Proceedings of the Think Tank for Osteosarcoma Medical Advisory Board.

A "Think Tank for Osteosarcoma" medical advisory board meeting was held in Santa Monica, CA, USA on February 2-3, 2024. The goal was to develop a strategic approach to prevent recurrence of osteosarcoma. Osteosarcoma metabolism and the genomic instability of osteosarcoma, immunotherapy for osteosarcoma, CAR-T cell therapy, DeltaRex-G tumor-targeted gene therapy, repurposed drugs, alternative medicines, and personalized medicine were discussed. Only DeltaRex-G was voted on. The conclusions were the following: No intervention has been demonstrated to improve survival in a clinical trial. Additionally, the consensus (10/12 in favor) was that DeltaRex-G without immunotherapy may be administered for up to one year. Phase 2/3 randomized studies of DeltaRex-G should be performed to determine whether the incidence of recurrence could be reduced in high-risk individuals. Furthermore, a personalized approach using drugs with minimal toxicity could be attempted with the acknowledgement that there are no efficacy data to base this on. Repurposed drugs and alternative therapies should be tested in mouse models of osteosarcoma. Moreover, unmodified IL-2 primed Gamma Delta (NK) cell therapy may be used to prevent recurrence. Lastly, rapid development of CAR-T cell therapy is recommended, and an institute dedicated to the study of osteosarcoma is needed.

Computational Modelling of Glucose Uptake by SGLT1 and Apical GLUT2 in the Enterocyte.

It has been suggested that glucose absorption in the small intestine depends on both constitutively expressed SGLT1 and translocated GLUT2 in the brush border membrane, especially in the presence of high levels of luminal glucose. Here, we present a computational model of non-isotonic glucose uptake by small intestinal epithelial cells. The model incorporates apical uptake via SGLT1 and GLUT2, basolateral efflux into the blood via GLUT2, and cellular volume changes in response to non-isotonic conditions. The dependence of glucose absorption on luminal glucose, blood flow rate, and inlet blood glucose concentration is studied. Uptake via apical GLUT2 is found to be sensitive to all these factors. Under a range of conditions, the maximum apical GLUT2 flux is about half of the SGLT1 flux and is achieved at high luminal glucose (> 50 mM), high blood flow rates, and low inlet blood concentrations. In contrast, SGLT1 flux is less sensitive to these factors. When luminal glucose concentration is less than 10 mM, apical GLUT2 serves as an efflux pathway for glucose to move from the blood to the lumen. The model results indicate that translocation of GLUT2 from the basolateral to the apical membrane increases glucose uptake into the cell; however, the reduction of efflux capacity results in a decrease in net absorption. Recruitment of GLUT2 from a cytosolic pool elicits a 10-20% increase in absorption for luminal glucose levels in the a 20-100 mM range. Increased SGLT1 activity also leads to a roughly 20% increase in absorption. A concomitant increase in blood supply results in a larger increase in absorption. Increases in apical glucose transporter activity help to minimise cell volume changes by reducing the osmotic gradient between the cell and the lumen.

Computational Modeling of Glucose Uptake in the Enterocyte.

Absorption of glucose across the epithelial cells of the small intestine is a key process in human nutrition and initiates signaling cascades that regulate metabolic homeostasis. Validated and predictive mathematical models of glucose transport in intestinal epithelial cells are essential for interpreting experimental data, generating hypotheses, and understanding the contributions of and interactions between transport pathways. Here we report on the development of such a model that, in contrast to existing models, incorporates mechanistic descriptions of all relevant transport proteins and is implemented in the CellML framework. The model is validated against experimental and simulation data from the literature. It is then used to elucidate the relative contributions of the sodium-glucose cotransporter (SGLT1) and the glucose transporter type 2 (GLUT2) proteins in published measurements of glucose absorption from human intestinal epithelial cell lines. The model predicts that the contribution of SGLT1 dominates at low extracellular glucose concentrations (<20 mM) and short exposure times (<60 s) while the GLUT2 contribution is more significant at high glucose concentrations and long durations. Implementation in CellML permitted a modular structure in which the model was composed by reusing existing models of the individual transporters. The final structure also permits transparent changes of the model components and parameter values in order to facilitate model reuse, extension, and customization (for example, to simplify, or add complexity to specific transporter/pathway models, or reuse the model as a component of a larger framework) and carry out parameter sensitivity studies.

Point-of-Care Ultrasound Needs Assessment, Curriculum Design, and Curriculum Assessment in a Large Academic Internal Medicine Residency Program.

OBJECTIVES: Internal medicine (IM) residency point-of-care ultrasound (POCUS) curricula are being developed but often are limited in scope or components. In this article, we discuss the demonstration of a need for POCUS training in our large academic IM residency program; the development of a longitudinal curriculum; and the impact of the curriculum on POCUS knowledge, use, and confidence. METHODS: In 2014, we designed a cross-sectional POCUS survey and knowledge test for all IM residents at the University of California, San Francisco. The results of this assessment drove the design of a longitudinal POCUS curriculum that included a 2-hour workshop for all IM interns and a 1-month elective offered to all IM residents. Residents were tested on their POCUS knowledge and image interpretation before the elective and were given the same test 6 months after the elective. The posttest included a survey of self-reported POCUS use and confidence. RESULTS: In the needs assessment, residents scored a mean of 27% on the knowledge test, and across all applications the percentage of residents reporting confidence in their POCUS skills was lower than the percentage reporting use of the application in clinical practice. Residents scored a mean of 37% on the elective pretest and 74% on the posttest, an increase of 37% (95% confidence interval 31.6-42.8, P < 0.001), with improvements seen across all applications. After the elective, self-reported use of POCUS and confidence in POCUS skills were increased for the applications, using the needs assessment as an approximate baseline. For core cardiac and pulmonary applications, 76% to 95% of residents, depending on application, reported "high" or "very high" use and 79% to 100% reported "high" or "very high" confidence in their POCUS skills. CONCLUSIONS: We used a needs assessment to guide the development of a longitudinal, multidisciplinary POCUS curriculum. Residents who completed all components showed substantial long-term gains in knowledge in all major applications and high use of and confidence in cardiac and pulmonary applications.

Blood cultures for community-acquired pneumonia: are they worthy of two quality measures? A systematic review.

BACKGROUND: Obtaining blood cultures (BCs) for patients hospitalized with community-acquired-pneumonia (CAP) has been recommended by experts and used as a measure of quality of care. However, BCs are infrequently positive in these patients and their effect on clinical management has been questioned. PURPOSE: We performed a systematic review of the literature to determine the impact of BCs on clinical management in CAP requiring hospitalization and thus its appropriateness as a quality measure. DATA SOURCES: We searched MEDLINE, MEDLINE In-Process, and the Cochrane databases for English-language studies that reported the effect of BCs on management of adults hospitalized with CAP. We also searched the reference lists of included studies and background articles and asked experts to review our list for completeness. STUDY SELECTION: Studies were chosen if they included adults admitted to the hospital with CAP, BCs were obtained at admission, and BC-directed management changes were reported. DATA EXTRACTION: We abstracted study design, BC positivity, and frequency of BC-directed management changes. DATA SYNTHESIS: Fifteen studies, all with observational cohort design, were identified and reviewed. Two included only patients with BCs positive for pneumococcus, yielding 13 studies for the primary analysis. BCs were true-positive in 0% to 14% of cases. They led to antibiotic narrowing in 0% to 3% of patients and to antibiotic broadening ultimately associated with a resistant organism in 0% to 1% of patients. CONCLUSIONS: BCs have very limited utility in immunocompetent patients hospitalized with CAP. Pneumonia quality measures that include BCs should be reassessed.

Retrotranslocation of the chaperone calreticulin from the endoplasmic reticulum lumen to the cytosol.

Polypeptide folding and quality control in the endoplasmic reticulum (ER) are mediated by protein chaperones, including calreticulin (CRT). ER localization of CRT is specified by two types of targeting signals, an N-terminal hydrophobic signal sequence that directs insertion into the ER and a C-terminal KDEL sequence that is responsible for retention in the ER. CRT has been implicated in a number of cytoplasmic and nuclear processes, suggesting that there may be a pathway for generating cytosolic CRT. Here we show that CRT is fully inserted into the ER, undergoes processing by signal peptidase, and subsequently undergoes retrotranslocation to the cytoplasm. A transcription-based reporter assay revealed an important role for the C-terminal Ca(2+) binding domain in CRT retrotranslocation. Neither ubiquitylation nor proteasome activity was necessary for retrotranslocation, which indicates that the pathway is different from that used by unfolded proteins targeted for destruction. Forced expression of cytosolic CRT is sufficient to rescue a cell adhesion defect observed in mouse embryo fibroblasts from crt(-/-) mice. The ability of CRT to retrotranslocate from the ER lumen to the cytosol explains how CRT can change compartments and modulate cell adhesion, transcription, and translation.

Transient, ligand-dependent arrest of the androgen receptor in subnuclear foci alters phosphorylation and coactivator interactions.

Here we report that mutations within the DNA-binding domain of AR, shown previously to inhibit nuclear export to the cytoplasm, cause an androgen-dependent defect in intranuclear trafficking of AR. Mutation of two conserved phenylalanines within the DNA recognition helix (F582, 583A) results in androgen-dependent arrest of AR in multiple subnuclear foci. A point mutation in one of the conserved phenylalanines (DeltaF582, F582Y) is known to cause androgen insensitivity syndrome (AIS). Both AIS mutants (DeltaF582, F582Y) and the export mutant (F582, 583A) displayed androgen-dependent arrest in foci, and all three mutants promoted androgen-dependent accumulation of the histone acetyl transferase CREB binding protein (CBP) in the foci. The foci correspond to a subnuclear compartment that is highly enriched for the steroid receptor coactivator glucocorticoid receptor-interacting protein (GRIP)-1. Agonist-bound wild-type AR induces the redistribution of GRIP-1 from foci to the nucleoplasm. This likely reflects a direct interaction between these proteins because mutation of a conserved residue within the major coactivator binding site on AR (K720A) inhibits AR-dependent dissociation of GRIP-1 from foci. GRIP-1 also remains foci-associated in the presence of agonist-bound F582, 583A, DeltaF582, or F582Y forms of AR. Two-dimensional phospho-peptide mapping and analysis with a phospho-specific antibody revealed that mutant forms of AR that arrest in the subnuclear foci are hypophosphorylated at Ser81, a site that normally undergoes androgen-dependent phosphorylation. Our working model is that the subnuclear foci are sites where AR undergoes ligand-dependent engagement with GRIP-1 and CBP, a recruitment step that occurs before Ser81 phosphorylation and association with promoters of target genes.