Differences in estimated persistent inward currents between ankle flexors and extensors in humans.

Persistent inward currents (PICs) are responsible for amplifying motoneuronal synaptic inputs and contribute to generating normal motoneuron activation. Delta-F (ΔF) is a well-established method that estimates PICs in humans indirectly from firing patterns of individual motor units. Traditionally, motor unit firing patterns are obtained by manually decomposing electromyography (EMG) signals recorded through intramuscular electrodes (iEMG). A previous iEMG study has shown that in humans the elbow extensors have higher ΔF than the elbow flexors. In this study, EMG signals were collected from the ankle extensors and flexors using high-density surface array electrodes during isometric sitting and standing at 10-30% maximum voluntary contraction. The signals were then decomposed into individual motor unit firings. We hypothesized that comparable to the upper limb, the lower limb extensor muscles (soleus) would have higher ΔF than the lower limb flexor muscles [tibialis anterior (TA)]. Contrary to our expectations, ΔF was higher in the TA than the soleus during sitting and standing despite the difference in cohort of participants and body positions. The TA also had significantly higher maximum discharge rate than the soleus while there was no difference in rate increase. When only the unit pairs with similar maximum discharge rates were compared, ∆F was still higher in the TA than the soleus. Future studies will focus on investigating the functional significance of the findings.NEW & NOTEWORTHY With the use of high-density surface array electrodes and convolutive blind source separation algorithm, thousands of motor units were decomposed from the soleus and tibialis anterior muscles. Persistent inward currents were estimated under seated and standing conditions via delta-F (∆F) calculation, and the results showed that unlike the upper limb, the flexor has higher ∆F than the extensor in the lower limb. Future studies will focus on functional significance of the findings.

Genetic disruption of KCC cotransporters in a mouse model of thalassemia intermedia.

ß-thalassemia (ß-Thal) is caused by defective ß-globin production leading to globin chain imbalance, aggregation of free alpha chain in developing erythroblasts, reticulocytes, and mature circulating red blood cells. The hypochromic thalassemic red cells exhibit increased cell dehydration in association with elevated K+ leak and increased K-Cl cotransport activity, each of which has been linked to globin chain imbalance and related oxidative stress. We therefore tested the effect of genetic inactivation of K-Cl cotransporters KCC1 and KCC3 in a mouse model of ß-thalassemia intermedia. In the absence of these transporters, the anemia of ß-Thal mice was ameliorated, in association with increased MCV and reductions in CHCM and hyperdense cells, as well as in spleen size. The resting K+ content of ß-Thal red cells was greatly increased, and Thal-associated splenomegaly slightly decreased. Lack of KCC1 and KCC3 activity in Thal red cells reduced red cell density and improved ß-Thal-associated osmotic fragility. We conclude that genetic inactivation of K-Cl cotransport can reverse red cell dehydration and partially attenuate the hematologic phenotype in a mouse model of ß-thalassemia.

Combined genetic disruption of K-Cl cotransporters and Gardos channel KCNN4 rescues erythrocyte dehydration in the SAD mouse model of sickle cell disease.

Excessive red cell dehydration contributes to the pathophysiology of sickle cell disease (SCD). The densest fraction of sickle red cells (with the highest corpuscular hemoglobin concentration) undergoes the most rapid polymerization of deoxy-hemoglobin S, leading to accelerated cell sickling and increased susceptibility to endothelial activation, red cell adhesion, and vaso-occlusion. Increasing red cell volume in order to decrease red cell density can thus serve as an adjunct therapeutic goal in SCD. Regulation of circulating mouse red cell volume and density is mediated largely by the Gardos channel, KCNN4, and the K-Cl cotransporters, KCC3 and KCC1. Whereas inhibition of the Gardos channel in subjects with sickle cell disease increased red cell volume, decreased red cell density, and improved other hematological indices in subjects with SCD, specific KCC inhibitors have not been available for testing. We therefore investigated the effect of genetic inactivation of KCC3 and KCC1 in the SAD mouse model of sickle red cell dehydration, finding decreased red cell density and improved hematological indices. We describe here generation of mice genetically deficient in the three major red cell volume regulatory gene products, KCNN4, KCC3, and KCC1 in C57BL6 non-sickle and SAD sickle backgrounds. We show that combined loss-of-function of all three gene products in SAD mice leads to incrementally increased MCV, decreased CHCM and % hyperchromic cells, decreased red cell density (phthalate method), increased resistance to hypo-osmotic lysis, and increased cell K content. The data show that combined genetic deletion of the Gardos channel and K-Cl cotransporters in a mouse SCD model decreases red cell density and improves several hematological parameters, supporting the strategy of combined pharmacological inhibition of these ion transport pathways in the adjunct treatment of human SCD.

Homeostatic regulation of extracellular signal-regulated kinase 1/2 activity and axonal Kv7.3 expression by prolonged blockade of hippocampal neuronal activity.

Homeostatic plasticity encompasses the mechanisms by which neurons stabilize their synaptic strength and excitability in response to prolonged and destabilizing changes in their network activity. Prolonged activity blockade leads to homeostatic scaling of action potential (AP) firing rate in hippocampal neurons in part by decreased activity of N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes including KCNQ3 which encodes Kv7.3. Neuronal Kv7 channels are mostly heterotetramers of Kv7.2 and Kv7.3 subunits and are highly enriched at the axon initial segment (AIS) where their current potently inhibits repetitive and burst firing of APs. However, whether a decrease in Kv7.3 expression occurs at the AIS during homeostatic scaling of intrinsic excitability and what signaling pathway reduces KCNQ3 transcript upon prolonged activity blockade remain unknown. Here, we report that prolonged activity blockade in cultured hippocampal neurons reduces the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) followed by a decrease in the activation of brain-derived neurotrophic factor (BDNF) receptor, Tropomyosin receptor kinase B (TrkB). Furthermore, both prolonged activity blockade and prolonged pharmacological inhibition of ERK1/2 decrease KCNQ3 and BDNF transcripts as well as the density of Kv7.3 and ankyrin-G at the AIS. Collectively, our findings suggest that a reduction in the ERK1/2 activity and subsequent transcriptional down-regulation may serve as a potential signaling pathway that links prolonged activity blockade to homeostatic control of BDNF-TrkB signaling and Kv7.3 density at the AIS during homeostatic scaling of AP firing rate.

The Cellular Basis for the Generation of Firing Patterns in Human Motor Units.

Motor units, which comprise a motoneuron and the set of muscle fibers it innervates, are the fundamental neuromuscular transducers for all motor commands. The one to one relationship between a motoneuron and its innervated muscle fibers allow motoneuron firing patterns to be readily measured in humans. In this chapter, we summarize the current understanding of the cellular basis for the generation of firing patterns in human motor units. We provide a brief review of landmark insights from classic studies and then proceed to consider the features of motor unit firing patterns that are most likely to be sensitive estimators of motoneuron inputs and properties. In addition, we discuss recent advances in technology for recording human motor unit firing patterns and highly realistic computer simulations of motoneurons. The final section presents our recent efforts to use the power of supercomputers for implementation of the motoneuron models, with a goal of achieving a true "reverse engineering" approach that maximizes the insights from motor unit firing patterns into the synaptic structure of motor commands.

Third year medical student knowledge gaps after a virtual surgical rotation.

INTRODUCTION: This study describes perceived knowledge gaps of third-year medical students after participating in a virtual surgical didactic rotation (EMLR) and shortened in-person surgery rotation during the COVID-19 Pandemic. METHODS: Open-ended and Likert questions were administered at the end of the virtual rotation and inperson-surgical rotation to medical students. Three blinded coders identified themes by semantic analysis. RESULTS: 82 students (51% of all MS3s) participated in the EMLR. Semantic analysis revealed gaps in perioperative management (Post-EMLR:18.4%, Post-Inpatient:26.5%), anatomy (Post-EMLR:8.2%, PostInpatient:26.5%). and surgical skills (Post-EMLR: 43.0%, Post-Inpatient: 44.1%). Students also described gaps related to OR etiquette (Post-EMLR: 12.2%, Post-Inpatient: 8.8%) and team dynamics/the hidden curriculum (Post- Inpatient:26.6%). There was a significant improvement in perceived confidence to perform inpatient tasks after completing the inpatient clinical experience (p ≤ 0.01). CONCLUSION: Virtual interactive didactics for cognitive skills development cannot replace a full clinical surgical experience for third-year medical students. Future curricula should address perceived gaps.

Surgery Clerkship Curriculum Changes at an Academic Institution during the COVID-19 Pandemic.

PROBLEM: The COVID-19 pandemic has suspended the surgery clinical clerkship for third-year medical students at numerous institutions across the world. As a result, educators and students have adapted rapidly. There is a paucity of precedents regarding urgent and brusque formal curricular changes for medical students enrolled in surgical clinical rotations. APPROACH: The University of California, San Francisco Department of Surgery created a surgically focused extended mastery learning rotation (EMLR). The surgery clerkship leadership designed a curriculum consisting of multiple learning strategies compatible with virtual learning environments. The primary aims of the newly developed EMLR were to help students consolidate their foundational science knowledge before their return to clinical medicine in an altered learning environment. The EMLR is currently underway, and further studies are necessary to evaluate its effectiveness.

Where Do We Go From Here? Assessing Medical Students' Surgery Clerkship Preparedness During COVID-19.

INTRODUCTION: The impact of COVID-19 on surgical education has been profound, and clinical learning experiences transitioned to virtual formats. This study investigated the impact of virtual experiences created to facilitate learning during the pandemic for medical students. METHODS: We performed a cohort study to determine the perceived clinical preparedness for medical students enrolled in the preclinical surgery pilot course, surgical Extended Mastery Learning Rotation (EMLR), and longitudinal surgical clerkship (LC). The preclinical surgery pilot course took place before COVID-19 disruptions, and the EMLR and LC experiences took place virtually. Specialty choice was examined in the EMLR and LC cohorts. Performance on the NBME surgical assessments was analyzed among students enrolled in the traditional clerkship and pandemic-disrupted courses and compared to national data using a two-sample t-test. RESULTS: Compared to preclinical students, EMLR and LC students demonstrated improvements in their perceived surgical clerkship readiness. After the 3-week EMLR course, in the setting of completing only one-third of the clerkship year, students had an average NBME Surgical Self-Assessment Exam score of 72 (SD 12), comparable to the national average of 71 (SD 9) p = 0.33. The average shelf exam score for students (N = 24) enrolled in the traditional clerkship (block 1), prior to COVID-19, disruptions was 66 (SD 9) compared to an average score of 69 (SD 9) for the longitudinal clerkship students (N = 20) that took the shelf exam later in the year (p = 0.36). COVID-19 disruptions did not affect specialty choice. All LC students have decided on a specialty; 50% nonsurgical and 50% surgical. From the EMLR cohort, 36% and 38% plan to pursue surgical and nonsurgical specialties, respectively, with 26% still undecided. CONCLUSIONS: Courses were well-liked and will be implemented in future clerkships. Surgical educators demonstrated flexibility and creativity in the development of the EMLR. Despite COVID-19 disruptions, medical students made progress in their clinical skills and foundational science knowledge. COVID-19 disruptions did not appear to impact specialty choice.

Near-Peer Learning During the Surgical Clerkship: A Way to Facilitate Learning After a 15-Month Preclinical Curriculum.

OBJECTIVES: To investigate the performance and perspectives of third-year medical students (MS3s) participating in near-peer learning (NPL) sessions during their core surgical clerkship following a 15-month preclerkship curriculum. DESIGN: An evaluation study of 7 NPL sessions developed and implemented by fourth-year medical students (MS4s) held from March 2019 to February 2020. MS4s taught 1-2 sessions per rotation that included test taking strategies, illness script development, radiology review, case-based multiple-choice questions, and rapid review. Participants completed a questionnaire with 11 seven-point Likert and open-ended questions after each session. Analyses included quantitative comparison of shelf score averages between NPL participants and nonparticipants and qualitative content analysis for open-ended questions. SETTING: Surgical clerkship at the University of California, San Francisco. PARTICIPANTS: Forty-eight (32%) MS3s participated, with an average attendance of 10 students per rotation. Thirty-three (69%) participants completed the questionnaire. RESULTS: MS3s enjoyed the session (6.9 ± 0.4), improved their knowledge (6.8 ± 0.5), and felt more prepared for the surgery shelf examination (6.5 ± 0.6). MS4 leaders found that MS3s always wanted radiology review, and their interest in test taking strategies and illness script development declined across the clerkship year. Participants had lower shelf exam scores compared to nonparticipants (68.1 vs 71.4, respectively; p = 0.04, ES = 0.03). Shelf exam scores increased over time in both cohorts. Each group had 2 shelf exam failures. Qualitative analysis suggests that MS3s appreciated the NPL's tailored approach and exam demystification, with a desire for increased NPL integration into the clerkship. CONCLUSION: Students participating in NPL were satisfied with the sessions. Participants may have been students who struggled as indicated by shelf exam scores and appreciated the support. The shift in preferred topics across the blocks reflects the students' development during clerkships. Near-peer teachers should adjust sessions over time to fit students' evolving needs.

Properties of Motor Units of Elbow and Ankle Muscles Decomposed Using High-Density Surface EMG.

Analyses of motor unit activity provide a window to the neural control of motor output. In recent years, considerable advancements in surface EMG decomposition methods have allowed for the discrimination of dozens of individual motor units across a range of muscle forces. While these non-invasive methods show great potential as an emerging technology, they have difficulty discriminating a representative sample of the motor pool. In the present study, we investigate the distribution of recruitment thresholds and motor unit action potential waveforms obtained from high density EMG across four muscles: soleus, tibialis anterior, biceps brachii, and triceps brachii. Ten young and healthy control subjects generated isometric torque ramps between 10-50% maximum voluntary torque during elbow or ankle flexion and extension. Hundreds of motor unit spike trains were decomposed for each muscle across all trials. For lower contraction levels and speeds, surface EMG decomposition discriminated a large number of low-threshold units. However, during contractions of greater speed and torque level the proportion of low threshold motor units decomposed was reduced, resulting in a relatively uniform distribution of recruitment thresholds. The number of motor units decomposed decreased as the contraction level and speed increased. The decomposed units showed a wide range of recruitment thresholds and motor unit action potential amplitudes. In conclusion, although surface EMG decomposition is a useful tool to study large populations of motor units, results of such methods should be interpreted in the context of limitations in sampling of the motor pool.

Using Technological Advances to Improve Surgery Curriculum: Experience With a Mobile Application.

OBJECTIVE: Our previous home-video basic surgical skills curriculum required substantial faculty time and resources, and was limited by delayed feedback and technical difficulties. Consequently, we integrated that curriculum with a mobile application platform. Our purpose is to describe this application and learner satisfaction. MATERIALS AND METHODS: The mobile platform incorporates a patented pedagogical design based on Ericsson's deliberate practice and Bandura's social learning theory. Instructors built step-wise skills modules. During the challenge phase, learners watched a video of surgical tasks completed by experts and uploaded a video of themselves performing the same task. In the Peer Review phase, they used a grading rubric to provide feedback. In the Recap stage, learners received individual feedback and could review their own videos. Two groups of learners, graduating medical students and matriculating surgical residents, participated in this independent learning platform, along with 2 to 4 laboratory sessions, and completed a survey about their experience. Survey responses were summarized descriptively and comments analyzed using content analysis. RESULTS: Fifty learners submitted videos of assigned tasks and completed peer reviews. Learners reported positive experiences specifically for the Peer Review Stage, structured home practice, ease of mobile access to submit and review videos, and ongoing immediate feedback. Over half of the learners reported spending at least 10 to 30 minute practicing skills before recording their videos and over 80% rerecorded at least 2 times before submission. Content analysis revealed learners engaged with the educational concepts designed into the platform. CONCLUSION: Learners easily used and were satisfied with a mobile-technology teaching platform that maintained the fundamental content, educational theories, and organizational structure of our previously effective surgical skills curriculum. Prior challenges were directly addressed through the mobile application's ease of use, support of deliberate practice, and improved timeliness of feedback.

Evaluation of a Surgery-Based Adjunct Course for Senior Medical Students Entering Surgical Residencies.

BACKGROUND: Preparatory courses for senior medical students aim to ease the transition from medical school to residency. We designed a novel adjunct curriculum to enhance students' readiness for surgical internship. This study addresses the feasibility and outcomes of this course. MATERIALS AND METHODS: A curriculum was designed based on ACGME surgical milestones. Students participated in 8 (3h) sessions held over 4 weeks as an adjunct to a well-established intern preparatory course. Course activities involved interactive simulation cases to emphasize care of surgical patients, and skills sessions focused on knot tying and suturing, which were reinforced with home video assignments. Students rated confidence on 14 management skills using a 5-point Likert scale (5 = high confidence). Faculty graded students' technical performance using a global scale (0-10) for 5 suturing exercises. Comparisons between precourse and postcourse data collected for all measures were made using t-tests (α = 0.05). RESULTS: A total of 11 students entering 4 different surgical fields participated. Overall confidence in patient management improved from 2.41 to 3.89 (standard deviation = 0.49, 0.35; p < 0.05). Students' scores on all 5 suturing tasks increased (p < 0.05). CONCLUSIONS: We developed a surgery-specific component to the existing preparatory course at our institution. Students demonstrated increased confidence in ward management skills and increased technical scores in all exercises. Although only 3 sessions were dedicated to technical skills, improvements may highlight the benefit of home video assignments. This course serves as a specialty-specific model for schools with existing preparatory courses. Our curriculum highlights skills specific for surgical residency, while maximizing resources.

Polarized axonal surface expression of neuronal KCNQ potassium channels is regulated by calmodulin interaction with KCNQ2 subunit.

KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.

Mouse strain-specific coding polymorphism in the Slc4a2/Ae2 gene encodes Ae2c2 variants differing in isoform-specific dominant negative activity.

The Slc4a2/Ae2 gene encodes multiple polypeptides arising from alternate promoter usage. The Ae2c promoter gives rise to only one Ae2c transcript from the human Ae2 gene, but to two, alternatively spliced, Ae2c1 and Ae2c2 transcripts from the mouse and rat genes. Unlike in the rat, the mouse Ae2c2 transcript encodes a novel Ae2c2 polypeptide. Here we report that the Ae2c2 residue 9 can be either proline or serine in a mouse strain-specific manner. Both Ae2c2 polypeptides express low function in Xenopus oocytes secondary to reduced or absent surface expression. Ae2c2S, but not Ae2c2P, exerts a dominant negative effect when coexpressed with Ae2a polypeptide, has a less prominent effect when coexpressed with Ae2b1 or Ae2c1 polypeptides, but has no effect on the function of coexpressed Ae2b2 polypeptide. Coexpression of Ae2c2P does not reduce activity of any Ae2 polypeptide variant. Ae2c2S and Ae2c2P also express low functional activity in HEK-293 cells. Knowledge of strain-specific coding polymorphisms with potential functional consequences such as that of Ae2c2 should aid in interpretation of strain-specific phenotypes investigated in the mouse phenome project.

Alkaline-shifted pHo sensitivity of AE2c1-mediated anion exchange reveals novel regulatory determinants in the AE2 N-terminal cytoplasmic domain.

The mouse anion exchanger AE2/SLC4A2 Cl(-)/HCO(-)(3) exchanger is essential to post-weaning life. AE2 polypeptides regulate pH(i), chloride concentration, cell volume, and transepithelial ion transport in many tissues. Although the AE2a isoform has been extensively studied, the function and regulation of the other AE2 N-terminal variant mRNAs of mouse (AE2b1, AE2b2, AE2c1, and AE2c2) have not been examined. We now present an extended analysis of AE2 variant mRNA tissue distribution and function. We show in Xenopus oocytes that all AE2 variant polypeptides except AE2c2 mediated Cl(-) transport are subject to inhibition by acidic pH(i) and to activation by hypertonicity and NH(+)(4). However, AE2c1 differs from AE2a, AE2b1, and AE2b2 in its alkaline-shifted pH(o)((50)) (7.70 +/- 0.11 versus 6.80 +/- 0.05), suggesting the presence of a novel AE2a pH-sensitive regulatory site between amino acids 99 and 198. Initial N-terminal deletion mutagenesis restricted this site to the region between amino acids 120 and 150. Further analysis identified AE2a residues 127-129, 130-134, and 145-149 as jointly responsible for the difference in pH(o)((50)) between AE2c1 and the longer AE2a, AE2b1, and AE2b2 polypeptides. Thus, AE2c1 exhibits a unique pH(o) sensitivity among the murine AE2 variant polypeptides, in addition to a unique tissue distribution. Physiological coexpression of AE2c1 with other AE2 variant polypeptides in the same cell should extend the range over which changing pH(o) can regulate AE2 transport activity.

A community exposure to 2-chloro-6-fluorophenol.

A rural town in western New York was the site of a release of a mixture of 2-chloro-6-fluorophenol (CFP), toluene and water due to a pressure build-up at a nearby chemical facility. The regional poison control center received calls from physician offices and individual patients describing symptoms felt related to this exposure. Symptoms included headache, dry throat, cough, chest discomfort, nausea/vomiting, and diarrhea. Environmental sampling by the state health department confirmed soil and surface contamination; however, despite a noticeable odor during sampling, staff did not detect CFP in air samples. To our knowledge, although many individuals visited their primary care providers, none were hospitalized. This incident suggests that acute exposure to CFP is similar to other phenol exposures with relatively minor symptoms including headache, irritation of mucous membranes, and gastrointestinal symptoms.