulence factor expressed by some influenza viruses. Its deleterious action for the infected host is mostly described in mammals, while the available information is scarce in avian hosts. In this work, we compared the effects of PB1-F2 in avian and mammalian hosts by taking advantage of the zoonotic capabilities of an avian H7N1 virus. In vitro, the H7N1 virus did not behave differently when PB1-F2 was deficient while a H3N2 virus devoid of PB1-F2 was clearly less inflammatory. Likewise, when performing in vivo challenges of either chickens or embryonated eggs, with the wild-type or the PB1-F2 deficient virus, no difference could be observed in terms of mortality, host response or tropism. PB1-F2 therefore does not appear to play a major role as a virulence factor in the avian host. However, when infecting NF-κB-luciferase reporter mice with the H7N1 viruses, a massive PB1-F2-dependent inflammation was quantified, highlighting the host specificity of PB1-F2 virulence. Surprisingly, a chimeric 7:1 H3N2 virus harboring an H7N1-origin segment 2 (i.e. expressing the avian PB1-F2) induced a milder inflammatory response than its PB1-F2-deficient counterpart. This result shows that the pro-inflammatory activity of PB1-F2 is governed by complex mechanisms involving components from both the virus and its infected host. Thus, a mere exchange of segment 2 between strains is not sufficient to transmit the deleterious character of PB1-F2.

Nowadays, nanoscience is a leading modern science that has a major impact on the food, pharmaceutical, and agriculture sectors. Several nanomaterials show a great potential for use during vine growing and winemaking processes. In viticulture, nanotechnology can be applied to protect vines against phytopathogens and to improve grape yield and quality. Thus, nanotechnology may allow the use of lesser amounts of phytochemical compounds, reducing environmental impact and promoting a more sustainable agriculture. And in winemaking, nanomaterials and nanodevices can be used to control the growth of spoilage microorganisms and to reduce or remove undesirable compounds, such as ethyl phenols (4-ethylphenol and 4-ethylguaiacol), biogenic amines, and tartaric acid, and so on, as well as to facilitate some technological processes (i.e. in wine filtration to eliminate microorganisms). This review summarizes recent studies with applications of nanotechnology in viticulture in order to facilitate agronomic management and optimize grape production and in enology to improve wine quality and safety. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Aim to perform a systematic review of the literature on treatment of paediatric patients with MOG-IgG associated disease (MOGAD). Method We followed the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement. The search was conducted in Pubmed (MEDLINE) seeking articles of treatment of MOGAD in patients ≤ 18 years published between January 2012 and April 25th, 2020. Results We found 72 non-controlled studies (observational studies, case reports and expert recommendations). There were no randomized controlled trials (RCTs). The most commonly reported acute phase treatment was intravenous methylprednisolone in 88% followed by oral steroids in 67%, intravenous human immunoglobulin (IVIG) in 66% and plasma exchange in 33% of the studies. Long-term maintenance treatment was described by 53 studies mainly in relapsing disease course. The most frequently reported treatments were prolonged oral corticosteroids in 53% of the studies followed by azathioprine (51%), mycophenolate mofetil (45%), rituximab (41%) and periodic intravenous immunoglobulin (26%). Interpretation long-term treatment was reported mainly in relapsing MOGAD paediatric patients. However, the most frequently used medications are not those that have shown higher reduction in the annualised relapse rate in observational studies. RCTs with standardized outcomes are needed to confirm the safety and efficacy of current and new treatments., (Copyright © 2021. Published by Elsevier B.V.)

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Purpose: Severe pneumonia and acute respiratory distress syndrome (ARDS) have been described in patients with severe coronavirus disease 2019 (COVID-19). Recently, early clinical data reported the feasibility of low doses of radiation therapy (RT) in the treatment of ARDS in patients with severe COVID-19. However, the involved mechanisms remained unknown., Methods and Materials: Here, we used airways-instilled lipopolysaccharide (LPS) and influenza virus (H1N1) as murine models of pneumonia, and toll-like receptor (TLR)-3 stimulation in human lung macrophages., Results: Low doses of RT (0.5-1 Gray) decreased LPS-induced pneumonia, and increased the percentage of nerve- and airway-associated macrophages producing interleukin (IL) 10. During H1N1 viral infection, we observed decreased lung tissue damage and immune cell infiltration in irradiated animals. Low doses of RT increased IL-10 production by infiltrating immune cells into the lung. Irradiation of TLR-3 ligand-stimulated human lung macrophages ex vivo increased IL-10 secretion and decreased interferon γ production in the culture supernatant. The percentage of human lung macrophages producing IL-6 was also decreased., Conclusions: Our data highlight a mechanism by which low doses of RT regulate lung inflammation and skew lung macrophages toward an anti-inflammatory profile. These data provide a preclinical mechanistic support to clinical trials evaluating low doses of RT, such as COVID-19-induced ARDS., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

PB1-F2 is a virulence factor of influenza A virus known to increase viral pathogenicity in mammalian hosts. PB1-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers. However, the correlation between PB1-F2 structures and the resulting inflammatory response is unknown. Here, we used synchrotron-coupled Fourier transform-IR and deep UV microscopies to determine the presence of PB1-F2 fibers in influenza A virus-infected mice. In order to study the correlation between PB1-F2 structure and the inflammatory response, transgenic mice expressing luciferase under the control of an NF-κB promotor, allowing in vivo monitoring of inflammation, were intranasally instilled with monomeric, fibrillated, or truncated forms of recombinant PB1-F2. Our intravital NF-κB imaging, supported by cytokine quantification, clearly shows the proinflammatory effect of PB1-F2 fibers compared with N-terminal region of PB1-F2 unable to fibrillate. It is noteworthy that instillation of monomeric PB1-F2 of H5N1 virus induced a stronger inflammatory response when compared with prefibrillated PB1-F2 of H1N1 virus, suggesting mechanisms of virulence depending on PB1-F2 sequence. Finally, using whole-body plethysmography to measure volume changes in the lungs, we quantified the effects of the different forms of PB1-F2 on respiratory parameters. Thus, we conclude that PB1-F2-induced inflammation and respiratory distress are tightly correlated with sequence polymorphism and oligomerization status of the protein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: The Pilates method is an approach to body and mind exercises that has as its foundation the gain of stability, strength and flexibility, and the work of muscular control, posture and breathing, which can generate repercussions on oxidative stress and ROS production, it is expected that Pilates can satisfactorily influence glycemic and oxidative stress reduction in elderly diabetes., Aim: To analyze the effect of a Pilates protocol on variables indicative of metabolic control and oxidative stress in patients with Type 2 Diabetes Mellitus., Method: Randomized clinical trial in type 2 diabetics enrolled in Hiperdia Parnaíba. A Pilates protocol was performed for 8 weeks, with 2 weekly consultations. The tested variables were: blood glucose, glycated hemoglobin, lipid profile, C-reactive protein and malondialdehyde. ANOVA tests, correlation of Wilcoxon, Friedman and Spearman, were used, with a significance level of 5%., Results: 44 diabetics participated in the study (intervention group: 22; control: 22), with a mean age of 61.23 ± 8.49years, the majority being female (77.3%), married (59.1%), literate (31.8%), with an average BMI of 26.96 ± 4.35 kg/m 2 . When analyzing the effects of the protocol, there was a significant reduction in glycated hemoglobin (p = 0.002) and oxidative stress (p = 0.004) in the intervention group, however, there were no differences in fasting glucose (p = 0.055) and in the profile lipid, expressed by the total cholesterol (p = 0.654), HDL (p = 0.591), LDL (p = 0.564) and triglycerides (0.192). There was a moderate positive correlation between oxidative stress and glycated hemoglobin (r = 0.44, p = 0.000)., Conclusion: The exercise protocol based on the Pilates method produced a reduction in glycated hemoglobin and oxidative stress., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest, according to the attached document., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Dermatophytosis is a superficial fungal infection that affects humans and is very common in small animals. The treatment using the most commonly used antifungals is failing, and new therapeutic alternatives are required to combat the resistance of these fungal infections. Previous studies by the group have shown that clioquinol is an important therapeutic alternative in the treatment of dermatophytosis. The object was to conduct studies of antidermatophytic activity and the irritant potential from the double and triple combinations of clioquinol, terbinafine and ciclopirox in ex vivo and in vivo alternative models. To evaluate the irritant potential of antifungal combinations, the alternative HET-CAM method (chicken egg test chorioallantoic membrane) was used. Ex vivo models were used to assess the effectiveness of antifungal combinations, using pig hooves and veterinary fur. Any possible tissue damage was to assess through in histopathology of swine ears. HET-CAM results showed that all combinations can be classified as non-irritating, corroborated by the results of the histopathological evaluation of the pig's ear skin. Only the double combinations managed to remove 100% of the colony-forming units (CFU) formed on the pig's hooves. The clioquinol + terbinafine combination and the triple combination were more effective than clioquinol + ciclopirox in eradicating the preformed biofilm in fur of veterinary origin. These results show the potential of formulations of clioquinol in combination with antifungals for use in humans and in the veterinary field to combat dermatophytosis, as an important alternative therapy, for use in the near future., (© 2021 Wiley-VCH GmbH.)

Objectives: Different screen time activities may be related to sleep, physical activity, and sedentary behavior. The objective was to examine the association between self-reported screen time activities and accelerometer-measured 24-h movement behaviors., Study Design: This was a cross-sectional study., Methods: Adolescents' (n = 718, 50.4% girls, 16 years) sleep duration, sedentary behavior, light-intensity physical activity (LPA), and moderate-to-vigorous physical activity (MVPA) were estimated with wrist-worn accelerometry. Time spent on screen time activities related to studying, working, watching videos, playing video games, and using social media was self-reported. Multilevel linear regressions were used to test the association between screen time with sleep, sedentary behavior, and physical activity., Results: Boys and girls slept 6.4 and 6.7 h per night, spent 10.4 and 10.1 h/d in sedentary behavior, spent 4.0 and 4.4 h/d in LPA, and spent 34.7 and 29.2 min/d in MVPA, respectively. Studying was inversely related to LPA and MVPA. Working was inversely related to sleep and positively related to LPA. Watching videos was associated with lower LPA and MVPA. For boys, videogames were associated with increased sedentary behavior and lower LPA and MVPA. For girls, studying and/or using social media were associated with lower LPA and MVPA., Conclusions: Indicators of screen time were associated with different accelerometer-measured 24-h movement behaviors in this sample of Brazilian adolescents., (Copyright © 2021 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.)

The number of deaths due to systemic fungal infections is increasing alarmingly, which is aggravated by the limitations of traditional treatments and multidrug resistance. Therefore, the research and development of new therapeutic options against pathogenic fungi is an urgent need. To evaluate the fungicidal activity of a synthetic compound, 1,3-bis-(3,4-dichlorophenoxy)propan-2-aminium chloride (2j), through time-kill studies and pharmacokinetics/pharmacodynamics (PK/PD) modeling. The protective effect of the compound was also evaluated using the Drosophila melanogaster minihost model of candidiasis. Mathematical modeling of time-kill data of compound 2j was performed to obtain PD characteristics. Additionally, Toll-deficient D. melanogaster flies were infected with a Candida albicans strain and treated with 2j. We observed that compound 2j demonstrated a time- and dose-dependent fungicidal effect against Candida spp. and dermatophytes, even at low concentrations, and rapidly achieved kill rates reaching the maximum effect in less than one hour. The efficacy of the compound against systemic candidiasis in D. melanogaster flies was comparable to that achieved by fluconazole. These results support the potential of compound 2j as a systemic antifungal agent candidate and serve as a starting point for further studies involving mammalian animal models., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Influenza is one of the most widespread viral infections worldwide and represents a major public health problem. The risk that one of the next pandemics is caused by an influenza strain is high. It is important to develop broad-spectrum influenza antivirals to be ready for any possible vaccine shortcomings. Anti-influenza drugs are available but they are far from ideal. Arguably, an ideal antiviral should target conserved viral domains and be virucidal, that is, irreversibly inhibit viral infectivity. Here, a new class of broad-spectrum anti-influenza macromolecules is described that meets these criteria and display exceedingly low toxicity. These compounds are based on a cyclodextrin core modified on its primary face with long hydrophobic linkers terminated either in 6'sialyl- N -acetyllactosamine (6'SLN) or in 3'SLN. SLN enables nanomolar inhibition of the viruses while the hydrophobic linkers confer irreversibility to the inhibition. The combination of these two properties allows for efficacy in vitro against several human or avian influenza strains, as well as against a 2009 pandemic influenza strain ex vivo. Importantly, it is shown that, in mice, one of the compounds provides therapeutic efficacy when administered 24 h post-infection allowing 90% survival as opposed to no survival for the placebo and oseltamivir., Competing Interests: O.K., V.C., C.T., and F.S. are inventors on patent number EP18192559.5. All the other authors declare no conflict of interest., (© 2020 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Specific therapies targeting B lymphocytes in multiple sclerosis (MS) have demonstrated reductions in disease activity and disability progression. Several observational studies have also shown the effects of targeting B lymphocytes in other rare CNS inflammatory diseases, such as neuromyelitis optica spectrum disorder (NMOSD) and autoimmune encephalitis (AE). However, some drugs targeting cytokine receptors involved in B-lymphocyte maturation and proliferation resulted in negative outcomes in MS. These apparently conflicting findings have stimulated research on the pathophysiologic mechanisms of B lymphocytes in CNS inflammatory diseases. It has been demonstrated that B lymphocytes participate in the pathogenesis of these conditions as antigen-presenting cells, producing proinflammatory cytokines that induce Th1 and Th17 responses and producing antibodies. However, they are also able to produce anti-inflammatory cytokines, such as interleukin-10, functioning as regulators of autoimmunity. Understanding these diverse effects is essential for the development of focused treatments. In this review, we discuss the possible mechanisms that underlie B-lymphocyte involvement in MS, NMOSD, and AE and the outcomes obtained by treatments targeting B lymphocytes., (© 2020 American Academy of Neurology.)

Hematological and solid cancers catabolize the semiessential amino acid arginine to drive cell proliferation. However, the resulting low arginine microenvironment also impairs chimeric antigen receptor T cells (CAR-T) cell proliferation, limiting their efficacy in clinical trials against hematological and solid malignancies. T cells are susceptible to the low arginine microenvironment because of the low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC). We demonstrate that T cells can be reengineered to express functional ASS or OTC enzymes, in concert with different chimeric antigen receptors. Enzyme modifications increase CAR-T cell proliferation, with no loss of CAR cytotoxicity or increased exhaustion. In vivo, enzyme-modified CAR-T cells lead to enhanced clearance of leukemia or solid tumor burden, providing the first metabolic modification to enhance CAR-T cell therapies., (© 2020 by The American Society of Hematology.)

Background: Dermatophytoses are the most frequent fungal infections worldwide and there have been described clinical resistance to the commonly used antifungals. Clioquinol is an antimicrobial that had the oral formulations withdrawn from the market in the 70s due to the report of neurotoxicity and recently has been considered as an effective alternative for the treatment of dermatophytosis., Objectives: To evaluate the effect of the double and triple association between clioquinol with terbinafine and ciclopirox on clinical isolates of dermatophytes. The cytotoxicity of these associations on human leukocytes was also verified., Methods: Checkerboard method was used to evaluate the interaction between antifungal agents. Time-kill assay was used to verify fungicidal action and evaluate the combination with greater effect for TRU47 isolate. Cell viability was assessed by loss of integrity of the leukocyte membrane in order to verify the toxicity., Results: Synergistic interaction was observed in 42% of isolates when terbinafine was associated with clioquinol and in 50% of isolates when ciclopirox was associated with clioquinol. The triple association resulted in synergistic interaction for 75% of the isolates. Clioquinol + terbinafine and triple combination were more effective for TRU47 isolate, and the combinations exhibited a time-dependent fungicidal effect. Furthermore, the results of cell viability demonstrated that clioquinol and terbinafine combination is not cytotoxic to human leukocytes., Conclusions: Clioquinol in combination with antifungals in the treatment of dermatophytosis can be a therapeutic strategy to overcome problems related to resistance, action spectrum and toxicity of the antifungal drugs used in the clinic., (© 2020 Blackwell Verlag GmbH.)

Polymyxins are important therapeutic options for treating infections, mainly those caused by carbapenem-resistant Klebsiella pneumoniae . Specific chemical characteristics of polymyxins make it difficult to perform antimicrobial susceptibility testing, especially within the clinical laboratory. Here we aimed to evaluate the performance of three phenotypic methods: Rapid NP Polymyxin Test, ColiSpot test and the SuperPolymyxin medium. To accomplish this, 170 non-duplicate clinical K. pneumoniae isolates were analysed (123 colistin-resistant and 47 susceptible). The sensitivity and specificity obtained for Rapid Polymyxin NP Test, Colispot and SuperPolymyxin medium were, respectively, 90% and 94%, 74% and 100%, and 82% and 85%. Very major errors occurred more frequently in low-level colistin-resistant isolates (MICs 4 and 8 μg/mL). Rapid Polymyxin NP proved to be a method capable of identifying colistin-resistant strains in acceptable categorical agreement. However, major errors and very major errors of this method were considered unacceptable for colistin-resistance screening. Although the Colispot test is promising and easy to perform and interpret, the results did not reproduce well in the isolates tested. The colistin-containing selective medium (SuperPolymyxin) showed limitations, including quantification of mucoid colonies and poor stability. Nevertheless, Colispot and SuperPolymyxin medium methods did not present acceptable sensitivity, specificity and categorical agreement. It is essential to use analytical tools that faithfully reproduce bacterial resistance in vitro , especially in last-line drugs, such as polymyxins, when misinterpretation of a test can result in therapeutic ineffectiveness., (© 2020 The Authors.)

Here, we describe the crystal structures of two distinct isoforms of ligand-free human karyopherin RanBP5 and investigate its global propensity to interact with influenza A virus polymerase. Our results confirm the general architecture and mechanism of the IMB3 karyopherin-β subfamily whilst also highlighting differences with the yeast orthologue Kap121p. Moreover, our results provide insight into the structural flexibility of β-importins in the unbound state. Based on docking of a nuclear localisation sequence, point mutations were designed, which suppress influenza PA-PB1 subcomplex binding to RanBP5 in a binary protein complementation assay., (Copyright © 2020 Elsevier Ltd. All rights reserved.)