A petavoxel fragment of human cerebral cortex reconstructed at nanoscale resolution.

To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.

Introduction to Tobacco Cessation and Motivational Interviewing: Evaluation of a Lecture and Case-Based Learning Activity for Medical Students.

Introduction Despite the fact that tobacco use continues to have significant public health impacts, most healthcare providers are not adequately trained to counsel patients on their tobacco use or to provide cessation resources. Although all healthcare providers have a role in providing tobacco cessation assistance, physicians and advanced practice providers are generally the only practitioners able to furnish tobacco cessation medications and bill insurance for their cessation services. Therefore, ensuring these practitioners are properly trained to offer tobacco cessation to their patients is critical to addressing this public health threat. In line with this goal, this study outlines the curriculum evaluation for an innovative student-facilitated tobacco cessation activity for medical students. Methods A lecture and case-based learning activity was created and piloted with a class of first-year medical students. The activity was facilitated by fourth-year medical students. Students took a pre-session survey to establish baseline experience and beliefs and a post-session survey to ascertain their confidence in applying what was covered in the session. Descriptive statistics were utilized to analyze the data. Results One hundred and twenty-eight students completed both surveys. Prior to the activity, students reported low levels of confidence in their ability to counsel patients and knowledge of cessation resources. Following the activity, more than 90% reported improvement in their ability to assess a patient's willingness to quit and counsel those ready to quit. Greater than 80% reported an improvement in their ability to counsel patients not ready to quit and to establish a quit plan. More than 90% of students reported that the session increased their self-efficacy in helping patients quit and that it was worth their time, with 96% committing to increasing their tobacco cessation efforts with their patients. Discussion Students valued the training and almost all reported that it increased their ability to help patients quit smoking. The use of student-facilitated case-based learning provided both an opportunity for students to practice cessation techniques and a low-stakes introduction to the OSCE format without the need for extensive faculty resources. Although this session was run with first-year medical students, the curriculum presented can be used for residents, nurses, and other healthcare professionals.

Brachytherapy Workflow Practices: Analysis of Different Workflow Scenarios in Patients With Cervical Cancer and Impact on IGBT Implementation-An IAEA Study.

PURPOSE: The workflow of brachytherapy (BT) is an essential aspect of treatment to consider in image-guided brachytherapy (IGBT). It has an overarching effect influencing patient throughput and the number of cancer treatments that can be performed as it occupies equipment, space, and personnel. There is limited research addressing this issue. Under the International Atomic Energy Agency's Coordinated Research Activity titled IGBT for cervix cancer: An implementation study, our study analyzes various scenarios in the clinical workflow of BT delivery for cervical cancer. It aims to determine the extent to which these scenarios allow the routine implementation of IGBT. With this information, current barriers and individualized adaptations to efficient workflows can be identified to enhance the global application of IGBT, leading to better cervical cancer treatment. MATERIALS AND METHODS: A web-based poll of questions regarding practices in BT workflow was presented to 62 participants from low-, lower middle-, upper middle-, and high-income countries (19 countries). RESULTS: This study highlighted diversity in BT practices across countries, income levels, and regions. It identified variations in workflow, patient throughput, and resource availability, which can have implications for the efficiency and quality of BT treatments. Scenario A, utilizing multiple locations for the steps of the BT procedure, was the most commonly used. The availability of resources, such as imaging devices and trained personnel, varied among the participating centers and remained challenging for IGBT implementation and sustainability. CONCLUSION: The design of the BT facility plays a vital role in improving efficiency, with a dedicated BT suite contributing to an efficient workflow but limiting patient throughput, especially for high-volume centers. Although IGBT is effective, its implementation requires consideration of various logistical challenges and should be individualized.

Mapping of neuronal and glial primary cilia contactome and connectome in the human cerebral cortex.

Primary cilia act as antenna receivers of environmental signals and enable effective neuronal or glial responses. Disruption of their function is associated with circuit disorders. To understand the signals these cilia receive, we comprehensively mapped cilia's contacts within the human cortical connectome using serial-section EM reconstruction of a 1 mm3 cortical volume, spanning the entire cortical thickness. We mapped the "contactome" of cilia emerging from neurons and astrocytes in every cortical layer. Depending on the layer and cell type, cilia make distinct patterns of contact. Primary cilia display cell-type- and layer-specific variations in size, shape, and microtubule axoneme core, which may affect their signaling competencies. Neuronal cilia are intrinsic components of a subset of cortical synapses and thus a part of the connectome. This diversity in the structure, contactome, and connectome of primary cilia endows each neuron or glial cell with a unique barcode of access to the surrounding neural circuitry.

Prevalence of Social Needs & Social Risks Among EMS Providers.

Background: A national shortage of Emergency Medical Service (EMS) providers is a critical issue for the profession that has only worsened during the COVID-19 pandemic. Researchers have identified possible causes, including difficult workplace conditions, low wages, and burnout. However, the impact of EMS providers' social needs and social risks has yet to be thoroughly explored. Methods: Demographic data for 1,112 EMS providers who responded to the 2021 national Social Needs in the Pre-hospital Setting (SNIPS) Study were analyzed to produce descriptive statistics and test for differences in social needs using chi square tests. Results: EMS providers reported experiencing housing insecurity (23.0%), food insecurity (27.4%), struggles with substance use (20.9%), mental health concerns (41.5%), domestic violence (18.5%), and healthcare affordability concerns (30.8%) during their EMS career. Almost 90% of study participants screened positive for burnout. Both women and emergency medical technicians (EMTs) were more likely than men and paramedics respectively to suffer from food and housing insecurity, mental health issues, and domestic violence at some point in their careers. Discussion: EMS providers reported high levels of burnout as well as a variety of social needs. Social needs may necessitate overtime work, increasing risk of burnout and negatively impacting providers' well-being and contributing to the provider shortage. Barriers to entry for paramedic training and gender differences in promotion rates may exacerbate the disparities experienced by EMTs and female providers, respectively. Conclusion: With high levels of burnout, staffing shortages, and EMS professionals leaving the profession, more must be done to support EMS providers. This includes programs to facilitate entry to and advancement within the EMS profession, ability to make ends meet without reliance on overtime pay, and mental health support.

Association of MGMT Promoter and Enhancer Methylation with Genetic Variants, Clinical Parameters, and Demographic Characteristics in Glioblastoma.

The response of glioblastoma (GBM) patients to the alkylating agent temozolomide (TMZ) vitally depends on the expression level of the repair protein O6-methylguanine-DNA methyltransferase (MGMT). Since MGMT is strongly regulated by promoter methylation, the methylation status of the MGMT promoter has emerged as a prognostic and predictive biomarker for GBM patients. By determining the methylation levels of the four enhancers located within or close to the MGMT gene, we recently found that enhancer methylation contributes to MGMT regulation. In this study, we investigated if methylation of the four enhancers is associated with SNP rs16906252, TERT promoter mutations C228T and C250T, TERT SNP rs2853669, proliferation index Ki-67, overall survival (OS), age, and sex of the patients. In general, associations with genetic variants, clinical parameters, and demographic characteristics were caused by a complex interplay of multiple CpGs in the MGMT promoter and of multiple CpGs in enhancer regions. The observed associations for intragenic enhancer 4, located in intron 2 of MGMT, differed from associations observed for the three intergenic enhancers. Some findings were restricted to subgroups of samples with either methylated or unmethylated MGMT promoters, underpinning the relevance of the MGMT promoter status in GBMs.

A large-scale volumetric correlated light and electron microscopy study localizes Alzheimer's disease-related molecules in the hippocampus.

Connectomics is a nascent neuroscience field to map and analyze neuronal networks. It provides a new way to investigate abnormalities in brain tissue, including in models of Alzheimer's disease (AD). This age-related disease is associated with alterations in amyloid-ß (Aß) and phosphorylated tau (pTau). These alterations correlate with AD's clinical manifestations, but causal links remain unclear. Therefore, studying these molecular alterations within the context of the local neuronal and glial milieu may provide insight into disease mechanisms. Volume electron microscopy (vEM) is an ideal tool for performing connectomics studies at the ultrastructural level, but localizing specific biomolecules within large-volume vEM data has been challenging. Here we report a volumetric correlated light and electron microscopy (vCLEM) approach using fluorescent nanobodies as immuno-probes to localize Alzheimer's disease-related molecules in a large vEM volume. Three molecules (pTau, Aß, and a marker for activated microglia (CD11b)) were labeled without the need for detergents by three nanobody probes in a sample of the hippocampus of the 3xTg Alzheimer's disease model mouse. Confocal microscopy followed by vEM imaging of the same sample allowed for registration of the location of the molecules within the volume. This dataset revealed several ultrastructural abnormalities regarding the localizations of Aß and pTau in novel locations. For example, two pTau-positive post-synaptic spine-like protrusions innervated by axon terminals were found projecting from the axon initial segment of a pyramidal cell. Three pyramidal neurons with intracellular Aß or pTau were 3D reconstructed. Automatic synapse detection, which is necessary for connectomics analysis, revealed the changes in density and volume of synapses at different distances from an Aß plaque. This vCLEM approach is useful to uncover molecular alterations within large-scale volume electron microscopy data, opening a new connectomics pathway to study Alzheimer's disease and other types of dementia.

Multi-layered maps of neuropil with segmentation-guided contrastive learning.

Maps of the nervous system that identify individual cells along with their type, subcellular components and connectivity have the potential to elucidate fundamental organizational principles of neural circuits. Nanometer-resolution imaging of brain tissue provides the necessary raw data, but inferring cellular and subcellular annotation layers is challenging. We present segmentation-guided contrastive learning of representations (SegCLR), a self-supervised machine learning technique that produces representations of cells directly from 3D imagery and segmentations. When applied to volumes of human and mouse cortex, SegCLR enables accurate classification of cellular subcompartments and achieves performance equivalent to a supervised approach while requiring 400-fold fewer labeled examples. SegCLR also enables inference of cell types from fragments as small as 10 µm, which enhances the utility of volumes in which many neurites are truncated at boundaries. Finally, SegCLR enables exploration of layer 5 pyramidal cell subtypes and automated large-scale analysis of synaptic partners in mouse visual cortex.

Resident Physicians' Knowledge of Emergency Medical Services: A Comparison Between Emergency Medicine and Non-Emergency Medicine Resident Physicians.

Background and objective Emergency medical services (EMS) are often assumed to only involve bringing patients to physicians for treatment in the emergency department. However, EMS staff are also responsible for responding to physicians in the primary care setting when medical emergencies arise. While emergency medicine (EM) residents are exposed to EMS as part of their curriculum, little is known about the knowledge of other resident physicians who may interact with EMS. In light of this, we conducted this study to address the scarcity of data related to this topic. Methods A quantitative cross-sectional knowledge assessment was conducted among resident physicians in emergency medicine, internal medicine, family medicine, pediatric, and combined medicine and pediatric residencies at the Penn State Milton S. Hershey Medical Center. Results Eighteen EM residents and 26 non-EM residents completed the assessment. The EM residents had a higher average score when compared to non-emergency medicine residents (69.2% vs. 53.8%, p=0.0012). Conclusion Variations in scores between EM and other specialties that interact with EMS highlight the need for further training and familiarization related to EMS for residents in non-EM specialties.

Social Needs in the Prehospital Setting (SNIPS): EMS Attitudes Toward Addressing Patient Social Needs.

Introduction: There has been interest in utilizing EMS to address patients' social determinants of health, which are thought to be the cause of many unnecessary transports, particularly for "super-utilizing" patients. However, existing research is limited regarding EMS clinicians' understanding of social determinants of health and attitudes toward potential interventions. Methods: This cross-sectional study was conducted using an internet-based survey of EMS clinicians across the United States with multiple methods of recruitment. Descriptive statistics and Chi Square Tests analyzed the data. Results: A total of 1,112 EMTs and paramedics completed the survey with 43.4% reporting familiarity with the term, "social determinants of health," and 87.7% screening positive for burnout. Greater than 60% reported willingness to use proposed interventions to address patient social needs. Those who reported familiarity with the term, "social determinants of health," were more likely to indicate willingness to utilize interventions and to believe they were responsible for addressing their patients' social needs. Burnout had no effect on clinicians' willingness to use resources. Discussion: Respondents showed substantial interest in using the proposed resources to address patient social needs, suggesting that EMS clinicians may be receptive to expanding their scope of responsibility to include socioeconomic interventions. EMS clinicians familiar with the term "social determinants of health" were more likely to believe they were responsible for addressing patient social needs and more willing to use interventions, suggesting a potential benefit to more education on the topic. Burnout among EMS clinicians may not be a barrier to implementing such interventions. Conclusion: Our survey suggests that EMS clinicians may be interested in helping to address their patients' social needs. EMS clinicians should be offered education on social determinants of health in their initial training and through continuing education. Partnerships with human services agencies will be important to ensure the effectiveness of prehospital interventions.

A Scalable Staining Strategy for Whole-Brain Connectomics.

Mapping the complete synaptic connectivity of a mammalian brain would be transformative, revealing the pathways underlying perception, behavior, and memory. Serial section electron microscopy, via membrane staining using osmium tetroxide, is ideal for visualizing cells and synaptic connections but, in whole brain samples, faces significant challenges related to chemical treatment and volume changes. These issues can adversely affect both the ultrastructural quality and macroscopic tissue integrity. By leveraging time-lapse X-ray imaging and brain proxies, we have developed a 12-step protocol, ODeCO, that effectively infiltrates osmium throughout an entire mouse brain while preserving ultrastructure without any cracks or fragmentation, a necessary prerequisite for constructing the first comprehensive mouse brain connectome.

The American Brachytherapy Society and Indian Brachytherapy Society consensus statement for the establishment of high-dose-rate brachytherapy programs for gynecological malignancies in low- and middle-income countries.

PURPOSE: The global cervical cancer burden is disproportionately high in low- and middle-income countries (LMICs), and outcomes can be governed by the accessibility of appropriate screening and treatment. High-dose-rate (HDR) brachytherapy plays a central role in cervical cancer treatment, improving local control and overall survival. The American Brachytherapy Society (ABS) and Indian Brachytherapy Society (IBS) collaborated to provide this succinct consensus statement guiding the establishment of brachytherapy programs for gynecological malignancies in resource-limited settings. METHODS AND MATERIALS: ABS and IBS members with expertise in brachytherapy formulated this consensus statement based on their collective clinical experience in LMICs with varying levels of resources. RESULTS: The ABS and IBS strongly encourage the establishment of HDR brachytherapy programs for the treatment of gynecological malignancies. With the consideration of resource variability in LMICs, we present 15 minimum component requirements for the establishment of such programs. Guidance on these components, including discussion of what is considered to be essential and what is considered to be optimal, is provided. CONCLUSIONS: This ABS/IBS consensus statement can guide the successful and safe establishment of HDR brachytherapy programs for gynecological malignancies in LMICs with varying levels of resources.

Connectomics of the Octopus vulgaris vertical lobe provides insight into conserved and novel principles of a memory acquisition network.

Here, we present the first analysis of the connectome of a small volume of the Octopus vulgaris vertical lobe (VL), a brain structure mediating the acquisition of long-term memory in this behaviorally advanced mollusk. Serial section electron microscopy revealed new types of interneurons, cellular components of extensive modulatory systems, and multiple synaptic motifs. The sensory input to the VL is conveyed via~1.8 × 106 axons that sparsely innervate two parallel and interconnected feedforward networks formed by the two types of amacrine interneurons (AM), simple AMs (SAMs) and complex AMs (CAMs). SAMs make up 89.3% of the~25 × 106VL cells, each receiving a synaptic input from only a single input neuron on its non-bifurcating primary neurite, suggesting that each input neuron is represented in only~12 ± 3.4SAMs. This synaptic site is likely a 'memory site' as it is endowed with LTP. The CAMs, a newly described AM type, comprise 1.6% of the VL cells. Their bifurcating neurites integrate multiple inputs from the input axons and SAMs. While the SAM network appears to feedforward sparse 'memorizable' sensory representations to the VL output layer, the CAMs appear to monitor global activity and feedforward a balancing inhibition for 'sharpening' the stimulus-specific VL output. While sharing morphological and wiring features with circuits supporting associative learning in other animals, the VL has evolved a unique circuit that enables associative learning based on feedforward information flow.

Multiplexed volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

Multiplexed volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

Returns After Discharge From the Emergency Department Observation Unit: Who, What, When, and Why?

INTRODUCTION: The number of emergency department observation units (EDOU) and observation stays has continued to increase. Despite this, there is limited data on the characteristics of patients who return unexpectedly to the ED after EDOU discharge. METHODS: We identified the charts of all patients who were admitted to the EDOU of an academic medical center between January 2018-June 2020 and had a return to the ED within 14 days of discharge from the EDOU. Patients were excluded if they were admitted to the hospital from the EDOU, left against medical advice, or died in the EDOU. We manually extracted selected demographic factors, comorbidities, and healthcare utilization data from the charts. Physician reviewers identified return visits thought to be related to the index visit or potentially avoidable. RESULTS: During the study period, there were 176,471 ED visits, 4,179 admissions to the EDOU, and 333 return visits to the ED within 14 days from discharge from the EDOU, representing 9.4% of all patients discharged from the EDOU. We identified a higher rate of return for patients treated for asthma and lower rates of return for patients treated for chest pain or syncope than the overall return rate. Physician reviewers determined that 64.6% of unplanned returns were related to the index visit, and 4.5% were potentially avoidable. Of potentially avoidable visits, 53.3% occurred within 48 hours of discharge, supporting the use of this period as a potential quality metric. While there was no significant difference in the percentage of related return visits between males and females, there was a higher rate of potentially avoidable visits for male patients. CONCLUSION: This study adds to the limited body of literature on EDOU returns, finding an overall return rate of under 10%, with about two-thirds of returns determined to be related to the index visit and <5% considered to be potentially avoidable.

Association between MGMT Enhancer Methylation and MGMT Promoter Methylation, MGMT Protein Expression, and Overall Survival in Glioblastoma.

The repair protein O6-methylguanine-DNA methyltransferase (MGMT) is regulated epigenetically, mainly by the methylation of the MGMT promoter. MGMT promoter methylation status has emerged as a prognostic and predictive biomarker for patients with newly diagnosed glioblastoma (GBM). However, a strong negative correlation between MGMT promoter methylation and MGMT protein expression cannot be applied as a rule for all GBM patients. In order to investigate if the DNA methylation status of MGMT enhancers is associated with MGMT promoter methylation, MGMT expression, and the overall survival (OS) of GBM patients, we established assays based on high-resolution melting analysis and pyrosequencing for one intragenic and three intergenic MGMT enhancers. For CpGs in an enhancer located 560 kb upstream of the MGMT promoter, we found a significant negative correlation between the methylation status and MGMT protein levels of GBM samples expressing MGMT. The methylation status of CpGs in the intragenic enhancer (hs696) was strongly negatively correlated with MGMT promoter methylation and was significantly higher in MGMT-expressing GBM samples than in MGMT-non-expressing GBM samples. Moreover, low methylation of CpGs 01-03 and CpGs 09-13 was associated with the longer OS of the GBM patients. Our findings indicate an association between MGMT enhancer methylation and MGMT promoter methylation, MGMT protein expression, and/or OS.

FEASIBILITY AND SAFETY OF A FIELD CARE CLINIC AS AN ALTERNATIVE AMBULANCE DESTINATION DURING THE COVID-19 PANDEMIC.

Background: Anticipating an increased utilization of healthcare facilities during the COVID-19 surge, the San Francisco Department of Public Health developed a plan to deploy neighborhood-based Field Care Clinics (FCCs) that would decompress emergency departments by serving patients with low acuity complaints. These clinics would receive patients directly from the Emergency Medical Services (EMS) system. Transports were initiated by a paramedic-driven protocol, originally by EMS crews and later by the Centralized Ambulance Destination Determination (CADDiE) System. In this study, we evaluated the outcomes of EMS patients who were transported to the FCC, specifically as to whether they required subsequent transfer to the emergency department. Methods: We performed a retrospective study of all patients transported to the Bayview-Hunters Point (BHP) neighborhood FCC by EMS between April 11th, 2020, and December 16th, 2020. Descriptive statistics and Chi-Square Tests were used to analyze patient data. Results: In total, 35 patients (20 men, 15 women, average age of 50.9 years) were transported to the FCC. Of these, 16 were Black/African American, 7 were White, 3 were Asian, with 9 identifying as of other races and 9 of Hispanic ethnicity. Twenty-three of these transports resulted from a CADDiE recommendation. Approximately half (n=20) of calls originated within the BHP neighborhood. The most frequent patient complaint was "Pain." Of patients transported to the FCC, 23 were treated and discharged. The 12 remaining patients required hospital transfer, with 3 being discharged after receiving treatment in the emergency department and 9 requiring hospital admission, psychiatric, or sobering services. The likelihood of hospital transfer did not significantly vary by sex (p=0.41), 9-1-1 call origination relative to BHP neighborhood (p=0.92), or CADDiE recommendation (p=0.51). Conclusion: Three-fourths of patients who required subsequent hospital transfer were admitted or required specialized services, suggesting that the FCC was viable for managing low acuity conditions. However, the underutilization of the FCC by EMS as a transport destination and a high hospital transfer rate indicates training and protocol refinement opportunities. Despite the small cohort size, this study demonstrates that an FCC alternative care site can act as a viable source for urgent and emergency care during a pandemic.

Comparing the Perceptions of Reciprocal- and Near-Peer Objective Structured Clinical Examinations (OSCEs) in Medical Students.

INTRODUCTION: The Objective Structured Clinical Examination (OSCE) is utilized by medical schools to assess students' competency in clinical skills. Literature has shown that first-year students who were tutored by fourth-year students (MS4s; near-peer) in practice OSCEs reported self-perceived improvement in OSCE skills. There is a lack of research regarding the effectiveness of first-year (MS1) pairs for reciprocal-peer practice OSCEs. This study aims to assess if virtual reciprocal-peer OSCEs provide comparable learning opportunities to virtual near-peer OSCEs. METHODS: MS1 students were assigned to work with a near-peer or a reciprocal-peer for one week, and then switched protocols the second week. One student in each reciprocal-peer pair was assigned to act as a standardized patient (SP). Their partner took a history, interpreted physical exam findings, prepared a note, and gave an oral presentation. The pair then switched roles using a second case. The near-peer group followed the same procedure, without the reversal of roles. RESULTS: A total of 135 MS1s participated in the first week and 129 in the second week. Students agreed that working with a near-peer was more valuable than a reciprocal-peer in the following parameters: peer feedback (N=113, 89%), history-taking skills (N=101, 80%), physical exam skills (N=102, 81%), and note-writing skills (N=109, 89%). Pairwise comparison utilizing Wilcoxon signed-rank test indicated participants preferred the choice of a fourth-year student partner over an MS1 partner (Z=1.436, p<0.001). CONCLUSION: Participants found working with a near-peer increased confidence in their clinical skills and near-peer feedback was more valuable. Although MS1s found that watching and evaluating their peers in a reciprocal-peer exercise was beneficial, students overwhelmingly preferred working with MS4s due to more valuable feedback.

Discovery of novel amino acid production traits by evolution of synthetic co-cultures.

BACKGROUND: Amino acid production features of Corynebacterium glutamicum were extensively studied in the last two decades. Many metabolic pathways, regulatory and transport principles are known, but purely rational approaches often provide only limited progress in production optimization. We recently generated stable synthetic co-cultures, termed Communities of Niche-optimized Strains (CoNoS), that rely on cross-feeding of amino acids for growth. This setup has the potential to evolve strains with improved production by selection of faster growing communities. RESULTS: Here we performed adaptive laboratory evolution (ALE) with a CoNoS to identify mutations that are relevant for amino acid production both in mono- and co-cultures. During ALE with the CoNoS composed of strains auxotrophic for either L-leucine or L-arginine, we obtained a 23% growth rate increase. Via whole-genome sequencing and reverse engineering, we identified several mutations involved in amino acid transport that are beneficial for CoNoS growth. The L-leucine auxotrophic strain carried an expression-promoting mutation in the promoter region of brnQ (cg2537), encoding a branched-chain amino acid transporter in combination with mutations in the genes for the Na+/H+-antiporter Mrp1 (cg0326-cg0321). This suggested an unexpected link of Mrp1 to L-leucine transport. The L-arginine auxotrophic partner evolved expression-promoting mutations near the transcriptional start site of the yet uncharacterized operon argTUV (cg1504-02). By mutation studies and ITC, we characterized ArgTUV as the only L-arginine uptake system of C. glutamicum with an affinity of KD = 30 nM. Finally, deletion of argTUV in an L-arginine producer strain resulted in a faster and 24% higher L-arginine production in comparison to the parental strain. CONCLUSION: Our work demonstrates the power of the CoNoS-approach for evolution-guided identification of non-obvious production traits, which can also advance amino acid production in monocultures. Further rounds of evolution with import-optimized strains can potentially reveal beneficial mutations also in metabolic pathway enzymes. The approach can easily be extended to all kinds of metabolite cross-feeding pairings of different organisms or different strains of the same organism, thereby enabling the identification of relevant transport systems and other favorable mutations.