April

You might not know it by looking at her ­with her luxurious black curly fur, rust colored stockings and eyebrows, white paws, broad regal white chest, white muzzle and blaze and crowning tip of white fur on the tail, a tail that seems to be perpetual wagging, in a medium slow relaxed way, in an “I’m just hanging out and loving every moment of life,” an easy way kind of wag ­for most people not in the know would just look at her and say, she’s a dog, or she’s “just” a dog, or, in a rather frightened, anxious, way, she’s a BIG dog ­ while others, quite embarrassingly, might even drop down on their knees, exclaiming, “What a BEAUTIFUL dog” or even “She’s the most beautiful dog I have ever seen!

From There to Here: Evaluating the Transition of an International Advanced Pharmacy Practice Experience in London

Purdue University College of Pharmacy offers an international advanced pharmacy practice experience (APPE) in London, England, where student pharmacists have the opportunity to work with pharmacy specialists at St. Bartholomew’s Hospital. The COVID-19 pandemic has significantly impacted travel across the world and created the need to transition this experience from in-person to virtual. Virtual interaction with international preceptors and revised research and clinical activities can provide students with a valuable learning experience

In the Absence of Frazzled Over-Expression of Abelson Tyrosine Kinase Disrupts Commissure Formation and Causes Axons to Leave the Embryonic CNS

BACKGROUND: In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl's role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures. METHODOLOGY/PRINCIPAL FINDINGS: The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS. CONCLUSION/SIGNIFICANCE: Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border

ProxECAT: Proxy External Controls Association Test. A new case-control gene region association test using allele frequencies from public controls.

A primary goal of the recent investment in sequencing is to detect novel genetic associations in health and disease improving the development of treatments and playing a critical role in precision medicine. While this investment has resulted in an enormous total number of sequenced genomes, individual studies of complex traits and diseases are often smaller and underpowered to detect rare variant genetic associations. Existing genetic resources such as the Exome Aggregation Consortium (>60,000 exomes) and the Genome Aggregation Database (~140,000 sequenced samples) have the potential to be used as controls in these studies. Fully utilizing these and other existing sequencing resources may increase power and could be especially useful in studies where resources to sequence additional samples are limited. However, to date, these large, publicly available genetic resources remain underutilized, or even misused, in large part due to the lack of statistical methods that can appropriately use this summary level data. Here, we present a new method to incorporate external controls in case-control analysis called ProxECAT (Proxy External Controls Association Test). ProxECAT estimates enrichment of rare variants within a gene region using internally sequenced cases and external controls. We evaluated ProxECAT in simulations and empirical analyses of obesity cases using both low-depth of coverage (7x) whole-genome sequenced controls and ExAC as controls. We find that ProxECAT maintains the expected type I error rate with increased power as the number of external controls increases. With an accompanying R package, ProxECAT enables the use of publicly available allele frequencies as external controls in case-control analysis

The Vaginal Microbiome: Disease, Genetics and the Environment

The vagina is an interactive interface between the host and the environment. Its surface is covered by a protective epithelium colonized by bacteria and other microorganisms. The ectocervix is nonsterile, whereas the endocervix and the upper genital tract are assumed to be sterile in healthy women. Therefore, the cervix serves a pivotal role as a gatekeeper to protect the upper genital tract from microbial invasion and subsequent reproductive pathology. Microorganisms that cross this barrier can cause preterm labor, pelvic inflammatory disease, and other gynecologic and reproductive disorders. Homeostasis of the microbiome in the vagina and ectocervix plays a paramount role in reproductive health. Depending on its composition, the microbiome may protect the vagina from infectious or non-infectious diseases, or it may enhance its susceptibility to them. Because of the nature of this organ, and the fact that it is continuously colonized by bacteria from birth to death, it is virtually certain that this rich environment evolved in concert with its microbial flora. Specific interactions dictated by the genetics of both the host and microbes are likely responsible for maintaining both the environment and the microbiome. However, the genetic basis of these interactions in both the host and the bacterial colonizers is currently unknown. _Lactobacillus_ species are associated with vaginal health, but the role of these species in the maintenance of health is not yet well defined. Similarly, other species, including those representing minor components of the overall flora, undoubtedly influence the ability of potential pathogens to thrive and cause disease. Gross alterations in the vaginal microbiome are frequently observed in women with bacterial vaginosis, but the exact etiology of this disorder is still unknown. There are also implications for vaginal flora in non-infectious conditions such as pregnancy, pre-term labor and birth, and possibly fertility and other aspects of women’s health. Conversely, the role of environmental factors in the maintenance of a healthy vaginal microbiome is largely unknown. To explore these issues, we have proposed to address the following questions:

*1.	Do the genes of the host contribute to the composition of the vaginal microbiome?* We hypothesize that genes of both host and bacteria have important impacts on the vaginal microbiome. We are addressing this question by examining the vaginal microbiomes of mono- and dizygotic twin pairs selected from the over 170,000 twin pairs in the Mid-Atlantic Twin Registry (MATR). Subsequent studies, beyond the scope of the current project, may investigate which host genes impact the microbial flora and how they do so.
*2.	What changes in the microbiome are associated with common non-infectious pathological states of the host?* We hypothesize that altered physiological (e.g., pregnancy) and pathologic (e.g., immune suppression) conditions, or environmental exposures (e.g., antibiotics) predictably alter the vaginal microbiome. Conversely, certain vaginal microbiome characteristics are thought to contribute to a woman’s risk for outcomes such as preterm delivery. We are addressing this question by recruiting study participants from the ~40,000 annual clinical visits to women’s clinics of the VCU Health System.
*3.	What changes in the vaginal microbiome are associated with relevant infectious diseases and conditions?* We hypothesize that susceptibility to infectious disease (e.g. HPV, _Chlamydia_ infection, vaginitis, vaginosis, etc.) is impacted by the vaginal microbiome. In turn, these infectious conditions clearly can affect the ability of other bacteria to colonize and cause pathology. Again, we are exploring these issues by recruiting participants from visitors to women’s clinics in the VCU Health System.

Three kinds of sequence data are generated in this project: i) rDNA sequences from vaginal microbes; ii) whole metagenome shotgun sequences from vaginal samples; and iii) whole genome shotgun sequences of bacterial clones selected from vaginal samples. The study includes samples from three vaginal sites: mid-vaginal, cervical, and introital. The data sets also include buccal and perianal samples from all twin participants. Samples from these additional sites are used to test the hypothesis of a per continuum spread of bacteria in relation to vaginal health. An extended set of clinical metadata associated with these sequences are deposited with dbGAP. We have currently collected over 4,400 samples from ~100 twins and over 450 clinical participants. We have analyzed and deposited data for 480 rDNA samples, eight whole metagenome shotgun samples, and over 50 complete bacterial genomes. These data are available to accredited investigators according to NIH and Human Microbiome Project (HMP) guidelines. The bacterial clones are deposited in the Biodefense and Emerging Infections Research Resources Repository ("http://www.beiresources.org/":http://www.beiresources.org/). 

In addition to the extensive sequence data obtained in this study, we are collecting metadata associated with each of the study participants. Thus, participants are asked to complete an extensive health history questionnaire at the time samples are collected. Selected clinical data associated with the visit are also obtained, and relevant information is collected from the medical records when available. This data is maintained securely in a HIPAA-compliant data system as required by VCU’s Institutional Review Board (IRB). The preponderance of these data (i.e., that judged appropriate by NIH staff and VCU’s IRB are deposited at dbGAP ("http://www.ncbi.nlm.nih.gov/gap":http://www.ncbi.nlm.nih.gov/gap). Selected fields of this data have been identified by NIH staff as ‘too sensitive’ and are not available in dbGAP. Individuals requiring access to these data fields are asked to contact the PI of this project or NIH Program Staff. 
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Equity, diversity, and inclusion at the Global Alliance for Genomics and Health

A lack of diversity in genomics for health continues to hinder equitable leadership and access to precision medicine approaches for underrepresented populations. To avoid perpetuating biases within the genomics workforce and genomic data collection practices, equity, diversity, and inclusion (EDI) must be addressed. This paper documents the journey taken by the Global Alliance for Genomics and Health (a genomics-based standard-setting and policy-framing organization) to create a more equitable, diverse, and inclusive environment for its standards and members. Initial steps include the creation of two groups: the Equity, Diversity, and Inclusion Advisory Group and the Regulatory and Ethics Diversity Group. Following a framework that we call "Reflected in our Teams, Reflected in our Standards," both groups address EDI at different stages in their policy development process. [Abstract copyright: © 2023 The Author(s).

Perceived and objective neighborhood support for outside of school physical activity in South African children.

The neighborhood environment has the potential to influence children's participation in physical activity. However, children's outdoor play is controlled by parents to a great extent. This study aimed to investigate whether parents' perceptions of the neighborhood environment and the objectively measured neighborhood environment were associated with children's moderate-to-vigorous intensity physical activity (MVPA) outside of school hours; and to determine if these perceptions and objective measures of the neighborhood environment differ between high and low socio-economic status (SES) groups.In total, 258 parents of 9-11 year-old children, recruited from the South African sample of the International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE), completed a questionnaire concerning the family and neighborhood environment. Objective measures of the environment were also obtained using Geographic Information Systems (GIS). Children wore an Actigraph (GT3X+) accelerometer for 7 days to measure levels of MVPA. Multilevel regression models were used to determine the association between the neighborhood environment and MVPA out of school hours.Parents' perceptions of the neighborhood physical activity facilities were positively associated with children's MVPA before school (β = 1.50 ± 0.51, p = 0.003). Objective measures of neighborhood safety and traffic risk were associated with children's after-school MVPA (β = -2.72 ± 1.35, p = 0.044 and β = -2.63 ± 1.26, p = 0.038, respectively). These associations were significant in the low SES group (β = -3.38 ± 1.65, p = 0.040 and β = -3.76 ± 1.61, p = 0.020, respectively), but unrelated to MVPA in the high SES group.This study found that several of the objective measures of the neighborhood environment were significantly associated with children's outside-of-school MVPA, while most of the parents' perceptions of the neighborhood environment were unrelated

BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression

For DNA replication to occur, chromatin must be remodeled. Yet, we know very little about which proteins alter nucleosome occupancy at origins and replication forks and for what aspects of replication they are required. Here, we demonstrate that the BRG1 catalytic subunit of mammalian SWI/SNF-related complexes co-localizes with origin recognition complexes, GINS complexes, and proliferating cell nuclear antigen at sites of DNA replication on extended chromatin fibers. The specific pattern of BRG1 occupancy suggests it does not participate in origin selection but is involved in the firing of origins and the process of replication elongation. This latter function is confirmed by the fact that Brg1 mutant mouse embryos and RNAi knockdown cells exhibit a 50% reduction in replication fork progression rates, which is associated with decreased cell proliferation. This novel function of BRG1 is consistent with its requirement during embryogenesis and its role as a tumor suppressor to maintain genome stability and prevent cancer

Urine Gastrin Releasing Peptide in the First Week Correlates with BPD and Post-Prematurity Respiratory Disease

Rationale: Bronchopulmonary dysplasia (BPD) is associated with post-prematurity respiratory disease (PRD) in survivors of extreme preterm birth. Identifying early biomarkers that correlate with later development of BPD and PRD may provide insights for intervention. In a preterm baboon model, elevated gastrin-releasing peptide (GRP) is associated with BPD, and GRP inhibition mitigates BPD occurrence. Objective: We performed a prospective cohort study to investigate whether urine GRP levels obtained in the first postnatal week were associated with BPD, PRD, and other urinary biomarkers of oxidative stress. Methods: Extremely low gestational age infants (23-28 completed weeks) were enrolled in a US multicenter observational study, The Prematurity and Respiratory Outcomes Program (http://clinicaltrials.gov/ct2/show/NCT01435187). We used multivariable logistic regression to examine the association between urine GRP in the first postnatal week and multiple respiratory outcomes: BPD, defined as supplemental oxygen use at 36 + 0 weeks postmenstrual age, and post-PRD, defined by positive quarterly surveys for increased medical utilization over the first year (PRD score). Results: A total of 109 of 257 (42%) infants had BPD, and 120 of 217 (55%) had PRD. On adjusted analysis, GRP level more than 80 was associated with BPD (adjusted odds ratio [aOR], 1.83; 95% confidence interval [CI], 1.03-3.25) and positive PRD score (aOR, 2.46; 95% CI, 1.35-4.48). Urine GRP levels correlated with duration of NICU ventilatory and oxygen support and with biomarkers of oxidative stress: allantoin and 8-hydroxydeoxyguanosine. Conclusions: Urine GRP in the first postnatal week was associated with concurrent urine biomarkers of oxidative stress and with later diagnoses of BPD and PRD

The development and assessment of a scale to measure the experience of an anorexic voice in anorexia nervosa

The anorexic voice (AV) is defined as a critical internal dialogue which has been implicated in the development and maintenance of anorexia nervosa (AN). Systematic research to explore this further requires a valid and reliable measurement tool. This study aimed to develop and assess the validity of the Experience of an Anorexic VoicE Questionnaire (EAVE-Q). EAVE-Q items were developed and checked for face and content validity through cognitive interviews with seven individuals diagnosed with AN. Participants with a diagnosis of AN (N = 148) completed the EAVE-Q, sociodemographic questions and measures of mood and quality of life to assess internal consistency and construct 3 validity. Forty-nine participants completed the EAVE-Q twice more to assess test-retest reliability. The EAVE-Q had good face and content validity and good acceptability. Principal axis factoring resulted in an 18-item scale organised into five domains with high internal consistency (α = .70 to α = .85). Domains correlated significantly with eating disorder symptoms, psychological distress and quality of life. The EAVE-Q did not discriminate between participants on the basis of body mass index. Test-retest reliability was moderate. Although the factor structure of the EAVE-Q requires replication in other AN samples, the EAVE-Q is the first measure of a critical internal dialogue in AN. It is hoped that it will aid future research to increase understanding of AN and the continued development of person-centred treatments