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Abstract Plasmacytoid dendritic cells (pDCs) are multifaceted immune cells executing various innate immunological functions. Their first line of defence consists in type I interferons (I-IFN) production upon nucleic acids sensing through endosomal Toll-like receptor (TLR) 7- and 9-dependent signalling pathways. Type I IFNs are a class of proinflammatory cytokines that have context-dependent functions on cancer immunosurveillance and immunoediting. In the last few years, different studies have reported that pDCs are also able to sense cytosolic DNA through cGAS–STING (stimulator of interferon genes) pathway eliciting a potent I-IFN production independently of TLR7/9. Human pDCs are also endowed with direct effector functions via the upregulation of TRAIL and production of granzyme B, the latter modulated by cytokines abundant in cancer tissues. pDCs have been detected in a wide variety of human malignant neoplasms, including virus-associated cancers, recruited by chemotactic stimuli. Although the role of pDCs in cancer immune surveillance is still uncompletely understood, their spontaneous activation has been rarely documented; moreover, their presence in the tumor microenvironment (TME) has been associated with a tolerogenic phenotype induced by immunosuppressive cytokines or oncometabolites. Currently tested treatment options can lead to pDCs activation and disruption of the immunosuppressive TME, providing a relevant clinical benefit. On the contrary, the antibody–drug conjugates targeting BDCA-2 on immunosuppressive tumor-associated pDCs (TA-pDCs) could be proposed as novel immunomodulatory therapies to achieve disease control in patients with advance stage hematologic malignancies or solid tumors. This Review integrate recent evidence on the biology of pDCs and their pharmacological modulation, suggesting their relevant role at the forefront of cancer immunity.

Abstract Background Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. Conclusions Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

ObjectivesAn increased number of elderly individuals affected by rheumatoid arthritis (RA) has been reported, including both patients with RA onset in advanced age and patients aged with the disease. In this registry-based study, we aimed to analyze the retention rate and cause of discontinuation of biologic (b) and targeted synthetic (ts)-disease-modifying anti-rheumatic drugs (DMARDs) in RA patients over 65 year old.MethodsRA patients enrolled in the Italian GISEA registry and starting a b- or a ts-DMARD over 65 years of age were included. Demographic, clinical, serologic, and therapeutic features were collected.ResultsA total of 1,221 elderly RA patients were analyzed (mean age 71.6 ± 5.2 years). RA was diagnosed before 65 years in 72.5% of cases, a 60.6% of patients experienced a previous b- or ts-DMARD. In patients older than 65 initiating a new b- or ts-DMARDS, tumor necrosis factor alpha inhibitors (TNFi) were prescribed in 29.6% of patients, abatacept in 24.8%, anti-interleukin 6 receptor antagonists (anti-IL6R) in 16.3%, Janus kinases inhibitors (JAKi) in 24.9%, and rituximab in 4.4%. The main causes of discontinuation were primary or secondary inadequate responses (66.1%). The median retention rate for all treatments was 181.3 weeks. A statistically higher retention rate was observed for abatacept when compared to TNFi (p = 0.02), JAKi (p

Genomic surveillance based on sequencing the entire genetic code of SARS-CoV-2 involves monitoring and studying genetic changes and variations in disease-causing organisms such as viruses and bacteria. By tracing the virus, it is possible to prevent epidemic spread in the community, ensuring a ‘precision public health’ strategy. A peptide-based design was applied to provide an efficacious strategy that is able to counteract any emerging viral variant of concern dynamically and promptly to affect the outcomes of a pandemic at an early stage while waiting for the production of the anti-variant-specific vaccine, which require longer times. The inhibition of the interaction between the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and one of the cellular receptors (DPP4) that its receptors routinely bind to infect human cells is an intriguing therapeutic approach to prevent the virus from entering human cells. Among the other modalities developed for this purpose, peptides surely offer unique advantages, including ease of synthesis, serum stability, low immunogenicity and toxicity, and small production and distribution chain costs. Here, we obtained a potent new inhibitor based on the rearrangement of a previously identified peptide that has been rationally designed on a cell dipeptidyl peptidase 4 (DPP4) sequence, a ubiquitous membrane protein known to bind the RBD-SPIKE domain of the virus. This novel peptide (named DPP4-derived), conceived as an endogenous “drug”, is capable of targeting the latest tested variants with a high affinity, reducing the VSV* DG-Fluc pseudovirus Omicron’s infection capacity by up to 14%, as revealed by in vitro testing in human Calu-3 cells. Surface plasmon resonance (SPR) confirmed the binding affinity of the new DPP4-derived peptide with Omicron variant RBD.

Abstract Background A proof-of-concept study to evaluate the feasibility and safety of minimally invasive ultrasound (US)-guided synovial biopsy of the radiocarpal (RC) joint using the anatomical snuffbox as an access route. Methods Twenty consecutive patients with active chronic arthritis of the wrist underwent minimally invasive US-guided synovial biopsy of the RC joint using the anatomical snuffbox as the access route. Samples were retrieved from 3 predetermined biopsy target sites of the RC synovia (proximal, vault, and distal site), aiming for a minimum of 12 samples. The procedure’s feasibility was evaluated based on the number and histological quality of retrieved tissue fragments tested on pre-defined histometric parameters. The safety and tolerability of the procedure were assessed through 1-week and 1-month follow-up clinical evaluations. Results A median number of 17 fragments (≥ 1 mm diameter size at macroscopic evaluation) per procedure was processed for histopathology (range 9–24) and dedicated to the study. At the histopathologic evaluation, a gradable tissue (visible lining layer and ≥ 4 fragments with IST) was recognized in 19/20 biopsies (95%), and all pre-defined histometric parameters were judged applicable and successfully measured in 19/19 gradable biopsies. All three biopsy target sites showed sampling accessibility. The entire procedure was generally well tolerated. At the 1-month follow-up, no patients showed infectious complications. Conclusions The access route through the anatomical snuff box in US-guided synovial biopsies of the RC joint allows for a safe and targeted collection of adequate tissue samples. This modification of the traditional access route may allow easier, repeatable, and safer sampling of anatomically distinct areas of the wrist in the course of arthritis.

IntroductionPlasmacytoid dendritic cells (pDCs) infiltrate a large set of human cancers. Interferon alpha (IFN-α) produced by pDCs induces growth arrest and apoptosis in tumor cells and modulates innate and adaptive immune cells involved in anti-cancer immunity. Moreover, effector molecules exert tumor cell killing. However, the activation state and clinical relevance of pDCs infiltration in cancer is still largely controversial. In Primary Cutaneous Melanoma (PCM), pDCs density decreases over disease progression and collapses in metastatic melanoma (MM). Moreover, the residual circulating pDC compartment is defective in IFN-α production.MethodsThe activation of tumor-associated pDCs was evaluated by in silico and microscopic analysis. The expression of human myxovirus resistant protein 1 (MxA), as surrogate of IFN-α production, and proximity ligation assay (PLA) to test dsDNA-cGAS activation were performed on human melanoma biopsies. Moreover, IFN-α and CXCL10 production by in vitro stimulated (i.e. with R848, CpG-A, ADU-S100) pDCs exposed to melanoma cell lines supernatants (SN-mel) was tested by intracellular flow cytometry and ELISA. We also performed a bulk RNA-sequencing on SN-mel-exposed pDCs, resting or stimulated with R848. Glycolytic rate assay was performed on SN-mel-exposed pDCs using the Seahorse XFe24 Extracellular Flux Analyzer.ResultsBased on a set of microscopic, functional and in silico analyses, we demonstrated that the melanoma milieu directly impairs IFN-α and CXCL10 production by pDCs via TLR-7/9 and cGAS-STING signaling pathways. Melanoma-derived immunosuppressive cytokines and a metabolic drift represent relevant mechanisms enforcing pDC-mediated melanoma escape.DiscussionThese findings propose a new window of intervention for novel immunotherapy approaches to amplify the antitumor innate immune response in cutaneous melanoma (CM).

Giacomo M Bruno,1,2 Maria Chiara Valentino,2 Alessandra Brunetti,3 Sergio Di Matteo,2 Ivan Begovic,2 Edoardo Croce,4 Garifallia Sakellariou,5 Serena Bugatti,6 Pietro Perotti,7 Silvia Vecchio,7 Simona Migliazza,7 Roberto Langella,8 Giorgio L Colombo1,2 1Department of Drug Sciences, Center of Pharmaceuticals Economics and Medical Technologies Evaluation, CEFAT.Unipv - University of Pavia at Centro di Ricerca SAVE Studi, Milano, Italy; 2S.A.V.E. Research Center - Studi Analisi Valutazioni Economiche, Milano, Italy; 3Research and Innovation Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 4Scientific Direction - IRCCS istituto Ortopedico Galeazzi, Milan, Italy; 5Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy; 6Department of Internal Medicine and Therapeutics, Division of Rheumatology, University of Pavia, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 7U.O.C. Epidemiological Observatory – Health Protection Agency of Pavia (ATS Pavia), Pavia, Italy; 8SIFO Regional Secretariat - Società Italiana di Farmacia Ospedaliera e dei Servizi Farmaceutici delle Aziende Sanitarie, Milano, ItalyCorrespondence: Giacomo M Bruno, Department of Drug Sciences, Center of Pharmaceuticals Economics and Medical Technologies Evaluation, CEFAT.Unipv - University of Pavia at Centro di Ricerca SAVE Studi, Milano, 20149, Italy, Email giacomomatteo.bruno@unipv.itBackground: The current flows of the SSN represent the set of interest whose interconnection alone justifies the current study. These flows can be interconnected with other sources, institutional or otherwise, in order to answer well-defined questions.Objective: The objective of the study is to verify, through the analysis of administrative databases, any differences in the consumption of health resources between biological off-patent originator drugs and biosimilars in real clinical practice, with particular reference to the rheumatology area.Methods: Through the use of assisted databases (BDA) of ATS Pavia we evaluated the differences in terms of consumption of health resources related to the different drugs under analysis. Annual and daily costs were calculated by total patient cost, stratified for different treatments, considering the sum of total costs for the prescriptions of drugs subject to the analysis. Another objective was to evaluate the adherence of the drugs of interest, by utilizing specific indicators (MPR).Results: A total of 145 patients were analyzed. Among enrolled patients, 26.9% of users were treated with a biosimilar drug, while 73.1% with a biologic originator. Adherence is higher if it is considered the population treated with biosimilar drugs (82.1%). Total cost (including drug prescriptions, hospitalizations, outpatient services, tests for any cause) during the observation period of 1 year is 14,274.08. 87.7% of the total is attributable to drugs. Non-hospitalized patients are the least expensive, whether they were treated with biologics or biosimilars.Conclusion: In our sample, biosimilar drugs tend to be underused: the treatment of a patient with a chronic autoimmune disease is a clinical process that involves many health professionals, and a criticality could also derive from the difficult communication between the various professional figures who get involved with the whole patient treatment.Keywords: real world data, biosimilar, rheumatic diseases, PDTA, EULAR recommendations, rheumatoid arthritis

Abstract Prostate cancer (PCa) is a leading cause of death in the male population commonly treated with androgen deprivation therapy that often relapses as androgen-independent and aggressive castration-resistant prostate cancer (CRPC). Ferroptosis is a recently described form of cell death that requires abundant cytosolic labile iron to promote membrane lipid peroxidation and which can be induced by agents that inhibit the glutathione peroxidase-4 activity such as RSL3. Exploiting in vitro and in vivo human and murine PCa models and the multistage transgenic TRAMP model of PCa we show that RSL3 induces ferroptosis in PCa cells and demonstrate for the first time that iron supplementation significantly increases the effect of RSL3 triggering lipid peroxidation, enhanced intracellular stress and leading to cancer cell death. Moreover, the combination with the second generation anti-androgen drug enzalutamide potentiates the effect of the RSL3 + iron combination leading to superior inhibition of PCa and preventing the onset of CRPC in the TRAMP mouse model. These data open new perspectives in the use of pro-ferroptotic approaches alone or in combination with enzalutamide for the treatment of PCa.

Standardized treatment options are lacking for patients with unresectable or multifocal follicular dendritic cell sarcoma (FDCS) and disease-related mortality is as high as 20%. Applying whole-genome sequencing (WGS) in one case and whole-exome sequencing (WES) in additional twelve cases, this study adds information on the molecular landscape of FDCS, expanding knowledge on pathobiological mechanisms and identifying novel markers of potential theragnostic significance. Massive parallel sequencing showed high frequency of mutations on oncosuppressor genes, particularly in RB1, CARS and BRCA2 and unveiled alterations on homologous recombination DNA damage repair-related genes in 70% (9/13) of cases. This indicates that patients with high-stage FDCS may be eligible for poly ADP ribose polymerase inhibition protocols. Low tumor mutational burden was confirmed in this study despite common PDL1 expression in FDCS arguing on the efficacy of immune checkpoint inhibitors. CDKN2A deletion, detected by WGS and confirmed by fluorescence in situ hybridization in 41% of cases (9/22) indicates that impairment of cell cycle regulation may sustain oncogenesis in FDCS. Absence of mutations in the RAS/RAF/MAPK pathway and lack of clonal hematopoiesis-related mutations in FDCS sanction its differences from dendritic cell-derived neoplasms of hematopoietic derivation. WGS and WES in FDCS provides additional information on the molecular landscape of this rare tumor, proposing novel candidate genes for innovative therapeutical approaches to improve survival of patients with multifocal disease.

Background Different patient clusters were preliminarily suggested to dissect the clinical heterogeneity in Still’s disease. Thus, we aimed at deriving and validating disease clusters in a multicentre, observational, prospective study to stratify these patients.Methods Patients included in GIRRCS AOSD-study group and AIDA Network Still Disease Registry were assessed if variables for cluster analysis were available (age, systemic score, erythrocyte sedimentation rate (ESR), C reactive protein (CRP) and ferritin). K-means algorithm with Euclidean metric and Elbow plot were used to derive an adequate number of clusters.Results K-means clustering assessment provided four clusters based on means standardised according to z-scores on 349 patients. All clusters mainly presented fever, skin rash and joint involvement. Cluster 1 was composed by 115 patients distinguished by lower values of age and characterised by skin rash myalgia, sore throat and splenomegaly. Cluster 2 included 128 patients identified by lower levels of ESR, ferritin and systemic score; multiorgan manifestations were less frequently observed. Cluster 3 comprised 31 patients categorised by higher levels of CRP and ferritin, they were characterised by fever and joint involvement. Cluster 4 contained 75 patients derived by higher values of age and systemic score. Myalgia, sore throat, liver involvement and life-threatening complications, leading to a high mortality rate, were observed in these patients.Conclusions Four patient clusters in Still’s disease may be recognised by a multidimensional characterisation (‘Juvenile/Transitional’, ‘Uncomplicated’, ‘Hyperferritinemic’ and ‘Catastrophic’). Of interest, cluster 4 was burdened by an increased rate of life-threatening complications and mortality, suggesting a more severe patient group.

Background: Major improvements in the management of rheumatoid arthritis (RA) have made clinical remission an achievable and desirable goal but, despite the relevance gained by a profound disease suppression, many patients with RA still miss clinical remission due to several factors influencing disease activity, including treatment adherence. Objective: To evaluate the effect of adherence to conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) on the achievement of clinical remission in a cohort of patients with new-onset inflammatory arthritis. Study design: A prospective cohort study was conducted using the ELECTRA database, which consists of clinical data from patients followed at the IRCCS Policlinico San Matteo Foundation (Pavia, Italy), linked to regional administrative healthcare databases. Methods: We enrolled patients with new-onset active disease between January 2006 and December 2013 and followed them until their first clinical remission or end of follow-up (December 2015). To assess the association of csDMARD adherence with clinical remission, we estimated the csDMARD proportion of days covered (PDC) during follow-up. PDC was added to the main clinical adjustment covariates as a time-dependent variable in a proportional hazard Cox regression model. Results: The cohort included 324 patients with a mean (SD) age of 58 (13.9) and predominantly female (74.5%). A total of 219 patients (67.6%) achieved clinical remission during follow-up and 85 (26.2%) in the first 6 months (early clinical remission). Cox regression models showed that a 10% increment of PDC increased the probability of achieving clinical remission by 10% ( p

Abstract Adenoid cystic carcinoma (ACC) of salivary gland is a slowly growing tumor showing a propensity for delayed recurrence, with decreased survival rates. The identification of poor prognosis patients may help in defining molecular-based targeted strategies in this rare disease orphan of new treatments. Through a gene expression microarray-based approach followed by GSE functional analysis the expression profile of 46 primary untreated ACC samples and of ACC (h-TERT) tumor cells was analyzed. Patients who experienced early relapse showed enrichment in proliferation-related gene sets, including the G2-M checkpoint, E2F and myc targets, and in gene sets related to IFN signaling and aberrant proteostasis (FDR

We focus the attention on the problem of audio classification in speech and music for multimedia applications. In particular, we present a comparison between two different techniques for speech/music discrimination. The first method is based on Zero crossing rate and Bayesian classification. It is very simple from a computational point of view, and gives good results in case of pure music or speech. The simulation results show that some performance degradation arises when the music segment contains also some speech superimposed on music, or strong rhythmic components. To overcome these problems, we propose a second method, that uses more features, and is based on neural networks (specifically a multi-layer Perceptron). In this case we obtain better performance, at the expense of a limited growth in the computational complexity. In practice, the proposed neural network is simple to be implemented if a suitable polynomial is used as the activation function, and a real-time implementation is possible even if low-cost embedded systems are used.

Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1-KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium–induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.

Abstract Chemotherapy-induced peripheral neuropathy (CIPN) and hypersensitivity reactions (HSRs) are among the most frequent and impairing side effects of the antineoplastic agent paclitaxel. Here, we demonstrated that paclitaxel can bind and activate complement component 5a receptor 1 (C5aR1) and that this binding is crucial in the etiology of paclitaxel-induced CIPN and anaphylaxis. Starting from our previous data demonstrating the role of interleukin (IL)-8 in paclitaxel-induced neuronal toxicity, we searched for proteins that activate IL-8 expression and, by using the Exscalate platform for molecular docking simulations, we predicted the high affinity of C5aR1 with paclitaxel. By in vitro studies, we confirmed the specific and competitive nature of the C5aR1-paclitaxel binding and found that it triggers intracellularly the NFkB/P38 pathway and c-Fos. In F11 neuronal cells and rat dorsal root ganglia, C5aR1 inhibition protected from paclitaxel-induced neuropathological effects, while in paclitaxel-treated mice, the absence (knock-out mice) or the inhibition of C5aR1 significantly ameliorated CIPN symptoms—in terms of cold and mechanical allodynia—and reduced the chronic pathological state in the paw. Finally, we found that C5aR1 inhibition can counteract paclitaxel-induced anaphylactic cytokine release in macrophages in vitro, as well as the onset of HSRs in mice. Altogether these data identified C5aR1 as a key mediator and a new potential pharmacological target for the prevention and treatment of CIPN and HSRs induced by paclitaxel.

Abstract The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug’s ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.

Soy is the main product of Brazilian agriculture and the fourth most cultivated bean globally. Since soy cultivation tends to increase and due to this large market, the guarantee of product quality is an indispensable factor for enterprises to stay competitive. Industries perform vigor tests to acquire information and evaluate the quality of soy planting. The tetrazolium test, for example, provides information about moisture damage, bedbugs, or mechanical damage. However, the verification of the damage reason and its severity are done by an analyst, one by one. Since this is massive and exhausting work, it is susceptible to mistakes. Proposals involving different supervised learning approaches, including active learning strategies, have already been used, and have brought significant results. Therefore, this paper analyzes the performance of non-supervised techniques for classifying soybeans. An extensive experimental evaluation was performed, considering (9) different clustering algorithms (partitional, hierarchical, and density-based) applied to 5 image datasets of soybean seeds submitted to the tetrazolium test, including different damages and/or their levels. To describe those images, we considered 18 extractors of traditional features. We also considered four metrics (accuracy, FOWLKES, DAVIES, and CALINSKI) and two-dimensionality reduction techniques (principal component analysis and t-distributed stochastic neighbor embedding) for validation. Results show that this paper presents essential contributions since it makes it possible to identify descriptors and clustering algorithms that shall be used as preprocessing in other learning processes, accelerating and improving the classification process of key agricultural problems.

Abstract Objectives Laryngeal squamous cell carcinomas (LSCCs) typically have an excellent prognosis for stage I tumors but a significant risk of locoregional and distant recurrence for intermediate to advanced disease. This study will investigate the clinical relevance of the tumor microenvironment in a large cohort of treatment‐naïve patients affected by stage II–IV LSCC. Methods Whole slide‐based digital pathology analysis was applied to measure six immune cell populations identified by immunohistochemistry (IHC) staining for CD3, CD8, CD20, CD66b, CD163 and CD38. Survival analysis was performed by Cox proportional hazards models and unsupervised hierarchical clustering using the k‐means method. Double IHC staining and in‐situ hybridisation by RNAscope allowed further analysis of a protumoral B cell population. Results A cohort of 98 patients was enrolled and analysed. The cluster of immune‐infiltrated LSCCs demonstrated a significantly worse disease‐specific survival rate. We also discovered a new association between high CD20+ B cells and a greater risk of distant recurrence. The phenotypic analysis of infiltrating CD20+ B cells showed a naïve (BCL6−CD27−Mum1−) regulatory phenotype, producing TGFβ but not IL10, according to an active TGFβ pathway, as proved by positive pSMAD2 staining. Conclusion The identification of regulatory B cells in the context of LSCC, along with the activation of the TGFβ pathway, could provide the basis for new trials investigating the efficacy of already available molecules targeting the TGFβ pathway in the treatment of LSCC.

O presente artigo tem por objetivo analisar um conjunto de charges publicadas na revista Cadernos do Terceiro Mundo entre os anos de 1977 e 1979. A proposta é fazer uma introdução ao debate da charge enquanto fonte e objeto para uma pesquisa histórica, considerando o humor e a ironia como elementos constitutivos para análises históricas, a partir de uma revista que tinha uma circulação em vários países do continente e com forte caráter político. Como estudo de caso, será analisada a abordagem das charges da Cadernos acerca da política dos direitos humanos do governo de Jimmy Carter (1977-1981) para os países da América Latina. Palavras-chave: Cadernos do Terceiro Mundo; charge; governo Carter; política de direitos humanos.

Powder bed homogeneity, contaminations, and printed surface quality are crucial in powder bed-based AM processes to obtain a defect-free part, but the scale at which these defects are seen is not compatible with the resolution of current industrial image-based monitoring solutions. In this work, we explore the implementation of an optical scanner in an industrial laser powder bed fusion (L-PBF) machine to detect powder bed and part-related defects. The sensor is mounted ”parasitically” on the recoater and exploits its movement to scan across the build platform before and after powder deposition to obtain high-resolution images. The acquisition seamlessly integrates with the process, without delaying the production as the acquisition occurs in parallel with the new layer deposition. The system was used to monitor test builds as well as longer builds (1000+ layers) to prove its robustness to the challenging L-PBF chamber environment. The in-situ powder bed images of the new monitoring system were compared to the acquisitions of a standard external camera setup. The improved image quality and resolution of the new system were demonstrated on both large-scale (>1 mm) and small-scale features. The new system proved to be capable of capturing printed surface topography anomalies and powder bed contaminations (

Context.--SARS-CoV-2 can undergo maternal-fetal transmission, heightening interest in the placental pathology findings from this infection. Transplacental SARS-CoV-2 transmission is typically accompanied by chronic histiocytic intervillositis together with necrosis and positivity of syncytiotrophoblast for SARS-CoV-2. Hofbauer cells are placental macrophages that have been involved in viral diseases, including HIV and Zika virus, but their involvement in SARS-CoV-2 is unknown. Objective.--To determine whether SARS-CoV-2 can extend beyond the syncytiotrophoblast to enter Hofbauer cells, endothelium, and other villous stromal cells in infected placentas of liveborn and stillborn infants. Design.--Case-based retrospective analysis by 29 perinatal and molecular pathology specialists of placental findings from a preselected cohort of 22 SARS-CoV-2infected placentas delivered to pregnant women testing positive for SARS-CoV-2 from 7 countries. Molecular pathology methods were used to investigate viral involvement of Hofbauer cells, villous capillary endothelium, syncytiotrophoblast, and other fetal-derived cells. Results.--Chronic histiocytic intervillositis and trophoblast necrosis were present in all 22 placentas (100%). SARS-CoV-2 was identified in Hofbauer cells from 4 of 22 placentas (18.2%). Villous capillary endothelial staining was positive in 2 of 22 cases (9.1%), both of which also had viral positivity in Hofbauer cells. Syncytiotrophoblast staining occurred in 21 of 22 placentas (95.5%). Hofbauer cell hyperplasia was present in 3 of 22 placentas (13.6%). In the 7 cases having documented transplacental infection of the fetus, 2 (28.6%) occurred in placentas with Hofbauer cell staining positive for SARS-CoV-2. Conclusions.--SARS-CoV-2 can extend beyond the trophoblast into the villous stroma, involving Hofbauer cells and capillary endothelial cells, in a small number of infected placentas. Most cases of SARS-CoV-2 transplacental fetal infection occur without Hofbauer cell involvement. (Arch Pathol Lab Med. 2021;145:1328-1340; doi: 10.5858/arpa.2021-0296-SA), At the start of the COVID-19 pandemic the medical and public health communities were concerned that the etiologic agent, a novel coronavirus that was eventually termed SARS-CoV-2, would be transmissible [...]

Abstract Esophageal cancer is an aggressive tumor that has a high rate of incidence and mortality worldwide. It is the 10th most frequent type in Brazil, being squamous cell carcinoma (ESCC) the predominant subtype. There is currently an incessant search to identify the frequently altered genes associated with esophageal squamous cell carcinoma biology that could be druggable. This study aimed to analyze the somatic mutation profile of a large panel of cancer-related genes in Brazilian ESCC. In a series of 46 ESCC diagnoses at Barretos Cancer Hospital, DNA isolated from paired fresh-frozen and blood tissue, a panel of 150 cancer-related genes was analyzed by next-generation sequencing. The genes with the highest frequency of mutations were TP53 (39/46, 84.8%), followed by NOTCH1 (7/46, 15.2%), NFE2L2 (5/46, 10.8%), RB1 (3/46, 6.5%), PTEN (3/46, 6.5%), CDKN2A (3/46, 6.5%), PTCH1 (2/46, 4.3%) and PIK3CA (2/46, 4.3%). There was no significant association between molecular and patients’ clinicopathological features. Applying an evolutionary action score of p53 (EAp53), we observed that 14 (35.9%) TP53 mutations were classified as high-risk, yet no association with overall survival was observed. Concluding, this the largest mutation profile of Brazilian ESCC patients, which helps in the elucidation of the major cancer-related genes in this population.

IntroductionSeveral studies and meta-analyses suggested the role of vitamin D 25OH in preventing severe forms of coronavirus disease 2019 (COVID-19). However, the evidence on the clinical benefits of vitamin D 25OH adequacy in patients hospitalized for COVID-19 remain conflicting and speculative. We aimed to investigate the association between vitamin D 25OH serum levels and mortality in hospitalized patients with moderate to severe COVID-19.MethodThis prospective observational multicentre study included 361 consecutive patients with moderate to severe COVID-19 admitted to the Italian hospitals involved in the NUTRI-COVID19 trial from March to August 2020. For each patient, serum vitamin D 25OH levels were assessed 48 h since admission and classified as deficient (

BackgroundThe role of working alliance remains unclear for many forms of internet-based interventions (IBIs), a set of effective psychotherapy alternatives that do not require synchronous interactions between patients and therapists. ObjectiveThis study examined the conceptual invariance, trajectories, and outcome associations of working alliance across an unguided IBI and guided IBIs that incorporated clinician support through asynchronous text messaging or video messaging. MethodsAdults with high education attainment (n=145) with subclinical levels of anxiety, stress, or depressive symptoms were randomized to 1 of 3 treatment conditions for 7 weeks. All participants received treatments from MyCompass, an unguided IBI using cognitive behavior therapy. Participants in condition 2 and 3 received supplemental, asynchronous clinician support through text and video, respectively. Working alliance with the IBIs was measured weekly using select items from the 12-item version of the Agnew Relationship Measure. Symptom and functional outcomes were assessed at baseline, at the end of treatment, and 1-month follow-up. ResultsWorking alliance with the IBIs was conceptually invariant across the 3 conditions. Working alliance followed a quadratic pattern of change over time for all conditions and declined significantly only in the text-support condition. After controlling for baseline symptoms, higher baseline levels of working alliance predicted less depression and less functional impairment at follow-up, whereas faster increases in working alliance predicted less worry at the end of treatment and at follow-up, all of which only occurred in the video-support condition. ConclusionsWorking alliance with the IBIs was generally established in the initial sessions. Although working alliance is conceptually invariant across IBIs with or without clinician support, the associations between working alliance and treatment outcomes among IBIs may differ depending on clinician involvement and the modalities of support. Trial RegistrationClinicalTrials.gov NCT05122429; https://clinicaltrials.gov/ct2/show/NCT05122429

* Context.--The number of neonates with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is increasing, and in a few there are reports of intrauterine infection. Objective.--To characterize the placental pathology findings in a preselected cohort of neonates infected by transplacental transmission arising from maternal infection with SARS-CoV-2, and to identify pathology risk factors for placental and fetal infection. Design.--Case-based retrospective analysis by a multinational group of 19 perinatal specialists of the placental pathology findings from 2 cohorts of infants delivered to mothers testing positive for SARS-CoV-2: live-born neonates infected via transplacental transmission who tested positive for SARS-CoV-2 after delivery and had SARS-CoV2 identified in cells of the placental fetal compartment by molecular pathology, and stillborn infants with syncytiotrophoblast positive for SARS-CoV-2. Results.--In placentas from all 6 live-born neonates acquiring SARS-CoV-2 via transplacental transmission, the syncytiotrophoblast was positive for coronavirus using immunohistochemistry, RNA in situ hybridization, or both. All 6 placentas had chronic histiocytic intervillositis and necrosis of the syncytiotrophoblast. The 5 stillborn/ terminated infants had placental pathology findings that were similar, including SARS-CoV-2 infection of the syncytiotrophoblast, chronic histiocytic intervillositis, and syncytiotrophoblast necrosis. Conclusions.--Chronic histiocytic intervillositis together with syncytiotrophoblast necrosis accompanies SARS-CoV2 infection of syncytiotrophoblast in live-born and stillborn infants. The coexistence of these 2 findings in all placentas from live-born infants acquiring their infection prior to delivery indicates that they constitute a pathology risk factor for transplacental fetal infection. Potential mechanisms of infection of the placenta and fetus with SARS-CoV-2, and potential future studies, are discussed. doi: 10.5858/arpa.2020-0771-SA, Following the identification of an outbreak of respiratory tract disease caused by a novel coronavirus infection, later termed coronavirus disease 2019 (COVID-19), in Wuhan, China, in December 2019, (1-5) there [...]

Though the bulk of the COVID-19 pandemic is behind, the search for effective and safe anti-SARS-CoV-2 drugs continues to be relevant. A highly pursued approach for antiviral drug development involves targeting the viral spike (S) protein of SARS-CoV-2 to prevent its attachment to the cellular receptor ACE2. Here, we exploited the core structure of polymyxin B, a naturally occurring antibiotic, to design and synthesize unprecedented peptidomimetics (PMs), intended to target contemporarily two defined, non-overlapping regions of the S receptor-binding domain (RBD). Monomers 1, 2, and 8, and heterodimers 7 and 10 bound to the S-RBD with micromolar affinity in cell-free surface plasmon resonance assays (KD ranging from 2.31 μM to 2.78 μM for dimers and 8.56 μM to 10.12 μM for monomers). Although the PMs were not able to fully protect cell cultures from infection with authentic live SARS-CoV-2, dimer 10 exerted a minimal but detectable inhibition of SARS-CoV-2 entry in U87.ACE2+ and A549.ACE2.TMPRSS2+ cells. These results validated a previous modeling study and provided the first proof-of-feasibility of using medium-sized heterodimeric PMs for targeting the S-RBD. Thus, heterodimers 7 and 10 may serve as a lead for the development of optimized compounds, which are structurally related to polymyxin, with improved S-RBD affinity and anti-SARS-CoV-2 potential.

During the COVID-19 pandemic, drug repurposing represented an effective strategy to obtain quick answers to medical emergencies. Based on previous data on methotrexate (MTX), we evaluated the anti-viral activity of several DHFR inhibitors in two cell lines. We observed that this class of compounds showed a significant influence on the virus-induced cytopathic effect (CPE) partly attributed to the intrinsic anti-metabolic activity of these drugs, but also to a specific anti-viral function. To elucidate the molecular mechanisms, we took advantage of our EXSCALATE platform for in-silico molecular modelling and further validated the influence of these inhibitors on nsp13 and viral entry. Interestingly, pralatrexate and trimetrexate showed superior effects in counteracting the viral infection compared to other DHFR inhibitors. Our results indicate that their higher activity is due to their polypharmacological and pleiotropic profile. These compounds can thus potentially give a clinical advantage in the management of SARS-CoV-2 infection in patients already treated with this class of drugs.

Angiogenesis is crucial for cancer progression. While several anti-angiogenic drugs are in use for cancer treatment, their clinical benefits are unsatisfactory. Thus, a deeper understanding of the mechanisms sustaining cancer vessel growth is fundamental to identify novel biomarkers and therapeutic targets. Alternative splicing (AS) is an essential modifier of human proteome diversity. Nevertheless, AS contribution to tumor vasculature development is poorly known. The Neuro-Oncological Ventral Antigen 2 (NOVA2) is a critical AS regulator of angiogenesis and vascular development. NOVA2 is upregulated in tumor endothelial cells (ECs) of different cancers, thus representing a potential driver of tumor blood vessel aberrancies. Here, we identified novel AS transcripts generated upon NOVA2 upregulation in ECs, suggesting a pervasive role of NOVA2 in vascular biology. In addition, we report that NOVA2 is also upregulated in ECs of gastric cancer (GC), and its expression correlates with poor overall survival of GC patients. Finally, we found that the AS of the Rap Guanine Nucleotide Exchange Factor 6 (RapGEF6), a newly identified NOVA2 target, is altered in GC patients and associated with NOVA2 expression, tumor angiogenesis, and poor patient outcome. Our findings provide a better understanding of GC biology and suggest that AS might be exploited to identify novel biomarkers and therapeutics for anti-angiogenic GC treatments.

Genomic surveillance based on sequencing the entire genetic code of SARS-CoV-2 involves monitoring and studying genetic changes and variations in disease-causing organisms such as viruses and bacteria. By tracing the virus, it is possible to prevent epidemic spread in the community, ensuring a 'precision public health' strategy. A peptide-based design was applied to provide an efficacious strategy that is able to counteract any emerging viral variant of concern dynamically and promptly to affect the outcomes of a pandemic at an early stage while waiting for the production of the anti-variant-specific vaccine, which require longer times. The inhibition of the interaction between the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and one of the cellular receptors (DPP4) that its receptors routinely bind to infect human cells is an intriguing therapeutic approach to prevent the virus from entering human cells. Among the other modalities developed for this purpose, peptides surely offer unique advantages, including ease of synthesis, serum stability, low immunogenicity and toxicity, and small production and distribution chain costs. Here, we obtained a potent new inhibitor based on the rearrangement of a previously identified peptide that has been rationally designed on a cell dipeptidyl peptidase 4 (DPP4) sequence, a ubiquitous membrane protein known to bind the RBD-SPIKE domain of the virus. This novel peptide (named DPP4-derived), conceived as an endogenous "drug", is capable of targeting the latest tested variants with a high affinity, reducing the VSV* DG-Fluc pseudovirus Omicron's infection capacity by up to 14%, as revealed by in vitro testing in human Calu-3 cells. Surface plasmon resonance (SPR) confirmed the binding affinity of the new DPP4-derived peptide with Omicron variant RBD. [ABSTRACT FROM AUTHOR]

Abstract Background Prevalence and outcomes of coronavirus disease (COVID)-19 in relation to immunomodulatory medications are still unknown. The aim of the study is to investigate the impact of glucocorticoids and immunosuppressive agents on COVID-19 in a large cohort of patients with chronic immune-mediated inflammatory arthritis. Methods The study was conducted in the arthritis outpatient clinic at two large academic hospitals in the COVID-19 most endemic area of Northern Italy (Lombardy). We circulated a cross-sectional survey exploring the prevalence of severe acute respiratory syndrome-coronavirus-2 nasopharyngeal swab positivity and the occurrence of acute respiratory illness (fever and/or cough and/or dyspnea), administered face-to-face or by phone to consecutive patients from 25 February to 20 April 2020. COVID-19 cases were defined as confirmed or highly suspicious according to the World Health Organization criteria. The impact of medications on COVID-19 development was evaluated. Results The study population included 2050 adults with chronic inflammatory arthritis receiving glucocorticoids, conventional-synthetic (cs), or targeted-synthetic/biological (ts/b) disease-modifying drugs (DMARDs). Laboratory-confirmed COVID-19 and highly suspicious infection were recorded in 1.1% and 1.4% of the population, respectively. Treatment with glucocorticoids was independently associated with increased risk of COVID-19 (adjusted OR [95% CI] ranging from 1.23 [1.04–1.44] to 3.20 [1.97–5.18] depending on the definition used). Conversely, patients treated with ts/bDMARDs were at reduced risk (adjusted OR ranging from 0.46 [0.18–1.21] to 0.47 [0.46–0.48]). No independent effects of csDMARDs, age, sex, and comorbidities were observed. Conclusions During the COVID-19 outbreak, treatment with immunomodulatory medications appears safe. Conversely, glucocorticoids, even at low-dose, may confer increased risk of infection. Trial registration Retrospectively registered. Not applicable.