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Abstract The PD-1/PDL-1 immune checkpoint inhibitors revolutionized cancer treatment, yet osteosarcoma remains a therapeutic challenge. In some types of cancer, PD-1 receptor is not solely expressed by immune cells but also by cancer cells, acting either as a tumor suppressor or promoter. While well-characterized in immune cells, little is known about the role and interactome of the PD-1 pathway in cancer. We investigated PD-1 expression in human osteosarcoma cells and studied PD-1 protein–protein interactions in cancer. Using U2OS cells as a model, we confirmed PD-1 expression by western blotting and characterized its intracellular as well as surface localization through flow cytometry and immunofluorescence. High-throughput analysis of PD-1 interacting proteins was performed using a pull-down assay and quantitative mass spectrometry proteomic analysis. For validation and molecular modeling, we selected tyrosine kinase receptor AXL—a recently reported cancer therapeutic target. We confirmed the PD-1/AXL interaction by immunoblotting and proximity ligation assay (PLA). Molecular dynamics (MD) simulations uncovered binding affinities and domain-specific interactions between extracellular (ECD) and intracellular (ICD) domains of PD-1 and AXL. ECD complexes exhibited strong binding affinity, further increasing for the ICD complexes, emphasizing the role of ICDs in the interaction. PD-1 phosphorylation mutant variants (Y223F and Y248F) did not disrupt the interaction but displayed varying strengths and binding affinities. Using bemcentinib, a selective AXL inhibitor, we observed reduced binding affinity in the PD-1/AXL interaction, although it was not abrogated. To facilitate the future translation of this finding into clinical application, we sought to validate the interaction in canine osteosarcoma. Osteosarcoma spontaneously occurs at significantly higher frequency in dogs and shares close genetic and pathological similarities with humans. We confirmed endogenous expression of PD-1 and AXL in canine osteosarcoma cells, with PD-1/AXL interaction preserved in the dog cells. Also, the interacting residues remain conserved in both species, indicating an important biological function of the interaction. Our study shed light on the molecular basis of the PD-1/AXL interaction with the implication for its conservation across species, providing a foundation for future research aimed at improving immunotherapy strategies and developing novel therapeutic approaches.

Prosody includes the pitch, timing and loudness in speech, which can convey meaning and emotion. This study examines whether prosodic categories affect novel noun learning and whether the referent characteristic influence learning. Previous research showed that emotional prosody interfered with adults’ noun learning (West et al., 2017), but it had no effect on children (West et al., 2022). However, these researchers varied their method across ages, including animacy and complexity of the referent, and it is unclear if the results extend beyond the three emotional prosodies tested. Participants in the current set of studies heard novel words presented in five prosodic categories (within-subject) in order to learn the label for either animate or inanimate objects (between-subject). Study 1 compared inanimate objects and aliens, with better noun learning performance for inanimate objects. Study 2 compared inanimate objects with the same objects with faces added, but there was no difference in noun learning by object type. Both studies showed differences in noun learning by the prosodic category, with warning less accurate than naming. These results demonstrate how extralinguistic factors like prosody, attention and referent complexity influence noun learning.

Introduction: In patients with acute spinal cord injury (SCI) and degenerative cervical myelopathy (DCM), spinal cord compression is considered a main contributor to spinal cord damage, associated with cerebrospinal fluid (CSF) space obstruction. CSF pressure (CSFP) dynamics are studied as a potential indirect biomechanical marker for spinal cord compression, and as a proxy to estimate spinal cord perfusion pressure (SCPP). Research question: Evidence for safety and feasibility of CSFP dynamics in clinical trials as well as interrelations with neuroimaging and intraspinal pressure, and relation to preclinical CSFP models. Material and methods: Systematic review. This review followed PRISMA guidelines, risk of bias assessment with ROBINS-I tool, PROSPERO registration (CRD42024545629). Results: 11 relevant papers were identified (n = 212 patients, n = 194 intraoperative, n = 18 bedside). Risk of bias for safety reporting was low-moderate. Intraoperative CSFP assessments were commonly performed in acute SCI. CSFP was assessed to calculate SCPP (7/11), to evaluate effects from surgical decompression (5/11) and for therapeutic CSF drainage (3/11). The adverse event rate associated with the intrathecal catheter was 8% (n = 15/194). Discussion and conclusion: The preliminary safety and feasibility profile of CSFP assessments in spinal cord compression encourages clinical application. However, a deeper risk-benefit analysis is limited as the clinical value is not yet determined, given challenges of defining disease specific critical CSFP and SCPP thresholds. The interrelation between measures of CSFP and neuroimaging is yet to be proven. Targeted preclinical studies are essential to improve our understanding of complex CSFP-cord compression interrelations.

The Diagnostic Immunology and Flow Cytometry Committee (DIFCC) acts as an expert scientific and educational resource for the College of American Pathologists in diagnostic immunology and flow cytometry clinical testing. [...], * Context.--In 2018 the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee designed and implemented a new plasma cell neoplasia flow cytometry proficiency testing program--PCNEO--to allow clinical flow cytometry laboratories to monitor and assess their performance compared with a peer group. Objective.--To report the results from the first 4 years of the PCNEO program. Design.--Program participants were sent 2 sets of challenges per year, each including 1 wet challenge and 2 dry challenges, with associated clinical and laboratory findings. The wet challenges were composed of myeloma cell line specimens (with or without dilution in preserved whole blood) for flow cytometric analysis. The dry (paper) challenges were composed of clinical case summaries and images of flow cytometric test results from various flow cytometry laboratories of committee members. Results.--A total of 116 to 145 laboratories from 17 countries enrolled in the proficiency testing program. For the wet challenges, almost all participants (97%-100%; cumulative, 98.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of undiluted myeloma cell lines. Slightly fewer participants (89.0%-97.4%; cumulative, 95.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of diluted myeloma cell lines (10% or 50% dilutions into peripheral blood) intended to better represent a typical clinical sample. There was generally agreement among 80% or more of participants for positive or negative staining for CD38, CD138, CD19, CD20, and surface and cytoplasmic k and X light chains. Similarly, 84% to 100% of participants were able to correctly identify the presence of neoplastic plasma cell populations in paper challenges, including the presence of small, neoplastic plasma cell populations (0.01%-5.0% clonal plasma cells) and the presence of nonneoplastic plasma cell populations (correctly identified by 91 %-96% of participants). Conclusions.--Participant performance in the new proficiency testing program was excellent overall, with the vast majority of participants able to perform flow cytometric analysis and identify neoplastic plasma cell populations and to identify small plasma cell clones or expanded populations of reactive plasma cells in dry challenge flow cytometry results. This program will allow laboratories to verify the accuracy of their testing program and test interpretations for the assessment of patients suspected of having a plasma cell neoplasm. (Arch Pathol Lab Med. 2024;148:699-704; doi: 10.5858/arpa. 2023-0035-CP)

Health science graduate programs are intensive experiences in students' lives which can contribute to perceived symptoms of anxiety and/or depression. The consequent impact of these symptoms can have an overall effect on the graduate students' ability to participate in their daily lives, along with their program of study. The purpose of this study was to understand the lived experiences of health science graduate students who experience perceived signs and symptoms of anxiety and/or depression. An open-ended, qualitative phenomenological survey was used to understand the experiences of 56 Doctor of Occupational Therapy, Doctor of Physical Therapy, and Doctor of Pharmacy students at a university in the southeastern United States. Three themes were identified from the data: the cyclic effect of symptoms and triggers; the importance of support systems; and the benefit of coping skill utilization. Future research is needed to understand the effects of preventive and rehabilitative measures to address health science graduate students' mental health.

Este artigo propõe uma abordagem baseada nos pressupostos da Teoria Histórico-Cultural de Vigotski e seus interlocutores, objetivando uma perspectiva humanizante e plural no processo educativo do componente curricular Ensino Religioso. Aponta-se para a inclusão e a valorização da pluralidade cultural religiosa brasileira, considerando a inconclusão e o inacabamento da pessoa, que é histórica, social e cultural. Para tanto, desenvolvemos uma pesquisa bibliográfica, documental de natureza qualitativa. De antemão podemos dizer que o componente curricular ensino religioso a partir de uma concepção plural e inclusiva vislumbra a construção de uma sociedade justa e equânime, baseados nos princípios da dignidade humana.

Readthrough of a translation termination codon is regulated by ribosomal A site recognition and insertion of near-cognate tRNAs. Small molecules exist that mediate incorporation of amino acids at the stop codon and production of full-length, often functional protein but defining the actual amino acid that is incorporated remains a challenging area. Herein, we report on the development a human cell model that can be used to determine whether rules can be developed using mass spectrometry that define the type of amino acid that is placed at a premature termination codon (PTC) during readthrough mediated by an aminoglycoside. The first PTC we analyzed contained the relatively common cancer-associated termination signal at codon 213 in the p53 gene. Despite of identifying a tryptic peptide with the incorporation of an R at codon 213 in the presence of the aminoglycoside, there were no other tryptic peptides detected across codon 213 that could be recovered; hence we constructed a more robust artificial PTC model. P53 expression plasmids were developed that incorporate a string of single synthetic TGA (opal) stop codons at S127P128A129 within the relatively abundant tryptic p53 peptide 121-SVTCTYSPALNK-132. The treatment of cells stably expressing the p53-TGA129 mutation, treated with Gentamicin, followed by immunoprecipitation and trypsinization of p53, resulted in the identification R, W, or C within the tryptic peptide at codon-TGA129; as expected based on the two-base pairing of the respective anticodons in the tRNA to UGA, with R being the most abundant. By contrast, incorporating the amber or ochre premature stop codons, TAA129 or TAG129 resulted in the incorporation of a Y or Q amino acid, again as expected based on the two base pairings to the anticodons, with Q being the most abundant. A reproducible non-canonical readthrough termination codon-skip event at the extreme C-terminus at codon 436 in the SBP-p53 fusion protein was detected which provided a novel assay for non-canonical readthrough at an extreme C-terminal PTC. The incorporation of amino acids at codons 127, 128, or 129 generally result in a p53 protein that is predicted to be ‘unfolded’ or inactive as defined by molecular dynamic simulations presumably because the production of mixed wild-type p53 and mutant oligomers are known to be inactive through dominant negative effects of the mutation. The data highlight the need to not only produce novel small molecules that can readthrough PTCs or C-terminal termination codons, but also the need to design methods to insert the required amino acid at the position that could result in a ‘wild-type’ functional protein.

In this paper, a problem set activity focused on understanding and applying unsupervised and supervised chemometric methods for the analysis of biodiesel-diesel blended fuels is described. This problem set was utilized in two upper-level analytical elective courses aimed for junior and senior level students. The data set consists of peak areas taken from gas chromatograms comprising various biodiesel concentrations and feedstock types. Students are tasked with analyzing the data set using principal component analysis (PCA), hierarchical cluster analysis (HCA), and k-nearest neighbors (kNN). The primary purpose of this problem set is to gain hands-on experience manipulating and drawing conclusions about large data sets using chemometric methods. The problem set activity utilizes a large GCMS data set with many varieties of sample types and can be included at any institution with no need for a GCMS instrument on campus.

Osteosarcomas are the most common primary malignant bone tumors and mostly affect children, adolescents, and young adults. Despite current treatment options such as surgery and polychemotherapy, the survival of patients with metastatic disease remains poor. In recent studies, punicalagin has reduced the cell viability, angiogenesis, and invasion in cell culture trials. The aim of this study was to examine the effects of punicalagin on osteosarcomas in a 3D in vivo tumor model. Human osteosarcoma biopsies and SaOs-2 and MG-63 cells, were grown in a 3D in vivo chorioallantoic membrane (CAM) model. After a cultivation period of up to 72 h, the tumors received daily treatment with punicalagin for 4 days. Weight measurements of the CAM tumors were performed, and laser speckle contrast imaging (LSCI) and a deep learning-based image analysis software (CAM Assay Application v.3.1.0) were used to measure angiogenesis. HE, Ki-67, and Caspase-3 staining was performed after explantation. The osteosarcoma cell lines SaOs-2 and MG-63 and osteosarcoma patient tissue displayed satisfactory growth patterns on the CAM. Treatment with punicalagin decreased tumor weight, proliferation, and tumor-induced angiogenesis, and the tumor tissue showed pro-apoptotic characteristics. These results provide a robust foundation for the implementation of further studies and show that punicalagin offers a promising supplementary treatment option for osteosarcoma patients. The 3D in vivo tumor model represents a beneficial model for the testing of anti-cancer therapies.

IntroductionNew diagnostic techniques are a substantial research focus in degenerative cervical myelopathy (DCM). This cross-sectional study determined the significance of cardiac-related spinal cord motion and the extent of spinal stenosis as indicators of mechanical strain on the cord.MethodsEighty-four DCM patients underwent MRI/clinical assessments and were classified as MRI+ [T2-weighted (T2w) hyperintense lesion in MRI] or MRI− (no T2w-hyperintense lesion). Cord motion (displacement assessed by phase-contrast MRI) and spinal stenosis [adapted spinal canal occupation ratio (aSCOR)] were related to neurological (sensory/motor) and neurophysiological readouts [contact heat evoked potentials (CHEPs)] by receiver operating characteristic (ROC) analysis.ResultsMRI+ patients (N = 31; 36.9%) were more impaired compared to MRI− patients (N = 53; 63.1%) based on the modified Japanese Orthopedic Association (mJOA) subscores for upper {MRI+ [median (Interquartile range)]: 4 (4–5); MRI−: 5 (5–5); p

Monoclonal antibodies targeting immune checkpoints have revolutionized oncology. Yet, the effectiveness of these treatments varies significantly among patients, and they are associated with unexpected adverse events, including hyperprogression. The murine research model used in drug development fails to recapitulate both the functional human immune system and the population heterogeneity. Hence, a novel model is urgently needed to study the consequences of immune checkpoint blockade. Dogs appear to be uniquely suited for this role. Approximately 1 in 4 companion dogs dies from cancer, yet no antibodies are commercially available for use in veterinary oncology. Here we characterize two novel antibodies that bind canine PD-1 with sub-nanomolar affinity as measured by SPR. Both antibodies block the clinically crucial PD-1/PD-L1 interaction in a competitive ELISA assay. Additionally, the antibodies were tested with a broad range of assays including Western Blot, ELISA, flow cytometry, immunofluorescence and immunohistochemistry. The antibodies appear to bind two distinct epitopes as predicted by molecular modeling and peptide phage display. Our study provides new tools for canine oncology research and a potential veterinary therapeutic.

Esse artigo desdobra o debate pautado na relevância de entender a educação proposta por Paulo Freire, tendo como partida o documentário “Menino 23 Infâncias perdidas no Brasil”, fundamentando como o corpo infantil negro (preto e pardo) cresce condenado a exploração e invisibilidade. É uma pesquisa qualitativa, de cunho documental, que parte da análise do documentário relacionando de modo crítico com a necropolítica dos corpos infantis negros (pretos e pardos). Descortinando e denunciando as negligências perante a sociedade, inclusive no espaço escolar. Desta forma, o presente trabalho tem como objetivo sulear as infâncias negras (pretas e pardas), além de discutir o racismo e a necessidade latente de práticas pedagógicas antirracistas que promovam representatividade no cotidiano escolar. Articulando e reforçando necessidade de romper com o biopoder e a necropolítica e visibilizar no espaço escolar, a história e cultura das infâncias negras (preta e parda), retirando os discursos de ódio e condenação historicamente aplicados e ressaltando a necessidade de perceber a importância que carrega a representatividade no período escolar, momento importante na constituição da identidade dos estudantes, enfatizando a proposta de articular o documentário aos dias atuais, os resultados descrevem que para reverter ou minimizar o racismo nos corpos infantis negros (pretos e pardos) é primordial a implementação/intervenção da pedagogia antirracista no âmbito educacional.

Subcutaneous panniculitis-like T-cell lymphoma is a cutaneous lymphoma characterized by CD8+ T-cell infiltrate in the subcutis that is rare in children. Acute lymphoblastic lymphoma is the most common pediatric malignancy and often presents with fevers and pancytopenia. Herein, we report 2 pediatric patients presenting with subcutaneous panniculitis-like T-cell lymphoma and B-cell acute lymphoblastic lymphoma, distinct hematologic malignancies arising from different lymphoid lineages, with no identifiable germline cancer predisposition.

BackgroundSafely and effectively discharging a patient from the hospital requires working within a multidisciplinary team. However, little is known about how perceptions of responsibility among the team impact discharge communication practices.ObjectiveOur study attempts to understand residents' perceptions of who is primarily responsible for discharge education, how these perceptions affect their own reported communication with patients, and how residents envision improving multidisciplinary communication around discharges.DesignA multi-institutional cross-sectional survey.ParticipantsInternal medicine (IM) residents from seven US residency programs at academic medical centers were invited to participate between March and May 2019, via email of an electronic link to the survey.Main measuresData collected included resident perception of who on the multidisciplinary team is primarily responsible for discharge communication, their own reported discharge communication practices, and open-ended comments on ways discharge multidisciplinary team communication could be improved.Key resultsOf the 613 resident responses (63% response rate), 35% reported they were unsure which member of the multidisciplinary team is primarily responsible for discharge education. Residents who believed it was either the intern's or the resident's primary responsibility had 4.28 (95% CI, 2.51-7.30) and 3.01 (95% CI, 1.66-5.71) times the odds, respectively, of reporting doing discharge communication practices frequently compared to those who were not sure who was primarily responsible. To improve multidisciplinary discharge communication, residents called for the following among team members: (1) clarifying roles and responsibilities for communication with patients, (2) setting expectations for communication among multidisciplinary team members, and (3) redefining culture around discharges.ConclusionsResidents report a lack of understanding of who is responsible for discharge education. This diffusion of ownership impacts how much residents invest in patient education, with more perceived responsibility associated with more frequent discharge communication.