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Competing Interests: S.M.S. received research grants (to the institution) from CAN-FITE, AstraZeneca, Bioline RX, BMS, Halozyme, Clovis Oncology, CTG Pharma, Exelexis, Geicam, Halozyme, Incyte, Lilly, Moderna, Teva Pharmaceuticals, and Roche and owns stocks and options in CTG Pharma, DocBoxMD, Tyrnovo, VYPE, Cytora, and CAN-FITE (none related to this study). The other authors declare no conflicts of interest.

Purpose: To evaluate the association between the HER2 score as provided by the Oncotype DX Recurrence Score (RS) assay, tumor characteristics, and outcomes in early-stage, ER + HER2-negative breast cancer (BC)., Methods: All women insured by the Clalit Health Services, with early-stage, ER + HER2-negative BC who underwent RS testing between 2008 and 2011 were included. Patient/tumor characteristics and Kaplan-Meier estimates for distant recurrence-free survival (DRFS) and overall survival (OS) were compared by HER2 category, based on the HER2 score provided by the RS assay: lower HER2 score group representing the lower third of the HER2 score range (≤ 8.5); higher HER2 score group representing the upper 2 thirds of the HER2 score range (8.6-10.7)., Results: 1535 patients were included (948 node negative, 587 node positive); 330 (21.5%) were categorized as lower HER2 score and 1205 (78.5%) as higher HER2 score. Compared to the higher HER2 score group, the lower score group included a significantly higher proportion of patients with RS ≥ 26 in both node-negative (41% vs. 13.6%, P < .001) and node-positive diseases (36% vs. 19.4%, P < .001). Compared to the higher HER2 score group, the lower score group had significantly lower Oncotype ER and PR scores and lower proportion of lobular disease. Age and tumor size were comparable between the HER2 score groups. Within each RS category, DRFS and OS were not associated with the HER2 score., Conclusion: Lower HER2 score was associated with higher RS results. Further study is desired to elucidate the role and significance of HER2 expression in early-stage, ER + HER2-negative., Competing Interests: Declarations. Competing interests: Author HG reports personal fee from: AstraZeneca (Honorarium), Gilead (Honorarium and consulting), Eli-Lilly (Honorarium and consulting), MSD (Honorarium and consulting), Novartis (Honorarium and consulting), Pfizer (Honorarium and consulting), Roche (Honorarium), Rhenium Oncotest (Honorarium and consulting), all not related to the submitted manuscript. Author RY reports personal fees from: Roche (consulting, invited speaker, Research grant), Pfizer (consulting), Novartis (consulting, invited speaker), Rhenium (consulting), Medison (invited speaker), MSD (consulting, invited speaker), Astra-Zeneca (consulting, invited speaker), Eli Lilly (consulting, invited speaker), Gilead (consulting), Stemline (invited speaker, consulting) all not related to submitted manuscript. Author SPS reports: Roche (consultancy, advisory board, speaker's bureau, travel grant), Novartis (consultancy, advisory board, speaker's bureau), Pfizer (consultancy, advisory board, speaker's bureau, travel grant, Institutional independent research grant), Astra-Zeneca (consultancy, advisory board, speaker's bureau), Gilead (consultancy, advisory board, speaker's bureau, travel grant), Eli Lily (consultancy, advisory board, speaker's bureau), MSD (consultancy, advisory board, speaker's bureau), Stemline (consultancy), all via institutional fees and not related to the submitted manuscript. Author AS reports: Roche(consultancy, advisory board, speaker's bureau, travel grant), Novartis (consultancy, advisory board, speaker's bureau), Pfizer (consultancy, advisory board, speaker's bureau, travel grant, institutional independent research grant), Astra-Zeneca (consultancy, advisory board, speaker's bureau), Gilead (consultancy, advisory board, speaker's bureau, travel grant), Lily (consultancy, advisory board, speaker's bureau), MSD (consultancy, advisory board, speaker's bureau), Stemline (consultancy), all via institutional fees and not related to the submitted manuscript. Author AS reports: Eli Lilly (consulting, advisory board, speakers bureau), Pfizer (consulting, advisory board, speakers bureau), Roche (consulting, advisory board, speakers bureau, research grant), Novartis (consulting, advisory board, speakers bureau, research grant), Gilead (consulting, advisory board), MSD (consulting, advisory board, speakers bureau, travel grant), Astra-Zenca (consulting, advisory board), Progenetics (consulting, advisory board), Rhenium (consulting, advisory board), Neopharm (travel grant), Celgene (travel grant), Medison (travel grant), all not related to the submitted work. Author ABS reports being a consultant for Oncotest Rhenium, and Exact Sciences, related to the submitted manuscript, and to Pfizer, Can-Fite, and MDI, not related to the submitted manuscript. Author SMS reports: research grant from Can-Fite, AstraZeneca, Bioline RX, BMS, Halozyme, Clovis Oncology, CTG Pharma, Exelexis, Geicam, Halozyme, Incyte, Lilly, Moderna, Teva pharmaceuticals, and Roche, and owning stocks and options in CTG Pharma, DocBoxMD, Tyrnovo, VYPE, Cytora, and CAN-FITE, all not related to the submitted manuscript. All other authors have no conflicts of interest. This study was funded by Oncotest-Rhenium. The funder played no role in the design and conduct of the analysis or its interpretation, and the decision to submit the manuscript for publication., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This study offers an unprecedented valuation of the French surface waters WFD chemical monitoring dataset, covering 101 substances (metals, industrial and persistent organic pollutants (POPs), plant protection product (PPP) and biocides active substances, combustion residues) measured monthly on 4000 sites of the 6 main continental river basins, during 12 years (2009-2020). The concentration data were first made comparable through an original process removing the bias induced by the space-and-time heterogeneity of the monitoring labs performance, to gather a reference workable set of monthly contamination indicators. These were then used to display the substances' seasonal and interannual timeseries, revealing, e.g. the succession of PPP active substances contamination peaking periods in the 6 basins, or the long-term trends of the concentrations of the various chemicals, sometimes evidencing insufficiencies in the monitoring performance. These environmental observations were put in regard of the knowledge of the substances ban, restriction or reduction measures, to assess how streams' chemical quality responds to them. Additionally, the observed contamination features and their variations over the years are discussed in terms of changes in their usages, product substitution, emission sources, and linked to environmental processes like runoff, river dilution and physicochemical conditions. Some original findings and interpretation are provided on glyphosate and AMPA wide-scale data inter-relation, and some light is cast on the efficacy of the recent national policies restricting pesticides use in populated areas. For PPPs, the developed water contamination indicators were compared to tonnage data. We assessed their degree of linear relationship, which we propose to quantitatively express through a substance specific basin-to-river contamination coefficient. The interannual variations of this coefficient appear to be related to the changes in the water contamination seasonal patterns. We were also able to describe and validate the dependency of this coefficient on the molecular properties of the substances, conferring some capabilities for predicting the relative environmental risk induced by non-yet monitored compounds. We finally discuss the relevance of the developed indicators to complement the national chemical pollutants management system currently in place., Competing Interests: Declarations. Ethics approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Purpose: Clinicopathological factors and the 21-gene Oncotype DX Breast Recurrence Score (RS) test both influence prognosis. Our goal was to develop a new tool, RSClinN+, to individualize recurrence risk and chemotherapy benefit predictions by menopausal status for patients with HR+/human epidermal growth factor receptor 2-negative, lymph node-positive breast cancer by integrating the RS result with clinicopathological factors (grade, tumor size, age)., Methods: We used patient-level data from 5,283 patients treated with chemoendocrine therapy (CET) versus endocrine therapy alone (ET) in the S1007 (N = 4,916) and S8814 (N = 367) trials to develop the tool. Cox proportional hazards regression models stratified by trial were used to estimate 5-year invasive disease-free survival for pre- and postmenopausal woman, respectively. The integrated RSClinN+ model was compared with RS alone and clinicopathological models using likelihood ratio tests. Absolute CET benefit was estimated as the difference between ET and CET risk estimates. Validation of RSClinN+ was performed in 592 patients with node-positive disease in the Clalit Health Services registry., Results: RSClinN+ provides better prognostic information than RS model alone (premenopausal P = .034; postmenopausal P < .001) or clinicopathological model alone (premenopausal P = .002; postmenopausal, P < .001). In postmenopausal women, RS showed interaction with CET benefit ( P = .016), with RSClinN+ absolute CET benefit ranging from <0.1% to 21.5% over RS ranges 0-50. In premenopausal patients with RS ≤25, there was no significant interaction between RS and CET benefit. In external validation, RSClinN+ risk estimates were prognostic (hazard ratio, 1.75 [95% CI, 1.38 to 2.20]) and concordant with observed risk (Lin's concordance, 0.92)., Conclusion: RSClinN+ provides improved estimates of prognosis and absolute CET benefit for individual patients compared with RS or with clinical data alone and could be used in patient counseling.

Despite their intensive use and their impact on ecosystems, biocides and surfactants are still poorly regulated and poorly monitored at large scale. In the frame of the revision of the national regulatory surveillance plan of surface waters, France planned in 2018 a monitoring campaign at national scale focused on these two types of substances of very emerging concern. Forty-nine contaminants (32 biocides and 17 surfactants) were investigated in surface water and sediment samples from 91 sampling sites, and in effluent and sludge samples of 7 wastewater treatment plants (WWTP), in mainland France and overseas regions. Between 33 and 52 % of the target contaminants were quantified at least once in water and sediment. High frequencies of quantification were observed for the surfactants (up to 91 % in water samples and up to 57 % in sediment samples for LAS C10-C13) and for the biocides (up to 64 % for fipronil in water samples and up to 90 % for methyl nonyl ketone in sediment samples). The median concentrations of surfactants were up to 2 μg/L in mainland surface water samples and up to 528 μg/kg in sediment samples, and for biocides, the median concentrations were up to 0.18 μg/L in mainland surface water samples and up to 104 μg/kg in sediment samples. PNEC exceedances in water and sediment were determined for both types of substances. The analysis of effluent and sludge suggested significant but not total removal of these substances in the WWTP. Temporal and spatial variations of the concentrations of both types of substances in surface water samples were also observed, suggesting both punctual and diffuse contamination sources of the surface water investigated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background and Aims: Loss of activity of the inflammatory bowel disease (IBD) susceptibility gene, protein tyrosine phosphatase non-receptor type 2 ( PTPN2 ), is associated with altered microbiome composition in both human subjects and mice. Further, expansion of the bacterial pathobiont, adherent-invasive E. coli (AIEC), is strongly linked to IBD pathogenesis. The mechanism by which intestinal epithelial cells (IEC) maintain equilibrium between commensal microbiota and immune cells to restrict invading pathobionts is poorly understood. Here, we investigated the role of IEC-specific PTPN2 in regulating AIEC colonization., Methods: Tamoxifen-inducible, intestinal epithelial cell-specific Ptpn2 knockout mice ( Ptpn2 ΔIEC ) and control Ptpn2 fl/fl mice were infected with either non-invasive E. coli K12, or fluorescent-tagged m AIEC ( m AIEC red ) for four consecutive days or administered PBS. Subsequently, bacterial colonization in mouse tissues was quantified. mRNA and protein expression were assayed in intestinal epithelial cells (IECs) or whole tissue lysates by PCR and Western blot. Tissue cytokine expression was determined by ELISA. Intestinal barrier function was determined by in vivo administration of 4 kDa FITC-dextran (FD4) or 70kDa Rhodamine-B dextran (RD70) fluorescent probes. Confocal microscopy was used to determine the localization of tight-junction proteins., Results: Ptpn2 ΔIEC mice exhibited increased m AIEC red - but not K12 - bacterial load in the distal colon compared to infected Ptpn2 fl/fl mice. The higher susceptibility to m AIEC red infection was associated with altered levels of antimicrobial peptide (AMPs). Ileal RNA expression of the alpha-defensin AMPs, Defa5 and Defa6 , as well as MMP7, was significantly lower in Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice, after m AIEC red but not K12 infection. Further, we observed increased tight junction-regulated permeability determined by elevated in vivo FD4 but not RD70 permeability in Ptpn2 ΔIEC -K12 mice compared to their respective controls. This effect was further exacerbated in Ptpn2 ΔIEC m AIEC-infected mice. Further, Ptpn2 ΔIEC mice displayed lower IL-22, IL-6, IL-17A cytokine expression post m AIEC infection compared to Ptpn2 fl/fl controls. Recombinant IL-22 reversed the FD4 permeability defect and reduced bacterial burden in Ptpn2 ΔIEC mice post m AIEC challenge., Conclusion: Our findings highlight that intestinal epithelial PTPN2 is crucial for mucosal immunity and gut homeostasis by promoting anti-bacterial defense mechanisms involving coordinated epithelial-immune responses to restrict pathobiont colonization., Competing Interests: Conflict of interest: The authors declare no conflict of interest.

Competing Interests: OE declares that in the past 3 years he has received the following fees in connection with issues raised by this practice guideline: Fees for lecture events (from companies in the healthcare industry); fees for publications (from publishers or companies in the healthcare industry); as a partner of REK Rechtsanwälte (Stuttgart, Balingen) for legal advice or representation of affected patients. WW has held continuing education courses for the North Rhine Medical Association for the additional qualification in social medicine, he has given lectures at the Federal Medical Service/Medizinischer Dienst Bund on statutory pension vs. statutory health insurance, he has given lectures on rehabilitation in occupational medicine at the universities of Mainz and Düsseldorf for a fee, and he has published on rehabilitation in oncology – without a fee – in ecomed-verlag. EK declares no conflicts of interest. RWH declares no conflicts of interest.

Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 10 10  GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity., (© 2024 The Authors. Electrophoresis published by Wiley‐VCH GmbH.)

Hematopoiesis is a highly dynamical and stochastic process, challenging our understanding of homeostasis. Clinical studies of leukemia or neutropenic patients revealed that multiple blood cell types fluctuate spontaneously with large yet regular oscillations of their frequencies. Yet the stability of hematopoiesis in healthy individuals remains understudied. Here we report on both cross-sectional and longitudinal studies of dozens of healthy mice, through high-dimensional mass and spectral cytometry, to understand hematopoiesis at homeostasis. We found that all cell types in the bone marrow, blood, and spleen exhibit large variations of frequency (e.g., with coefficients of variation larger than 1). While the frequencies of individual cell type fluctuate, there existed extensive and robust correlations/anti-correlations between cell types, exemplified by the pronounced anti-correlation between blood neutrophils and B cells. Through longitudinal study of the blood content of healthy mice, we found that leukocyte fluctuations are ergodic yet subject to chaotic behaviors characterized by a broad spectrum of characteristic timescales. We then built a minimal mathematical model to capture these dynamical features of hematopoiesis (fluctuations, correlations, and chaos) and explain how the accumulation of B cells (e.g. during lymphoma development) would transition the blood cell dynamics from chaos to oscillations (as observed clinically). Finally, we demonstrated the ubiquity and consistency of the correlated fluctuations in hematopoiesis by comparing mouse cohorts of different genetic backgrounds and ages. To conclude, we discuss how study of hematopoiesis must factor in the newfound chaotic dynamics at homeostasis, towards better modeling the responses to perturbations.

Background: The 21-gene recurrence score (RS) assay (Oncotype DX) is used to guide adjuvant chemotherapy use for patients with hormone receptor-positive, HER2 (human epidermal growth factor receptor 2)-negative, axillary node-negative breast cancer. Its role, however, in providing prognostic information for late distant recurrence when added to clinicopathologic prognostic factors is unknown., Methods: A patient-specific meta-analysis including 10,004 women enrolled in three trials was updated using extended follow-up data from TAILORx, integrating the RS with histologic grade, tumor size, and age at surgery for the RSClin tool. Cox models integrating clinicopathologic factors and the RS were compared by using likelihood ratio (LR) tests. External validation of prognosis for distant recurrence in years 0 to 10 and 5 to 10 was performed in an independent cohort of 1098 women in a real-world registry., Results: RSClin provided significantly more prognostic information than either the clinicopathologic factors (ΔLR chi-square, 86.2; P<0.001) or RS alone (ΔLR chi-square, 131.0; P<0.001). The model was prognostic in an independent cohort for distant recurrence by 10 years after diagnosis (standardized hazard ratio, 1.56; 95% confidence interval, 1.25 to 1.94), was associated with late distant recurrence risk between 5 and 10 years after diagnosis (standardized hazard ratio, 1.78; 95% confidence interval, 1.25 to 2.55), and approximated the observed 10-year distant recurrence risk (Lin concordance, 0.87) and 5- to 10-year distant recurrence risk (Lin concordance, 0.92)., Conclusions: The 21-gene RS is prognostic for distant recurrence and overall survival in early breast cancer. A model integrating the 21-gene RS and clinicopathologic factors improved estimates of distant recurrence risk compared with either used individually and stratified late distant recurrence risk. (Funded by the National Cancer Institute, National Institutes of Health [U10CA180820, U10CA180794, UG1CA189859, U10CA180868, and U10CA180822] and others.).

Objective: Frozen Shoulder (FS) is a musculoskeletal pathology that leads to disability, functional decline, and a worsening in quality of life. Physiotherapists are the primary professionals involved in the treatment of FS, and it is essential to determine if their practice aligns with evidence-based suggestions., Aim: The aim is to assess the knowledge, skills, and operational strategies of Italian physiotherapists regarding FS and compare them with the existing literature., Methods: A web-based, anonymous, and voluntary cross-sectional survey was developed and administered to Italian physiotherapists to evaluate their clinical practices., Results: A total of 501 physiotherapists (38.5% female), completed the survey. More than half were under 35 years old (67.8%), declared working in private practice settings or being self-employed (57.1%), and were primarily engaged with musculoskeletal patients (81.8%). For subjects with FS at their first access, 21.4% identified X-rays as the most useful imaging technique to recognize pathologies beyond rehabilitation competence. In terms of general management, the majority reported working with an orthopaedic or physiatrist (47.5%) or in a multidisciplinary team (33.5%). Regarding manual therapy techniques, 63.3% of physiotherapists preferred intense degree mobilization, posterior direction, and moderate pain at the end of the range of motion for low irritable/high stiffness FS; however, there is a lack of consensus for managing very irritable/low stiffness FS. The majority of physiotherapists (57.7%) concurred that stretching improves the balance between metalloproteinase and its inhibitors. Additionally, 48.3% of physiotherapists selected mobile phone videos and messages to improve patients' compliance with exercises at home and for motivational/educational purposes., Discussion and Conclusion: The clinical practices of Italian physiotherapists in FS subjects sometimes deviate from evidence-based recommendations. While some discrepancies may be attributed to the existing uncertainties in the literature regarding knowledge and management strategies for FS patients, the authors recommend a stronger adherence to evidence-based practice., (© 2024. The Author(s).)

Fibroblast heterogeneity remains undefined in eosinophilic esophagitis (EoE), an allergic inflammatory disorder complicated by fibrosis. We utilized publicly available single-cell RNA sequencing data (GSE201153) of EoE esophageal biopsies to identify fibroblast sub-populations, related transcriptomes, disease status-specific pathways and cell-cell interactions. IL13-treated fibroblast cultures were used to model active disease. At least 2 fibroblast populations were identified, F&#95;A and F&#95;B. Several genes including ACTA2 were more enriched in F&#95;A. F&#95;B percentage was greater than F&#95;A and epithelial-mesenchymal transition upregulated in F&#95;B vs. F&#95;A in active and remission EoE. Epithelial-mesenchymal transition was also upregulated in F&#95;B in active vs. remission EoE and TNF-α signaling via NFKB was downregulated in F&#95;A. IL-13 treatment upregulated ECM-related genes more profoundly in ACTA2- fibroblasts than ACTA2+ myofibroblasts. After proliferating epithelial cells, F&#95;B and F&#95;A contributed most to cell-cell communication networks. ECM-Receptor interaction strength was stronger than secreted or cell-cell contact signaling in active vs. remission EoE and significant ligand-receptor pairs were driven mostly by F&#95;B. This unbiased analysis identifies at least 2 fibroblast sub-populations in EoE in vivo, distinguished in part by ACTA2 . Fibroblasts play a critical role in cell-cell interactions in EoE, most profoundly via ECM-receptor signaling via the F&#95;B sub-group.

Commensal intestinal bacteria shape our microbiome and have decisive roles in preserving host metabolic and immune homeostasis. They conspicuously impact disease development and progression, including amyloid-beta (Aβ) and alpha (α)-synuclein pathology in neurodegenerative diseases, conveying the importance of the brain-gut-microbiome axis in such conditions. However, little is known about the longitudinal microbiome landscape and its potential clinical implications in other protein misfolding disorders, such as prion disease. We investigated the microbiome architecture throughout prion disease course in mice. Fecal specimens were assessed by 16S ribosomal RNA sequencing. We report a temporal microbiome signature in prion disease and uncovered alterations in Lachnospiraceae, Ruminococcaceae, Desulfovibrionaceae, and Muribaculaceae family members in this disease. Moreover, we determined the enrichment of Bilophila, a microorganism connected to cognitive impairment, long before the clinical manifestation of disease symptoms. Based on temporal microbial abundances, several associated metabolic pathways and resulting metabolites, including short-chain fatty acids, were linked to the disease. We propose that neuroinflammatory processes relate to perturbations of the intestinal microbiome and metabolic state by an interorgan brain-gut crosstalk. Furthermore, we describe biomarkers possibly suitable for early disease diagnostics and anti-prion therapy monitoring. While our study is confined to prion disease, our discoveries might be of equivalent relevance in other proteinopathies and central nervous system pathologies., 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 © 2024 Losa, Morsy, Emmenegger, Manz, Schwarz, Aguzzi and Scharl.)

As clinical trials in oncology require substantial efforts, maximizing the insights gained from them by conducting subgroup analyses is often attempted. The goal of these analyses is to identify subgroups of patients who are likely to benefit, as well as the subgroups of patients who are unlikely to benefit from the studied intervention. International guidelines occasionally include or exclude novel medications and technologies for specific subpopulations based on such analyses of pivotal trials without requiring confirmatory trials. This Perspective discusses the importance of providing a complete dataset of clinical information when reporting subgroup analyses and explains why such transparency is key for better clinical interpretation of the results and the appropriate application to clinical care, by providing examples of transparent reporting of clinical studies and examples of incomplete reporting of clinical studies., Competing Interests: OR reports being a speaker for AstraZeneca, Boehringer Ingelheim, BMS, MSD, Novartis, Pfizer, Roche, Takeda, and Teva, and being a consultant for Rhenium, NucleaiMD, and Edocate. MM reports reported receiving a research grant from AstraZeneca, being a speaker for AstraZeneca, Roche, MSD, BMS, Pfizer, Novartis, Abbvie, and Takeda, and being a consultant for MSD and Takeda. DR reports being a speaker for BMS. RY reports receiving research grant from Roche, being a speaker for Roche, Novartis, MSD, AstraZeneca, and Eli Lilly, and being a consultant for Roche, Pfizer, Novartis, Medison, AstraZeneca, Gilead, and Eli Lilly. SS reports receiving a research grant from Can-Fite, AstraZeneca, Bioline RX, BMS, Halozyme, Clovis Oncology, CTG Pharma, Exelexis, Geicam, Halozyme, Incyte, Lilly, Moderna, Teva pharmaceuticals, and Roche, and owning stocks and options in CTG Pharma, DocBoxMD, Tyrnovo, VYPE, Cytora, and CAN-FITE. Author KG was employed by Leumit Health Services. 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 © 2024 Rotem, Geiger, Hanovich, Moskovitz, Kurman, Reinhorn, Peretz, Yerushalmi and Stemmer.)

Established treatments for advanced hepatocellular carcinoma (HCC) with Child-Pugh cirrhosis B (CPB, moderate hepatic dysfunction) are lacking. A recently published randomized phase 2 study in CPB HCC investigating the safety and efficacy of namodenoson (25 mg BID), an A3 adenosine-receptor agonist vs. placebo, suggested a favorable safety profile and a positive efficacy signal in patients with HCC with a CPB score of 7 (CPB7). The present study reports a 61-year-old woman with CPB7 HCC who received namodenoson for over 6 years through this study and its open-label extension. Computed tomography scans demonstrated partial and complete responses after 7 weeks and 4 years of treatment, respectively. Low albumin levels (31 g/l) and elevated baseline levels of alanine transaminase and aspartate aminotransferase (68 U/l and 44 U/l, respectively) were reported. After 4 weeks of treatment, these levels normalized and were stable for over 6 years. No treatment-emergent adverse events were noted. At the time of reporting, the response is ongoing as manifested by imaging studies and liver function evaluation., Competing Interests: MF, ZH, MHS, and PF are Can-Fite BioPharma, Ltd employees. SMS received research grant and stock options from Can-Fite BioPharma Ltd. AB-S is a consultant for Can-Fite BioPharma Ltd. The remaining authors declare no competing interests. Can-Fite BioPharma sponsored the phase 2 study; however, this sponsorship had no bearing on the results of the study or their scientific interpretation., (Copyright: © 2024 Ciurescu et al.)

Background: The hamstring muscles have a key function in the stability of the knee, limiting the anterior translation of the tibia. Therefore, to better perform rehabilitation after anterior cruciate ligament (ACL) surgery, it is important to develop a specific program based on hamstring strength recovery. It is possible to increase strength and muscle hypertrophy through high load exercises (HL); the recommended load is about 60%-80% of a maximum repetition (MR). Although low-load resistance training (LL) is ineffective at reproducing these values, the use of Blood Flow Restriction (BFR) with LL exercises appears to allow athletes to increase strength and muscle hypertrophy. This could limit functional decline and mitigate muscle atrophy allowing to optimize the recovery path and load management in post-operative patients. Recent scientific evidence, as far as the increasingly frequent use of BFR in rehabilitation and sports rehabilitation is concerned, suggests that these devices could represent one of the most significant innovations in the physiotherapy field. The aim of this study was to increase the strength of the hamstrings in the early phases of ACL rehabilitation with an LL-BFR training protocol for speeding up the development of adequate muscle strength., Case Descriptions: The patient, a 25-year-old male professional footballer, suffered from ACL injury during a football match, and after three months, he underwent a reconstruction ACL surgery with medial Hamstring tendon autograft. The athlete engaged a pre-operative program to restore a full active and passive knee range of motion and increase muscular strength. The first rehabilitation phase was supported by the adoption of BFR for hamstring strengthening, starting from the sixth week post-surgery (T0). A complete assessment of posterior hamstring muscles was performed through a hand-held dynamometer and load detection platforms. Three different types of exercises, focusing on the hamstring muscles, were chosen. Two further assessments were performed over time (T1 ant T2), highlighting different changes that occurred., Results: Interesting results showed a significant increase between T0 and T1 for all the assessed outcomes; in this case an average increase in strength of 59.87% between the beginning and the end of 4 weeks rehabilitation protocol was obtained in the first interval (T0-T1), while only 25.26% resulted in the second interval (T1-T2). However, the collected data should be considered with caution due to some limitations: the single experience of a single patient can hardly be generalized. Moreover, the reliance on isometric measurement of maximal strength and the absence of a direct strength measurement of the hamstrings during squat remain questionable., Conclusion: The final results suggest the capacity of the LL-BFR exercises to recreate a condition of a high intensity muscular effort with respect to load management, especially after surgery. This highlights the need to further investigate BFR adoption as it allows the patients to speed up their rehabilitation goals in developing adequate muscle strength., (© 2023 John Wiley & Sons Ltd.)

Data on using the 21-gene Recurrence Score (RS) testing on second breast cancer (BC; second primary or local recurrence) are lacking. This cohort study examined patients with first and second BC, who underwent 21-gene testing both times. It included a 'study-cohort' (60 N0/N1mi/N1 ER + HER2‒ BC patients with ≥2 RS results >1 year apart) and a 'general 21-gene-tested BC-cohort' (2044 previously described N0/N1mi/N1 patients). The median time between the first and second BC was 5.2 (IQR, 3.1-7.1) years; the second BC was ipsilateral in 68%. Patient/tumor characteristics of the first- and second-BC in the 'study-cohort' were similar, except for the RS which was higher in the second BC (median [IQR]: 23 [17-30] vs 17 [14-22], p < 0.001). Overall, 56 patients had follow-up data, of whom 5 experienced distant recurrence (2 RS 11-25 patients and 3 RS 26-100 patients). Studies exploring the prognostic utility of the RS in this setting are warranted., (© 2023. The Author(s).)

The A3 adenosine receptor (A3AR) is over-expressed in human hepatocellular carcinoma (HCC) cells. Namodenoson, an A3AR agonist, induces de-regulation of the Wnt and NF-kB signaling pathways resulting in apoptosis of HCC cells. In a phase I healthy volunteer study and in a phase I/II study in patients with advanced HCC, namodenoson was safe and well tolerated. Preliminary evidence of antitumor activity was observed in the phase I/II trial in a subset of patients with advanced disease, namely patients with Child-Pugh B (CPB) hepatic dysfunction, whose median overall survival (OS) on namodenoson was 8.1 months. A phase II blinded, randomized, placebo-controlled trial was subsequently conducted in patients with advanced HCC and CPB cirrhosis. The primary endpoint of OS superiority over placebo was not met. However, subgroup analysis of CPB7 patients (34 namodenoson-treated, 22 placebo-treated) showed nonsignificant differences in OS/progression-free survival and a significant difference in 12-month OS (44% vs 18%, p = 0.028). Partial response was achieved in 9% of namodenoson-treated patients vs 0% in placebo-treated patients. Based on the positive efficacy signal in HCC CPB7 patients and the favorable safety profile of namodenoson, a phase III study is underway., (© 2023. The Author(s).)

Background: There is a clinical association between migraine and multiple sclerosis., Main Body: Migraine and MS patients share similar demographics, with the highest incidence among young, female and otherwise healthy patients. The same hormonal constellations/changes trigger disease exacerbation in both entities. Migraine prevalence is increased in MS patients, which is further enhanced by disease-modifying treatment. Clinical data show that onset of migraine typically starts years before the clinical diagnosis of MS, suggesting that there is either a unidirectional relationship with migraine predisposing to MS, and/or a "shared factor" underlying both conditions. Brain imaging studies show white matter lesions in both MS and migraine patients. Neuroinflammatory mechanisms likely play a key role, at least as a shared downstream pathway. In this review article, we provide an overview of the literature about 1) the clinical association between migraine and MS as well as 2) brain MRI studies that help us better understand the mechanistic relationship between both diseases with implications on their underlying pathophysiology., Conclusion: Studies suggest a migraine history predisposes patients to develop MS. Advanced brain MR imaging may shed light on shared and distinct features, while helping us better understand mechanisms underlying both disease entities., (© 2023. Springer-Verlag Italia S.r.l., part of Springer Nature.)

Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations during development, as well as the quality of different batches and the consistency of the processes. Here, we compare biophysical methods to characterize purity and DNA content of viral capsids from five different serotypes (AAV2, AAV5, AAV6, AAV8, and AAV9). For this purpose, we apply multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to obtain the species' contents and to derive the wavelength-specific correction factors for the respective insert-size. In an orthogonal manner we perform anion exchange chromatography (AEX) and UV-spectroscopy and the three methods yield comparable results on empty/filled capsid contents with these correction factors. Whereas AEX and UV-spectroscopy can quantify empty and filled AAVs, only SV-AUC could identify the low amounts of partially filled capsids present in the samples used in this study. Finally, we employ negative-staining transmission electron microscopy and mass photometry to support the empty/filled ratios with methods that classify individual capsids. The obtained ratios are consistent throughout the orthogonal approaches as long as no other impurities and aggregates are present. Our results show that the combination of selected orthogonal methods can deliver consistent empty/filled contents on non-standard genome sizes, as well as information on other relevant critical quality attributes, such as AAV capsid concentration, genome concentration, insert size length and sample purity to characterize and compare AAV preparations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Interventions using split belt treadmills (SBTM) aim to improve gait symmetry (GA) in Parkinson's disease (PD). Comparative effects in conjugated SBTM conditions were not studied systematically despite potentially affecting intervention outcomes. We compared gait adaptation effects instigated by SBTM walking with respect to the type (increased\decreased speed) and the side (more/less affected) of the manipulated belt in PD., Methods: Eight individuals with PD performed four trials of SBTM walking, each consisted of baseline tied belt configuration, followed by split belt setting - either WS or BS belt's speed increased or decreased by 50% from baseline, and final tied belt configuration. Based on the disease's motor symptoms, a 'worst' side (WS) and a 'best' side (BS) were defined for each participant., Results: SB initial change in GA was significant regardless of condition (p ≤ 0.02). This change was however more pronounced for BS-decrease compared with its matching condition WS-increase (p = 0.016). Similarly, the same was observed for WS-decrease compared to BS-increase (p = 0.013). Upon returning to tied belt condition, both BS-decrease and WS-increased resulted in a significant change in GA (p = 0.04). Upper limb asymmetry followed a similar trend of GA reversal, although non-significant., Conclusions: Stronger effects on GA were obtained by decreasing the BS belt's speed of the best side, rather than increasing the speed of the worst side. Albeit a small sample size, which limits the generalisability of these results, we propose that future clinical studies would benefit from considering such methodological planning of SBTM intervention, for maximising of intervention outcomes. Larger samples may reveal arm swinging asymmetries alterations to match SBTM adaptation patterns. Finally, further research is warranted to study post-adaption effects in order to define optimal adaptation schemes to maximise the therapeutic effect of SBTM based interventions., (© 2023. The Author(s).)

Background and Aims: Relative roles of HSCs and portal fibroblasts in alcoholic hepatitis (AH) are unknown. We aimed to identify subpopulations of collagen type 1 alpha 1 (Col1a1)-expressing cells in a mouse AH model by single-cell RNA sequencing (scRNA-seq) and filtering the cells with the HSC (lecithin retinol acyltransferase [Lrat]) and portal fibroblast (Thy-1 cell surface antigen [Thy1] and fibulin 2 [Fbln2]) markers and vitamin A (VitA) storage., Approach and Results: Col1a1-green fluorescent protein (GFP) mice underwent AH, CCl 4 , and bile duct ligation (BDL) procedures to have comparable F1-F2 liver fibrosis. Col1a1-expressing cells were sorted via FACS by VitA autofluorescence and GFP for single-cell RNA sequencing. In AH, approximately 80% of Lrat+Thy1-Fbln2- activated HSCs were VitA-depleted (vs. ~13% in BDL and CCl 4 ). Supervised clustering identified a subset co-expressing Lrat and Fbln2 (Lrat+Fbln2+), which expanded 44-fold, 17-fold, and 1.3-fold in AH, BDL, and CCl 4 . Lrat+Fbln2+ cells had 3-15-times inductions of profibrotic, myofibroblastic, and immunoregulatory genes versus Lrat+Fbln2- cells, but 2-4-times repressed HSC-selective genes. AH activated HSCs had up-regulated inflammatory (chemokine [C-X-C motif] ligand 2 [Cxcl2], chemokine [C-C motif] ligand 2), antimicrobial (Il-33, Zc3h12a), and antigen presentation (H2-Q6, H2-T23) genes versus BDL and CCl 4 . Computational deconvolution of AH versus normal human bulk-liver RNA-sequencing data supported an expansion of LRAT+FBLN2+ cells in AH; AH patient liver immunohistochemistry showed FBLN2 staining along fibrotic septa enriched with LRAT+ cells; and in situ hybridization confirmed co-expression of FBLN2 with CXCL2 and/or human leukocyte antigen E in patient AH. Finally, HSC tracing in Lrat-Cre;Rosa26mTmG mice detected GFP+FBLN2+ cells in AH., Conclusion: A highly profibrotic, inflammatory, and immunoregulatory Lrat+Fbln2+ subpopulation emerges from HSCs in AH and may contribute to the inflammatory and immunoreactive nature of AH., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The incidence of Total Shoulder Arthroplasty (TSA) and Reverse Total Shoulder Arthroplasty (RTSA) is constantly increasing. As a result, the interest in post-surgical rehabilitation has grown, since it is crucial in order to achieve full recovery and successful outcomes. The first aim of this study is to investigate the Italian physiotherapists (PTs) clinical practice in the management of patients with TSA and RTSA and to compare it with the best evidence available in the literature. The second purpose of this study is to assess any existing difference between the survey answers and the different sample subgroups., Materials and Methods: This cross-sectional observation study was designed following the CHERRIES checklist and the STROBE guidelines. A 4-sections survey with a total of 30 questions was developed for investigating post-surgery rehabilitation management in patient with TSA and RTSA. The survey was sent to Italian PTs from December 2020 until February 2021., Results: Six-hundred and seven PTs completed the survey regarding both TSA and RTSA; 43.5% of participants (n = 264/607) stated that TSA is more likely to dislocate during abduction and external rotation. Regarding reverse prosthesis, 53.5% (n = 325/607) affirmed RTSA is more likely to dislocate during internal rotation, adduction and extension. In order to recover passive Range of Motion (pROM), 62.1% (n = 377/607) of participants reported that they gain anterior flexion, abduction, internal rotation, external rotation up to 30°, with full pROM in all directions granted at 6-12 weeks. Regarding the active ROM (aROM), 44.2% (n = 268/607) of participants stated that they use active-assisted procedures within a range under 90° of elevation and abduction at 3-4 weeks and higher than 90° at 6-12 weeks, with full recovery at a 3-month mark. Sixty-five point seven percent of the sample (n = 399/607) declared that, during the rehabilitation of patients with TSA, they tend to focus on strengthening the scapular and rotator cuff muscles, deltoid, biceps and triceps. Conversely, 68.0% (n = 413/607) of participants stated that, for the rehabilitation of patients with RTSA, they preferably focus on strengthening the periscapular and deltoid muscles. Finally, 33.1% (n = 201/607) of participants indicated the instability of the glenoid prosthetic component as the most frequent complication in patients with TSA, while 42.5% (n = 258/607) of PTs identified scapular neck erosion as the most frequent post-RTSA surgery complication., Conclusions: The clinical practice of Italian PTs effectively reflects the indications of the literature as far as the strengthening of the main muscle groups and the prevention of movements, which may result in a dislocation, are concerned. Some differences emerged in the clinical practice of Italian PTs, regarding the restoration of active and passive movement, the starting and progression of muscle strengthening and the return to sport (RTS). These differences are actually quite representative of the current knowledge in post-surgical rehabilitation for shoulder prosthesis in the rehabilitation field., Level of Evidence: V., (© 2023. The Author(s).)

Data on adjuvant chemotherapy (CT) benefit in ER + HER2‒ early-stage breast cancer (EBC) patients with Recurrence Score (RS) 26-30 are limited. This real-world study evaluated the relationships between the RS, adjuvant treatments, and outcomes in 534 RS 26-30 patients tested through Clalit Health Services (N0: n = 394, 49% CT-treated; N1mi/N1: n = 140, 62% CT-treated). The CT-treated and untreated groups were imbalanced (more high-risk clinicopathologic characteristics in CT-treated patients). With median follow-up of 8 years, Kaplan-Meier estimates for overall survival (OS), distant recurrence-free survival (DRFS), and BC-specific mortality (BCSM) were not significantly different between CT-treated and untreated N0 patients. Seven-year rates (95% CI) in CT-treated vs untreated: OS, 97.9% (94.4-99.2%) vs 97.9% (94.6-99.2%); DRFS, 91.5% (86.6-94.7%) vs 91.2% (86.0-94.6%); BCSM, 0.5% (0.1-3.7%) vs 1.6% (0.5-4.7%). For N1mi/N1 patients, OS/DRFS did not differ significantly between treatment groups; whereas BCSM did (1.3% [0.2-8.6%] vs 6.2% [2.0-17.7%] for CT-treated and untreated patients, respectively, p = 0.024)., (© 2023. The Author(s).)

Background: In acute cardiovascular disease management, the delay between the admission in a hospital emergency department and the assessment of the disease from a Delayed Enhancement cardiac MRI (DE-MRI) scan is one of the barriers for an immediate management of patients with suspected myocardial infarction or myocarditis., Objectives: This work targets patients who arrive at the hospital with chest pain and are suspected of having a myocardial infarction or a myocarditis. The main objective is to classify these patients based solely on clinical data in order to provide an early accurate diagnosis., Methods: Machine learning (ML) and ensemble approaches have been used to construct a framework to automatically classify the patients according to their clinical conditions. 10-fold cross-validation is used during the model's training to avoid overfitting. Approaches such as Stratified, Over-sampling, Under-sampling, NearMiss, and SMOTE were tested in order to address the imbalance of the data (i.e. proportion of cases per pathology). The ground truth is provided by a DE-MRI exam (normal exam, myocarditis or myocardial infarction)., Results: The stacked generalization technique with Over-sampling seems to be the best one providing more than 97% of accuracy corresponding to 11 wrong classifications among 537 cases. Generally speaking, ensemble classifiers such as Stacking provided the best prediction. The five most important features are troponin, age, tobacco, sex and FEVG calculated from echocardiography., Conclusion: Our study provides a reliable approach to classify the patients in emergency department between myocarditis, myocardial infarction or other patient condition from only clinical information, considering DE-MRI as ground-truth. Among the different machine learning and ensemble techniques tested, the stacked generalization technique is the best one providing an accuracy of 97.4%. This automatic classification could provide a quick answer before imaging exam such as cardiovascular MRI depending on the patient's condition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rahman 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.)

Proteinoids-simple polymers composed of amino acids-were suggested decades ago by Fox and coworkers to form spontaneously by heat. These special polymers may self-assemble in micrometer structures called proteinoid microspheres, presented as the protocells of life on earth. Interest in proteinoids increased in recent years, in particular for nano-biomedicine. They were produced by stepwise polymerization of 3-4 amino acids. Proteinoids based on the RGD motif were prepared for targeting tumors. Nanocapsules form by heating proteinoids in an aqueous solution and slowly cooling to room temperature. Proteinoid polymers and nanocapsules suit many biomedical applications owing to their non-toxicity, biocompatibility and immune safety. Drugs and/or imaging reagents for cancer diagnostic, therapeutic and theranostic applications were encapsulated by dissolving them in aqueous proteinoid solutions. Here, recent in vitro and in vivo studies are reviewed.

HER2 mutations are infrequent genomic events in biliary tract cancers (BTCs). Neratinib, an irreversible, pan-HER, oral tyrosine kinase inhibitor, interferes with constitutive receptor kinase activation and has activity in HER2-mutant tumours. SUMMIT is an open-label, single-arm, multi-cohort, phase 2, 'basket' trial of neratinib in patients with solid tumours harbouring oncogenic HER2 somatic mutations (ClinicalTrials.gov: NCT01953926). The primary objective of the BTC cohort, which is now complete, is first objective response rate (ORR) to neratinib 240 mg orally daily. Secondary objectives include confirmed ORR, clinical benefit rate, progression-free survival, duration of response, overall survival, safety and tolerability. Genomic analyses were exploratory. Among 25 treatment-refractory patients (11 cholangiocarcinoma, 10 gallbladder, 4 ampullary cancers), the ORR is 16% (95% CI 4.5-36.1%). The most common HER2 mutations are S310F (n = 11; 48%) and V777L (n = 4; 17%). Outcomes appear worse for ampullary tumours or those with co-occurring oncogenic TP53 and CDKN2A alterations. Loss of amplified HER2 S310F and acquisition of multiple previously undetected oncogenic co-mutations are identified at progression in one responder. Diarrhoea is the most common adverse event, with any-grade diarrhoea in 14 patients (56%). Although neratinib demonstrates antitumour activity in patients with refractory BTC harbouring HER2 mutations, the primary endpoint was not met and combinations may be explored., (© 2023. The Author(s).)

Parsonage-Turner Syndrome or neuralgic amyotrophy is a peripheral neuropathy typically characterized by an abrupt onset of pain, followed by progressive neurological deficits (e.g. weakness, atrophy, occasionally sensory abnormalities) that involve the upper limb, mainly the shoulder, encompassing an extensive spectrum of clinical manifestations, somehow difficult to recognize. This case report describes the proper management of a 35-year-old, bank employee and sports amateur who reported subtle and progressive upper limb disorder with previous history of neck pain. SARS-CoV-2 pandemic era made patient's access to the healthcare system more complicated. Nevertheless, proper management of knowledge, relevant aspects of telerehabilitation-based consultation for musculoskeletal pain, advanced skills, tools and technologies led the physiotherapist to suspect an atypical presentation of Parsonage-Turner Syndrome. Further, neurologist consultation and electromyography suggested signs of denervation in the serratus anterior and supraspinatus muscle. Therefore, an appropriate physiotherapist's screening for referral is conducted to correct diagnosis and thorough treatment.

A major effort in cancer research is to organize the complexities of the disease into fundamental traits. Despite conceptual progress in the last decades and the synthesis of hallmark features, no organizing principles governing cancer beyond cellular features exist. We analyzed experimentally determined structures harboring the most significant and prevalent driver missense mutations in human cancer, covering 73% ( n = 168178) of the Catalog of Somatic Mutation in Cancer tumor samples (COSMIC). The results reveal that a single structural element-κ-helix (polyproline II helix)-lies at the core of driver point mutations, with significant enrichment in all major anatomical sites, suggesting that a small number of molecular traits are shared by most and perhaps all types of cancer. Thus, we uncovered the lowest possible level of organization at which carcinogenesis takes place at the protein level. This framework provides an initial scheme for a mechanistic understanding underlying the development of tumors and pinpoints key vulnerabilities., (© The Author(s) 2023. Published by Oxford University Press on behalf of NAR Cancer.)

Background & Aims: Hepatocellular carcinoma (HCC) is a model of a diverse spectrum of cancers because it is induced by well-known etiologies, mainly hepatitis C virus (HCV) and hepatitis B virus. Here, we aimed to identify HCV-specific mutational signatures and explored the link between the HCV-related regional variation in mutations rates and HCV-induced alterations in genome-wide chromatin organization., Methods: To identify an HCV-specific mutational signature in HCC, we performed high-resolution targeted sequencing to detect passenger mutations on 64 HCC samples from 3 etiology groups: hepatitis B virus, HCV, or other. To explore the link between the genomic signature and genome-wide chromatin organization we performed chromatin immunoprecipitation sequencing for the transcriptionally permissive H3K4Me3, H3K9Ac, and suppressive H3K9Me3 modifications after HCV infection., Results: Regional variation in mutation rate analysis showed significant etiology-dependent regional mutation rates in 12 genes: LRP2, KRT84, TMEM132B, DOCK2, DMD, INADL, JAK2, DNAH6, MTMR9, ATM, SLX4, and ARSD. We found an enrichment of C->T transversion mutations in the HCV-associated HCC cases. Furthermore, these cases showed regional variation in mutation rates associated with genomic intervals in which HCV infection dictated epigenetic alterations. This signature may be related to the HCV-induced decreased expression of genes encoding key enzymes in the base excision repair pathway., Conclusions: We identified novel distinct HCV etiology-dependent mutation signatures in HCC associated with HCV-induced alterations in histone modification. This study presents a link between cancer-causing mutagenesis and the increased predisposition to liver cancer in chronic HCV-infected individuals, and unveils novel etiology-specific mechanisms leading to HCC and cancer in general., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

Shoulder pain is often attributable to a musculoskeletal disorder, but in some instances, it may be linked to pathologies outside the physiotherapist's area of expertise. Specifically, some intracranial problems can cause pain and disability to the shoulder complex. This case report aims to describe the clinical presentation, history taking, physical examination, and clinical decision-making procedures in a patient with an intracranial epidermoid cyst mimicking a musculoskeletal disorder of the shoulder girdle. A 42-year-old man complained of pain and disability in his left shoulder. Sudden, sharp pain was reported during overhead movements, associated with intermittent tingling of the left upper trapezius and left scapular area. Moreover, the patient reported reduced hearing in his left ear and left facial dysesthesia. The physical examination led the physiotherapist to hypothesize a pathology outside the physiotherapist's scope of practice and to refer the patient to another health professional to further investigate the patient through imaging. It is essential for the physiotherapist to recognize when the patient's clinical condition requires a referral to another healthcare professional. Therefore, the physiotherapist must be able to, in a timely manner, identify signs and symptoms suggesting the presence of medical pathology beyond his expertise, through appropriate medical history collection and physical evaluation.

Many conditions could potentially cause pain in the lower limbs. One of these is peripheral arterial disease (PAD). PAD is often a real challenge to be recognized for clinicians due to symptoms that commonly mimic musculoskeletal conditions. PAD is defined as a total or partial blockage of the vessels that supply blood from the heart to the periphery. Its prevalence is around 7 percent in subjects between 55 and 59, reaching almost 25% in individuals between 95 and 99 years old. The most dominant symptom of PAD is lower limb pain. Also, PAD can produce other symptoms such as discoloration, altered skin temperature, and, when arterial blood flow is insufficient to meet the metabolic demands of resting muscle or tissue, focal areas of ischemia. In our view, physical therapists should be capable of triaging for PAD in a direct access setting. Therefore, in this Professional Issue, we present the main characteristics of PAD and the physiotherapy role in its management. A supplementary step-by-step guide will provide further resources for testing PAD., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: To investigate the efficacy of manipulation under anesthesia (MUA) compared to other non-surgical therapeutic strategies for patients with frozen shoulder contracture syndrome (FSCS). Methods: A systematic review of literature was conducted. A literature search was performed in MEDLINE, EMBASE, PEDro, Cochrane Central Library and Scopus. Only randomized controlled trials were included and assessed for critical appraisal through the Cochrane Collaborations tools. Results: Five randomized controlled trials were included. The overall risk of bias (RoB) was high in 4 out of 5 of the included studies. MUA was found to be not superior in terms of reduction of pain and improvement of function when compared to cortisone injections with hydrodilatation (mean regression coefficient MUA −2.77 vs. injection −2.75; 95% CI (−1.11 to 1.15)) and home exercise (mean difference 95% CI: 0.2 (−0.64 to 1.02)) in the short term (3 months), and cortisone injections with hydrodilatation (mean regression coefficient MUA 3.13 vs. injection 3.23; 95% CI (−0.90 to 1.11)) in the long term (>6 months). Moreover, if compared to structured physiotherapy, MUA highlighted a higher Oxford Shoulder Score at final 1-year follow up (mean difference 95% CI: 1.05 (−1.28 to 3.39); p = 0.38). Similar results were obtained for disability, with statistically no significant long-term (>12 months) differences between MUA and home exercise (mean difference 95% CI: 0 (−3.2 to 3.2)) or structured physiotherapy (mean difference 95% CI: −0.50 (−5.70 to 4.70); p = 0.85)). Only two trials reported adverse events. Conclusions: This review suggested that limited and inconsistent evidence currently exists on the efficacy of MUA compared to other non-surgical strategies in the management of patients with FSCS. Future research should focus on clinical trials with higher methodological quality.

Objective: To evaluate the durability of response 3 months after the third BNT162b2 vaccine in adults aged 60 years and older., Design: Prospective cohort study., Setting: Single tertiary centre., Participants: Healthcare workers/family members aged ≥60 years old who received the third BNT162b2 dose., Interventions: Blood samples were drawn immediately before (T0), 10-19 days (T1) and 74-103 days (T2) after the third dose., Primary and Secondary Outcome Measures: Anti-spike IgG titres were determined using a commercial assay and seropositivity was defined as ≥50 arbitrary units (AU)/mL. Neutralising antibody titres were determined at T2. Adverse events, COVID-19 infections and Clinical Frailty Scale (CFS) levels were documented., Results: The analysis included 97 participants (median age, 70 years (IQR, 66-74), 58% CFS level 2). IgG titres, which increased significantly from T0 to T1 (median, 440 AU/mL (IQR, 294-923) and median, 25 429 AU/mL (IQR, 14 203-36 114), respectively; p<0.001), decreased significantly by T2, but all remained seropositive (median, 8306 AU/mL (IQR, 4595-14 701), p<0.001 vs T1). In a multivariable analysis, only time from the second vaccine was significantly associated with lower IgG levels at T2 (p=0.017). At T2, 60 patients were evaluated for neutralising antibodies; all were seropositive (median, 1294 antibody titres; IQR, 848-2072). Neutralising antibody and anti-spike IgG levels were correlated (r=0.6, p<0.001). No major adverse events or COVID-19 infections were reported., Conclusions: Anti-spike IgG and neutralising antibody levels remain adequate 3 months after the third BNT162b2 vaccine in healthy adults aged ≥60 years, although the decline in IgG is concerning. A third dose of vaccine in this population should be top priority., Competing Interests: Competing interests: SMS received research grants (to the institution) from CAN-FITE, AstraZeneca, BiolineRx, BMS, Halozyme, Clovis Oncology, CTG Pharma, Exelixis, Geicam, Incyte, Lilly, Moderna, Teva Pharmaceuticals and Roche, and owns stocks and options in CTG Pharma, DocBoxMD, TyrNovo, VYPE, Cytora and CAN-FITE. AB-S is employed by BioInsight., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

A key factor for assessing the state of the heart after myocardial infarction (MI) is to measure whether the myocardium segment is viable after reperfusion or revascularization therapy. Delayed enhancement-MRI or DE-MRI, which is performed 10 min after injection of the contrast agent, provides high contrast between viable and nonviable myocardium and is therefore a method of choice to evaluate the extent of MI. To automatically assess myocardial status, the results of the EMIDEC challenge that focused on this task are presented in this paper. The challenge's main objectives were twofold. First, to evaluate if deep learning methods can distinguish between non-infarct and pathological exams, i.e. exams with or without hyperenhanced area. Second, to automatically calculate the extent of myocardial infarction. The publicly available database consists of 150 exams divided into 50 cases without any hyperenhanced area after injection of a contrast agent and 100 cases with myocardial infarction (and then with a hyperenhanced area on DE-MRI), whatever their inclusion in the cardiac emergency department. Along with MRI, clinical characteristics are also provided. The obtained results issued from several works show that the automatic classification of an exam is a reachable task (the best method providing an accuracy of 0.92), and the automatic segmentation of the myocardium is possible. However, the segmentation of the diseased area needs to be improved, mainly due to the small size of these areas and the lack of contrast with the surrounding structures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

The efficacy/safety of combining palbociclib (a CDK4/6 inhibitor) and sunitinib (a multi-targeted receptor tyrosine kinase inhibitor) was evaluated, using patient-derived xenograft (PDX) models. Twenty-three PDX mice models were developed from patients with various solid tumors. The mice were randomized to 4 groups (5-6 mice in each): control/palbociclib (100 mg/kg)/sunitinib (50 mg/kg)/combination. Drugs were administered orally, 5 days/week. In 17/23 PDX models (74%), the combination demonstrated a synergistic inhibitory effect vs the monotherapies ("responder" models) with no unexpected toxicities. In 13/17 responder models, where standard-of-care (SOC) was an additional comparator, the combination was more effective than SOC in 7 models, as effective in 4, and less effective in 2. The mean ± SEM experiment duration in 15/17 responder models (2/17 were excluded due to technical issues) was 86 ± 12 and 31 ± 5 days for the combination and control groups, respectively (p = 0.0002). The effect of the combination was dose-dependent. Cell-viability experiments in A549/MDA-MB-231/HT-29 cell lines and experiments using tumor-derived primary cell spheroids supported the PDX findings. In conclusion, combination of palbociclib and sunitinib exerts a synergistic anti-tumor effect without adding unexpected toxicity. A clinical trial assessing this combination is underway., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In response to viral infection, neutrophils release inflammatory mediators as part of the innate immune response, contributing to pathogen clearance through virus internalization and killing. Pre-existing co- morbidities correlating to incidence of severe COVID-19 are associated with chronic airway neutrophilia. Furthermore, examination of COVID-19 explanted lung tissue revealed a series of epithelial pathologies associated with the infiltration and activation of neutrophils, indicating neutrophil activity in response to SARS- CoV-2 infection. To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory responses to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. SARS-CoV-2 infection of the airway epithelium alone does not result in a notable pro-inflammatory response from the epithelium. The addition of neutrophils induces the release of proinflammatory cytokines and stimulates a significantly augmented pro-inflammatory response subsequent SARS-CoV-2 infection. The resulting inflammatory response is polarized with differential release from the apical and basolateral side of the epithelium. Additionally, the integrity of the epithelial barrier is impaired with notable epithelial damage and infection of basal stem cells. This study reveals a key role for neutrophil-epithelial interactions in determining inflammation and infectivity in response to SARS-CoV-2 infection.

Both humoral and cellular anamnestic responses are significant for protective immunity against SARS-CoV-2. In the current study, the responses in elderly people before and after a fourth vaccine dose of BNT162b2 were compared to those of individuals immunized with three vaccine doses. Although a boost effect was observed, the high response following the third administration questions the necessity of an early fourth boost.

Climate change is one of the greatest public health threats of the twenty-first century. Recent surveys of veterinary students and practicing veterinarians have highlighted their concerns about the impacts of climate change on animal health and a strong desire to be knowledgeable about the practice and promotion of environmental sustainability within clinical practice. Most American Veterinary Medical Association (AVMA)-accredited veterinary schools have a veterinary teaching hospital (VTH) where veterinary students receive their core clinical education. Given this, VTHs may provide opportunities for students to learn how veterinary clinics can decrease their environmental footprint and actions they could incorporate into their future clinical work. To assess the feasibility of and support for introducing environmentally sustainable practices into VTHs, we distributed an anonymous online survey to all AVMA-accredited veterinary schools with an associated VTH. Responses were received from 843 individuals representing 23 VTHs in 7 countries. While the overwhelming majority of responding personnel believe this is an important topic, there is little evidence that sustainable behaviors are being practiced or showcased within VTHs. Respondents were most interested in working to increase recycling and reduce general waste and energy consumption within their hospitals. In addition to a lack of educational resources, funding was a commonly identified barrier to incorporating more environmentally sustainable practices. These results add to the growing evidence that enhanced incorporation of sustainability into veterinary medical education at all stages is needed and that VTHs provide a unique opportunity to lead by example.

This study proposes machine learning-based models to automatically evaluate the severity of myocardial infarction (MI) from physiological, clinical, and paraclinical features. Two types of machine learning models are investigated for the MI assessment: the classification models classify the presence of the infarct and the persistent microvascular obstruction (PMO), and the regression models quantify the Percentage of Infarcted Myocardium (PIM) of patients suspected of having an acute MI during their reception in the emergency department. The ground truth labels for these supervised models are derived from the corresponding Delayed Enhancement MRI (DE-MRI) exams and manual annotations of the myocardium and scar tissues. Experiments were conducted on 150 cases and evaluated with cross-validation. Results showed that for the MI (PMO inclusive) and the PMO (infarct exclusive), the best models obtained respectively a mean error of 0.056 and 0.012 for the quantification, and 88.67 and 77.33% for the classification accuracy of the state of the myocardium. The study of the features' importance also revealed that the troponin value had the strongest correlation to the severity of the MI among the 12 selected features. For the proposal's translational perspective, in cardiac emergencies, qualitative and quantitative analysis can be obtained prior to the achievement of MRI by relying only on conventional tests and patient features, thus, providing an objective reference for further treatment by physicians., Competing Interests: TD works for the Cardiac Simulation and Imaging Software Company (CASIS) that developed the QIR software used in this study. 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 Chen, Shi, Pommier, Cottin, Salomon, Decourselle, Lalande and Couturier.)

Background: In a previous analysis of this phase 3 trial, first-line ribociclib plus letrozole resulted in significantly longer progression-free survival than letrozole alone among postmenopausal patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. Whether overall survival would also be longer with ribociclib was not known., Methods: Here we report the results of the protocol-specified final analysis of overall survival, a key secondary end point. Patients were randomly assigned in a 1:1 ratio to receive either ribociclib or placebo in combination with letrozole. Overall survival was assessed with the use of a stratified log-rank test and summarized with the use of Kaplan-Meier methods after 400 deaths had occurred. A hierarchical testing strategy was used for the analysis of progression-free survival and overall survival to ensure the validity of the findings., Results: After a median follow-up of 6.6 years, 181 deaths had occurred among 334 patients (54.2%) in the ribociclib group and 219 among 334 (65.6%) in the placebo group. Ribociclib plus letrozole showed a significant overall survival benefit as compared with placebo plus letrozole. Median overall survival was 63.9 months (95% confidence interval [CI], 52.4 to 71.0) with ribociclib plus letrozole and 51.4 months (95% CI, 47.2 to 59.7) with placebo plus letrozole (hazard ratio for death, 0.76; 95% CI, 0.63 to 0.93; two-sided P = 0.008). No new safety signals were observed., Conclusions: First-line therapy with ribociclib plus letrozole showed a significant overall survival benefit as compared with placebo plus letrozole in patients with HR-positive, HER2-negative advanced breast cancer. Median overall survival was more than 12 months longer with ribociclib than with placebo. (Funded by Novartis; MONALEESA-2 ClinicalTrials.gov number, NCT01958021.)., (Copyright © 2022 Massachusetts Medical Society.)

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage and is, therefore, treated with systemic drugs, such as tyrosine-kinase inhibitors (TKIs). These drugs, however, offer only modest survival benefits due to the rapid development of drug resistance. To identify genes implicated in TKI resistance, a cluster of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 activation screen was performed in hepatoma cells treated with regorafenib, a TKI used as second-line therapy for advanced HCC. The screen results show that Hexokinase 1 (HK1), catalyzing the first step in glucose metabolism, is a top candidate for conferring TKI resistance. Compatible with this, HK1 was upregulated in regorafenib-resistant cells. Using several experimental approaches, both in vitro and in vivo, we show that TKI resistance correlates with HK1 expression. Furthermore, an HK inhibitor resensitized resistant cells to TKI treatment. Together, our data indicate that HK1 may function as a critical factor modulating TKI resistance in hepatoma cells and, therefore, may serve as a biomarker for treatment success., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Human adipose-derived stem cells (hADSCs) have the capacity for osteogenic differentiation and, in combination with suitable biomaterials and growth factors, the regeneration of bone defects. In order to differentiate hADSCs into the osteogenic lineage, bone morphogenetic proteins (BMPs) have been proven to be highly effective, especially when expressed locally by route of gene transfer, providing a constant stimulus over an extended period of time. However, the creation of genetically modified hADSCs is laborious and time-consuming, which hinders clinical translation of the approach. Instead, expedited single-surgery gene therapy strategies must be developed. Therefore, in an in vitro experiment, we evaluated a novel growth factor delivery system, comprising adenoviral BMP-2 transduced fascia tissue in terms of BMP-2 release kinetics and osteogenic effects, on hADSCs seeded on an innovative biomimetic spongiosa-like scaffold. As compared to direct BMP-2 transduction of hADSCs or addition of recombinant BMP-2, overexpressing fascia provided a more uniform, constant level of BMP-2 over 30 days. Despite considerably higher BMP-2 peak levels in the comparison groups, delivery by overexpressing fascia led to a strong osteogenic response of hADSCs. The use of BMP-2 transduced fascia in combination with hADSCs may evolve into an expedited single-surgery gene transfer approach to bone repair.

Osteoid osteoma (OO) is a relatively common, benign bone-forming tumour, which mainly occurs on the long tubular bones of the limbs in adolescents. Usually, the OO is classified based on its localisation. Night-time pain is the major symptom of OO, which is commonly relieved using non-steroidal anti-inflammatory drugs, while surgery is required only for those patients with severe pain or in case of failure of previous conservative treatments. Our case report describes a 56-year-old male basketball player who self-referred to our outpatient physical therapy with a shoulder pain complaint. Considering the anamnesis and the physical examination, the physical therapist referred the patient to an orthopaedic surgeon, who suggested a detailed imaging investigation. The peculiarity of this clinical case is the overlapping of two clinical presentations: the symptomatology of the OO and the concurrent mechanical disorder due to a rotator cuff tendinopathy.

Background: During embryogenesis lateral symmetry is broken, giving rise to Left/Right (L/R) breast tissues with distinct identity. L/R-sided breast tumors exhibit consistently-biased incidence, gene expression, and DNA methylation. We postulate that a differential L/R tumor-microenvironment crosstalk generates different tumorigenesis mechanisms., Methods: We performed in-silico analyses on breast tumors of public datasets, developed xenografted tumors, and conditioned MDA-MB-231 cells with L/R mammary extracts., Results: We found L/R differential DNA methylation involved in embryogenic and neuron-like functions. Focusing on ion-channels, we discovered significant L/R epigenetic and bioelectric differences. Specifically, L-sided cells presented increased methylation of hyperpolarizing ion channel genes and increased Ca 2+ concentration and depolarized membrane potential, compared to R-ones. Functional consequences were associated with increased proliferation in left tumors, assessed by KI67 expression and mitotic count., Conclusions: Our findings reveal considerable L/R asymmetry in cancer processes, and suggest specific L/R epigenetic and bioelectric differences as future targets for cancer therapeutic approaches in the breast and many other paired organs., (© 2022. The Author(s).)

Purpose: Immunogenicity of Covid-19 vaccines may be negatively impacted by anti-cancer treatment. The management of primary brain tumors (PBTs) routinely includes temozolomide and steroids, which are immune-suppressive. We aimed to determine the rate of seropositivity in PBT patients following receipt of two doses of the BNT162b2 vaccine., Methods: We prospectively evaluated IgG levels against SARS-CoV-2 spike protein in 17 PBT patients following two doses of the BNT162b2 vaccine. IgG levels were collected at two time points: T1-after a median of 44 days from the second vaccine dose and T2-after a median of 130 days from the second dose. Titers were compared against a group of healthy controls (HC) comprised of patients' family members., Results: At T1, 88.2% (15/17) of PBT patients achieved seroconversion, compared with 100% (12/12) of HCs. Median IgG titer was significantly lower in the PBT group (1908 AU/mL vs 8,198 AU/mL; p = 0.002). At T2, 80% (12/15) of PBT patients seroconverted, compared to 100% (10/10) of HCs. Median IgG titer remained significantly lower in the PBT group (410 AU/mLvs 1687 AU/mL; p = 0.002). During the peri-vaccination period, 15 patients received systemic treatment and 8 patients were treated with corticosteroids. All 3 patients who failed to seroconvert at T2 were treated with corticosteroids. In a univariate analysis, steroid use was negatively associated with antibody titer., Conclusion: Most PBT patients successfully seroconvert following two doses of the BNT162b2 vaccine, albeit with lower antibody titer compared to HCs. Steroid use during the vaccination period is associated with lower titer., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)