Scalable purification of extracellular vesicles with high yield and purity using multimodal flowthrough chromatography.

Extracellular vesicles (EVs) are cell derived membranous nanoparticles. EVs are important mediators of cell-cell communication via the transfer of bioactive content and as such they are being investigated for disease diagnostics as biomarkers and for potential therapeutic cargo delivery to recipient cells. However, existing methods for isolating EVs from biological samples suffer from challenges related to co-isolation of unwanted materials such as proteins, nucleic acids, and lipoproteins. In the pursuit of improved EV isolation techniques, we introduce multimodal flowthrough chromatography (MFC) as a scalable alternative to size exclusion chromatography (SEC). The use of MFC offers significant advantages for purifying EVs, resulting in enhanced yields and increased purity with respect to protein and nucleic acid co-isolates from conditioned 3D cell culture media. Compared to SEC, significantly higher EV yields with similar purity and preserved functionality were also obtained with MFC in 2D cell cultures. Additionally, MFC yielded EVs from serum with comparable purity to SEC and similar apolipoprotein B content. Overall, MFC presents an advancement in EV purification yielding EVs with high recovery, purity, and functionality, and offers an accessible improvement to researchers currently employing SEC.

Musculoskeletal Issues in Pediatric Burn Patients.

A burn injury affects virtually every organ system. The purpose of this article is to review musculoskeletal issues in children with burn injuries. Both acute and long-term problems will be discussed. A low threshold to consult a pediatric orthopaedist is recommended.

Storylines of family medicine V: ways of thinking-honing the therapeutic self.

Storylines of Family Medicine is a 12-part series of thematically linked essays with accompanying illustrations that explore the many dimensions of family medicine, as interpreted by individual family physicians and medical educators in the USA and elsewhere around the world. In 'V: ways of thinking-honing the therapeutic self', authors present the following sections: 'Reflective practice in action', 'The doctor as drug-Balint groups', 'Cultivating compassion', 'Towards a humanistic approach to doctoring', 'Intimacy in family medicine', 'The many faces of suffering', 'Transcending suffering' and 'The power of listening to stories.' May readers feel a deeper sense of their own therapeutic agency by reflecting on these essays.

Cellular mechanisms of cooperative context-sensitive predictive inference.

We argue that prediction success maximization is a basic objective of cognition and cortex, that it is compatible with but distinct from prediction error minimization, that neither objective requires subtractive coding, that there is clear neurobiological evidence for the amplification of predicted signals, and that we are unconvinced by evidence proposed in support of subtractive coding. We outline recent discoveries showing that pyramidal cells on which our cognitive capabilities depend usually transmit information about input to their basal dendrites and amplify that transmission when input to their distal apical dendrites provides a context that agrees with the feedforward basal input in that both are depolarizing, i.e., both are excitatory rather than inhibitory. Though these intracellular discoveries require a level of technical expertise that is beyond the current abilities of most neuroscience labs, they are not controversial and acclaimed as groundbreaking. We note that this cellular cooperative context-sensitivity greatly enhances the cognitive capabilities of the mammalian neocortex, and that much remains to be discovered concerning its evolution, development, and pathology.

Dysfunctions of cellular context-sensitivity in neurodevelopmental learning disabilities.

Pyramidal neurons have a pivotal role in the cognitive capabilities of neocortex. Though they have been predominantly modeled as integrate-and-fire point processors, many of them have another point of input integration in their apical dendrites that is central to mechanisms endowing them with the sensitivity to context that underlies basic cognitive capabilities. Here we review evidence implicating impairments of those mechanisms in three major neurodevelopmental disabilities, fragile X, Down syndrome, and fetal alcohol spectrum disorders. Multiple dysfunctions of the mechanisms by which pyramidal cells are sensitive to context are found to be implicated in all three syndromes. Further deciphering of these cellular mechanisms would lead to the understanding of and therapies for learning disabilities beyond any that are currently available.

Variation in Access to Palliative Radiotherapy in Prostate Cancer: A Population-Based Study in Canada.

BACKGROUND: As a result of improvements in cancer therapies, patients with metastatic malignancies are living longer, and the role of palliative radiotherapy has become increasingly recognized. However, access to adequate palliative radiotherapy may continue to be a challenge, as is evident from the high proportion of patients dying of prostate cancer who never receive palliative radiotherapy. The main objective of this investigation is to identify and describe the factors associated with the receipt of palliative radiation treatment in a decedent cohort of prostate cancer patients in Ontario. METHODOLOGY:  Population-based administrative databases from Ontario, Canada, were used to identify prostate cancer decedents, 65 years or older who received androgen deprivation therapy between January 1, 2013, and December 31, 2018. Baseline and treatment characteristics were analyzed using univariate and multivariate logistic regression models for association with receipt of radiotherapy in a two-year observation period before death. RESULTS: We identified 3,788 prostate cancer decedents between 2013 and 2018; among these, 49.9% received radiotherapy in the two years preceding death. There were statistically significant positive associations between receipt of radiotherapy and younger age at diagnosis (odds ratio [OR] 1.6, 95% confidence interval [CI] 1.1-2.3); higher stage at diagnosis (OR 1.3, 95% CI 1.1-1.7); receipt of care at a regional cancer center (OR 1.8, 95% CI 1.3-2.4); and involvement of radiation oncologists (OR 155.1, 95% CI 83.3-288.7) or medical oncologists (OR 1.4, 95% CI 1.1-1.8). However, there were no associations between receipt of radiotherapy and income, distance to the nearest cancer center, involvement of urologists in cancer care, healthcare administrative region, home-care involvement, or number of hospitalizations in the observation period. CONCLUSIONS: We found the utilization of palliative radiotherapy for prostate cancer patients in Ontario varies depending on age, stage at diagnosis, number of comorbidities, registration at regional cancer centers, and involvement of oncologists. There were no differences detected based on income or distance from a cancer center. The findings of this study represent an important opportunity to facilitate better access to palliative radiotherapy and referrals to multidisciplinary regional cancer centers, to improve the quality of life of this patient population.

Development of a mentor training curriculum to support LGBTQIA+ health professionals.

While mentors can learn general strategies for effective mentoring, existing mentorship curricula do not comprehensively address how to support marginalized mentees, including LGBTQIA+ mentees. After identifying best mentoring practices and existing evidence-based curricula, we adapted these to create the Harvard Sexual and Gender Minority Health Mentoring Program. The primary goal was to address the needs of underrepresented health professionals in two overlapping groups: (1) LGBTQIA+ mentees and (2) any mentees focused on LGBTQIA+ health. An inaugural cohort (N = 12) of early-, mid-, and late-career faculty piloted this curriculum in spring 2022 during six 90-minute sessions. We evaluated the program using confidential surveys after each session and at the program's conclusion as well as with focus groups. Faculty were highly satisfied with the program and reported skill gains and behavioral changes. Our findings suggest this novel curriculum can effectively prepare mentors to support mentees with identities different from their own; the whole curriculum, or parts, could be integrated into other trainings to enhance inclusive mentoring. Our adaptations are also a model for how mentorship curricula can be tailored to a particular focus (i.e., LGBTQIA+ health). Ideally, such mentor trainings can help create more inclusive environments throughout academic medicine.

Real-World Evaluation of Primary Versus Secondary Prevention of Skeletal-Related Events in Metastatic Castration-Resistant Prostate Cancer.

INTRODUCTION: Anti-osteoclast treatment with denosumab or zoledronate is known to effectively reduce the need for radiotherapy to bone and other skeletal-related events (SREs) in patients with metastatic castration-resistant prostate cancer (mCRPC). In this study, we analyze primary versus secondary initiation of bone-targeting agents (BTAs) relative to first palliative bone radiotherapy in patients dying of mCRPC. METHODS: Provincial administrative databases from Ontario, Canada identified patients with prostate cancer (2007-2018, n = 98 646) who received continuous androgen deprivation therapy (n = 29 453), died of prostate cancer (2013-2018, n = 3864), and received life-prolonging therapy for mCRPC (n = 1850). Variables were collected looking back 3 years from death. Multivariable analysis explored the relationship between clinical variables and BTAs. RESULTS: Of the 58% (1066/1850) patients with mCRPC who received BTA, only 289 (25.4%) started BTA prior to first palliative bone radiotherapy as primary prevention. Eight hundred and forty-eight (74.6%) patients either never received BTA before death (n = 447) or started BTA only after first bone radiotherapy (n = 401). More patients received denosumab (n = 825, 77%) than zoledronic acid (n = 241, 23%). 51.2% (582/1137) of palliative bone radiotherapy was initiated in the last 12 months of life. Factors associated with the use of BTA included elevated alkaline phosphatase (OR = 1.0, P = .023), de novo metastases (OR = 1.4, P = .005), medical oncologist involvement (OR = 2.0, P = .007), diagnosis 2012-2017 versus 2007-2011 (OR = 0.75, P = .034), and academic center (OR = 0.061, P = .007). CONCLUSION: A majority of patients with mCRPC never receive BTAs prior to first SRE, despite universal access and availability of these agents in Ontario. These results highlight an opportunity to improve outcomes by emphasizing early introduction of BTA in patients with mCRPC being started on systemic therapy.

Virtual Care for Patients with Advanced Well Differentiated Gastroenteropancreatic Neuroendocrine Tumor (GEP-NET).

INTRODUCTION: The COVID-19 pandemic resulted in an unprecedent shift towards virtual cancer care, including the care of gastroenteropancreatic neuroendocrine tumors (GEP-NETs). The aim of this study was to evaluate the use of virtual care for GEP-NETs during the COVID-19 pandemic at a high-volume academic cancer center. METHODS: This retrospective, observational study performed at the Ottawa Hospital Cancer Center in Canada evaluated adult patients with GEP-NETs seen in consultation by medical oncology between 1 June 2019 and 31 December 2022. Demographic, clinicopathologic, cancer treatment and visit data were collected. Univariable and multivariable analyses assessed the relationship between patient characteristics and virtual care use. RESULTS: A total of 103 patients with well-differentiated GEP-NETS were included. Overall, 18/103 (17.5%) consults and 594/781 (76.1%) follow-ups were performed virtually. All consultation visits returned to in-person assessment by 2022, while 67.0% and 41.4% follow-ups remained virtual in 2022 and 2023, respectively. The year of follow-up, sex, employment and Charlston comorbidity index were associated with virtual follow-up use in the multivariable analysis. DISCUSSION: Virtual care remained a predominant method of GEP-NET patient assessment in the peri-pandemic period. These results highlight an opportunity to improve access to subspecialty neuroendocrine cancer care through the continued use of virtual care.

Wellbeing Impact Study of High-Speed 2 (WISH2): Protocol for a mixed-methods examination of the impact of major transport infrastructure development on mental health and wellbeing.

Although research has demonstrated that transport infrastructure development can have positive and negative health-related impacts, most of this research has not considered mental health and wellbeing separately from physical health. There is also limited understanding of whether and how any effects might be experienced differently across population groups, whether this differs according to the stage of development (e.g. planning, construction), and how changes to planned infrastructure may affect mental health and wellbeing. This paper presents a protocol for the Wellbeing Impact Study of HS2 (WISH2), which seeks to address these questions using a high-speed rail development in the UK as an applied example. WISH2 is a 10-year, integrated, longitudinal, mixed-methods project using general practices (primary medical care providers in the UK) as an avenue for participant recruitment and for providing a geographically defined population for which aggregated data on mental health indicators are available. The research comprises: (i) a combined longitudinal and repeated cross-sectional cohort study involving multiple waves of survey data collection and data from medical records; (ii) longitudinal, semi-structured interviews and focus groups with residents and community stakeholders from exposed areas; (iii) analysis of administrative data aggregated at the general practice population level; and (iv) health economic analysis of mental health and wellbeing impacts. The study findings will support the development of strategies to reduce negative impacts and/or enhance positive mental health and wellbeing impacts of high-speed rail developments and other large-scale infrastructure projects.

Efficacy of PD-1/PD-L1 immunotherapy on brain metastatic non-small-cell lung cancer and treatment-related adverse events: A systematic review.

BACKGROUND: Recent evidence suggests that PD-1/PD-L1 immunotherapy improves outcomes in patients with brain metastatic non-small cell lung cancer. METHODS: Records were searched electronically on MEDLINE, Embase and BIOSIS. Hazard ratios and their 95% confidence intervals for overall survival and progression free survival, and treatment-related adverse events data were extracted. Risk of bias was assessed in included studies using the Cochrane Collaboration's revised tool to assess risk of bias in randomized trials. RESULTS: PD-1/PD-L1 immunotherapy increased overall survival by 33% and progression free survival by 47% compared with chemotherapy. Two studies had a high risk of bias. Treatment-related adverse events were reported in 95%, 89% and 65% of patients receiving chemoimmunotherapy,chemotherapy and single agent immunotherapy, respectively. CONCLUSION: PD-1/PD-L1 inhibitors alone or in addition to chemotherapy increase overall and progression free survival when compared with chemotherapy alone. Chemoimmunotherapy and chemotherapy patients experienced the most treatment-related adverse events.

HYENA detects oncogenes activated by distal enhancers in cancer.

Somatic structural variations (SVs) in cancer can shuffle DNA content in the genome, relocate regulatory elements, and alter genome organization. Enhancer hijacking occurs when SVs relocate distal enhancers to activate proto-oncogenes. However, most enhancer hijacking studies have only focused on protein-coding genes. Here, we develop a computational algorithm "HYENA" to identify candidate oncogenes (both protein-coding and non-coding) activated by enhancer hijacking based on tumor whole-genome and transcriptome sequencing data. HYENA detects genes whose elevated expression is associated with somatic SVs by using a rank-based regression model. We systematically analyze 1,146 tumors across 25 types of adult tumors and identify a total of 108 candidate oncogenes including many non-coding genes. A long non-coding RNA TOB1-AS1 is activated by various types of SVs in 10% of pancreatic cancers through altered 3-dimensional genome structure. We find that high expression of TOB1-AS1 can promote cell invasion and metastasis. Our study highlights the contribution of genetic alterations in non-coding regions to tumorigenesis and tumor progression.

Rapid Gastric emptying in spontaneously hypertensive rats.

OBJECTIVE: To assess the rate of gastric emptying in spontaneously hypertensive rats (SHR) and to evaluate rapid gastric emptying as a possible predisposing factor for hypertension. Rapid gastric emptying of carbohydrates, known to elevate postprandial serum glucose, has been reported to occur in many insulin-resistant states, including hypertension. SHR exhibit insulin resistance similar to human hypertensive patients. No prior studies have assessed gastric emptying of an oral glucose solution in SHR as compared with control Wistar Kyoto rats (WKY). METHODS: Using scintigraphic imaging, gastric emptying of a physiologic, orally consumed glucose solution was assessed in 12 SHR and 12 control WKY at 5 weeks of age, prior to the development of hypertension, and at 12 weeks of age after hypertension was fully established. RESULTS: At 5 weeks, the gastric half-emptying time (GHET) was 67.8 ±â€Š9.8 min for the SHR vs. 109.3 ±â€Š18 ( P  = 0.042) minutes for the WKY controls. At 12 weeks, the GHET was 37.29 ±â€Š10.3 min for the SHR vs. 138.53 ±â€Š37.6 ( P  = 0.016) min for the WKY controls. CONCLUSION: Gastric emptying was significantly more rapid in the SHR before and after the development of hypertension. Even though SHR are known to have increased sympathetic activity associated with their development of hypertension, this increased sympathetic activity does not inhibit gastric emptying. SHR are a promising animal model for investigating therapeutic agents for treating hypertension aimed at slowing the rate of gastric emptying.

Simplified homology-assisted CRISPR for gene editing in Drosophila.

In vivo genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 generates powerful tools to study gene regulation and function. We revised the homology-assisted CRISPR knock-in method to convert Drosophila GAL4 lines to LexA lines using a new universal knock-in donor strain. A balancer chromosome-linked donor strain with both body color (yellow) and eye red fluorescent protein (RFP) expression markers simplified the identification of LexA knock-in using light or fluorescence microscopy. A second balancer chromosome-linked donor strain readily converted the second chromosome-linked GAL4 lines regardless of target location in the cis-chromosome but showed limited success for the third chromosome-linked GAL4 lines. We observed a consistent and robust expression of the yellow transgene in progeny harboring a LexA knock-in at diverse genomic locations. Unexpectedly, the expression of the 3xP3-RFP transgene in the "dual transgene" cassette was significantly increased compared with that of the original single 3xP3-RFP transgene cassette in all tested genomic locations. Using this improved screening approach, we generated 16 novel LexA lines; tissue expression by the derived LexA and originating GAL4 lines was similar or indistinguishable. In collaboration with 2 secondary school classes, we also established a systematic workflow to generate a collection of LexA lines from frequently used GAL4 lines.

Pilot test of Consensus Reporting Items for Studies in Primary care (CRISP).

The Consensus Reporting Items for Studies in Primary care (CRISP) provides a new research reporting guideline to meet the needs of the producers and users of primary care (PC) research. Developed through an iterative program of research, including investigators, practicing clinicians, patients, community representatives, and educators, the CRISP Checklist guides PC researchers across the spectrum of research methods, study designs, and topics. This pilot test included a variety of team members using the CRISP Checklist for writing, revising, and reviewing PC research reports. All or most of the 15 participants reported that the checklist was easy to use, improved research reports, and should be recommended by PC research journals. The checklist is adaptable to different study types; not all items apply to all reports. The CRISP Checklist can help meet the needs of PC research when used in parallel with existing guidelines that focus on specific methods and limited topics.

Improving the Reporting of Primary Care Research: Consensus Reporting Items for Studies in Primary Care-the CRISP Statement.

Primary care (PC) is a unique clinical specialty and research discipline with its own perspectives and methods. Research in this field uses varied research methods and study designs to investigate myriad topics. The diversity of PC presents challenges for reporting, and despite the proliferation of reporting guidelines, none focuses specifically on the needs of PC. The Consensus Reporting Items for Studies in Primary Care (CRISP) Checklist guides reporting of PC research to include the information needed by the diverse PC community, including practitioners, patients, and communities. CRISP complements current guidelines to enhance the reporting, dissemination, and application of PC research findings and results. Prior CRISP studies documented opportunities to improve research reporting in this field. Our surveys of the international, interdisciplinary, and interprofessional PC community identified essential items to include in PC research reports. A 2-round Delphi study identified a consensus list of items considered necessary. The CRISP Checklist contains 24 items that describe the research team, patients, study participants, health conditions, clinical encounters, care teams, interventions, study measures, settings of care, and implementation of findings/results in PC. Not every item applies to every study design or topic. The CRISP guidelines inform the design and reporting of (1) studies done by PC researchers, (2) studies done by other investigators in PC populations and settings, and (3) studies intended for application in PC practice. Improved reporting of the context of the clinical services and the process of research is critical to interpreting study findings/results and applying them to diverse populations and varied settings in PC.Annals "Online First" article.

A pilot clinical skills coaching program to reimagine remediation: a cohort study.

Background New approaches are needed to improve and destigmatize remediation in undergraduate medical education (UME).  The COVID-19 pandemic magnified the need to support struggling learners to ensure competency and readiness for graduate medical education (GME).  Clinical skills (CS) coaching is an underutilized approach that may mitigate the stigma of remedial learning. Methods A six-month CS coaching pilot was conducted at Harvard Medical School (HMS) as a destigmatized remedial learning environment for clerkship and post-clerkship students identified as 'at risk' based on objective structured clinical examinations (OSCE).  The pilot entailed individual and group coaching with five faculty, direct bedside observation of CS, and standardized patient encounters with video review. Strengths-based coaching principles and appreciative inquiry were emphasized.  Results Twenty-three students participated in the pilot: 14 clerkship students (cohort 1) and 9 post-clerkship students (cohort 2).  All clerkship students (cohort 1) demonstrated sustained improvement in CS across three OSCEs compared to baseline: at pilot close, at 6-months post pilot, and at 21-24 months post-pilot all currently graduating students (10/10, 100%) passed the summative OSCE, an HMS graduation requirement. All post-clerkship students (cohort 2) passed the HMS graduation OSCE (9/9,100%). Feedback survey results included clerkship students (9/14; 64%) and post-clerkship students (7/9; 78%); all respondents unanimously agreed that individual coaching was "impactful to my clinical learning and practice". Faculty and leadership fully supported the pilot as a destigmatized and effective approach to remediation.  Conclusion Remediation has an essential and growing role in medical schools.  CS coaching for remedial learning can reduce stigma, foster a growth mindset, and support sustained progress for 'at risk' early clerkship through final year students. An "implementation template" with suggested tools and timelines can be locally adapted to guide CS coaching for UME remediation. The CS coaching pilot model is feasible and can be generalized to many UME programs.