IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.

Cross-Cutting Lessons Learned During the COVID-19 Pandemic-the Walter Reed Army Institute of Research Experience.

INTRODUCTION: At the start of the coronavirus disease 2019 (COVID-19) pandemic, Walter Reed Army Institute of Research (WRAIR) mobilized to rapidly conduct medical research to detect, prevent, and treat the disease in order to minimize the impact of the pandemic on the health and readiness of U.S. Forces. WRAIR's major efforts included the development of the Department of Defense (DoD) COVID-19 vaccine candidate, researching novel drug therapies and monoclonal antibodies, refining and scaling-up diagnostic capabilities, evaluating the impact of viral diversity, assessing the behavioral health of Soldiers, supporting U.S. DoD operational forces overseas, and providing myriad assistance to allied nations. WRAIR personnel have also filled key roles within the whole of government response to the pandemic. WRAIR had to overcome major pandemic-related operational challenges in order to quickly execute a multimillion-dollar portfolio of COVID-19 research. Consequently, the organization learned lessons that could benefit other leaders of medical research organizations preparing for the next pandemic. MATERIALS AND METHODS: We identified lessons learned using a qualitative thematic analysis of 76 observation/recommendation pairs from across the organization. These lessons learned were organized under the Army's four pillars of readiness (staffing, training, equipping, and leadership development). To this framework, we added organizing and leading to best capture our experiences within the context of pandemic response. RESULTS: The major lessons learned for organizing were: (1) the pandemic created a need to rapidly pivot to new scientific priorities; (2) necessary health and safety precautions disrupted the flow of normal science and put programs at risk of missing milestones; (3) relationships with partners and allies facilitated medical diplomacy and advancement of U.S. national military and economic goals; and (4) a successful response required interoperability within and across multiple organizations. For equipping: (1) existing infrastructure lacked sufficient capacity and technical capability to allow immediate countermeasure development; (2) critical supply chains were strained; and (3) critical information system function and capacity were suddenly insufficient under maximum remote work. For staffing and training: (1) successful telework required rapid shifts in management, engagement, and accountability methods; and (2) organizational policies and processes had to adapt quickly to support remote staffing. For leading and leadership development (1) engaged, hopeful, and empathetic leadership made a difference; and (2) the workforce benefitted from concerted leadership communication that created a shared understanding of shifting priorities as well as new processes and procedures. CONCLUSIONS: An effective pandemic response requires comprehensive institutional preparedness that facilitates flexibility and surge capacity. The single most important action leaders of medical research organizations can take to prepare for the next pandemic is to develop a quick-reaction force that would activate under prespecified criteria to manage reprioritization of all science and support activities to address pandemic response priorities at the velocity of relevance.

Commercial Speech and Unhealthy Food Products: Conceptual Foundations.

This article provides a critical and philosophical assessment of arguments invoked for and against the constitutional protection of commercial expression and the regulation of commercial speech with a focus on the commercialization of unhealthy food products.

Commercial Speech and the Prohibition of Tobacco Advertising: The Colombian Constitutional Court Approach.

This article argues that the decision by the Columbian high court to totally ban the advertising and promotion of tobacco products is sound and could indeed be applied to other types of harmful products.

INTRODUCTION Commercial Speech and the Commercial Determinants of Health.

This article introduces a symposium that aims to identify and critically assess the legal strategies of the tobacco, alcohol, and food and beverage industries which rest on freedom of expression arguments.

Order effects in PTSD network analysis: important implications for diagnostic conceptualization, treatment refinement, and research.

BACKGROUND: For decades confirmatory factor analysis (CFA) has been the preeminent method to study the underlying structure of posttraumatic stress disorder (PTSD); however, methodological limitations of CFA have led to the emergence of other analytic approaches. In particular, network analysis has become a gold standard to investigate the structure and relationships between PTSD symptoms. A key methodological limitation, however, which has significant clinical implications, is the lack of data on the potential impact of item order effects on the conclusions reached through network analyses. METHODS: The current study, involving a large sample (N = 5055) of active duty army soldiers following deployment to Iraq, assessed the vulnerability of network analyses and prevalence rate to item order effects. This was done by comparing symptom networks of the DSM-IV PTSD checklist items to these same items distributed in random order. Half of the participants rated their symptoms on traditionally ordered items and half the participants rated the same items, but in random order and interspersed between items from other validated scales. Differences in prevalence rate and network composition were examined. RESULTS: The prevalence rate differed between the ordered and random item samples. Network analyses using the ordered survey closely replicated the conclusions reached in the existing network analyses literature. However, in the random item survey, network composition differed considerably. CONCLUSION: Order effects appear to have a significant impact on conclusions reached from PTSD network analysis. Prevalence rates were also impacted by order effects. These findings have important diagnostic and clinical treatment implications.

A human rights-based approach to non-communicable diseases: mandating front-of-package warning labels.

Across the globe, the consumption of energy-dense and nutrient-poor foods and beverages has escalated rates of diet-related non-communicable diseases (NCDs), driven by deceptive marketing tactics from the food and beverage industry. The international community has increasingly recognized the need to provide consumers with accurate health information on food and beverage products as part of their right to health. In July 2020, the U.N. Special Rapporteur on the right to health released a powerful Statement calling for the adoption of front-of-package warning labeling to tackle NCDs. Just a few weeks after the Statement's release, the Pan American Health Organization published a report highlighting the relevance of front-of-package labeling as a policy tool for the prevention of NCDs in the Americas, demonstrating further support to this regulatory intervention.In this piece, we explain why front-of-package warning labeling should be part of a comprehensive strategy to promote healthier lives, delving into the human-rights aspects of front-of-package labels. In particular, we explore the role the food and beverage industry play in increasing the consumption of unhealthy foods and beverages, and the relevance of scientific evidence free from conflicts of interest to adequately protect the right to health and health-related rights.

E3 ubiquitin ligase Wwp1 regulates ciliary dynamics of the Hedgehog receptor Smoothened.

The Hedgehog pathway, critical to vertebrate development, is organized in primary cilia. Activation of signaling causes the Hedgehog receptor Ptch1 to exit cilia, allowing a second receptor, Smo, to accumulate in cilia and activate the downstream steps of the pathway. Mechanisms regulating the dynamics of these receptors are unknown, but the ubiquitination of Smo regulates its interaction with the intraflagellar transport system to control ciliary levels. A focused screen of ubiquitin-related genes identified nine required for maintaining low ciliary Smo at the basal state. These included cytoplasmic E3s (Arih2, Mgrn1, and Maea), a ciliary localized E3 (Wwp1), a ciliary localized E2 (Ube2l3), a deubiquitinase (Bap1), and three adaptors (Kctd5, Skp1a, and Skp2). The ciliary E3, Wwp1, binds Ptch1 and localizes to cilia at the basal state. Activation of signaling removes both Ptch1 and Wwp1 from cilia, thus providing an elegant mechanism for Ptch1 to regulate ciliary Smo levels.

Psychological distress across the deployment cycle: exploratory growth mixture model.

BACKGROUND: Prior research has identified behavioural health outcomes as key sequelae to combat deployment. However, relatively little is known about differential patterns of change in depression or generalised anxiety linked to deployment to a combat zone. In this paper, we add to the existing trajectory literature and examine key predictive factors of behavioural health risk. AIMS: The primary aim is to leverage growth mixture modelling to ascertain trajectories of psychological distress, operationalised as a coherent construct combining depression and generalised anxiety, and to identify factors that differentiate adaptive and maladaptive patterns of change. METHOD: Data were collected from a brigade combat team prior to a combat deployment to Afghanistan, during deployment, at immediate re-integration and approximately 2-3 months thereafter. The main outcome was measured using the Patient Health Questionnaire Anxiety and Depression Scale (PHQ-ADS). RESULTS: Three latent trajectories were identified: a low-stable trajectory, a declining trajectory and a rising trajectory. Most individuals aligned with the low-stable trajectory. A conditional model using covariates measured during deployment showed that the low-stable trajectory differed consistently from the remaining trajectories on self-reported loneliness and non-combat deployment stressors. CONCLUSIONS: The examination of differential patterns of adaptation, to identify individuals at higher risk, is critical for the efficient targeting of resources. Our findings further indicate that loneliness may be a useful leverage point for clinical and organisational intervention.

Military suicide prevention: The importance of leadership behaviors as an upstream suicide prevention target.

OBJECTIVE: To evaluate the longitudinal relationships between unit cohesion, Army leader behaviors, and subordinate suicidal/death ideation. Recent cross-sectional research indicates that subordinates who perceive that their leaders instill a sense of purpose regarding military service demonstrate less frequent suicidal ideation. METHOD: Five hundred fifty-nine soldiers completed self-report measures of perceptions of leadership behaviors, unit cohesion, and suicidal/death ideation during deployment as well as one and three months following deployment. Latent change score modeling was conducted to evaluate the course and direction of study variables as well as the relationship between them. RESULTS: Although lower levels of suicidal/death ideation were related to leader-provided purpose, leader-provided meaning, and unit cohesion at baseline, only leader-provided purpose and unit cohesion prospectively predicted changes in suicidal/death ideation. CONCLUSIONS: Consistent with the goal of military leadership to augment effective clinical interventions that reduce suicide risk, prevention programs that reach a broader population of personnel should be considered. Enhanced leadership training may be an important primary prevention tool to reduce suicide risk that warrants further research.

Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations.

The programmable sequence specificity of CRISPR has found uses in gene editing and diagnostics. This manuscript describes an additional application of CRISPR through a family of novel DNA enrichment technologies. CAMP (CRISPR Associated Multiplexed PCR) and cCAMP (chimeric CRISPR Associated Multiplexed PCR) utilize the sequence specificity of the Cas9/sgRNA complex to target loci for the ligation of a universal adapter that is used for subsequent amplification. cTRACE (chimeric Targeting Rare Alleles with CRISPR-based Enrichment) also applies this method to use Cas9/sgRNA to target loci for the addition of universal adapters, however it has an additional selection for specific mutations through the use of an allele-specific primer. These three methods can produce multiplex PCR that significantly reduces the optimization required for every target. The methods are also not specific to any downstream analytical platform. We additionally will present a mutation specific enrichment technology that is non-amplification based and leaves the DNA in its native state: TRACE (Targeting Rare Alleles with CRISPR-based Enrichment). TRACE utilizes the Cas9/sgRNA complex to sterically protect the ends of targeted sequences from exonuclease activity which digests both the normal variant as well as any off-target sequences.

Sleep disturbance mediates the association of adverse childhood experiences with mental health symptoms and functional impairment in US soldiers.

Adverse childhood experiences (ACEs) can have long-term impacts on a person's mental health, which extend into adulthood. There is a high prevalence of ACEs among service members. Further, service members also report frequently experiencing disrupted sleep. We hypothesized that disrupted sleep may serve a mechanistic function connecting ACEs to functional impairment and poorer mental health. In a cross-sectional sample (n = 759), we found evidence for an indirect effect of ACEs on mental health outcomes through disrupted sleep. In a different sample using two time-points (n = 410), we found evidence for an indirect effect of ACEs on changes in mental health outcomes and functional impairment during a reset period, through changes in disrupted sleep during the same period. Implications, limitations and future research directions are discussed.

Assessing the Well-being of Sexual Minority Soldiers at a Military Academic Institution.

INTRODUCTION: The goal of the present study was to characterize behavioral health rates, behavioral health care utilization, loneliness, and perceived prejudice and support among sexual minority soldiers. MATERIALS AND METHODS: Cross-sectional survey data were obtained from 640 active-duty U.S. soldiers enrolled in an academic training institute who provided information on their sexual orientation. Survey topics included demographics, behavioral health, behavioral health care utilization, and mitigating factors (eg, perceived prejudice, perceived support, and loneliness). Chi-square analyses were utilized to determine any differences between groups for behavioral health rates and behavioral health care utilization. Robust regression was used for analysis of self-reported loneliness. RESULTS: A higher proportion of lesbian, gay, bisexual (LGB) soldiers than heterosexual soldiers screened positive for anxiety, post-traumatic stress disorder, and suicidality. No between-group differences in behavioral health care utilization were found; however, a higher proportion of LGB soldiers sought help from military family life counselors. No between-group differences for loneliness were found. Finally, perceived prejudice was higher for LGB soldiers and perceived support was lower. CONCLUSION: Organizational barriers, such as perceived prejudice and lack of support, appear to still exist for sexual minority soldiers. Increasing organizational support and implementing training and education for health care providers in order to better support the LGB soldier community may mitigate these barriers.

Leader provided purpose: Military leadership behavior and its association with suicidal ideation.

Suicide in U.S. Army Soldiers is of major concern as it is estimated that over 100 Soldiers die by suicide each year. Examining risk and protective factors is essential to develop both an understanding of Soldier suicide as well as inform systemic interventions to reduce suicide. One potential systemic approach is to embed preventive mechanisms within the structure of the military rather than the typical administration of primary intervention through mandatory training. To examine potential mechanisms of leader-based interventions, several leadership behaviors were assessed in a cross-sectional sample of n = 1,096 active duty Soldiers. Soldiers completed self-report measures of interpersonal predictors of suicide, suicidal ideation (SI), leadership behaviors, and unit cohesion. Logistic regression was used to identify leadership behaviors related to SI. Only the leader behavior attempting to foster a sense of purpose predicted SI. Leader provided purpose (LPP) was then entered into indirect effect analyses to evaluate the mechanisms of this relationship. Analyses revealed that LPP predicted SI through unit cohesion, thwarted belongingness, and perceived burdensomeness. Results demonstrate that specific aspects of military leadership such as fostering Soldier purpose may enhance resilience and reduce risk for SI.

Allelic Diversity in the Serum Amyloid A2 Gene and Amyloid A Amyloidosis in a Breeding Colony of Zebra Finches (Taeniopygia guttata).

A high incidence of amyloid A (AA) amyloidosis was observed in the research breeding colony of zebra finches at our institution. Some birds with hepatic AA amyloidosis were asymptomatic for comorbid conditions frequently associated with the development of AA amyloidosis, whereas other birds with comorbid conditions failed to develop AA amyloidosis, suggesting a potential genetic component to the disease. Sequencing the SAA2 gene from 20 birds yielded 18 distinct sequences that coded for 5 isoforms of the protein. Most of the amino acid substitutions are unlikely to affect the protein's structure or function, but 2 changes-R52L and V84M-were predicted to be disruptive. In particular, R52 is highly conserved across vertebrates, with only arginine or lysine found at this position in reported sequences to date. The atypical R52L substitution occurred in 2 otherwise healthy birds with hepatic AA amyloidosis, supporting the idea that this change is pathogenic.

U.S. Soldiers and Foreign Language School: Stressors and Health.

INTRODUCTION: While previous studies have examined the stress of the military training environment, studies have not systematically examined the stress associated with attending the Defense Language Institute Foreign Language Center (DLIFLC). Service members assigned to DLIFLC endure intense academic pressure to succeed while meeting military requirements. Thus, not only are traditional academic stressors likely to be of concern but there are other academic and military-related stressors that have to managed by students. The goal of the present study was to characterize the stressors facing military students, document their mental health status and well-being, and identify mitigating factors such as coping, social support, time management, and the classroom environment. MATERIALS AND METHODS: Data were obtained from a cross-sectional survey administered in March of 2016. Study participants were 759 active-duty U.S. soldiers enrolled in DLIFLC, with a consent rate of 87.7%. Surveys were administered in classroom settings. Survey topics included demographics, student experience (e.g., classroom hours, stressors), mental health (e.g., depression, anxiety, hazardous alcohol use) and burnout, and mitigating factors (e.g., coping, social support, time management, classroom environment). Multiple logistic regressions were used to identify which variables in the predictor set were significantly associated with each of the five outcomes while controlling for the presence of all other variables. RESULTS: In terms of behavioral health, 7.2% met screening criteria for depression, 9.4% for anxiety, and 17.1% for hazardous alcohol use; 43.4% reported high/very high levels of burnout. About one-third of the sample who had taken a test failed at least one (32.2%). In terms of common stressors more than half reported high or very high-stress levels from meeting academic expectations, not getting enough sleep, and pressure to succeed from civilian language instructors. For depression and anxiety, regression results found that denial coping was a risk factor whereas positive social interaction and classroom climate were protective factors. For hazardous alcohol use, denial coping and higher rank were risk factors and acceptance and time management were protective factors. In terms of academic burnout, in-class and military work hours were risk factors, whereas time management and classroom climate were protective. Finally, lower educational attainment, time spent in the classroom and times spent on military duties predicted exam failure. CONCLUSION: Individual coping, social connection, and classroom climate are each associated with better DLIFLC student adjustment. Denial coping appears to impede individuals from assembling the personal resources needed to study a foreign language. In contrast, acceptance appears to support healthier adjustment, perhaps freeing individuals to focus on the task at hand rather than expend valuable energy resisting the demands being placed on them. Positive social interaction also appears to provide an important resource for students, and positive classroom climate is also associated with better mental health. These findings suggest that there are measures that individuals and the school can take to improve the DLIFLC experience and support students as they manage a myriad of stressors given the significance of their success to individual students and to the larger organization.

La-related protein 6 controls ciliated cell differentiation.

BACKGROUND: La-related protein 6 (LARP6) is an evolutionally conserved RNA-binding protein. Vertebrate LARP6 binds the 5' stem-loop found in mRNAs encoding type I collagen to regulate their translation, but other target mRNAs and additional functions for LARP6 are unknown. The aim of this study was to elucidate an additional function of LARP6 and to evaluate the importance of its function during development. METHODS: To uncover the role of LARP6 in development, we utilized Morpholino Oligos to deplete LARP6 protein in Xenopus embryos. Then, embryonic phenotypes and ciliary structures of LAPR6 morphants were examined. To identify the molecular mechanism underlying ciliogenesis regulated by LARP6, we tested the expression level of cilia-related genes, which play important roles in ciliogenesis, by RT-PCR or whole mount in situ hybridization (WISH). RESULTS: We knocked down LARP6 in Xenopus embryos and found neural tube closure defects. LARP6 mutant, which compromises the collagen synthesis, could rescue these defects. Neural tube closure defects are coincident with lack of cilia, antenna-like cellular organelles with motility- or sensory-related functions, in the neural tube. The absence of cilia at the epidermis was also observed in LARP6 morphants, and this defect was due to the absence of basal bodies which are formed from centrioles and required for ciliary assembly. In the process of multi-ciliated cell (MCC) differentiation, mcidas, which activates the transcription of genes required for centriole formation during ciliogenesis, could partially restore MCCs in LARP6 morphants. In addition, LARP6 likely controls the expression of mcidas in a Notch-independent manner. CONCLUSIONS: La-related protein 6 is involved in ciliated cell differentiation during development by controlling the expression of cilia-related genes including mcidas. This LARP6 function involves a mechanism that is distinct from its established role in binding to collagen mRNAs and regulating their translation.

Growth mixture modeling of post-combat aggression: Application to soldiers deployed to Iraq.

Prior research has found substantial heterogeneity in the course of key post-deployment outcomes, such as PTSD. The current paper employs growth mixture modeling to identify differential trajectories of change in the course of post-combat aggression. A Brigade Combat Team completed surveys within 72h of return from an Iraq deployment, 4 months later, and at 12 months after return. Based on model fit indices, analyses yielded four latent aggression trajectories: "low-stable", "delayed", "recovery", and "chronic". In addition, most individuals aligned with a "low-stable" trajectory indicative of minimal aggression in the first year following return from a combat deployment. A conditional model showed that lower posttraumatic stress and lower combat exposure characterized individuals aligned with the "low-stable" aggression trajectory relative to individuals aligned with "chronic" and "delayed" aggression trajectories. Implications for targeted intervention and future research are discussed.

Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila.

Cilia are essential for cell signaling and sensory perception. In many cell types, a cytoskeletal structure called the ciliary rootlet links the cilium to the cell body. Previous studies indicated that rootlets support the long-term stability of some cilia. Here we report that Drosophila melanogaster Rootletin (Root), the sole orthologue of the mammalian paralogs Rootletin and C-Nap1, assembles into rootlets of diverse lengths among sensory neuron subtypes. Root mutant neurons lack rootlets and have dramatically impaired sensory function, resulting in behavior defects associated with mechanosensation and chemosensation. Root is required for cohesion of basal bodies, but the cilium structure appears normal in Root mutant neurons. We show, however, that normal rootlet assembly requires centrioles. The N terminus of Root contains a conserved domain and is essential for Root function in vivo. Ectopically expressed Root resides at the base of mother centrioles in spermatocytes and localizes asymmetrically to mother centrosomes in neuroblasts, both requiring Bld10, a basal body protein with varied functions.