Why and How to Account for Sex and Gender in Brain and Behavioral Research.

Long overlooked in neuroscience research, sex and gender are increasingly included as key variables potentially impacting all levels of neurobehavioral analysis. Still, many neuroscientists do not understand the difference between the terms "sex" and "gender," the complexity and nuance of each, or how to best include them as variables in research designs. This TechSights article outlines rationales for considering the influence of sex and gender across taxa, and provides technical guidance for strengthening the rigor and reproducibility of such analyses. This guidance includes the use of appropriate statistical methods for comparing groups as well as controls for key covariates of sex (e.g., total intracranial volume) and gender (e.g., income, caregiver stress, bias). We also recommend approaches for interpreting and communicating sex- and gender-related findings about the brain, which have often been misconstrued by neuroscientists and the lay public alike.

Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter protein.

Noncoding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq-sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA-protein interactions can be more reliably detected. We find that the concentration of the RNA-DNA adapter is an important parameter in determining the signal in the system and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq-RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein-RNA interactions with the B3H system and will facilitate exploration of the binding mechanisms of these interactions.

The Protein Gap: The Rise and Fall of a Charismatic Nutrient in International Public Health.

From the early 1950s to the early 1970s, international nutritionists considered childhood protein malnutrition the world's most serious public health threat. By 1974, many believed that this "protein gap" had been exaggerated. Two questions remain: why protein, and why this period? Four converging developments created a network that maintained protein's "charisma": new food technology, a growing international health infrastructure, the nominal demise of eugenics, and new geopolitical priorities in a world shaped by both the Cold War and decolonization struggles. A transnational network of nutrition experts argued that protein deficiencies could explain bodily and population differences that would have, in an earlier era, been attributed to race or inheritance. Protein malnutrition could help explain "backward" economies and cultures, they claimed, and protein supplementation would help spur development. The protein gap theory thus framed difference in the language of modernization theory, but left intact older hierarchies of bodies, nations, and races.

Inpatient Rehabilitation After Pediatric and Adolescent Trauma: Outcomes and Discharge Needs.

INTRODUCTION: Traumatic injury is the leading cause of pediatric mortality and morbidity in the United States. Pediatric trauma survivors requiring inpatient rehabilitation (IPR) require coordinated, multispecialty follow-up. Knowledge of the nature and level of disability is necessary for planning this continued care that is specific to the needs of pediatric trauma patients. This study aims to describe the outcomes of pediatric and adolescent trauma patients using measures of functional progression. MATERIALS AND METHODS: A retrospective review of trauma patients aged ≤18 y admitted to IPR between January 2018 and December 2020 at the only certified pediatric rehabilitation center in the region was performed. RESULTS: Ninety five children and adolescents were admitted to IPR after traumatic injury with diagnoses of multitrauma (MT, N = 18), traumatic brain injury (TBI, N = 59), and spinal cord injury (SCI, N = 18). School aged children returned to school at high rates for all injury types (MT: 86.7%, TBI: 97.4%, SCI: 93.8%, P = ns). All groups had similar hospital and rehabilitation length of stay, and most patients required a durable medical equipment at discharge (79%). Using pediatric functional independence measure scoring progression from admission to discharge from IPR, SCI patients made significant improvement in bladder function and the least improvement in stair function. Patients sustaining a TBI made significant improvement in memory and comprehension tasks. CONCLUSIONS: Pediatric and adolescent trauma patients admitted to IPR had a positive progression during their therapy but required variable ongoing care depending on the mechanism of injury. Excellent rates of returning to school were seen across the three injury types.

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ.

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.

Cigarette Purchasing Patterns, Readiness to Quit, and Quit Attempts Among Homeless Smokers.

INTRODUCTION: Cigarette purchasing patterns may be linked with greater readiness to make a quit attempt and more quit attempts among domiciled samples. However, little is known about the cigarette purchasing patterns of homeless smokers or their potential relations to quitting intention and behaviors. This study redressed this gap among a convenience sample of homeless adult smokers from a large shelter in Dallas, Texas. METHODS: Participants (N = 207; Mage = 43; 71.5% male) smoked ≥100 cigarettes over the lifetime and endorsed current daily smoking. Variables assessed included cigarette dependence (time to first cigarette of the day), monthly income, quantity of cigarettes most recently purchased, average money spent on cigarettes weekly, readiness/motivation to quit smoking, and the number intentional quit attempts lasting ≥24h in the past year. Regression analyses were conducted to characterize associations of cigarette purchasing patterns with readiness to quit and quit attempts controlling for sex, age, cigarette dependence, and income. RESULTS: Most participants purchased cigarettes by the pack (61.4%), and more than half the sample spent ≤$20 on cigarettes per week. Results indicated that spending less money per week on cigarettes was associated with greater readiness to quit (P = .016), even when controlling for income, cigarette dependence, and other covariates. Stratified analyses indicated that this association was significant only for homeless smokers reporting no regular monthly income. CONCLUSIONS: Homeless daily smokers with no reported income who spend little money on cigarettes may make particularly apt targets for cessation interventions due to potential associations with quitting motivation. IMPLICATIONS: Adults who are homeless smoke at greater rates and quit at lower rates than domiciled adults, leading to significant smoking-related health disparities among this group. Findings suggest that cigarette purchasing patterns are linked with readiness to quit smoking among smokers who are homeless. Results elucidate one of the myriad factors that contribute to tobacco-related disparities among this group and findings may have implications for cessation interventions in homeless shelters and other contexts where resources are limited.

Improved constructs for bait RNA display in a bacterial three-hybrid assay.

We have previously developed a transcription-based bacterial three-hybrid (B3H) assay as a genetic approach to probe RNA-protein interactions inside of E. coli cells. This system offers a straightforward path to identify and assess the consequences of mutations in RBPs with molecular phenotypes of interest. One limiting factor in detecting RNA-protein interactions in the B3H assay is RNA misfolding arising from incorrect base-pair interactions with neighboring RNA sequences in a hybrid RNA. To support correct folding of hybrid bait RNAs, we have explored the use of a highly stable stem ("GC clamp") to isolate regions of a hybrid RNA as discrete folding units. In this work, we introduce new bait RNA constructs to 1) insulate the folding of individual components of the hybrid RNA with GC clamps and 2) express bait RNAs that do not encode their own intrinsic terminator. We find that short GC clamps (5 or 7 bp long) are more effective than a longer 13bp GC clamp in the B3H assay. These new constructs increase the number of Hfq-sRNA and -5'UTR interactions that are detectable in the B3H system and improve the signal-to-noise ratio of many of these interactions. We therefore recommend the use of constructs containing short GC clamps for the expression of future B3H bait RNAs. With these new constructs, a broader range of RNA-protein interactions are detectable in the B3H assay, expanding the utility and impact of this genetic tool as a platform to search for and interrogate mechanisms of additional RNA-protein interactions.

Gender-related variables for health research.

BACKGROUND: In this paper, we argue for Gender as a Sociocultural Variable (GASV) as a complement to Sex as a Biological Variable (SABV). Sex (biology) and gender (sociocultural behaviors and attitudes) interact to influence health and disease processes across the lifespan-which is currently playing out in the COVID-19 pandemic. This study develops a gender assessment tool-the Stanford Gender-Related Variables for Health Research-for use in clinical and population research, including large-scale health surveys involving diverse Western populations. While analyzing sex as a biological variable is widely mandated, gender as a sociocultural variable is not, largely because the field lacks quantitative tools for analyzing the influence of gender on health outcomes. METHODS: We conducted a comprehensive review of English-language measures of gender from 1975 to 2015 to identify variables across three domains: gender norms, gender-related traits, and gender relations. This yielded 11 variables tested with 44 items in three US cross-sectional survey populations: two internet-based (N = 2051; N = 2135) and a patient-research registry (N = 489), conducted between May 2017 and January 2018. RESULTS: Exploratory and confirmatory factor analyses reduced 11 constructs to 7 gender-related variables: caregiver strain, work strain, independence, risk-taking, emotional intelligence, social support, and discrimination. Regression analyses, adjusted for age, ethnicity, income, education, sex assigned at birth, and self-reported gender identity, identified associations between these gender-related variables and self-rated general health, physical and mental health, and health-risk behaviors. CONCLUSION: Our new instrument represents an important step toward developing more comprehensive and precise survey-based measures of gender in relation to health. Our questionnaire is designed to shed light on how specific gender-related behaviors and attitudes contribute to health and disease processes, irrespective of-or in addition to-biological sex and self-reported gender identity. Use of these gender-related variables in experimental studies, such as clinical trials, may also help us understand if gender factors play an important role as treatment-effect modifiers and would thus need to be further considered in treatment decision-making.

Fragile X granules are a family of axonal ribonucleoprotein particles with circuit-dependent protein composition and mRNA cargos.

Local axonal protein synthesis plays a crucial role in the formation and function of neuronal circuits. Understanding the role of this mechanism in specific circuits requires identifying the protein composition and mRNA cargos of the ribonucleoprotein particles (RNPs) that form the substrate for axonal translation. FXGs (Fragile X granules) are axonal RNPs present in a stereotyped subset of mature axons in the intact brain that contain one or more of the Fragile X related (FXR) proteins (FMRP, FXR2P, and FXR1P) along with mRNA and ribosomes. Here we performed a systematic survey of the FXR protein composition and mRNA association of FXGs in the brain. We have identified four FXG types that can be categorized based on their FXR protein complement. All FXGs contain FXR2P, with FMRP and/or FXR1P present in circuit-selective subsets. Individual neuronal cell types predominantly express a single FXG type, with FMRP-containing FXGs the most prevalent in forebrain neurons. All FXG types associate with ribosomes and mRNA, but the specific mRNA cargos are a function of FXG type, brain region and neuron class. Transcripts for ß-catenin and its regulator APC associate with a subset of forebrain FXGs. Moreover, both these transcripts can colocalize within individual FXGs, suggesting that the axonal translation of functionally related proteins may be coordinately regulated with high spatiotemporal resolution. Cell type-dependent expression of specific RNP types with distinct mRNA cargos, such as FXGs, presents a potential mechanism for regulating local translation and its output in a circuit-dependent manner.

Systematic mapping of fragile X granules in the mouse brain reveals a potential role for presynaptic FMRP in sensorimotor functions.

Loss of Fragile X mental retardation protein (FMRP) leads to Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism. Although the functions of FMRP and its homologs FXR1P and FXR2P are well studied in the somatodendritic domain, recent evidence suggests that this family of RNA binding proteins also plays a role in the axonal and presynaptic compartments. Fragile X granules (FXGs) are morphologically and genetically defined structures containing Fragile X proteins that are expressed axonally and presynaptically in a subset of circuits. To further understand the role of presynaptic Fragile X proteins in the brain, we systematically mapped the FXG distribution in the mouse central nervous system. This analysis revealed both the circuits and the neuronal types that express FXGs. FXGs are enriched in circuits that mediate sensory processing and motor planning-functions that are particularly perturbed in FXS patients. Analysis of FXG expression in the hippocampus suggests that CA3 pyramidal neurons use presynaptic Fragile X proteins to modulate recurrent but not feedforward processing. Neuron-specific FMRP mutants revealed a requirement for neuronal FMRP in the regulation of FXGs. Finally, conditional FMRP ablation demonstrated that FXGs are expressed in axons of thalamic relay nuclei that innervate cortex, but not in axons of thalamic reticular nuclei, striatal nuclei, or cortical neurons that innervate thalamus. Together, these findings support the proposal that dysregulation of axonal and presynaptic Fragile X proteins contribute to the neurological symptoms of FXS.