Epidermal keratinocyte polarity and motility require Ca²âº influx through TRPV1.

Ca(2+) has long been known to play an important role in cellular polarity and guidance. We studied the role of Ca(2+) signaling during random and directed cell migration to better understand whether Ca(2+) directs cell motility from the leading edge and which ion channels are involved in this function by using primary zebrafish keratinocytes. Rapid line-scan and time-lapse imaging of intracellular Ca(2+) (Ca(2+)i) during migration and automated image alignment enabled us to characterize and map the spatiotemporal changes in Ca(2+)i. We show that asymmetric distributions of lamellipodial Ca(2+) sparks are encoded in frequency, not amplitude, and that they correlate with cellular rotation during migration. Directed migration during galvanotaxis increases the frequency of Ca(2+) sparks over the entire lamellipod; however, these events do not give rise to asymmetric Ca(2+)i signals that correlate with turning. We demonstrate that Ca(2+)-permeable channels within these cells are mechanically activated and include several transient receptor potential family members, including TRPV1. Last, we demonstrate that cell motility and Ca(2+)i activity are affected by pharmacological agents that target TRPV1, indicating a novel role for this channel during cell migration.

Variability of microchip capillary electrophoresis with conductivity detection.

Microfluidic CE with conductivity detection platforms could have an impact on the future development of smaller, faster and portable devices. However, for the purpose of reliable identification and quantification, there is a need to understand the degree of irreproducibility associated with the analytical technique. In this study, a protocol was developed to remove baseline drift problems sometimes observed in such devices. The protocol, which consisted of pre-conditioning steps prior to analysis, was used to further assess measurement variability from 24 individual microchips fabricated from six separate batches of glass substrate. Results show acceptable RSD percentage for retention time measurements but large variability in their corresponding peak areas (with some microchips having variability of ∼50%). Sources of variability were not related to substrate batch but possibly to a number of factors such as applied voltage fluctuations or variations in microchannel quality, for example surface roughness that will subsequently affect microchannel dimensions.

Seasonal phenology of the cerambycid beetles of east-central Illinois.

We summarize field data on the species composition and seasonal phenology of the community of cerambycid beetles of east-central Illinois. Data were drawn from field bioassays conducted during 2009 - 2012 that tested attraction of adult beetles of diverse species to a variety of synthetic pheromones and host plant volatiles. A total of 34,086 beetles of 114 species were captured, including 48 species in the subfamily Cerambycinae, 41 species in the Lamiinae, 19 species in the Lepturinae, two species in the Spondylidinae, and one species each in the Necydalinae, Parandrinae, Prioninae, and the Disteniidae. Most of the best-represented species were attracted to pheromones that were included in field experiments, particularly species that use (R)-3-hydroxyhexan-2-one as a pheromone component. The species captured, and their patterns of abundance and seasonal phenology were similar to those in an earlier study conducted in Pennsylvania. The most abundant species identified in both studies included the cerambycines Elaphidion mucronatum (Say), Neoclytus a. acuminatus (F.), Neoclytus m. mucronatus (F.), and Xylotrechus colonus (F.). Cerambycine species became active in an orderly progression from early spring through late fall, whereas most lamiine species were active in summer and fall, and lepturine species were limited to summer. Potential cross attraction between some cerambycine species that shared pheromone components may have been averted by differences in seasonal activity period, and by minor pheromone components that acted as synergists for conspecifics and/or antagonists for heterospecifics. These results provide quantitative data on the abundance and seasonal phenology of a large number of species.

Evaluation of Inlet Temperature with Three Sprayer Designs for Desorption Electrospray Ionization Mass Spectrometry Tissue Analysis.

Mass spectrometry imaging (MSI) allows for the spatially resolved detection of endogenous and exogenous molecules and atoms in biological samples, typically prepared as thin tissue sections. Desorption electrospray ionization (DESI) is one of the most commonly utilized MSI modalities in preclinical research. DESI ion source technology is still rapidly evolving, with new sprayer designs and heated inlet capillaries having recently been incorporated in commercially available systems. In this study, three iterations of DESI sprayer designs are evaluated: (1) the first, and until recently only, commercially available Waters sprayer; (2) a developmental desorption electro-flow focusing ionization (DEFFI)-type sprayer; and (3) a prototype of the newly released Waters commercial sprayer. A heated inlet capillary is also employed, allowing for controlled inlet temperatures up to 500 °C. These three sprayers are evaluated by comparative tissue imaging analyses of murine testes across this temperature range. Single ion intensity versus temperature trends are evaluated as exemplar cases for putatively identified species of interest, such as lactate and glutamine. A range of trends are observed, where intensities follow either increasing, decreasing, bell-shaped, or other trends with temperature. Data for all sprayers show approximately similar trends for the ions studied, with the commercial prototype sprayer (sprayer version 3) matching or outperforming the other sprayers for the ions investigated. Finally, the mass spectra acquired using sprayer version 3 are evaluated by uniform manifold approximation and projection (UMAP) and k-means clustering. This approach is shown to provide valuable insight that is complementary to the presented univariate evaluation for reviewing the parameter space in this study. Full spectral temperature optimization data are provided as supporting data to enable other researchers to design experiments that are optimal for specific ions.

Healthcare Worker Mental Health and Wellbeing During COVID-19: Mid-Pandemic Survey Results.

Introduction/Background: HealthCare worker (HCW) mental health and wellbeing are uniquely affected by the complexities of COVID-19 due to exposure to the virus, isolation from family and friends, risk and uncertainty. Little if any inquiry has examined the effects on an entire healthcare system, particularly immediately post-surge. We sought to examine the prevalence of psychiatric symptoms and behavioral health difficulties as a healthcare system transitioned out of the first wave. We assessed the effects of work role, setting and individual diversity factors on employee distress and coping strategies. Materials and Methods: This was an Institutional Review Board approved, unfunded, voluntary survey sent via REDCap link, to all employees of Hartford HealthCare, a mid-sized healthcare system (N ≈ 29,900) between May 15th and June 26th, 2020. Two system-wide emails and two emails targeting managers were sent during this time frame. Eight thousand four hundred and ninety four employees (28.4% of all e-mails distributed) participated in the survey, representing clinical, support, administrative, and medical staff across hospital, outpatient, residential, and business settings. The survey contained items assessing personal background, work environment/culture, and formal measures, including: patient health questionnaire-9 (PHQ-9), general anxiety disorder-7 (GAD-7), primary care post-traumatic stress disorder screen for DSM-5 (PC-PTSD), alcohol use disorders identification test (AUDIT-C), and the insomnia severity index (ISI). Results: Almost 1/3 of respondents (31%) reported symptoms of clinically significant anxiety; 83% moderate to severe depression; and 51% moderate to severe insomnia. Thirteen percent screened positive for post-traumatic stress disorder. Frontline staff (p ≤ 0.001 vs. others) and females (p < 0.001 vs. males) endorsed the highest levels of distress, while race (p ≤ 0.005) and ethnicity (p < 0.03 for anxiety, PTSD and insomnia) had a complex and nuanced interaction with symptoms. Conclusion: Pandemic stress effects all healthcare employees, though not equally. The effects of work role and environment are intuitive though critical. These data suggest individual diversity factors also play an important role in mental health and wellbeing. All must be considered to optimize employee functioning.

Health Care Workers' Mental Health and Quality of Life During COVID-19: Results From a Mid-Pandemic, National Survey.

OBJECTIVE: The authors sought to quantify the rates of psychological distress among health care workers (HCWs) during the COVID-19 pandemic and to identify job-related and personal risk and protective factors. METHODS: From April 1 to April 28, 2020, the authors conducted a national survey advertised via e-mail lists, social media, and direct e-mail. Participants were self-selecting, U.S.-based volunteers. Scores on the Patient Health Questionnaire-9, General Anxiety Disorder-7, Primary Care Posttraumatic Stress Disorder Screen, and Alcohol Use Disorders Identification Test-C were used. The relationships between personal resilience and risk factors, work culture and stressors and supports, and COVID-19-related events were examined. RESULTS: Of 1,685 participants (76% female, 88% White), 31% (404 of 1,311) endorsed mild anxiety, and 33% (427 of 1,311) clinically meaningful anxiety; 29% (393 of 1,341) reported mild depressive symptoms, and 17% (233 of 1,341) moderate to severe depressive symptoms; 5% (64 of 1,326) endorsed suicidal ideation; and 14% (184 of 1,300) screened positive for posttraumatic stress disorder. Pediatric HCWs reported greater anxiety than did others. HCWs' mental health history increased risk for anxiety (odds ratio [OR]=2.78, 95% confidence interval [CI]=2.09-3.70) and depression (OR=3.49, 95% CI=2.47-4.94), as did barriers to working, which were associated with moderate to severe anxiety (OR=2.50, 95% CI=1.80-3.48) and moderate depressive symptoms (OR=2.15, 95% CI=1.45-3.21) (p<0.001 for all comparisons). CONCLUSIONS: Nearly half of the HCWs reported serious psychiatric symptoms, including suicidal ideation, during the COVID-19 pandemic. Perceived workplace culture and supports contributed to symptom severity, as did personal factors.

Air medical transport curriculum provides education for medical students.

INTRODUCTION: Increased regionalization of healthcare will most likely result in greater utilization of air medical transport (AMT). Education of providers regarding the utilization and capabilities of AMT will be important. This study was undertaken to determine whether our curriculum in AMT provided a useful and satisfying educational experience for medical students METHODS: The curriculum included three 6-hour fly-along sessions, 2 hours of lectures, 2 hours of case review, an 80-page syllabus listing articles and position papers, and a 3-page writing assignment. The study was a review of the participants' (1st- and 2nd-year medical students) test scores and postelective evaluation for 1 year. The setting was a two-helicopter AMT program based at an urban, university-affiliated level 1 trauma center. Twelve students (1/month) participated in the month-long elective. The students took a 20-item test before and after the elective and a 15-item postelective evaluation (5-point scale: very dissatisfied, dissatisfied, neutral, satisfied, very satisfied). A paired t-test was used to compare the mean pre-test and mean post-test scores. The evaluation data were analyzed descriptively. RESULTS: The mean number of correct responses on the post-test (M = 18.67, SD = 1.61) was significantly higher than the mean number of correct responses on the pretest (M = 13.83, SD = 2.17), P < .001. All students were either satisfied (56%) or very satisfied (44%) with the lectures and case review. Eleven (91%) were very satisfied with interaction with the crew, and nine (75%) were very satisfied overall (25% satisfied). Seven (58%) were very likely to use AMT in their career, two (16.7%) likely, two (16.7%) unsure, and one (8.3%) unlikely. CONCLUSIONS: Our curriculum in AMT provided a useful and satisfying educational opportunity for medical students. Their knowledge of AMT increased, and all were satisfied or very satisfied with the didactics and the crew interaction. Most thought that they would use AMT in their career.

MALDI-2 at Atmospheric Pressure-Parameter Optimization and First Imaging Experiments.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique for mapping the spatial distribution of biomolecules directly from tissue. However, like most other MSI techniques, it suffers from low ionization yields and ion suppression effects for biomolecules that might be of interest for a specific application at hand. Recently, a form of laser postionization was introduced (coined MALDI-2) that critically boosts the ion yield for many glyco- and phospholipids by several orders of magnitude and makes the detection of further biomolecular species possible. While the MALDI-2 technique is being increasingly applied by the MSI community, it is still only implemented in fine vacuum ion sources in a pressure range of about 1-10 mbar. Here, we show the first implementation of the technique to a custom-built atmospheric pressure ion source coupled to an Orbitrap Elite system. We present results from parameter optimization of MALDI-2 at atmospheric pressure, compare our findings to previously published fine vacuum data, and show first imaging results from mouse cerebellum with a 20 µm pixel size. Our findings broaden the feasibility of the technique to overall more flexible atmospheric pressure ion sources.

COVID-19 mortality risk assessment: An international multi-center study.

Timely identification of COVID-19 patients at high risk of mortality can significantly improve patient management and resource allocation within hospitals. This study seeks to develop and validate a data-driven personalized mortality risk calculator for hospitalized COVID-19 patients. De-identified data was obtained for 3,927 COVID-19 positive patients from six independent centers, comprising 33 different hospitals. Demographic, clinical, and laboratory variables were collected at hospital admission. The COVID-19 Mortality Risk (CMR) tool was developed using the XGBoost algorithm to predict mortality. Its discrimination performance was subsequently evaluated on three validation cohorts. The derivation cohort of 3,062 patients has an observed mortality rate of 26.84%. Increased age, decreased oxygen saturation (≤ 93%), elevated levels of C-reactive protein (≥ 130 mg/L), blood urea nitrogen (≥ 18 mg/dL), and blood creatinine (≥ 1.2 mg/dL) were identified as primary risk factors, validating clinical findings. The model obtains out-of-sample AUCs of 0.90 (95% CI, 0.87-0.94) on the derivation cohort. In the validation cohorts, the model obtains AUCs of 0.92 (95% CI, 0.88-0.95) on Seville patients, 0.87 (95% CI, 0.84-0.91) on Hellenic COVID-19 Study Group patients, and 0.81 (95% CI, 0.76-0.85) on Hartford Hospital patients. The CMR tool is available as an online application at covidanalytics.io/mortality_calculator and is currently in clinical use. The CMR model leverages machine learning to generate accurate mortality predictions using commonly available clinical features. This is the first risk score trained and validated on a cohort of COVID-19 patients from Europe and the United States.

The involvement of Ca2+ and integrins in directional responses of zebrafish keratocytes to electric fields.

Many cells respond directionally to small DC electrical fields (EFs) by an unknown mechanism, but changes in intracellular Ca(2+) are widely assumed to be involved. We have used zebrafish (Danio rerio) keratocytes in an effort to understand the nature of the EF-cell interaction. We find that the adult zebrafish integument drives substantial currents outward through wounds produced by scale removal, establishing that keratocytes near the wound will experience endogenous EFs. Isolated keratocytes in culture turn toward the cathode in fields as small as 7 mV mm(-1), and the response is independent of cell size. Epidermal sheets are similarly sensitive. The frequency of intracellular Ca(2+) spikes and basal Ca(2+) levels were increased by EFs, but the spikes were not a necessary aspect of migration or EF response. Two-photon imaging failed to detect a pattern of gradients of Ca(2+) across the lamellipodia during normal or EF-induced turning but did detect a sharp, stable Ca(2+) gradient at the junction of the lamellipodium and the cell body. We conclude that gradients of Ca(2+) within the lamellipodium are not required for the EF response. Immunostaining revealed an anode to cathode gradient of integrin beta1 during EF-induced turning, and interference with integrin function attenuated the EF response. Neither electrophoretic redistribution of membrane proteins nor asymmetric perturbations of the membrane potential appear to be involved in the EF response, and we propose a new model in which hydrodynamic forces generated by electro-osmotic water flow mediate EF-cell interactions via effects on focal adhesions.

Comparison of use of the the Airtraq with direct laryngoscopy by paramedics in the simulated airway.

INTRODUCTION: Paramedics often encounter patients with difficult airways requiring emergent airway management. OBJECTIVE: The purpose of this study was to compare intubation utilizing the Airtraq with direct laryngoscopy (DL) in the manikin model. We evaluated the number of attempts, the time to successful intubation, and the Airtraq's learning curve. METHODS: This was a randomized, crossover study involving paramedics. Each participant was given a standardized lecture and a demonstration of the Airtraq device. After a 5-minute practice session on a Laerdal Airway Management Trainer with the Airtraq and DL, participants managed the following four scenarios on a Laerdal SimMan manikin: 1) normal airway; 2) tongue edema; 3) cervical spine immobilization; and 4) repeated normal airway. Results were analyzed using the Wilcoxon signed rank test. RESULTS: Thirty paramedics participated in this study. For scenario 1, there were no significant differences in either the number of attempts or the time to ventilation between the devices. For scenario 2, the mean time to ventilation was significantly faster, and fewer intubation attempts were observed with the Airtraq when compared with DL. For scenario 3, there were no significant differences in number of attempts and time to ventilation. Scenario 4 demonstrated significantly less time to ventilation and fewer intubation attempts with the Airtraq. A significant decrease in time to ventilation was observed with the Airtraq when comparing scenarios 1 and 4. CONCLUSIONS: The Airtraq was shown to be equal to or faster than DL. The Airtraq has a rapid learning curve demonstrated by a significantly decreased time to ventilation between scenarios 1 and 4.

The Influence of MS Imaging Parameters on UV-MALDI Desorption and Ion Yield.

Ultraviolet matrix-assisted laser desorption/ionization mass spectrometry imaging (UV-MALDI MSI) is a widely used technique for imaging molecular distributions within biological systems. While much work exists concerning desorption in UV-MALDI MS, the effects of commonly varied parameters for imaging applications (repetition rate, use of continuous raster mode and raster speed), which determine spatial resolution and limits of detection for the technique, remain largely unknown. We use multiple surface characterization modalities to obtain quantitative measurements of material desorption and analyte ion yield in thin film model systems of two matrix compounds, arising from different UV-MALDI MSI sampling conditions. Observed changes in resulting ablation feature point to matrix-dependent spatial resolution and laser-induced matrix modification effects. Analyte ion yields of 10-9 to 10-6 are observed. Complex changes in ion yield, between spot and raster sampling and arising from varied laser repetition rate and raster speed, are observed. Graphical Abstract.

Construction and testing of an atmospheric-pressure transmission-mode matrix assisted laser desorption ionisation mass spectrometry imaging ion source with plasma ionisation enhancement.

Matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) at atmospheric pressure (AP) is, with a few notable exceptions, overshadowed by its vacuum based forms and AP transmission mode (TM) MALDI-MS lacks the up-take its potential benefits might suggest. The reasons for this are not fully understood and it is clear further development is required to realise the flexibility and power of this ionisation method and geometry. Here we report the build of a new AP-TM-MALDI-MSI ion source with plasma ionisation enhancement. This novel ion source is used to analyse a selection of increasingly complex systems from molecular standards to murine brain tissue sections. Significant enhancement of detected ion intensity is observed in both positive and negative ion mode in all systems, with up to 2000 fold increases observed for a range of tissue endogenous species. The substantial improvements conferred by the plasma enhancement are then employed to demonstrate the acquisition of proof of concept tissue images, with high quality spectra obtained down to 10 × 10 µm pixel size.

Novel electronic health record (EHR) education intervention in large healthcare organization improves quality, efficiency, time, and impact on burnout.

A novel approach to advanced electronic health record (EHR) skills training was developed in a large healthcare organization to improve high-quality EHR documentation, while reducing stressors linked to physician burnout.The 3-day intensive EHR education intervention covered best practices in EHR documentation and physician well-being. The specialty physician faculty used interactive teaching including demonstration, facilitation, and individual coaching. Laptops were provided for hands-on practice. Mixed-method evaluation included real-time feedback, daily surveys, and post-activity surveys to measure participant learning and satisfaction, and also collection of performance data from the EHR to measure use of order sets designed to improve quality of care.Since 2014, 46 trainings were held with 3500 physicians. Most physicians (85%-98% across all programs) reported improved quality, readability, and clinical accuracy of documentation; fewer medical errors; and increased efficiency in chart review and data retrieval due to the training. Seventy-eight per cent estimated a time savings of 4 to 5 minutes or more per hour. Physician performance data from the EHR showed significant improvement in use of order sets for several critical health conditions such as sepsis, stroke, and chest pain of possible cardiac cause.This advanced EHR training for physicians was well-received and improved physicians' use of several order sets designed to improve quality of care. EHR training programs such as this may have impact on the safety, quality, accuracy, and timeliness of care and may also help reduce physician burnout by improving critical skills and reducing time interfacing with all aspects of a patient's health record.

Matrix Optical Absorption in UV-MALDI MS.

In ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI MS) matrix compound optical absorption governs the uptake of laser energy, which in turn has a strong influence on experimental results. Despite this, quantitative absorption measurements are lacking for most matrix compounds. Furthermore, despite the use of UV-MALDI MS to detect a vast range of compounds, investigations into the effects of laser energy have been primarily restricted to single classes of analytes. We report the absolute solid state absorption spectra of the matrix compounds α-cyano-4-hydroxycinnamic acid (CHCA), para-nitroaniline (PNA), 2-mercaptobenzothiazole (MBT), 2,5-dihydroxybenzoic acid (2,5-DHB), and 2,4,6-trihydroxyacetophenone (THAP). The desorption/ionization characteristics of these matrix compounds with respect to laser fluence was investigated using mixed systems of matrix with either angiotensin II, PC(34:1) lipid standard, or haloperidol, acting as representatives for typical classes of analyte encountered in UV-MALDI MS. The first absolute solid phase spectra for PNA, MBT, and THAP are reported; additionally, inconsistencies between previously published spectra for CHCA are resolved. In light of these findings, suggestions are made for experimental optimization with regards to matrix and laser wavelength selection. The relationship between matrix optical cross-section and wavelength-dependant threshold fluence, fluence of maximum ion yield, and R, a new descriptor for the change in ion intensity with fluence, are described. A matrix cross-section of 1.3 × 10-17 cm-2 was identified as a potential minimum for desorption/ionization of analytes. Graphical Abstract ᅟ.

Embryonic zebrafish neuronal growth is not affected by an applied electric field in vitro.

Naturally occurring electric fields (EFs) have been implicated in cell guidance during embryonic development and adult wound healing. Embryonic Xenopus laevis neurons sprout preferentially towards the cathode, turn towards the cathode, and migrate faster towards the cathode in the presence of an external EF in vitro. A recent Phase 1 clinical trial has investigated the effects of oscillating EFs on human spinal cord regeneration. The purpose of this study was to investigate whether embryonic zebrafish neurons respond to an applied EF, and thus extend this research into another vertebrate system. Neural tubes of zebrafish embryos (16-17 somites) were dissected and dissociated neuroblasts were plated onto laminin-coated glass. A 100 mV/mm EF was applied to cell cultures for 4 or 20 h and the responses of neurons to the applied EFs were investigated. After 4h in an EF neurites were significantly shorter than control neurites. No other statistically significant effects were observed. After 20 h, control and EF-exposed neurites were no different in length. No length difference was seen between cathodally- and anodally-sprouted neurites. Application of an EF did not affect the average number of neurons in a chamber. Growth cones did not migrate preferentially towards either pole of the EF and no asymmetry was seen in neurite sprout sites. We conclude that zebrafish neurons do not respond to a 100 mV/mm applied EF in vitro. This suggests that neurons of other vertebrate species may not respond to applied EFs in the same ways as Xenopus laevis neurons.

Suitability of analytical methods to measure solubility for the purpose of nanoregulation.

Solubility is an important physicochemical parameter in nanoregulation. If nanomaterial is completely soluble, then from a risk assessment point of view, its disposal can be treated much in the same way as "ordinary" chemicals, which will simplify testing and characterisation regimes. This review assesses potential techniques for the measurement of nanomaterial solubility and evaluates the performance against a set of analytical criteria (based on satisfying the requirements as governed by the cosmetic regulation as well as the need to quantify the concentration of free (hydrated) ions). Our findings show that no universal method exists. A complementary approach is thus recommended, to comprise an atomic spectrometry-based method in conjunction with an electrochemical (or colorimetric) method. This article shows that although some techniques are more commonly used than others, a huge research gap remains, related with the need to ensure data reliability.

Pulsating ion fluxes and growth at the pollen tube tip.

Reproduction in higher plants requires the directed growth of pollen tubes in order to transmit sperm cells to the ovules at the base of the style. Many signaling processes have been implicated in polarized pollen tube growth. Here, changes in the concentration of calcium, potassium, hydrogen (pH), and chloride are discussed, as they may all contribute to the process of oscillatory growth and guidance observed in pollen tubes.