Hippocampal aggregation signatures of pathogenic UBQLN2 in amyotrophic lateral sclerosis and frontotemporal dementia.

Pathogenic variants in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis and/or frontotemporal dementia characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial amyotrophic lateral sclerosis and/or frontotemporal dementia cases not caused by UBQLN2 pathogenic variants, particularly C9orf72-linked cases. This makes the mechanistic role of mutant ubiquilin 2 protein and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 44 genotypically diverse amyotrophic lateral sclerosis cases with or without frontotemporal dementia, including eight cases with UBQLN2 variants (resulting in p.S222G, p.P497H, p.P506S, p.T487I (two cases), and p.P497L (three cases)). Using multiplexed (5-label) fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43, dipeptide repeat aggregates, and p62, in the hippocampus of controls (n = 6), or amyotrophic lateral sclerosis with or without frontotemporal dementia in sporadic (n = 20), unknown familial (n = 3), SOD1-linked (n = 1), FUS-linked (n = 1), C9orf72-linked (n = 5), and UBQLN2-linked (n = 8) cases. We differentiate between i) ubiquilin 2 aggregation together with phosphorylated TDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation promoted by UBQLN2 pathogenic variants that cause amyotrophic lateral sclerosis/and frontotemporal dementia. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in amyotrophic lateral sclerosis with or without frontotemporal dementia, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.

Whole-Cell MALDI-ToF MS Coupled with Untargeted Metabolomics Facilitates Investigations of Microbial Chemical Interactions.

The emergence of drug-resistant pathogens necessitates the development of new countermeasures. In this regard, the introduction of probiotics to directly attack or competitively exclude pathogens presents a useful strategy. Application of this approach requires an understanding of how a probiotic and its target pathogen interact. A key means of probiotic-pathogen interaction involves the production of small molecules called natural products (NPs). Here, we report the use of whole-cell matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry to characterize NP production by candidate probiotics (mouse airway microbiome isolates) when co-cultured with the respiratory pathogen Burkholderia. We found that a Bacillus velezensis strain inhibits growth of and elicits NP production by Burkholderia thailandensis. Dereplication of known NPs detected in the metabolome of this B. velezensis strain suggests that a previously unannotated bioactive compound is involved. Thus, we present the use of whole-cell MALDI as a broadly applicable method for screening the NP composition of microbial co-cultures; this can be combined with other -omics methods to characterize probiotic-pathogen and other microbe-microbe interactions.

Identity-by-descent analysis of CMTX3 links three families through a common founder.

A large 78 kb insertion from chromosome 8q24.3 into Xq27.1 was identified as the cause of CMTX3 in three families of European descent from Australia (CMT193, CMT180) and New Zealand/United Kingdom (CMT623). Using the relatedness tool XIBD to perform genome-wide identity-by-descent (IBD) analysis on 16 affected individuals from the three families demonstrated they all share the CMTX3 disease locus identical-by-descent, confirming the mutation arose in a common ancestor. Relationship estimation from IBD segment data has genetically linked all three families through 6th and 7th degree relatives.

RNA sequencing of peripheral blood in amyotrophic lateral sclerosis reveals distinct molecular subtypes: Considerations for biomarker discovery.

AIM: Amyotrophic lateral sclerosis (ALS) is a heterogeneous neurodegenerative disease with limited therapeutic options. A key factor limiting the development of effective therapeutics is the lack of disease biomarkers. We sought to assess whether biomarkers for diagnosis, prognosis or cohort stratification could be identified by RNA sequencing (RNA-seq) of ALS patient peripheral blood. METHODS: Whole blood RNA-seq data were generated for 96 Australian sporadic ALS (sALS) cases and 48 healthy controls (NCBI GEO accession GSE234297). Differences in sALS-control gene expression, transcript usage and predicted leukocyte proportions were assessed, with pathway analysis used to predict the activity state of biological processes. Weighted Gene Co-expression Network Analysis (WGCNA) and machine learning algorithms were applied to search for diagnostic and prognostic gene expression patterns. Unsupervised clustering analysis was employed to determine whether sALS patient subgroups could be detected. RESULTS: Two hundred and forty-five differentially expressed genes were identified in sALS patients relative to controls, with enrichment of immune, metabolic and stress-related pathways. sALS patients also demonstrated switches in transcript usage across a small set of genes. We established a classification model that distinguished sALS patients from controls with an accuracy of 78% (sensitivity: 79%, specificity: 75%) using the expression of 20 genes. Clustering analysis identified four patient subgroups with gene expression signatures and immune cell proportions reflective of distinct peripheral effects. CONCLUSIONS: Our findings suggest that peripheral blood RNA-seq can identify diagnostic biomarkers and distinguish molecular subtypes of sALS patients however, its prognostic value requires further investigation.

Disproportionate impact of abortion restriction: Implications for emergency department clinicians.

Individuals experiencing intimate partner violence (IPV) and/or human trafficking (HT) are at increased risk of severe health consequences as a result of legislation criminalizing and/or restricting abortion, which is expected to increase as a result of the Supreme Court decision Dobbs v. Jackson. These risks are further stratified by race, socioeconomics, and other marginalizing demographic attributes. IPV and HT introduce barriers to maintaining physical and mental health, due to control of access to transportation and funds by the abuser, fear of retribution for seeking healthcare, and other barriers. Individuals experiencing IPV or HT often lack reproductive autonomy, as a result of facing reproductive coercion at the hands of their abusers. Following the Dobbs decision, these vulnerable patient populations will face further limitations on their reproductive autonomy and increased obstacles to obtaining an abortion if they medically need or desire one. This will likely result in more patients presenting to the emergency department due to complications from unsafe or unsupervised self-managed abortions, as well as patients being reluctant to report having obtained an unlawful abortion due to fear of legal consequences. This is particularly relevant to individuals experiencing IPV and HT, as they may be more likely to use these methods for obtaining an abortion due to numerous barriers. Emergency medicine clinicians are vital in providing care to these patients, as they frequently present to emergency departments. A multi-pronged approach to better support these patients is essential, involving an increased index of suspicion for IPV, HT or the complications of unsupervised abortion, improved organizational structures, specialized training for staff, improved screening methods, reflection on implicit bias, and recommendations for mindful documentation and legal considerations.

Scaling a Behavioral Health Home Delivery Model to Special Populations.

The present project utilized a Learning Collaborative (LC) to disseminate the Behavioral Health Home Plus (BHHP) physical-behavioral health integration model to providers serving two behavioral health populations at risk for adverse health conditions: youth psychiatric residential treatment facilities (five sites) and adult opioid treatment providers (seven sites). Following the positive results of a randomized controlled trial utilizing an LC to implement two behavioral health home models in community mental health provider organizations serving adults with serious mental illness, Community Care Behavioral Health Organization facilitated integration of the models to scale health and wellness supports to additional behavioral health care delivery settings. This paper presents provider results focused on BHHP implementation training, LC implementation, physical health and wellness promotion within sites, and BHHP model sustainment plans. Provider self-reported data indicate that the LC approach is a successful tool for integrating and sustaining BHHP model components in routine care.

New candidates for regulated gene integrity revealed through precise mapping of integrative genetic elements.

Integrative genetic elements (IGEs) are mobile multigene DNA units that integrate into and excise from host bacterial genomes. Each IGE usually targets a specific site within a conserved host gene, integrating in a manner that preserves target gene function. However, a small number of bacterial genes are known to be inactivated upon IGE integration and reactivated upon excision, regulating phenotypes of virulence, mutation rate, and terminal differentiation in multicellular bacteria. The list of regulated gene integrity (RGI) cases has been slow-growing because IGEs have been challenging to precisely and comprehensively locate in genomes. We present software (TIGER) that maps IGEs with unprecedented precision and without attB site bias. TIGER uses a comparative genomic, ping-pong BLAST approach, based on the principle that the IGE integration module (i.e. its int-attP region) is cohesive. The resultant IGEs from 2168 genomes, along with integrase phylogenetic analysis and gene inactivation tests, revealed 19 new cases of genes whose integrity is regulated by IGEs (including dut, eccCa1, gntT, hrpB, merA, ompN, prkA, tqsA, traG, yifB, yfaT and ynfE), as well as recovering previously known cases (in sigK, spsM, comK, mlrA and hlb genes). It also recovered known clades of site-promiscuous integrases and identified possible new ones.

Staff Perceptions of Barriers and Facilitators to Implementation of Behavioral Health Homes at Community Mental Health Provider Settings.

Individuals living with a serious mental illness are disproportionately affected by preventable and/or manageable chronic conditions. Integrated care and support for behavioral and physical health within community mental health provider (CMHP) settings, also known as behavioral health homes (BHH), can lead to improvements in care and cost outcomes. This study explored staff perceptions of barriers and facilitators to BHH implementation. We conducted semi-structured interviews with CMHP staff at baseline, 1, and 2 years after the start of implementation. We analyzed interviews to identify major themes. We conducted 65 total interviews with 30 unique staff members. Common barriers included staff turnover, hesitation to change care processes, and acute service user needs. Facilitators included agency-wide culture change, intervention champions, and integration of intervention processes into daily workflows. Despite common barriers, CMHP staff identified several elements related to successful BHH implementation, including the CMHP-wide cultural shift to comprehensively address health/wellness that benefitted service users and staff alike.

Health information diffusion on Twitter: The content and design of WHO tweets matter.

BACKGROUND: Micro-blogging services empower health institutions to quickly disseminate health information to many users. By analysing user data, infodemiology (i.e. improving public health using user contributed health related content) can be measured in terms of information diffusion. OBJECTIVES: Tweets by the WHO were examined in order to identify tweet attributes that lead to a high information diffusion rate using Twitter data collected between November 2019 and January 2020. METHODS: One thousand hundred and seventy-seven tweets were collected using Python's Tweepy library. Afterwards, k-means clustering and manual coding were used to classify tweets by theme, sentiment, length and count of emojis, pictures, videos and links. Resulting groups with different characteristics were analysed for significant differences using Mann-Whitney U- and Kruskal-Wallis H-tests. RESULTS: The topic of the tweet, the included links, emojis and (one) picture as well as the tweet length significantly affected the tweets' diffusion, whereas sentiment and videos did not show any significant influence on the diffusion of tweets. DISCUSSION: The findings of this study give insights on why specific health topics might generate less attention and do not showcase sufficient information diffusion. CONCLUSION: The subject and appearance of a tweet influence its diffusion, making the design equally essential to the preparation of its content.

LGBT+ Older Adults in Rural South Central Appalachia: Perceptions of Current and Future Formal Service Needs.

Older sexual and gender minority adults living in rural areas of the U.S. face challenges in accessing welcoming and inclusive formal health, mental health, social, and long-term care services. The intersection of the local rural sociocultural context with lesbian, gay, bisexual, and transgender (LGBT+) identities and aging presents both challenges and opportunities for improving formal services for LGBT+ older adults. Little is known about how the rural south central Appalachian context intersects with the identities of LGBT+ older adults with regard to accessing formal services. This paper presents the results of a qualitative study examining experiences, concerns, and recommendations regarding formal services among 11 LGBT+ older adults residing in rural south central Appalachia. Several of the participants described experiencing discrimination and/or marginalization while accessing formal services. A number of participants were fearful about the lack of LGBT+-inclusive services and expressed that they would consider leaving the area if their own or their partner's health declined or if they or their partner required long-term care services, particularly residential care. Many participants expressed the need for local provider education and training about the needs of LGBT+ older adults. Implications for practice, policy, and research are discussed in this paper.

Identifying rare, medically relevant variation via population-based genomic screening in Alabama: opportunities and pitfalls.

PURPOSE: To evaluate the effectiveness and specificity of population-based genomic screening in Alabama. METHODS: The Alabama Genomic Health Initiative (AGHI) has enrolled and evaluated 5369 participants for the presence of pathogenic/likely pathogenic (P/LP) variants using the Illumina Global Screening Array (GSA), with validation of all P/LP variants via Sanger sequencing in a CLIA-certified laboratory before return of results. RESULTS: Among 131 variants identified by the GSA that were evaluated by Sanger sequencing, 67 (51%) were false positives (FP). For 39 of the 67 FP variants, a benign/likely benign variant was present at or near the targeted P/LP variant. Variants detected within African American individuals were significantly enriched for FPs, likely due to a higher rate of nontargeted alternative alleles close to array-targeted P/LP variants. CONCLUSION: In AGHI, we have implemented an array-based process to screen for highly penetrant genetic variants in actionable disease genes. We demonstrate the need for clinical validation of array-identified variants in direct-to-consumer or population testing, especially for diverse populations.

Splicing factor proline and glutamine rich intron retention, reduced expression and aggregate formation are pathological features of amyotrophic lateral sclerosis.

AIM: Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts. METHODS: We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. RESULTS: At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. CONCLUSIONS: Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.

CYLD is a causative gene for frontotemporal dementia - amyotrophic lateral sclerosis.

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

Evidence for polygenic and oligogenic basis of Australian sporadic amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with phenotypic and genetic heterogeneity. Approximately 10% of cases are familial, while remaining cases are classified as sporadic. To date, >30 genes and several hundred genetic variants have been implicated in ALS. METHODS: Seven hundred and fifty-seven sporadic ALS cases were recruited from Australian neurology clinics. Detailed clinical data and whole genome sequencing (WGS) data were available from 567 and 616 cases, respectively, of which 426 cases had both datasets available. As part of a comprehensive genetic analysis, 853 genetic variants previously reported as ALS-linked mutations or disease-associated alleles were interrogated in sporadic ALS WGS data. Statistical analyses were performed to identify correlation between clinical variables, and between phenotype and the number of ALS-implicated variants carried by an individual. Relatedness between individuals carrying identical variants was assessed using identity-by-descent analysis. RESULTS: Forty-three ALS-implicated variants from 18 genes, including C9orf72, ATXN2, TARDBP, SOD1, SQSTM1 and SETX, were identified in Australian sporadic ALS cases. One-third of cases carried at least one variant and 6.82% carried two or more variants, implicating a potential oligogenic or polygenic basis of ALS. Relatedness was detected between two sporadic ALS cases carrying a SOD1 p.I114T mutation, and among three cases carrying a SQSTM1 p.K238E mutation. Oligogenic/polygenic sporadic ALS cases showed earlier age of onset than those with no reported variant. CONCLUSION: We confirm phenotypic associations among ALS cases, and highlight the contribution of genetic variation to all forms of ALS.

Examining the Effects of Music-Based Interventions on Pain and Anxiety in Hospitalized Children: An Integrative Review.

PROBLEM: Children often experience pain and anxiety during a hospital stay. Effective pain and anxiety management plays a crucial role in healing. However, recent literature has highlighted multiple barriers to managing pain and anxiety in children, such as parent and provider fears of the adverse effects of pain and anxiety medications. ELIGIBILITY CRITERIA: A database search was conducted for articles published between 2009 and 2019 to evaluate the impact of nurse-led, music-based interventions as an adjunct method of pain and anxiety management in hospitalized children. Articles were included if study subjects were ages 0-21 years old, the study used live or recorded music as an intervention, and occurred in an inpatient setting. SAMPLE: A total of seven randomized control trials and one quasi-experimental study were included for analysis. RESULTS: There is consistent and significant evidence that music can reduce anxiety in hospitalized children before and during procedures. Results with respect to pain and vital signs, often viewed as the physiologic analogs to pain, were mixed. CONCLUSIONS: Music-based interventions are safe for hospitalized children. Several studies highlighted the importance of patient preference in selecting music for children. A heavy reliance on pre-recorded audio, delivered via headphones illustrates the feasibility and cost-effectiveness of music-based interventions. IMPLICATIONS: Nurse-led, music-based interventions have been shown to be an affordable, safe, effective, and feasible alternative for managing anxiety in hospitalized children. Music should be considered as an adjunct therapy to traditional anxiety treatment. Further research is needed to determine the effects of music on pain.

Genetic and immunopathological analysis of CHCHD10 in Australian amyotrophic lateral sclerosis and frontotemporal dementia and transgenic TDP-43 mice.

OBJECTIVE: Since the first report of CHCHD10 gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia. METHODS: Whole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10. CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology. RESULTS: No causal, novel or disease-associated variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease. CONCLUSIONS: Genetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.

Quality Improvement in the Emergency Department: A Project to Reduce Door-to-Electrocardiography Times for Patients Presenting With Chest Pain.

INTRODUCTION: The American Heart Association/American College of Cardiology guidelines recommend obtaining electrocardiography for patients who present to the emergency department with chest pain in less than 10 minutes of arrival. Reducing door-to-electrocardiography time is an important step in adhering to the recommended door-to-balloon times (≤ 90 minutes) for patients who present with ST-segment elevation myocardial infarction. METHODS: Based on lean sigma principles, a protocol was implemented in an adult emergency department that included deferring nurse triage for patients with complaints of chest pain, chest tightness, and chest pressure and providing them with a red heart symbol as an indicator for clinical technicians to prioritize their electrocardiography order. Pre- and postintervention data were collected over a 12-month period. RESULTS: Before the intervention, the mean door-to-electrocardiography time was 17 minutes for patients with chest pain (n = 893). After the intervention, the mean door-to-electrocardiography time for patients with chest pain significantly decreased to 7 minutes (n = 1,057) (t = 10.47, P ≤ 0.001). Initially, the percentage of compliance with door-to-electrocardiography standard of 10 minutes was 31% and improved to 83% after implementation of the new protocol. DISCUSSION: Implementation of the optimized door-to-electrocardiography protocol decreased the time for obtaining diagnostics and improved compliance with the American Heart Association/American College of Cardiology guidelines, potentially decreasing door-to-balloon times for patients who presented with ST-segment elevation myocardial infarction.

Social Support and Social Networks among LGBT Older Adults in Rural Southern Appalachia.

While research has begun to examine social networks and social support among LGBT older adults living in rural contexts, no research to date has examined these issues within the unique context of rural southern Appalachia. Thus, the purpose of this qualitative study was to extend this emerging area of research by exploring the perspectives of LGBT older adults on their social networks and social support while living in rural southern Appalachia. In this study, 11 LGBT-identifying older adults were interviewed regarding their social networks and social support within the cultural context of rural Southern Appalachia. Participants generally described having rich informal social support networks that seemed to buffer and mitigate the deleterious effects of the wider culture of homophobia and transphobia. These networks, while varying from person to person, included families of choice (spouse / partner, close friends), neighbors, pets, biological family / families of origin, religious and spiritual communities, women's or men's social groups, and current or former coworkers. While six of the participants voiced that their support system was adequate for their needs, there were reports of mixed, tenuous, or insufficient support systems for five participants. After reviewing main findings, implications for research, practice, and policy are discussed.

Expression of ALS/FTD-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations.

Caffeine and energy drink use by combat arms soldiers in Afghanistan as a countermeasure for sleep loss and high operational demands.

OBJECTIVES: Combat deployments are characterized by high operational demands with limited opportunities for sleep leading to fatigue and degraded cognitive and operational performance. Caffeine in moderate doses is recognized as an effective intervention for physical and cognitive decrements associated with sleep loss. METHODS: This report is based on data collected by two separate, independently conducted surveys administered in Afghanistan in 2011-2012. It assessed caffeine use and sleep disruption among U.S. Army combat soldiers (J-MHAT 8; n = 518) and among deployed soldiers with different military assignments (USARIEM Deployment Survey; n = 260). RESULTS: Daily caffeine intake assessed in the J-MHAT 8 survey averaged 404 ± 18 mg. In the USARIEM Deployment Survey, intake was 303 ± 29 mg and was significantly higher among combat arms soldiers (483 ± 100 mg) compared to combat service support personnel (235 ± 23 mg). In both surveys, over 55% of total caffeine intake was from energy drinks. Additional sources of caffeine included coffee, tea, sodas, gum, candy, and over-the-counter medications. Higher caffeine intake was not associated with ability to fall asleep at night or wake-up in the morning (J-MHAT 8 survey). Higher caffeine consumption was associated with disrupted sleep from high operational tempo and nighttime duties of combat operations. DISCUSSION: Overall caffeine consumption and energy drink use in Afghanistan was greater than among non-deployed soldiers and civilians. Caffeine was frequently used as a countermeasure during night operations to offset adverse effects of sleep loss on physical and cognitive function, consistent with current Department of the Army recommendations.