Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Human Motor Neurons Elicit Pathological Hallmarks of ALS and Reveal Potential Biomarkers of the Disease in Response to Prolonged IFNγ Exposure.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder marked by progressive motor neuron degeneration and muscle denervation. A recent transcriptomic study integrating a wide range of human ALS samples revealed that the upregulation of p53, a downstream target of inflammatory stress, is commonly detected in familial and sporadic ALS cases by a mechanism linked to a transactive response DNA-binding protein 43 (TDP-43) dysfunction. In this study, we show that prolonged interferon-gamma (IFNγ) treatment of human induced pluripotent stem cell-derived spinal motor neurons results in a severe cytoplasmic aggregation of TDP-43. TDP-43 dysfunction resulting from either IFNγ exposure or an ALS-associated TDP-43 mutation was associated with the activation of the p53 pathway. This was accompanied by the hyperactivation of neuronal firing, followed by the complete loss of their electrophysiological function. Through a comparative single-cell transcriptome analysis, we have identified significant alterations in ALS-associated genes in motor neurons exposed to IFNγ, implicating their direct involvement in ALS pathology. Interestingly, IFNγ was found to induce significant levels of programmed death-ligand 1 (PD-L1) expression in motor neurons without affecting the levels of any other immune checkpoint proteins. This finding suggests a potential role of excessive PD-L1 expression in ALS development, given that PD-L1 was recently reported to impair neuronal firing ability in mice. Our findings suggest that exposing motor neurons to IFNγ could directly derive ALS pathogenesis, even without the presence of the inherent genetic mutation or functional glia component. Furthermore, this study provides a comprehensive list of potential candidate genes for future immunotherapeutic targets with which to treat sporadic forms of ALS, which account for 90% of all reported cases.

SOBA: Development and testing of a soluble oligomer binding assay for detection of amyloidogenic toxic oligomers.

The formation of toxic Amyloid ß-peptide (Aß) oligomers is one of the earliest events in the molecular pathology of Alzheimer's Disease (AD). These oligomers lead to a variety of downstream effects, including impaired neuronal signaling, neuroinflammation, tau phosphorylation, and neurodegeneration, and it is estimated that these events begin 10 to 20 y before the presentation of symptoms. Toxic Aß oligomers contain a nonstandard protein structure, termed α-sheet, and designed α-sheet peptides target this main-chain structure in toxic oligomers independent of sequence. Here we show that a designed α-sheet peptide inhibits the deleterious effects on neuronal signaling and also serves as a capture agent in our soluble oligomer binding assay (SOBA). Pre-incubated synthetic α-sheet-containing Aß oligomers produce strong SOBA signals, while monomeric and ß-sheet protofibrillar Aß do not. α-sheet containing oligomers were also present in cerebrospinal fluid (CSF) from an AD patient versus a noncognitively impaired control. For the detection of toxic oligomers in plasma, we developed a plate coating to increase the density of the capture peptide. The proof of concept was achieved by testing 379 banked human plasma samples. SOBA detected Aß oligomers in patients on the AD continuum, including controls who later progressed to mild cognitive impairment. In addition, SOBA discriminated AD from other forms of dementia, yielding sensitivity and specificity of 99% relative to clinical and neuropathological diagnoses. To explore the broader potential of SOBA, we adapted the assay for a-synuclein oligomers and confirmed their presence in CSF from patients with Parkinson's disease and Lewy body dementia.

Creating stem cell-derived neuromuscular junctions in vitro.

Recent development of novel therapies has improved mobility and quality of life for people suffering from inheritable neuromuscular disorders. Despite this progress, the majority of neuromuscular disorders are still incurable, in part due to a lack of predictive models of neuromuscular junction (NMJ) breakdown. Improvement of predictive models of a human NMJ would be transformative in terms of expanding our understanding of the mechanisms that underpin development, maintenance, and disease, and as a testbed with which to evaluate novel therapeutics. Induced pluripotent stem cells (iPSCs) are emerging as a clinically relevant and non-invasive cell source to create human NMJs to study synaptic development and maturation, as well as disease modeling and drug discovery. This review will highlight the recent advances and remaining challenges to generating an NMJ capable of eliciting contraction of stem cell-derived skeletal muscle in vitro. We explore the advantages and shortcomings of traditional NMJ culturing platforms, as well as the pioneering technologies and novel, biomimetic culturing systems currently in use to guide development and maturation of the neuromuscular synapse and extracellular microenvironment. Then, we will explore how this NMJ-in-a-dish can be used to study normal assembly and function of the efferent portion of the neuromuscular arc, and how neuromuscular disease-causing mutations disrupt structure, signaling, and function.

NanoMEA: A Tool for High-Throughput, Electrophysiological Phenotyping of Patterned Excitable Cells.

Matrix nanotopographical cues are known to regulate the structure and function of somatic cells derived from human pluripotent stem cell (hPSC) sources. High-throughput electrophysiological analysis of excitable cells derived from hPSCs is possible via multielectrode arrays (MEAs) but conventional MEA platforms use flat substrates and do not reproduce physiologically relevant tissue-specific architecture. To address this issue, we developed a high-throughput nanotopographically patterned multielectrode array (nanoMEA) by integrating conductive, ion-permeable, nanotopographic patterns with 48-well MEA plates, and investigated the effect of substrate-mediated cytoskeletal organization on hPSC-derived cardiomyocyte and neuronal function at scale. Using our nanoMEA platform, we found patterned hPSC-derived cardiac monolayers exhibit both enhanced structural organization and greater sensitivity to treatment with calcium blocking or conduction inhibiting compounds when subjected to high-throughput dose-response studies. Similarly, hPSC-derived neurons grown on nanoMEA substrates exhibit faster migration and neurite outgrowth speeds, greater colocalization of pre- and postsynaptic markers, and enhanced cell-cell communication only revealed through examination of data sets derived from multiple technical replicates. The presented data highlight the nanoMEA as a new tool to facilitate high-throughput, electrophysiological analysis of ordered cardiac and neuronal monolayers, which can have important implications for preclinical analysis of excitable cell function.

Nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology.

In this study, we report nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology. With the aim of defining sophisticated Nafion nanostructures with highly ionic conductivity, fabrication parameters such as Nafion concentration and curing temperature were optimized. By increasing curing temperature and Nafion concentration, we were able to control the replication fidelity of Nafion nanopatterns when copied from a PDMS master mold. We also found that cross-sectional morphology and ion current density of nanopatterned Nafion strongly depends on the fabrication parameters. To investigate this dependency, current-voltage analysis was conducted using organic electrochemical transistors (OECT) overlaid with patterned Nafion substrates. Nanopatterned Nafion was found to allow higher ion current densities than unpatterned surfaces. Furthermore, higher curing temperatures were found to render Nafion layers with higher ion/electrical transfer properties. To optimize nanopattern dimensions, electrical current flows, and film uniformity, a final configuration consisting of 5% nanopatterned Nafion cured at 65°C was chosen. Multielectrode arrays (MEAs) were then covered with optimized Nafion nanopatterns and used for electrophysiological analysis of two types of induced pluripotent stem cell-derived cardiomyocytes (iPSCs-CMs). These data highlight the suitability of nanopatterned Nafion, combined with MEAs, for enhancing the cellular environment of iPSC-CMs for use in electrophysiological analysis in vitro.

Effects of COVID-19 on College Students' Mental Health in the United States: Interview Survey Study.

BACKGROUND: Student mental health in higher education has been an increasing concern. The COVID-19 pandemic situation has brought this vulnerable population into renewed focus. OBJECTIVE: Our study aims to conduct a timely assessment of the effects of the COVID-19 pandemic on the mental health of college students. METHODS: We conducted interview surveys with 195 students at a large public university in the United States to understand the effects of the pandemic on their mental health and well-being. The data were analyzed through quantitative and qualitative methods. RESULTS: Of the 195 students, 138 (71%) indicated increased stress and anxiety due to the COVID-19 outbreak. Multiple stressors were identified that contributed to the increased levels of stress, anxiety, and depressive thoughts among students. These included fear and worry about their own health and of their loved ones (177/195, 91% reported negative impacts of the pandemic), difficulty in concentrating (173/195, 89%), disruptions to sleeping patterns (168/195, 86%), decreased social interactions due to physical distancing (167/195, 86%), and increased concerns on academic performance (159/195, 82%). To cope with stress and anxiety, participants have sought support from others and helped themselves by adopting either negative or positive coping mechanisms. CONCLUSIONS: Due to the long-lasting pandemic situation and onerous measures such as lockdown and stay-at-home orders, the COVID-19 pandemic brings negative impacts on higher education. The findings of our study highlight the urgent need to develop interventions and preventive strategies to address the mental health of college students.

Investigating Mental Health of US College Students During the COVID-19 Pandemic: Cross-Sectional Survey Study.

BACKGROUND: Evidence suggests that the COVID-19 pandemic has generally increased levels of stress and depression among the public. However, the impact on college students in the United States has not been well-documented. OBJECTIVE: This paper surveys the mental health status and severity of depression and anxiety of college students in a large university system in the United States during the COVID-19 pandemic. METHODS: An online survey was conducted among undergraduate and graduate students recruited from Texas A&M University via email. The survey consisted of two standardized scales-the Patient Health Questionnaire-9 and the General Anxiety Disorder-7-for depression and anxiety, and additional multiple-choice and open-ended questions regarding stressors and coping mechanisms specific to COVID-19. RESULTS: Among the 2031 participants, 48.14% (n=960) showed a moderate-to-severe level of depression, 38.48% (n=775) showed a moderate-to-severe level of anxiety, and 18.04% (n=366) had suicidal thoughts. A majority of participants (n=1443, 71.26%) indicated that their stress/anxiety levels had increased during the pandemic. Less than half of the participants (n=882, 43.25%) indicated that they were able to cope adequately with the stress related to the current situation. CONCLUSIONS: The proportion of respondents showing depression, anxiety, and/or suicidal thoughts is alarming. Respondents reported academic-, health-, and lifestyle-related concerns caused by the pandemic. Given the unexpected length and severity of the outbreak, these concerns need to be further understood and addressed.

Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease.

One of the most profound advances in the last decade of biomedical research has been the development of human induced pluripotent stem cell (hiPSC) models for identification of disease mechanisms and drug discovery. Human iPSC technology has the capacity to revolutionize healthcare and the realization of personalized medicine, but differentiated tissues derived from stem cells come with major criticisms compared to native tissue, including variability in genetic backgrounds, a lack of functional maturity, and differences in epigenetic profiles. It is widely believed that increasing complexity will lead to improved clinical relevance, so methods are being developed that go from a single cell type to various levels of 2-D coculturing and 3-D organoids. As this inevitable trend continues, it will be essential to thoroughly understand the strengths and weaknesses of more complex models and to develop criteria for assessing biological relevance. We believe the payoff of robust, high-throughput, clinically meaningful human stem cell models could be the elimination of often inadequate animal models. To facilitate this transition, we will look at the challenges and strategies of complex model development through the lens of neurodegeneration to encapsulate where the disease-in-a-dish field currently is and where it needs to go to improve.

Irrational exuberance and neural crash warning signals during endogenous experimental market bubbles.

Groups of humans routinely misassign value to complex future events, especially in settings involving the exchange of resources. If properly structured, experimental markets can act as excellent probes of human group-level valuation mechanisms during pathological overvaluations--price bubbles. The connection between the behavioral and neural underpinnings of such phenomena has been absent, in part due to a lack of enabling technology. We used a multisubject functional MRI paradigm to measure neural activity in human subjects participating in experimental asset markets in which endogenous price bubbles formed and crashed. Although many ideas exist about how and why such bubbles may form and how to identify them, our experiment provided a window on the connection between neural responses and behavioral acts (buying and selling) that created the bubbles. We show that aggregate neural activity in the nucleus accumbens (NAcc) tracks the price bubble and that NAcc activity aggregated within a market predicts future price changes and crashes. Furthermore, the lowest-earning subjects express a stronger tendency to buy as a function of measured NAcc activity. Conversely, we report a signal in the anterior insular cortex in the highest earners that precedes the impending price peak, is associated with a higher propensity to sell in high earners, and that may represent a neural early warning signal in these subjects. Such markets could be a model system to understand neural and behavior mechanisms in other settings where emergent group-level activity exhibits mistaken belief or valuation.

Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System.

Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.

Charcot-Marie-Tooth Disease Type 4H Resulting from Compound Heterozygous Mutations in FGD4 from Nonconsanguineous Korean Families.

Charcot-Marie-Tooth disease type 4H (CMT4H) is an autosomal recessive demyelinating subtype of peripheral enuropathies caused by mutations in the FGD4 gene. Most CMT4H patients are in consanguineous Mediterranean families characterized by early onset and slow progression. We identified two CMT4H patients from a Korean CMT cohort, and performed a detailed genetic and clinical analysis in both cases. Both patients from nonconsanguineous families showed characteristic clinical manifestations of CMT4H including early onset, scoliosis, areflexia, and slow disease progression. Exome sequencing revealed novel compound heterozygous mutations in FGD4 as the underlying cause in both families (p.Arg468Gln and c.1512-2A>C in FC73, p.Met345Thr and c.2043+1G>A (p.Trp663Trpfs*30) in FC646). The missense mutations were located in highly conserved RhoGEF and PH domains which were predicted to be pathogenic in nature by in silico modeling. The CMT4H occurrence frequency was calculated to 0.7% in the Korean demyelinating CMT patients. This study is the first report of CMT4H in Korea. FGD4 assay could be considered as a means of molecular diagnosis for sporadic cases of demyelinating CMT with slow progression.

Workers' Acceptance of Digital Procedures: An Application of the Technology Acceptance Model.

OCCUPATIONAL APPLICATIONSThere are increasing numbers of organizations that are implementing digital procedures (e.g., standard operating procedures). These efforts are often assumed to be a positive development but can be quite costly-both in terms of money and training for a digital rollout. As a result, organizations and practitioners may find themselves at risk for failure when implementing digital procedures. The results of the current study suggest that if workers perceive digital procedures as useful and easy to use, this perception translates into positive attitudes, which subsequently result in fewer deviations. Since acceptance is relatively easy to assess, practitioners can benefit from using these assessments prior to a digital transition/roll-out to both compare competing hardware and software applications, and to initiate and continuously monitor the development of digital procedures. We consider this approach as advantageous to having management develop a system and fully deploying digital procedures without any consideration of worker acceptance.

Background: There is increasing prevalence of digital procedures being introduced in the process safety industries. Presumably, this increase is due to a desire to take advantage of the technology afforded to workers that otherwise is not inherent to traditional paper-based procedures. A critical question that has not been addressed, though, is to what extent do workers accept this new technology in a new digital procedure rollout? Furthermore, does acceptance lead to procedure-related behavior, such as procedure deviations?Purpose: We used the technology acceptance model (TAM), which includes two dimensions of technology acceptance­perceived usefulness (PU) and perceived ease of use (PEU)­as the focal antecedent constructs. We hypothesized that these constructs would predict more proximal attitudes toward procedures, which in turn predict procedure deviations.Method: We used path analyses to test six study hypotheses developed from the TAM. Data were collected from 16 workers at a large, international chemical corporation that worked in logistics. Specific measures obtained were from multi-item, Likert-scale measures of the TAM-PU and PEU dimensions, utility and compliance attitudes toward procedures, and procedure deviation frequency.Results: Four of the six study hypotheses were supported. TAM-PU and TAM-PEU both significantly predicted (positively) utility attitudes toward procedures (71% variance explained), whereas only TAM-PU significantly predicted (positively) compliance attitudes toward procedures (63% variance explained). In turn, only compliance attitudes significantly predicted (negatively) how frequently workers deviated from procedures (27% variance explained).Conclusions: These results suggest that workers were generally accepting of the digital procedures and that worker perceptions of perceived usefulness perceptions likely have an indirect effect on procedure deviation frequency. We see this study as a novel contribution to the process safety and procedures research domain. Limitations and future research directions will be discussed.

Immunocal® limits gliosis in mouse models of repetitive mild-moderate traumatic brain injury.

Successive traumatic brain injuries (TBIs) exacerbate neuroinflammation and oxidative stress. No therapeutics exist for populations at high risk of repetitive mild TBIs (rmTBIs). We explored the preventative therapeutic effects of Immunocal®, a cysteine-rich whey protein supplement and glutathione (GSH) precursor, following rmTBI and repetitive mild-moderate TBI (rmmTBI). Populations that suffer rmTBIs largely go undiagnosed and untreated; therefore, we first examined the potential therapeutic effect of Immunocal® long-term following rmTBI. Mice were treated with Immunocal® prior to, during, and following rmTBI induced by controlled cortical impact until analysis at 2 weeks, 2 months, and 6 months following the last rmTBI. Astrogliosis and microgliosis were measured in cortex at each time point and edema and macrophage infiltration by MRI were analyzed at 2 months post-rmTBI. Immunocal® significantly reduced astrogliosis at 2 weeks and 2 months post-rmTBI. Macrophage activation was observed at 2 months post-rmTBI but Immunocal® had no significant effect on this endpoint. We did not observe significant microgliosis or edema after rmTBI. The dosing regimen was repeated in mice subjected to rmmTBI; however, using this experimental paradigm, we examined the preventative therapeutic effects of Immunocal® at a much earlier timepoint because populations that suffer more severe rmmTBIs are more likely to receive acute diagnosis and treatment. Increases in astrogliosis, microgliosis, and serum neurofilament light (NfL), as well as reductions in the GSH:GSSG ratio, were observed 72 h post-rmmTBI. Immunocal® only significantly reduced microgliosis after rmmTBI. In summary, we report that astrogliosis persists for 2 months post-rmTBI and that inflammation, neuronal damage, and altered redox homeostasis present acutely following rmmTBI. Immunocal® significantly limited gliosis in these models; however, its neuroprotection was partially overwhelmed by repetitive injury. Treatments that modulate distinct aspects of TBI pathophysiology, used in combination with GSH precursors like Immunocal®, may show more protection in these repetitive TBI models.

The Effects of Drowsiness Detection Technology and Education on Nurses' Beliefs and Attitudes toward Drowsy Driving.

Occupational ApplicationsNurses' perceived health threat from driving drowsy along with their attitude toward an intervention can be targeted to improve nurses' intentions to avoid this dangerous behavior. The evidence presented in this paper suggests that educational interventions that raise awareness of the risks of drowsy driving and its consequences (e.g., fatalities or injuries), as well as peer stories about their experiences, may positively affect nurses' perceived health threat and attitudes toward drowsy driving interventions.

Background Drowsy driving is prevalent among night-shift nurses, yet there is a gap in understanding nurses' beliefs and attitudes that may affect their intention to avoid drowsy driving.Objectives The objectives of the study were twofold: 1) investigate how behavioral constructs such as beliefs and attitudes may affect nurses' intention to avoid drowsy driving; and 2) assess changes in such beliefs and attitudes during a study that evaluated the effectiveness of educational and technological interventions.Methods Three-hundred night-shift nurses were recruited from a large hospital in Texas to participate in a randomized controlled trial. Participants were randomly assigned to three groups: 1) control; 2) educational intervention; and 3) combined educational and technological intervention. The study utilized an integrated model drawing from the constructs of the Theory of Planned Behavior and the Health Belief Model to elicit attitudes, beliefs, and intentions to use in-vehicle drowsiness detection technologies. Each group was surveyed pre- intervention and at post-intervention around 3 months later to assess changes in beliefs and attitudes. Structural equation models and path analysis were used to analyze changes in beliefs.Results Seventy-nine participants completed the pre-intervention questionnaire, and 44 nurses completed the pre- and post-intervention surveys. Intention was predicted primarily by attitude and perceived health threat. Perceived health threat also mediated the relationship between behavioral intention and the influence of subjective norms as well as perceived behavioral control. Participants who received education about drowsy driving had positive changes in beliefs.Conclusions Nurses' perceived health threat from driving drowsy and their attitude toward our intervention were important motivators to avoid drowsy driving. Interventions aiming at raising awareness of the risks associated with drowsy driving may be effective at motivating nurses to avoid drowsy driving.

Conformational sampling of CMT-2D associated GlyRS mutations.

During protein synthesis, aminoacyl-tRNA synthetases covalently link amino acids with their cognate tRNAs. Amino acid mutations in glycyl-tRNA synthetase can disrupt protein synthesis and lead to a neurological disorder known as Charcot-Marie-Tooth disease type 2D (CMT-2D). Several studies employing diverse techniques have identified potential disease mechanisms at the molecular level. The majority of CMT-2D mutations in glycyl-tRNA are found within its dimer interface. However, no atomic structures bearing these mutations have been solved. Consequently, the specific disease-causing structural changes that occur in glycyl-tRNA synthetase have not been definitively established. Here we use molecular dynamics simulations to probe conformational changes in glycyl-tRNA synthetase caused by one mutation within the dimer interface: G240R. Our results show that the mutation alters the number of native interactions at the dimer interface and also leads to altered dynamics of two regions of glycyl-tRNA synthetase associated with tRNA binding. Additionally, we use our simulations to make predictions about the effects of other clinically reported CMT-2D mutations. Our results identify a region of the glycyl-tRNA synthetase structure that may be disrupted in a large number of CMT-2D mutations. Structural changes in this region may be a common molecular mechanism in glycyl-tRNA synthetase CMT-2D pathologies. Statement of significance: In this study, we use molecular dynamics simulations to elucidate structural conformations accessible to glycyl-tRNA synthetase (GlyRS), an enzyme that ligates cytosolic glycine with tRNA-Gly. This protein contains multiple flexible regions with dynamics that elude in vitro structural characterization. Our computational approach provides unparalleled atomistic details of structural changes in GlyRS that contribute to its role in protein synthesis. A number of mutations in GlyRS are associated with a peripheral nerve disorder, Charcot-Marie-Tooth disease type 2D (CMT-2D). Mutation-induced structural and dynamic changes in GlyRS have similarity that elude in vitro structural characterization. Our simulations provide insights into disease mechanisms for one such mutation: G240R. Additionally, we leverage our computational data to identify regions of GlyRS critical to its function and to predict the effects of other disease-associated mutations. These results open up new directions for research into the molecular characterization of GlyRS and into hypothesis-driven studies of CMT-2D disease mechanisms.

HDAC6 Inhibition Corrects Electrophysiological and Axonal Transport Deficits in a Human Stem Cell-Based Model of Charcot-Marie-Tooth Disease (Type 2D).

Charcot-Marie-Tooth disease type 2D (CMT2D), is a hereditary peripheral neuropathy caused by mutations in the gene encoding glycyl-tRNA synthetase (GARS1). Here, human induced pluripotent stem cell (hiPSC)-based models of CMT2D bearing mutations in GARS1 and their use for the identification of predictive biomarkers amenable to therapeutic efficacy screening is described. Cultures containing spinal cord motor neurons generated from this line exhibit network activity marked by significant deficiencies in spontaneous action potential firing and burst fire behavior. This result matches clinical data collected from a patient bearing a GARS1P724H mutation and is coupled with significant decreases in acetylated α-tubulin levels and mitochondrial movement within axons. Treatment with histone deacetylase 6 inhibitors, tubastatin A and CKD504, improves mitochondrial movement and α-tubulin acetylation in these cells. Furthermore, CKD504 treatment enhances population-level electrophysiological activity, highlighting its potential as an effective treatment for CMT2D.

High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing.

Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.

The Impact of Commutes, Work Schedules, and Sleep on Near-Crashes during Nurses' Post Shift-Work Commutes: A Naturalistic Driving Study.

OCCUPATIONAL APPLICATIONSDriving and survey data were collected from nurses following the night-shift and analyzed with logistic regression and frequency analysis. The analyses showed that prior near-crashes and drive length contributed significantly to near-crashes. The frequency analysis showed that most near-crashes occurred on major roadways, including principal arterials, major collectors, and interstates, within the first 15 minutes of the drive. These results highlight the urgent need for countermeasures to prevent drowsy driving incidents among night-shift nurses. Specifically, nurses and hospital systems should focus on countermeasures that encourage taking a break on the post work commute and those that can intervene during the drive. This may include the use of educational programs to teach nurses the importance of adequate rest or taking a break to sleep during their drive home, or technology that can recognize drowsiness and alert nurses of their drowsiness levels, prompting them to take a break.

TECHNICAL ABSTRACTBackground Night-shift nurses are susceptible to drowsy driving crashes due to their long working hours, disrupted circadian rhythm, and reduced sleep hours. However, the extent to which work, sleep, and on-road factors impact the nurses' commutes and the occurrence of near-crash events is not well documented.Purpose A longitudinal naturalistic driving study with night-shift nurses from a large hospital in the United States was conducted to measure these factors and analyze the occurrence and location of near-crashes during post-shift commutes.Methods An on-board data recorder was used to record acceleration, speed, and GPS coordinates continuously. Nurses also completed daily surveys on their sleep, work, and commute. Near-crashes were identified from the data based on acceleration thresholds. Data from a total of 853 drives from 22 nurses and corresponding surveys were analyzed using Poisson and negative binomial regressions for swerve and hard brake near-crash events, respectively.Results Swerve events were increased by the length of the drive (RR = 2.59, LL = 1.62, UL = 4.16), and the occurrence of hard brakes (RR = 1.69, LL = 1.45, UL = 1.99), while hard brake events were increased by the occurrence of swerves (RR = 1.55, LL = 1.28, UL = 1.88). The majority of near-crashes occurred on principal arterials (n = 293), minor arterials (n = 71), and interstates (n = 51).Conclusions The results demonstrate the high risk of near-crashes during post-shift commutes, which may present danger to nurses and other drivers, and highlight the need for countermeasures that address shift structures, sleep quality, and taking breaks.

Gender Differences in Fear and Risk Perception During the COVID-19 Pandemic.

The COVID-19 pandemic has led many people to suffer from emotional distress. Previous studies suggest that women process and express affective experiences, such as fear, with a greater intensity compared to men. We administered an online survey to a sample of participants in the United States that measures fear of COVID-19, perceptions about health and financial risks, and preventative measures taken. Despite the empirical fact that men are more likely to experience adverse health consequences from COVID-19, women report greater fear and more negative expectations about health-related consequences of COVID-19 than men. However, women are more optimistic than men regarding the financial consequences of the pandemic. Women also report more negative emotional experiences generally during the pandemic, particularly in situations where other people or the government take actions that make matters worse. Though women report taking more preventative measures than men in response to the pandemic, gender differences in behavior are reduced after controlling for fear. These results shed light on how differences in emotional experiences of the pandemic may inform policy interventions.