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Globally, mosquito-borne diseases are a major source of mortality, with the burden focused primarily in the poorest nations. Central to reducing mosquito borne disease is an understanding of the biology of those species responsible for transmitting disease to humans. Accordingly, this work focuses on the ecology and evolutionary biology of two vectors of global importance: Aedes aegypti and Aedes albopictus. The first chapter examines the role of urbanization and species interactions in determining Aedes abundance across a 900km latitudinal range representing vast climatic gradients and the major ecotypes of Central Africa. Additionally, we performed pathogen screening on over 9,000 female mosquitoes for dengue, chikungunya, and Zika viruses.The second chapter focuses on the distribution and potential causes of insecticide resistance in Aedes across the same broad climatic and ecotype gradient in Cameroon. In this chapter we detected significant variation in insecticide resistance between cities, habitat types, species, and insecticide class. We applied land use data to show the significant effect that urbanization can have on determining insecticide efficacy across an area as small as a single city and further revealed the underlying genetic and metabolic mechanisms for insecticide resistance. The final chapter investigates viral fitness among the primary arboviruses transmitted by A. aegypti and A. albopictus: chikungunya, dengue (serotypes 1-4), yellow fever, and Zika. Our innovative approach to comparing relative viral fitness is multi-facted, combining field observations of human infection rates with laboratory studies of host and vector competence.

Breast parenchymal patterns on radiologic images are associated with breast cancer risk. Radiomic features have been proposed as quantitative measures of parenchymal patterns. We defined intrinsic imaging phenotypes of breast parenchymal patterns based on radiomic features extracted from full field digital mammography (FFDM) in breast screening populations and assessed whether these phenotypes are associated with breast cancer risk and masking. We selected 30,000 women with 4-view FFDM exams from Hologic machines from three institutions (Hospital of the University of Pennsylvania, Mayo Clinic, and San Francisco Mammography Registry), randomly split into a training (20,000 women) and test set (10,000 women). In total, 390 radiomic features were automatically extracted from each image using a validated software pipeline, standardized, and adjusted for site differences using ComBat. We used two methods, hierarchical clustering and Principal Components (PCs) analysis, to classify significant variation among the features in the training set and replicate among the test set. Next, we applied the replicated clusters and PCs to an independent nested case-control set [1082 invasive breast cancer (BC) cases (of which 151 were Black and 893 White women, 38 other race) matched to 2837 controls (411 Black and 2345 White women, 81 other race) on age, race, timing of images, and site]. We examined associations of the clusters and PCs with invasive breast cancer risk, as well as masking [defined as a false-negative (FN) screen (124 cases and 319 matched controls) and additionally the subset with symptomatic interval cancer (IC) within 12 months of negative screen (88 cases and 223 matched controls)] using conditional logistic regression. We evaluated their association with breast cancer alone, and with adjustment for age, body mass index (BMI) and breast density assessed by Breast Imaging Reporting and Data System (BI-RADS) using likelihood ratio tests. We estimated discrimination using area under the curve (AUC) and compared AUCs for models that included the radiomic clusters and PCs with the model that included only age, BMI and density. We also stratified analyses by race (Black/White). From hierarchical clustering, we defined six statistically significant phenotype clusters (each of at least 1000 women) in the training set which were replicated in the test set. For PC Analysis, we identified six PCs in the training set, explaining 85% of the variation in texture features and reproduced these in the test set. The six radiomic phenotype clusters (P< 0.001) and six PCs (P< 0.001) were both associated with invasive BC, including after adjusting for age, BMI, and density (cluster P=0.004; PCs P< 0.001). Improvements in discrimination of invasive BC with inclusion of PCs or clusters were more pronounced among Black women (Table). Further, the PCs (P< 0.001) and clusters (P< 0.001) were significantly associated with FN overall and for symptomatic IC (PCs P< 0.001; clusters P=0.001), but only PCs remained significant after adjusting for age, BMI and density (PCs P=0.004 for FN; PCs P=0.007 for symptomatic IC). Discrimination of masking also improved with inclusion of both clusters and PCs (Table). We identified reproducible radiomic phenotypes that are associated with invasive BC risk, above and beyond breast density with the strongest associations for invasive BC among Black women and symptomatic interval cancers. Citation Format: Stacey Winham, Anne Marie McCarthy, Aimilia Gastounioti, Christopher Scott, Aaron Norman, Walter C. Mankowski, Lauren Pantalone, Matthew Jensen, Eric A. Cohen, Hannah Horng, Kathleen Brandt, Emily F. Conant, Karla Kerlikowske, Despina Kontos, Celine Vachon. Radiomic phenotypes of breast texture and association with breast cancer risk and masking [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS4-06.

We examined more than 38,000 spouse pairs from four neurodevelopmental disease cohorts and the UK Biobank to identify phenotypic and genetic patterns in parents associated with neurodevelopmental disease risk in children. We identified correlations between six phenotypes in parents and children, including correlations of clinical diagnoses such as obsessive-compulsive disorder (R=0.31-0.49, p

Objective Suction-assisted laryngoscopy and airway decontamination (SALAD) was created to assist with the decontamination of a massively soiled airway. This study aims to investigate the usefulness of SALAD training to prehospital emergency providers to improve their ability to intubate a massively contaminated airway. Methods This was a prospective study conducted as a before and after teaching intervention. Participants were made up of prehospital providers who were present at regularly scheduled training sessions and were asked to intubate a high-fidelity mannequin simulating large-volume emesis before and after SALAD instruction. They were subsequently tested on 3-month skill retention. Twenty subjects participated in all stages of the study and were included in the analysis. Results The median time to successful intubation for all study participants before instruction was 60.5 seconds (interquartile range [IQR] = 44.0-84.0); post-training was 43.0 seconds (IQR = 38.0-57.5); and at the 3-month follow-up, it was 29.5 seconds (IQR = 24.5-39.0). The greatest improvement was seen on subgroup analysis of the slowest 50th percentile where the median time before instruction was 84.0 seconds (IQR = 68.0-96.0); post-instruction was 41.5 seconds (IQR = 36.0-65.0); and at the 3-month follow-up, it was 29.5 seconds (IQR = 25.0-39.0). Conclusion The implementation of the SALAD technique through a structured educational intervention improved time to intubation and the total number of attempts.

The Enterobacteriales order is composed of Gram-negative bacteria that range from harmless symbionts to well-studied pathogens. We announce complete genome sequences of five related SO-1-like Enterobacteriales bacteriophages (also known as the Dhillonvirus genus) isolated from wastewater that infect Escherichia coli (Opt-212, Over9000, Pubbukkers, and Teewinot) or Shigella boydii (StarDew).

There have been many tactics throughout the years aimed at reducing central line-associated bloodstream infections (CLABSI) in the healthcare setting. To reduce CLABSI rates at this facility, we employed cognitive science-based online training directed at nursing departments. Following implementation, we found significant reductions in CLABSI rates that were sustained for a minimum of 9 months. These results demonstrate that this learning methodology can be used to help decrease CLABSI and potentially other health care-associated infections.

ObjectiveDescribe a case of NMDAR encephalitis in a young Latino male patient, additionally the factors resulting in delayed preventative and diagnostic medical care, which contributed to the development of a preventable case of NMDAR encephalitis.BackgroundAdolescent undocumented immigrants in the United States face a history of prejudice and bias that perpetuates disparities and stigmas related to their healthcare. The lack of culturally informed practices among healthcare workers can create multiple lost opportunities to deliver standard of care practices, including routine testicular exams. The treatment of NMDAR encephalitis with immunotherapy, and resection of culpable tumors when present, can be lifesaving. Recognition of the germ cell tumor association has also renewed awareness of the importance of screening for such tumors.Design/MethodsN/A.ResultsCase: A 25-year-old male who immigrated from Mexico to the U.S. at age 13 presented to the hospital for concern of status epilepticus. His past medical history included atypical developmental delay beginning in late teenage years. A large abdominal mass was identified on imaging as a stage IIIC (pT1bN0M1bS2) NSGCT (70% teratoma/30% seminoma) tumor arising from an unresected, undescended left testicle. Autonomic instability in the setting of this malignancy prompted an evaluation for, and diagnosis of, NMDAR encephalitis. His course was complicated by altered mental status, seizures, sympathetic storming, and orofacial dystonia. After tumor resection, and initiation of immune therapy, the patient showed a remarkable recovery.ConclusionsThis patient's preventive healthcare was impacted at multiple timepoints by changing political policies and a lack of culturally informed practices that unpredictably disrupted reliable access to medical care. Recognition of care gaps allows us to expand our differential diagnoses, and enact a comprehensive approach to fill in gaps. Effective communication, incorporating focused discussions within culturally sensitive frameworks, requires ongoing education for clinicians regarding the populations they serve to prevent disease and minimize health care disparities.

Copy number variants (CNVs) are a major cause of several genetic disorders, making their detection an essential component of genetic analysis pipelines. Current methods for detecting CNVs from exome-sequencing data are limited by high false-positive rates and low concordance because of inherent biases of individual algorithms. To overcome these issues, calls generated by two or more algorithms are often intersected using Venn diagram approaches to identify “high-confidence” CNVs. However, this approach is inadequate, because it misses potentially true calls that do not have consensus from multiple callers. Here, we present CN-Learn, a machine-learning framework that integrates calls from multiple CNV detection algorithms and learns to accurately identify true CNVs using caller-specific and genomic features from a small subset of validated CNVs. Using CNVs predicted by four exome-based CNV callers (CANOES, CODEX, XHMM, and CLAMMS) from 503 samples, we demonstrate that CN-Learn identifies true CNVs at higher precision (∼90%) and recall (∼85%) rates while maintaining robust performance even when trained with minimal data (∼30 samples). CN-Learn recovers twice as many CNVs compared to individual callers or Venn diagram–based approaches, with features such as exome capture probe count, caller concordance, and GC content providing the most discriminatory power. In fact, ∼58% of all true CNVs recovered by CN-Learn were either singletons or calls that lacked support from at least one caller. Our study underscores the limitations of current approaches for CNV identification and provides an effective method that yields high-quality CNVs for application in clinical diagnostics.

Traumatic spinal cord injuries are significant contributors to the morbidity and mortality burden of trauma patients worldwide, and consume significant resources in both their acute and rehabilitative care. Another cause of mortality and morbidity is hypertrophic obstructive cardiomyopathy, which can cause syncope or sudden cardiac death in patients with no known prior cardiac disease. This case report describes a unique combination of these two high-risk pathologies in a scene trauma patient, and provides an overview of the pathophysiology and treatment of these high-risk disease processes.

Purpose: To assess the contribution of rare variants in the genetic background toward variability of neurodevelopmental phenotypes in individuals with rare copy-number variants (CNVs) and gene-disruptive variants. Methods: We analyzed quantitative clinical information, exome sequencing, and microarray data from 757 probands and 233 parents and siblings who carry disease-associated variants. Results: The number of rare likely deleterious variants in functionally intolerant genes (“other hits”) correlated with expression of neurodevelopmental phenotypes in probands with 16p12.1 deletion (n=23, p=0.004) and in autism probands carrying gene-disruptive variants (n=184, p=0.03) compared with their carrier family members. Probands with 16p12.1 deletion and a strong family history presented more severe clinical features (p=0.04) and higher burden of other hits compared with those with mild/no family history (p=0.001). The number of other hits also correlated with severity of cognitive impairment in probands carrying pathogenic CNVs (n=53) or de novo pathogenic variants in disease genes (n=290), and negatively correlated with head size among 80 probands with 16p11.2 deletion. These co-occurring hits involved known disease-associated genes such as SETD5, AUTS2, and NRXN1, and were enriched for cellular and developmental processes. Conclusion: Accurate genetic diagnosis of complex disorders will require complete evaluation of the genetic background even after a candidate disease-associated variant is identified.

Many birds undertake long biannual voyages during the night. During these times of the year birds drastically reduce their amount of sleep, yet curiously perform as well on tests of physical and cognitive performance than during non-migrating times of the year. This inherent physiological protection disappears when birds are forced to stay awake at other times of the year; thus these protective changes are only associated with the nocturnal migratory state. The goal of the current study was to identify the physiological mechanisms that confer protection against the consequences of sleep loss while simultaneously allowing for the increased physical performance required for migration. We performed RNA-seq analyses of heart and liver collected from birds at different times of day under different migratory states and analyzed these data using differential expression, pathway analysis and WGCNA. We identified changes in gene expression networks implicating multiple systems and pathways. These pathways regulate many aspects of metabolism, immune function, wound repair, and protection of multiple organ systems. Consequently, the circannual program controlling the appearance of the migratory phenotype involves the complex regulation of diverse gene networks associated with the physical demands of migration.

Spaceflight poses risks to the central nervous system (CNS), and understanding neurological responses is important for future missions. We report CNS changes in Drosophila aboard the International Space Station in response to spaceflight microgravity (SFμg) and artificially simulated Earth gravity (SF1g) via inflight centrifugation as a countermeasure. While inflight behavioral analyses of SFμg exhibit increased activity, postflight analysis displays significant climbing defects, highlighting the sensitivity of behavior to altered gravity. Multi-omics analysis shows alterations in metabolic, oxidative stress and synaptic transmission pathways in both SFμg and SF1g; however, neurological changes immediately postflight, including neuronal loss, glial cell count alterations, oxidative damage, and apoptosis, are seen only in SFμg. Additionally, progressive neuronal loss and a glial phenotype in SF1g and SFμg brains, with pronounced phenotypes in SFμg, are seen upon acclimation to Earth conditions. Overall, our results indicate that artificial gravity partially protects the CNS from the adverse effects of spaceflight.

Background Recent studies have suggested that individual variants do not sufficiently explain the variable expressivity of phenotypes observed in complex disorders. For example, the 16p12.1 deletion is associated with developmental delay and neuropsychiatric features in affected individuals, but is inherited in > 90% of cases from a mildly-affected parent. While children with the deletion are more likely to carry additional “second-hit” variants than their parents, the mechanisms for how these variants contribute to phenotypic variability are unknown. Methods We performed detailed clinical assessments, whole-genome sequencing, and RNA sequencing of lymphoblastoid cell lines for 32 individuals in five large families with multiple members carrying the 16p12.1 deletion. We identified contributions of the 16p12.1 deletion and “second-hit” variants towards a range of expression changes in deletion carriers and their family members, including differential expression, outlier expression, alternative splicing, allele-specific expression, and expression quantitative trait loci analyses. Results We found that the deletion dysregulates multiple autism and brain development genes such as FOXP1, ANK3, and MEF2. Carrier children also showed an average of 5323 gene expression changes compared with one or both parents, which matched with 33/39 observed developmental phenotypes. We identified significant enrichments for 13/25 classes of “second-hit” variants in genes with expression changes, where 4/25 variant classes were only enriched when inherited from the noncarrier parent, including loss-of-function SNVs and large duplications. In 11 instances, including for ZEB2 and SYNJ1, gene expression was synergistically altered by both the deletion and inherited “second-hits” in carrier children. Finally, brain-specific interaction network analysis showed strong connectivity between genes carrying “second-hits” and genes with transcriptome alterations in deletion carriers. Conclusions Our results suggest a potential mechanism for how “second-hit” variants modulate expressivity of complex disorders such as the 16p12.1 deletion through transcriptomic perturbation of gene networks important for early development. Our work further shows that family-based assessments of transcriptome data are highly relevant towards understanding the genetic mechanisms associated with complex disorders.

A neurodevelopmental disorder-associated gene, ISWI/SMARCA5, acts during development to coordinate sleep later in life., Sleep disruptions are among the most commonly reported symptoms across neurodevelopmental disorders (NDDs), but mechanisms linking brain development to normal sleep are largely unknown. From a Drosophila screen of human NDD-associated risk genes, we identified the chromatin remodeler Imitation SWItch/SNF (ISWI) to be required for adult fly sleep. Loss of ISWI also results in disrupted circadian rhythms, memory, and social behavior, but ISWI acts in different cells and during distinct developmental times to affect each of these adult behaviors. Specifically, ISWI expression in type I neuroblasts is required for both adult sleep and formation of a learning-associated brain region. Expression in flies of the human ISWI homologs SMARCA1 and SMARCA5 differentially rescues adult phenotypes, while de novo SMARCA5 patient variants fail to rescue sleep. We propose that sleep deficits are a primary phenotype of early developmental origin in NDDs and point toward chromatin remodeling machinery as critical for sleep circuit formation.

With more than a quarter of the population of the United States of America living greater than 60 minutes from the nearest level I or II trauma center, the utilization of helicopter emergency medical services provides a lifeline for the critically ill and injured. The use of prehospital ultrasound in the HEMS environment can provide improved diagnostic accuracy, accelerate a patient’s care timeline, guide therapy and procedures, and assist crew decision-making. These myriads of benefits, however, come at the potential cost of space limitations within the aircraft, training burdens for initial and refresher competencies, the need for physician-based QA/QI, and the cost of introducing such a program. Each aeromedical transport program needs to weigh the perceived advantages and challenges of utilizing prehospital ultrasound and make an informed decision as to whether its use would benefit the patient population they serve.

We previously identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. We hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while “second-hits” in the genetic background modulate the phenotypic trajectory. To test this model, we examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of “second-hit” genes inDrosophila melanogasterandXenopus laevis. We observed that knockdown of 16p12.1 homologs affect multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. Compared to genes within the 16p11.2 deletion, which has higherde novooccurrence, 16p12.1 homologs were less likely to interact with each other inDrosophilamodels or a human brain-specific interaction network, suggesting that interactions with “second-hit” genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 pairwise interactions inDrosophilabetween 16p12.1 homologs and 76 homologs of patient-specific “second-hit” genes (such asARID1BandCACNA1A), genes within neurodevelopmental pathways (such asPTENandUBE3A), and transcriptomic targets (such asDSCAMandTRRAP) identified genetic interactions in 63% of the tested pairs. In 11 out of 15 families, homologs of patient-specific “second-hits” enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs. In fact, homologs ofSETD5synergistically interacted with homologs ofMOSMOin bothDrosophilaandX. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. Our results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with “second-hit” genes determine the ultimate phenotypic manifestation.Author SummaryCopy-number variants, or deletions and duplications in the genome, are associated with multiple neurodevelopmental disorders. The developmental delay-associated 16p12.1 deletion is mostly inherited, and severely affected children carry an excess of “second-hits” variants compared to mildly affected carrier parents, suggesting that additional variants modulate the clinical manifestation. We studied this “two-hit” model usingDrosophilaandXenopus laevis, and systematically tested how homologs of “second-hit” genes modulate neurodevelopmental defects observed for 16p12.1 homologs. We observed that 16p12.1 homologs independently led to multiple neurodevelopmental features and weakly interacted with each other, suggesting that interactions with “second-hit” homologs potentially have a higher impact towards neurodevelopmental defects than interactions between 16p12.1 homologs. We tested 212 pairwise interactions of 16p12.1 homologs with “second-hit” homologs and genes within conserved neurodevelopmental pathways, and observed modulation of neurodevelopmental defects caused by 16p12.1 homologs in 11 out of 15 families, and 16/32 of these changes could be attributed to genetic interactions. Interestingly, we observed thatSETD5homologs interacted with homologs ofMOSMO, which conferred additional neuronal phenotypes not observed with knockdown of individual homologs. We propose that the 16p12.1 deletion sensitizes the genome to multiple neurodevelopmental defects, and complex interactions with “second-hit” genes determine the clinical trajectory of the disorder.

Sleep disruptions are among the most commonly-reported symptoms across neurodevelopmental disorders (NDDs), but mechanisms linking brain development to normal sleep are largely unknown. From aDrosophilascreen of human NDD-associated risk genes, we identified the chromatin remodelerImitation SWItch/SNF(ISWI) to be required for adult fly sleep. Loss ofISWIalso results in disrupted circadian rhythms, memory, and social behavior, butISWIacts in different cells and during distinct developmental times to affect each of these adult behaviors. Specifically,ISWIexpression in type I neuroblasts is required for both adult sleep and formation of a learning-associated brain region. Expression in flies of the humanISWIhomologsSMARCA1andSMARCA5differentially rescue adult phenotypes. We propose that sleep deficits are a primary phenotype of early developmental origin in NDDs, and point towards chromatin remodeling machinery as critical for sleep circuit formation.

Rural beneficiaries make up nearly one quarter of the Medicare population, yet rural providers and patients face specific challenges with health and health care delivery that remain inadequately understood. Health disparities between rural and urban residents are widespread, barriers to health care in rural communities persist, and the rural health care workforce is limited. To better understand and track the relationship between rurality and performance under Medicare's payment programs, researchers must be able to identify rural beneficiaries, providers, and hospitals. Although numerous definitions of rurality are applied across the Medicare program, empirical research is lacking comparing the different definitions of rurality and the impact of their application to quality, outcome, or costs. Definitions that recognize rurality as a graded concept, rather than a dichotomous one, hold promise. Understanding the strengths and limitations of different approaches to identifying rurality will help researchers choose the best method for their particular purpose, and help policymakers interpret studies using these approaches.

The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development., Author summary Rare copy-number variants, or large deletions and duplications in the genome, are associated with a wide range of neurodevelopmental disorders. The 3q29 deletion confers an increased risk for schizophrenia and autism. To understand the conserved biological mechanisms that are disrupted by this deletion, we systematically tested 14 individual homologs and 314 pairwise interactions of 3q29 genes for neuronal, cellular, and developmental phenotypes in Drosophila melanogaster and Xenopus laevis models. We found that multiple homologs of genes within the deletion region contribute towards developmental defects, such as larval lethality and disrupted cellular organization. Interestingly, we found that NCBP2 acts as a key modifier gene within the region, enhancing the developmental phenotypes of each of the homologs for other 3q29 genes and leading to disruptions in apoptosis and cell cycle pathways. Our results suggest that multiple genes within the 3q29 region interact with each other through shared mechanisms and jointly contribute to neurodevelopmental defects.