A mouse DRG genetic toolkit reveals morphological and physiological diversity of somatosensory neuron subtypes.

Dorsal root ganglia (DRG) somatosensory neurons detect mechanical, thermal, and chemical stimuli acting on the body. Achieving a holistic view of how different DRG neuron subtypes relay neural signals from the periphery to the CNS has been challenging with existing tools. Here, we develop and curate a mouse genetic toolkit that allows for interrogating the properties and functions of distinct cutaneous targeting DRG neuron subtypes. These tools have enabled a broad morphological analysis, which revealed distinct cutaneous axon arborization areas and branching patterns of the transcriptionally distinct DRG neuron subtypes. Moreover, in vivo physiological analysis revealed that each subtype has a distinct threshold and range of responses to mechanical and/or thermal stimuli. These findings support a model in which morphologically and physiologically distinct cutaneous DRG sensory neuron subtypes tile mechanical and thermal stimulus space to collectively encode a wide range of natural stimuli.

Bursting translation on single mRNAs in live cells.

Stochasticity has emerged as a mechanism of gene regulation. Much of this so-called "noise" has been attributed to bursting transcription. Although bursting transcription has been studied extensively, the role of stochasticity in translation has not been fully investigated due to the lack of enabling imaging technology. In this study, we developed techniques to track single mRNAs and their translation in live cells for hours, allowing the measurement of previously uncharacterized translation dynamics. We applied genetic and pharmacological perturbations to control translation kinetics and found that, like transcription, translation is not a constitutive process but instead cycles between inactive and active states, or "bursts." However, unlike transcription, which is largely frequency-modulated, complex structures in the 5'-untranslated region alter burst amplitudes. Bursting frequency can be controlled through cap-proximal sequences and trans-acting factors such as eIF4F. We coupled single-molecule imaging with stochastic modeling to quantitatively determine the kinetic parameters of translational bursting.

A DRG genetic toolkit reveals molecular, morphological, and functional diversity of somatosensory neuron subtypes.

Mechanical and thermal stimuli acting on the skin are detected by morphologically and physiologically distinct sensory neurons of the dorsal root ganglia (DRG). Achieving a holistic view of how this diverse neuronal population relays sensory information from the skin to the central nervous system (CNS) has been challenging with existing tools. Here, we used transcriptomic datasets of the mouse DRG to guide development and curation of a genetic toolkit to interrogate transcriptionally defined DRG neuron subtypes. Morphological analysis revealed unique cutaneous axon arborization areas and branching patterns of each subtype. Physiological analysis showed that subtypes exhibit distinct thresholds and ranges of responses to mechanical and/or thermal stimuli. The somatosensory neuron toolbox thus enables comprehensive phenotyping of most principal sensory neuron subtypes. Moreover, our findings support a population coding scheme in which the activation thresholds of morphologically and physiologically distinct cutaneous DRG neuron subtypes tile multiple dimensions of stimulus space.

Malignant hyperthermia in severe COVID-19: 2 case reports.

Malignant hyperthermia is a rare but potentially fatal condition. We present 2 cases of young patients with coronavirus disease 2019 (COVID-19) requiring intubation for hypoxic respiratory failure who both developed significant hyperthermia post intubation and were suspected to have malignant hyperthermia. However, the 2 patients had different responses to conservative management and dantrolene. These cases highlight the increased challenge imposed by intubation complications when managing patients with COVID-19.

Brain state flexibility accompanies motor-skill acquisition.

Learning requires the traversal of inherently distinct cognitive states to produce behavioral adaptation. Yet, tools to explicitly measure these states with non-invasive imaging - and to assess their dynamics during learning - remain limited. Here, we describe an approach based on a distinct application of graph theory in which points in time are represented by network nodes, and similarities in brain states between two different time points are represented as network edges. We use a graph-based clustering technique to identify clusters of time points representing canonical brain states, and to assess the manner in which the brain moves from one state to another as learning progresses. We observe the presence of two primary states characterized by either high activation in sensorimotor cortex or high activation in a frontal-subcortical system. Flexible switching among these primary states and other less common states becomes more frequent as learning progresses, and is inversely correlated with individual differences in learning rate. These results are consistent with the notion that the development of automaticity is associated with a greater freedom to use cognitive resources for other processes. Taken together, our work offers new insights into the constrained, low dimensional nature of brain dynamics characteristic of early learning, which give way to less constrained, high-dimensional dynamics in later learning.

Interdisciplinary Development of an Improved Emergency Department Procedural Work Surface Through Iterative Design and Use Testing in Simulated and Clinical Environments.

STUDY OBJECTIVE: A stable and readily accessible work surface for bedside medical procedures represents a valuable tool for acute care providers. In emergency department (ED) settings, the design and implementation of traditional Mayo stands and related surface devices often limit their availability, portability, and usability, which can lead to suboptimal clinical practice conditions that may affect the safe and effective performance of medical procedures and delivery of patient care. We designed and built a novel, open-source, portable, bedside procedural surface through an iterative development process with use testing in simulated and live clinical environments. METHODS: The procedural surface development project was conducted between October 2014 and June 2016 at an academic referral hospital and its affiliated simulation facility. An interdisciplinary team of emergency physicians, mechanical engineers, medical students, and design students sought to construct a prototype bedside procedural surface out of off-the-shelf hardware during a collaborative university course on health care design. After determination of end-user needs and core design requirements, multiple prototypes were fabricated and iteratively modified, with early variants featuring undermattress stabilizing supports or ratcheting clamp mechanisms. Versions 1 through 4 underwent 2 hands-on usability-testing simulation sessions; version 5 was presented at a design critique held jointly by a panel of clinical and industrial design faculty for expert feedback. Responding to select feedback elements over several surface versions, investigators arrived at a near-final prototype design for fabrication and use testing in a live clinical setting. This experimental procedural surface (version 8) was constructed and then deployed for controlled usability testing against the standard Mayo stands in use at the study site ED. Clinical providers working in the ED who opted to participate in the study were provided with the prototype surface and just-in-time training on its use when performing bedside procedures. Subjects completed the validated 10-point System Usability Scale postshift for the surface that they had used. The study protocol was approved by the institutional review board. RESULTS: Multiple prototypes and recursive design revisions resulted in a fully functional, portable, and durable bedside procedural surface that featured a stainless steel tray and intuitive hook-and-lock mechanisms for attachment to ED stretcher bed rails. Forty-two control and 40 experimental group subjects participated and completed questionnaires. The median System Usability Scale score (out of 100; higher scores associated with better usability) was 72.5 (interquartile range [IQR] 51.3 to 86.3) for the Mayo stand; the experimental surface was scored at 93.8 (IQR 84.4 to 97.5 for a difference in medians of 17.5 (95% confidence interval 10 to 27.5). Subjects reported several usability challenges with the Mayo stand; the experimental surface was reviewed as easy to use, simple, and functional. In accordance with experimental live environment deployment, questionnaire responses, and end-user suggestions, the project team finalized the design specification for the experimental procedural surface for open dissemination. CONCLUSION: An iterative, interdisciplinary approach was used to generate, evaluate, revise, and finalize the design specification for a new procedural surface that met all core end-user requirements. The final surface design was evaluated favorably on a validated usability tool against Mayo stands when use tested in simulated and live clinical settings.

Objective Assessment and Thematic Categorization of Patient-audible Information in an Emergency Department.

OBJECTIVES: The objective was to assess and categorize the understandable components of patient-audible information (e.g., provider conversations) in emergency department (ED) care areas and to initiate a baseline ED soundscape assessment. METHODS: Investigators at an academic referral hospital accessed 21 deidentified transcripts of recordings made with binaural in-ear microphones in patient rooms (n = 10) and spaces adjacent to nurses' stations (n = 11), during ED staff sign-outs as part of an approved quality management process. Transcribed materials were classified by speaker (health care provider, patient/family/friend, or unknown). Using qualitative analysis software and predefined thematic categories, two investigators then independently coded each transcript by word, phrase, clause, and/or sentence for general content, patient information, and HIPAA-defined patient identifiers. Scheduled reviews were used to resolve any data coding discrepancies. RESULTS: Patient room recordings featured a median of 11 (interquartile range [IQR] = 2 to 33) understandable words per minute (wpm) over 16.2 (IQR = 15.1 to 18.4) minutes; nurses' station recordings featured 74 (IQR = 47 to 109) understandable wpm over 17.0 (IQR = 15.4 to 20.3) minutes. Transcript content from patient room recordings was categorized as follows: clinical, 44.8% (IQR = 17.7% to 62.2%); nonclinical, 0.0% (IQR = 0.0% to 0.0%); inappropriate (provider), 0.0% (IQR = 0.0% to 0.0%); and unknown, 6.0% (IQR = 1.7% to 58.2%). Transcript content from nurses' stations was categorized as follows: clinical, 86.0% (IQR = 68.7% to 94.7%); nonclinical, 1.2% (IQR = 0.0% to 19.5%); inappropriate (provider), 0.1% (IQR = 0.0% to 2.3%); and unknown, 1.3% (IQR = 0.0% to 7.1%). Limited patient information was audible on patient room recordings. Audible patient information at nurses' stations was coded as follows (median words per sign-out sample): general patient history, 116 (IQR = 19 to 206); social history, 12 (IQR = 4 to 19); physical examination, 39 (IQR = 19 to 56); imaging results, 0 (IQR = 0 to 21); laboratory results, 7 (IQR = 0 to 22); other results, 0 (IQR = 0 to 3); medical decision-making, 39 (IQR = 10 to 69); management (general), 118 (IQR = 79 to 235); pain management, 4 (IQR = 0 to 53); and disposition, 42 (IQR = 22 to 60). Medians of 0 (IQR = 0 to 0) and 3 (IQR = 1 to 4) patient name identifiers were audible on in-room and nurses' station sign-out recordings, respectively. CONCLUSIONS: Sound recordings in an ED setting captured audible and understandable provider discussions that included confidential, protected health information and discernible quantities of nonclinical content.

Microvessel density as a molecular marker for identifying high-grade prostatic intraepithelial neoplasia precursors to prostate cancer.

BACKGROUND: Existing clinical data have shown that high-grade prostatic intraepithelial neoplasia (HGPIN) is the most likely precursor to prostate cancer (CaP). Criteria to distinguish HGPIN that progress to CaP from those that do not remain poorly defined. Our objective was to evaluate microvessel density as a molecular marker for distinguishing HGPINs that have the potential of progressing to cancer. MATERIALS AND METHODS: Human prostatic tissue samples were collected randomly from 50 prostatectomy and cystoprostatectomy patients. Formalin-fixed and paraffin-embedded sections were used for immunohistochemical localization of rabbit anti-human von Willebrand factor VIII (vWF) IgG, mouse anti-high molecular weight cytokeratin 34BE-12 in basal cells, and mouse anti-heparan sulphate proteoglycan (HSPG) IgGs in basement membranes associated with benign prostatic hyperplasia (BPH), PIN associated with some BPH (isolated PIN), and PIN associated with CaP. RESULTS: Analysis of immunostaining data showed that PINs could be categorized according to their distributions within and outside 2 standard deviations (SD) of the mean for microvessel density. The average number of microvessels was significantly higher (P < 0.0001) in PINs associated with Gleason score 7 tumors than those associated with Gleason scores 4-6 (P < 0.1328) or 8 and 9 tumors (P < 0.1708). Morphologically, PINs within 2 SD were composed of low- and high-grade type, whereas those outside 2 SD of microvessel density were predominantly of high-grade type. Cytokeratin and HSPG localization patterns also showed differences in PINs found within and outside 2 SD of microvessel density. We found localization of cytokeratin 34BE-12 in basal cells of specimens with BPH alone, isolated PIN, and PIN associated with CaP within 2 SD, whereas many PINs outside 2 SD showed disruptions in cytokeratin localization. The basement membranes of PINs within 2 SD of microvessel density were relatively intact, whereas those outside 2 SD were fragmented. CONCLUSIONS: Our immunostaining data indicates that once HGPIN is found in the initial prostatic biopsy, it should be evaluated for microvessel density by localization of vWF. Our data indicate that characteristics of HGPIN can be augmented by evaluations of cytokeratin and HSPG molecular markers to assess the potential of HGPIN progression to malignancy. When biopsy samples show HGPIN with increased microvessel density and disrupted cytokeratin and HSPG markers, the patient may be a candidate for repeat biopsy. Since our study is limited to 50 prostate tissue samples, we emphasize that our conclusion is tentative and ought to be confirmed in a study with a larger sample size. This is the first report to show that microvessel density may distinguish HGPIN that is a precursor to prostate cancer.