A Genomic and Genetic Analysis of Doublesex Targets and Function in Drosophila Sexual Dimorphism

Sex determination pathways are diverse throughout the animal kingdom, but converge upon conserved genes that encode products that regulate sexual dimorphism. One such downstream factor across many diverged sex determination pathways is the Drosophila doublesex (dsx) gene. The role of doublesex is highly conserved in different insects and dsx homologs (dsx, mab-3 related transcription factors, DMRTs) play roles in sexual differentiation in a diverse array of metazoans. In Drosophila, nearly all manifestations of sexual dimorphism between males and females are regulated by doublesex, yet there are only three known direct targets of DSX, which cannot account for the differences in regulation by DSX in sexually dimorphic tissues. To gain a comprehensive understanding of DSX targets, we undertook multiple experimental approaches that allowed us to identify genes that were bound by DSX, genes whose expression changed in response to DSX perturbation, and genes that function in dsx-expressing cells. DSX protein binding was assayed by ChIP-seq and DamID-seq on S2 cells expressing tagged DSX-M or DSX-F. We also examined DSX occupancy in adult fat body and gonads using DamID-seq or DamID-chip. These experiments identified 3,717 genes bound by DSX in at least one occupancy dataset. Strikingly, we found that genes with the highest levels of DSX occupancy were bound by DSX in all occupancy data sets. This suggests one main mechanism of DSX action would be binding to potential targets in all tissues/contexts rather than having context-dependent targets. In this model of DSX action, additional inputs (such as segmental identity) would be needed to enact transcriptional regulation of bound genes in the appropriate context. Further strengthening this model, although 2,668 genes are bound by DSX in our adult 
fat body occupancy data, less than 1% of these occupied genes show large and robust transcriptional changes in response to acute changes in DSX isoform. We found that predicted DSX targets are significantly enriched in genes that yield phenotypes in sexually dimorphic tissues after RNAi knockdown in dsx-expressing cells (p=0.002). 41 (70.7%) of high probability DSX targets had phenotypes in at least one sexual dimorphic tissue compared to 7 (31.8%) of low probability targets. Altogether, the occupancy, transcriptional profiling, and functional testing have provided a detailed description of how dsx regulates sexual development. New dsx-interacting genes include genes involved in insect hormone signaling. We have identified the Ecdysone receptor gene as a target of DSX. Since the Drosophila gonad represents an excellent model to dissect how DSX acts on a particular time and place to promote development of a sexually dimorphic tissue, we examined the Ecdysone receptor gene, which is involved in ecdysteroid signaling, for roles in gonad sexual development. My data supports the hypothesis that the steroid hormone ecdysone elicits a different response in the male vs. female gonad and that this difference is regulated by DSX and may be important for proper formation of the ovary vs. the testis. Rather than being strictly a genetic process, results from our experiments may demonstrate that sexual differentiation in the gonad occurs through a combination of signals that include sex specific hormone signaling. Since the formation of the gonad may represent processes that are conserved from flies to man, this research will provide insight into conserved genes that regulate developmentally similar pathways whose outcome generates major differences observed between the sexes

Decolonizing the U.S. Health Care System: Undocumented and Disabled after ACA

The Affordable Care Act (ACA) explicitly denies newly arrived documented and undocumented immigrants health insurance coverage, effectively making them the largest remaining uninsured segment of the U.S. population. Using mixed qualitative methods, our original research illustrates the health consequences experienced by uninsured, disabled undocumented immigrants as they navigate what they describe as an apartheid health care system. Critiquing the notion of immigrants as “public charges” or burdens on the system, our qualitative analysis focuses on Houston Health Action, a community-based organization led by and for undocumented, low-income disabled immigrants in Houston, Texas. Engaging a critical migration and critical disabilities studies framework, we use this valuable case to highlight contemporary contradictions in health care and immigration legislation and the embodied consequences of the intersecting oppressions of race, ability, immigration status, and health care access

An Undergraduate Laboratory Manual for Analyzing a CRISPR Mutant with a Predicted Role in Regeneration

Exposing students to undergraduate research has reportedly improved students’ development of knowledge and skills in the laboratory, self-efficacy, satisfaction with their research, retention, and perseverance when faced with obstacles. Furthermore, utilizing authentic course-based undergraduate research experiences (CUREs) includes all students enrolled in the class, giving those who may not otherwise have access to an independent undergraduate research project an opportunity to engage in the scientific process in context of an original, unanswered question. In the fall of 2016, second semester introductory biology students conducted a semester-long research project on the transcription factor Lin28a to determine the effect of Lin28a on regeneration in a CRISPR mutant. During ten laboratory periods, students completed four experiments: 1) genotyping mutants by PCR and RFLP, 2) neuromast regeneration after copper sulfate treatment, 3) measuring changes in gene expression by RT-PCR after fin clipping, and 4) swimming behavior. In the context of this class, students were challenged to design their own experiments, interpret their own data, and make connections among the experiments to draft a final paper presenting their results and conclusions. Here, we present a student laboratory manual that can be adapted to other relevant CRISPR mutants. Overall, this coursework aligns with Vision and Change, and these experiments gave students a taste of the questions, techniques, and experimental design currently used in the field of regenerative biology

BDNF val66met polymorphism is associated with modified experience-dependent plasticity in human motor cortex.

Motor training can induce profound physiological plasticity within primary motor cortex, including changes in corticospinal output and motor map topography. Using transcranial magnetic stimulation, we show that training-dependent increases in the amplitude of motor-evoked potentials and motor map reorganization are reduced in healthy subjects with a val66met polymorphism in the brain-derived neurotrophic factor gene (BDNF), as compared to subjects without the polymorphism. The results suggest that BDNF is involved in mediating experience-dependent plasticity of human motor cortex

Consequences of being phenotypically mismatched with the environment: Rapid muscle ultrastructural changes in cold-shocked black-capped chickadees (Poecile atricapillus)

Phenotypic flexibility has received considerable attention in the last decade; however, whereas many studies have reported amplitude of variation in phenotypic traits, much less attention has focused on the rate at which traits can adjust in response to sudden changes in the environment. We investigated whole animal and muscle phenotypic changes occurring in black-capped chickadees (Poecile atricapillus) acclimated to cold (-5°C) and warm (20°C) temperatures in the first 3 h following a 15°C temperature drop (over 3 h). Before the temperature change, cold-acclimated birds were consuming 95% more food, were carrying twice as much body fat, and had 23% larger pectoralis muscle fiber diameters than individuals kept at 20°C. In the 3 h following the temperature drop, these same birds altered their pectoralis muscle ultrastructure by increasing the number of capillaries per fiber area and the number of nuclei per millimeter of fiber by 22%, consequently leading to a 22% decrease in myonuclear domain (amount of cytoplasm serviced per nucleus), whereas no such changes were observed in the warm-acclimated birds. To our knowledge, this is the first demonstration of such a rapid adjustment in muscle fiber ultrastructure in vertebrates. These results support the hypothesis that chickadees maintaining a cold phenotype are better prepared than warm-phenotype individuals to respond to a sudden decline in temperature, such as what may be experienced in their natural wintering environment

Screening for and Diagnosing Malnutrition in Hospitalized Pediatric Patients

Background: Malnutrition is often underdiagnosed, and consequently undertreated, in hospitalized patients. A nationwide study is being conducted to validate indicators (the Malnutrition Clinical Characteristics [MCC]) to diagnose malnutrition in hospitalized patients. Methods: For the full study, sixty pediatric hospitals will collect patient medical history, patient STRONGKids malnutrition screening score, and nutrition intervention data. Six hundred pediatric patients will be randomly selected from the cohort to be assessed for the MCC and the Nutrition Focused Physical Exam (NFPE). Medical outcomes will be collected for all patients for a three-month period thereafter. Baseline data from a subset of sites that have started data collection were descriptively analyzed using Stata 15. Results: As of March 2020, 113 pediatric patients are enrolled in the study, with 50 children ages 1-24 months and 63 children and adolescents ages 2-17. Based on the STRONGkids screener, 73% (n = 82) of participants were “at risk” for malnutrition. A higher proportion of participants in the older age group screened at risk (n=54; 86%) compared to the younger group (n=28; 56%). Fifty-seven of the 113 participants were included in the MCC subgroup, of whom 35 (61%) screened at-risk for malnutrition. Based on the MCC criteria, 49% (n = 28) were diagnosed with malnutrition. Inadequate nutrient intake was the MCC indicator most often used to support a malnutrition diagnosis in younger participants, while weight loss was the most commonly used indicator for older participants. Across both age groups, muscle wasting and subcutaneous fat loss were the most commonly reported NFPE indicators that further supported a malnutrition diagnosis. Conclusion: Screening-based risk for malnutrition and malnutrition indicators differ for infants and young children compared to older children and teens. Differences in risk factors for malnutrition by age group and the validity of the MCC will be assessed as more data are collected

Growth Factor Binding Peptides in Poly (Ethylene Glycol) Diacrylate (PEGDA)-Based Hydrogels for an Improved Healing Response of Human Dermal Fibroblasts

Growth factors (GF) are critical cytokines in wound healing. However, the direct delivery of these biochemical cues into a wound site significantly increases the cost of wound dressings and can lead to a strong immunological response due to the introduction of a foreign source of GFs. To overcome this challenge, we designed a poly(ethylene glycol) diacrylate (PEGDA) hydrogel with the potential capacity to sequester autologous GFs directly from the wound site. We demonstrated that synthetic peptide sequences covalently tethered to PEGDA hydrogels physically retained human transforming growth factor beta 1 (hTGFβ1) and human vascular endothelial growth factor (hVEGF) at 3.2 and 0.6 ng/mm2, respectively. In addition, we demonstrated that retained hTGFβ1 and hVEGF enhanced human dermal fibroblasts (HDFa) average cell surface area and proliferation, respectively, and that exposure to both GFs resulted in up to 1.9-fold higher fraction of area covered relative to the control. After five days in culture, relative to the control surface, non-covalently bound hTGFβ1 significantly increased the expression of collagen type I and hTGFβ1 and downregulated vimentin and matrix metalloproteinase 1 expression. Cumulatively, the response of HDFa to hTGFβ1 aligns well with the expected response of fibroblasts during the early stages of wound healing

Collagen Based Multicomponent Interpenetrating Networks as Promising Scaffolds for 3D Culture of Human Neural Stem Cells, Human Astrocytes, and Human Microglia

This work describes for the first time the fabrication and characterization of multicomponent interpenetrating networks composed of collagen I, hyaluronic acid, and poly(ethylene glycol) diacrylate for the 3D culture of human neural stem cells, astrocytes, and microglia. The chemical composition of the scaffolds can be modulated while maintaining values of complex moduli within the range of the mechanical performance of brain tissue (∼6.9 kPa) and having cell viability exceeding 84%. The developed scaffolds are a promising new family of biomaterials that can potentially serve as 3D in vitro models for studying the physiology and physiopathology of the central nervous system

Heterogeneous N2O5 Uptake During Winter: Aircraft Measurements During the 2015 WINTER Campaign and Critical Evaluation of Current Parameterizations

Nocturnal dinitrogen pentoxide (N2O5) heterogeneous chemistry impacts regional air quality and the distribution and lifetime of tropospheric oxidants. Formed from the oxidation of nitrogen oxides, N2O5 is heterogeneously lost to aerosol with a highly variable reaction probability, γ(N2O5), dependent on aerosol composition and ambient conditions. Reaction products include soluble nitrate (HNO3 or NO3−) and nitryl chloride (ClNO2). We report the first‐ever derivations of γ(N2O5) from ambient wintertime aircraft measurements in the critically important nocturnal residual boundary layer. Box modeling of the 2015 Wintertime INvestigation of Transport, Emissions, and Reactivity (WINTER) campaign over the eastern United States derived 2,876 individual γ(N2O5) values with a median value of 0.0143 and range of 2 × 10−5 to 0.1751. WINTER γ(N2O5) values exhibited the strongest correlation with aerosol water content, but weak correlations with other variables, such as aerosol nitrate and organics, suggesting a complex, nonlinear dependence on multiple factors, or an additional dependence on a nonobserved factor. This factor may be related to aerosol phase, morphology (i.e., core shell), or mixing state, none of which are commonly measured during aircraft field studies. Despite general agreement with previous laboratory observations, comparison of WINTER data with 14 literature parameterizations (used to predict γ(N2O5) in chemical transport models) confirms that none of the current methods reproduce the full range of γ(N2O5) values. Nine reproduce the WINTER median within a factor of 2. Presented here is the first field‐based, empirical parameterization of γ(N2O5), fit to WINTER data, based on the functional form of previous parameterizations

Think Outside the Color Box: Probabilistic Target Selection and the SDSS-XDQSO Quasar Targeting Catalog

We present the SDSS-XDQSO quasar targeting catalog for efficient flux-based quasar target selection down to the faint limit of the Sloan Digital Sky Survey (SDSS) catalog, even at medium redshifts (2.5 <~ z <~ 3) where the stellar contamination is significant. We build models of the distributions of stars and quasars in flux space down to the flux limit by applying the extreme-deconvolution method to estimate the underlying density. We convolve this density with the flux uncertainties when evaluating the probability that an object is a quasar. This approach results in a targeting algorithm that is more principled, more efficient, and faster than other similar methods. We apply the algorithm to derive low-redshift (z < 2.2), medium-redshift (2.2 <= z 3.5) quasar probabilities for all 160,904,060 point sources with dereddened i-band magnitude between 17.75 and 22.45 mag in the 14,555 deg^2 of imaging from SDSS Data Release 8. The catalog can be used to define a uniformly selected and efficient low- or medium-redshift quasar survey, such as that needed for the SDSS-III's Baryon Oscillation Spectroscopic Survey project. We show that the XDQSO technique performs as well as the current best photometric quasar-selection technique at low redshift, and outperforms all other flux-based methods for selecting the medium-redshift quasars of our primary interest. We make code to reproduce the XDQSO quasar target selection publicly available