Bromine is an essential trace element for assembly of collagen IV scaffolds in tissue development and architecture.

Bromine is ubiquitously present in animals as ionic bromide (Br(-)) yet has no known essential function. Herein, we demonstrate that Br(-) is a required cofactor for peroxidasin-catalyzed formation of sulfilimine crosslinks, a posttranslational modification essential for tissue development and architecture found within the collagen IV scaffold of basement membranes (BMs). Bromide, converted to hypobromous acid, forms a bromosulfonium-ion intermediate that energetically selects for sulfilimine formation. Dietary Br deficiency is lethal in Drosophila, whereas Br replenishment restores viability, demonstrating its physiologic requirement. Importantly, Br-deficient flies phenocopy the developmental and BM defects observed in peroxidasin mutants and indicate a functional connection between Br(-), collagen IV, and peroxidasin. We establish that Br(-) is required for sulfilimine formation within collagen IV, an event critical for BM assembly and tissue development. Thus, bromine is an essential trace element for all animals, and its deficiency may be relevant to BM alterations observed in nutritional and smoking-related disease. PAPERFLICK:

The zinc-finger protein Z4 cooperates with condensin II to regulate somatic chromosome pairing and 3D chromatin organization.

Chromosome pairing constitutes an important level of genome organization, yet the mechanisms that regulate pairing in somatic cells and the impact on 3D chromatin organization are still poorly understood. Here, we address these questions in Drosophila, an organism with robust somatic pairing. In Drosophila, pairing preferentially occurs at loci consisting of numerous architectural protein binding sites (APBSs), suggesting a role of architectural proteins (APs) in pairing regulation. Amongst these, the anti-pairing function of the condensin II subunit CAP-H2 is well established. However, the factors that regulate CAP-H2 localization and action at APBSs remain largely unknown. Here, we identify two factors that control CAP-H2 occupancy at APBSs and, therefore, regulate pairing. We show that Z4, interacts with CAP-H2 and is required for its localization at APBSs. We also show that hyperosmotic cellular stress induces fast and reversible unpairing in a Z4/CAP-H2 dependent manner. Moreover, by combining the opposite effects of Z4 depletion and osmostress, we show that pairing correlates with the strength of intrachromosomal 3D interactions, such as active (A) compartment interactions, intragenic gene-loops, and polycomb (Pc)-mediated chromatin loops. Altogether, our results reveal new players in CAP-H2-mediated pairing regulation and the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions.

Modeling prevention behaviors during the COVID-19 pandemic using Bayesian belief networks and protection motivation theory.

Prevention behaviors are important in mitigating the transmission of COVID-19. The protection motivation theory (PMT) links perceptions of risk and coping ability with the act of adopting prevention behaviors. The goal of this research is to test the application of the PMT in predicting adoption of prevention behaviors during the COVID-19 pandemic. Two research objectives are achieved to explore motivating factors for adopting prevention behaviors. (1) The first objective is to identify variables that are strong predictors of prevention behavior adoption. A data-driven approach is used to train Bayesian belief network (BBN) models using results of a survey of N = 7797 $N=7797$ participants reporting risk perceptions and prevention behaviors during the COVID-19 pandemic. A large set of models are generated and analyzed to identify significant variables. (2) The second objective is to develop models based on the PMT to predict prevention behaviors. BBN models that predict prevention behaviors were developed using two approaches. In the first approach, a data-driven methodology trains models using survey data alone. In the second approach, expert knowledge is used to develop the structure of the BBN using PMT constructs. Results demonstrate that trust and experience with COVID-19 were important predictors for prevention measure adoption. Models that were developed using the PMT confirm relationships between coping appraisal, threat appraisal, and protective behaviors. Data-driven and PMT-based models perform similarly well, confirming the use of PMT in this context. Predicting adoption of social distancing behaviors provides insight for developing policies during pandemics.

Biallelic GTF2IRD1 variants in brothers with profound neurodevelopmental disorder: A possible novel disorder involving a critical gene for Williams syndrome.

GTF2IRD1, a gene on chromosome 7 which encodes a transcription factor, is of significant clinical interest due to its heterozygous loss as part of the classical deletion associated with Williams-Beuren syndrome (WBS). However, biallelic variants in GTF2IRD1 alone as part of an autosomal recessive disease have not been previously reported. Here, we present two full brothers with variants in trans of GTF2IRD1 at c.1231C > T (p.Arg411Trp) and c.2632C > G (p.Leu878Val). A detailed clinical phenotype is described, which includes severe neurodevelopmental disability, facial dysmorphology, and pectus excavatum. Importantly, out of eight full siblings, only these two brothers harboring both variants in trans present with the profound described phenotype. We present the possibility that these brothers represent the identification of a new syndrome characterized by biallelic variants in GTF2IRD1, which may also have important implications for the molecular etiology of WBS.

Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates.

Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands-typically camelid antibodies-that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH = 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (KD ~ 10-5 -10- 6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC10% > 1013 vp/mL of resin) and product yields (~50%-80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50%-80%), 80- to 400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.

Secondary Risk Theory: Validation of a Novel Model of Protection Motivation.

Protection motivation theory states individuals conduct threat and coping appraisals when deciding how to respond to perceived risks. However, that model does not adequately explain today's risk culture, where engaging in recommended behaviors may create a separate set of real or perceived secondary risks. We argue for and then demonstrate the need for a new model accounting for a secondary threat appraisal, which we call secondary risk theory. In an online experiment, 1,246 participants indicated their intention to take a vaccine after reading about the likelihood and severity of side effects. We manipulated likelihood and severity in a 2 × 2 between-subjects design and examined how well secondary risk theory predicts vaccination intention compared to protection motivation theory. Protection motivation theory performed better when the likelihood and severity of side effects were both low (R2 = 0.30) versus high (R2 = 0.15). In contrast, secondary risk theory performed similarly when the likelihood and severity of side effects were both low (R2 = 0.42) or high (R2 = 0.45). But the latter figure is a large improvement over protection motivation theory, suggesting the usefulness of secondary risk theory when individuals perceive a high secondary threat.

"Influenza" versus "Flu": Do Different Medical Terms Affect Vaccination Intention?

Confusion about disease terminology contributes to lower risk perceptions that may lead to lesser engagement in protective measures. Communication campaigns promoting influenza vaccination have become commonplace around the world. Such campaigns vary widely in their terminology used from depicting the disease and response formally as an "influenza vaccination" to more colloquial terms like "flu shot." This study gathered responses from 896 Singapore residents through an online experiment and employed a Chi-square test to assess if different medical terms describing the same preventive measure ("influenza vaccine" and "flu shot") influence vaccination intentions. Results indicate that the formal term "influenza vaccine" prompts significantly greater vaccination intention than the abbreviated colloquial term "flu shot," even when no further information about the disease or vaccine is provided. This finding suggests that the proclivity to use the less formal term "flu shot" in widespread campaigns is less advantageous in prompting intentions to vaccinate against the disease. This may be the result of an activated availability bias brought on by the distinct semantic frames. We conclude that in this instance the medical terms should not be used interchangeably and that "influenza vaccine" may be more advantageous in future communication to encourage adoption of advised health behavior.

Peroxidasin forms sulfilimine chemical bonds using hypohalous acids in tissue genesis.

Collagen IV comprises the predominant protein network of basement membranes, a specialized extracellular matrix, which underlie epithelia and endothelia. These networks assemble through oligomerization and covalent crosslinking to endow mechanical strength and shape cell behavior through interactions with cell-surface receptors. A recently discovered sulfilimine (S=N) bond between a methionine sulfur and hydroxylysine nitrogen reinforces the collagen IV network. We demonstrate that peroxidasin, an enzyme found in basement membranes, catalyzes formation of the sulfilimine bond. Drosophila peroxidasin mutants have disorganized collagen IV networks and torn visceral muscle basement membranes, pointing to a critical role for the enzyme in tissue biogenesis. Peroxidasin generates hypohalous acids as reaction intermediates, suggesting a paradoxically anabolic role for these usually destructive oxidants. This work highlights sulfilimine bond formation as what is to our knowledge the first known physiologic function for peroxidasin, a role for hypohalous oxidants in tissue biogenesis, and a possible role for peroxidasin in inflammatory diseases.

Lens capsule as a model to study type IV collagen.

The study of collagen IV has benefited greatly from the seminal work conducted by Arthur Veis and colleagues over three decades ago. Through a series of electron microscopy studies focused on lens basement membrane, an appreciation was gained for the distinct network-forming properties of collagen IV. Veis correctly suggested that network assembly is a phenomenon of the non-collagenous termini of the molecule. This review seeks to document how the field advanced following these seminal conclusions, including recent discoveries regarding the molecular reinforcement of networks that support Veis' conclusions.

Collateral flow and brain changes on computed tomography angiography predict infarct volume on early diffusion-weighted imaging.

BACKGROUND: We investigated whether a computed tomography (CT)-based score could predict a large infarct (≥ 80 mL) on early diffusion-weighted magnetic resonance imaging (DWI). METHODS: Acute stroke patients considered for endovascular therapy within 8 hours of the onset of symptoms were included. The Alberta Stroke Program Early Computed Tomography Score (ASPECTS) was determined on noncontrast CT and computed tomography angiography source images (CTA-SI). Limited collateral flow was defined as less than 50% collateral filling on CTA-SI. RESULTS: Fifty-six patients were analyzed. National Institutes of Health Stroke Scale score was 20 (15-24) in the large infarct group and 16 (11-20) in the small infarct group (P = .049). ASPECTS on noncontrast CT and CTA-SI was 5 (3-8) and 3 (2-6) in the large infarct group and 9 (8-10) and 8 (7-9) in the small infarct group (both P < .001), respectively. Limited collateral flow was frequent in the large infarct group than in the small infarct group (92% vs. 11%, P < .001). Multivariate analysis found that CTA-SI ASPECTS less than or equal to 5 (odds ratio [OR], 40.55; 95% confidence interval [CI], 1.10-1493.44; P = .044) and limited collateral flow (OR, 114.64; 95% CI, 1.93-6812.79; P = .023) were associated with a large infarct. Absence of ASPECTS less than or equal to 5 and limited collateral flow on CTA-SI predicted absence of a large infarct with a sensitivity of .89, specificity of 1.00, positive predictive value of 1.00, and negative predictive value of .71. CONCLUSIONS: Assessment of ASPECTS and collateral flow on CTA-SI may be able to exclude a patient with large infarct on early DWI.

HiCrayon reveals distinct layers of multi-state 3D chromatin organization.

The co-visualization of chromatin conformation with 1D 'omics data is key to the multi-omics driven data analysis of 3D genome organization. Chromatin contact maps are often shown as 2D heatmaps and visually compared to 1D genomic data by simple juxtaposition. While common, this strategy is imprecise, placing the onus on the reader to align features with each other. To remedy this, we developed HiCrayon, an interactive tool that facilitates the integration of 3D chromatin organization maps and 1D datasets. This visualization method integrates data from genomic assays directly into the chromatin contact map by coloring interactions according to 1D signal. HiCrayon is implemented using R shiny and python to create a graphical user interface (GUI) application, available in both web or containerized format to promote accessibility. HiCrayon is implemented in R, and includes a graphical user interface (GUI), as well as a slimmed-down web-based version that lets users quickly produce publication-ready images. We demonstrate the utility of HiCrayon in visualizing the effectiveness of compartment calling and the relationship between ChIP-seq and various features of chromatin organization. We also demonstrate the improved visualization of other 3D genomic phenomena, such as differences between loops associated with CTCF/cohesin vs. those associated with H3K27ac. We then demonstrate HiCrayon's visualization of organizational changes that occur during differentiation and use HiCrayon to detect compartment patterns that cannot be assigned to either A or B compartments, revealing a distinct 3rd chromatin compartment. Overall, we demonstrate the utility of co-visualizing 2D chromatin conformation with 1D genomic signals within the same matrix to reveal fundamental aspects of genome organization. Local version: https://github.com/JRowleyLab/HiCrayon Web version: https://jrowleylab.com/HiCrayon.

Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography.

Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. In particular, the AAV purification pipeline hinges on protein ligands for the affinity-based capture step. While featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product's activity and stability. Additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, (ii) enable product elution under near-physiological conditions (pH 6.0), and (iii) grant extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades - namely, AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. This corroborates the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (≈1014 vp mL-1 ) and product yields (≈60%-80%) on par with commercial adsorbents, and purifies AAV2 from HEK293 and Sf9 cell lysates with high recovery (up to 78%), reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%).

Biallelic variants in POPDC2 cause a novel autosomal recessive syndrome presenting with cardiac conduction defects and variable hypertrophic cardiomyopathy.

POPDC2 encodes for the Popeye domain-containing protein 2 which has an important role in cardiac pacemaking and conduction, due in part to its cAMP-dependent binding and regulation of TREK-1 potassium channels. Loss of Popdc2 in mice results in sinus pauses and bradycardia and morpholino knockdown of popdc2 in zebrafish results in atrioventricular (AV) block. We identified bi-allelic variants in POPDC2 in 4 families that presented with a phenotypic spectrum consisting of sinus node dysfunction, AV conduction defects and hypertrophic cardiomyopathy. Using homology modelling we show that the identified POPDC2 variants are predicted to diminish the ability of POPDC2 to bind cAMP. In in vitro electrophysiological studies we demonstrated that, while co-expression of wild-type POPDC2 with TREK-1 increased TREK-1 current density, POPDC2 variants found in the patients failed to increase TREK-1 current density. While patient muscle biopsy did not show clear myopathic disease, it showed significant reduction of the expression of both POPDC1 and POPDC2, suggesting that stability and/or membrane trafficking of the POPDC1-POPDC2 complex is impaired by pathogenic variants in any of the two proteins. Single-cell RNA sequencing from human hearts demonstrated that co-expression of POPDC1 and 2 was most prevalent in AV node, AV node pacemaker and AV bundle cells. Sinoatrial node cells expressed POPDC2 abundantly, but expression of POPDC1 was sparse. Together, these results concur with predisposition to AV node disease in humans with loss-of-function variants in POPDC1 and POPDC2 and presence of sinus node disease in POPDC2, but not in POPDC1 related disease in human. Using population-level genetic data of more than 1 million individuals we showed that none of the familial variants were associated with clinical outcomes in heterozygous state, suggesting that heterozygous family members are unlikely to develop clinical manifestations and therefore might not necessitate clinical follow-up. Our findings provide evidence for POPDC2 as the cause of a novel Mendelian autosomal recessive cardiac syndrome, consistent with previous work showing that mice and zebrafish deficient in functional POPDC2 display sinus and AV node dysfunction.

Exposure to climate change information predicts public support for solar geoengineering in Singapore and the United States.

Solar geoengineering is a controversial climate policy measure that could lower global temperature by increasing the amount of light reflected by the Earth. As scientists and policymakers increasingly consider this idea, an understanding of the level and drivers of public support for its research and potential deployment will be key. This study focuses on the role of climate change information in public support for research and deployment of stratospheric aerosol injection (SAI) in Singapore (n = 503) and the United States (n = 505). Findings were consistent with the idea that exposure to information underlies support for research and deployment. That finding was stronger in the United States, where climate change is a more contentious issue, than in Singapore. Cost concern was negatively related to support for funding and perceived risk was negatively related to support for deployment. Perceived government efficacy was a more positive predictor of support for funding in Singapore than in the United States. Additionally, relatively low support for local deployment was consistent with a NIMBY mindset. This was the first study to quantify the role of climate change information in SAI policy support, which has practical implications for using the media and interpersonal channels to communicate about SAI policy measures.

The worsening divergence of biotechnology: the importance of risk culture.

In the last 20 years, the field of biotechnology has made significant progress and attracted substantial investments, leading to different paths of technological modernization among nations. As a result, there is now an international divide in the commercial and intellectual capabilities of biotechnology, and the implications of this divergence are not well understood. This raises important questions about why global actors are motivated to participate in biotechnology modernization, the challenges they face in achieving their goals, and the possible future direction of global biotechnology development. Using the framework of prospect theory, this paper explores the role of risk culture as a fundamental factor contributing to this divergence. It aims to assess the risks and benefits associated with the early adoption of biotechnology and the regulatory frameworks that shape the development and acceptance of biotechnological innovations. By doing so, it provides valuable insights into the future of biotechnology development and its potential impact on the global landscape.

Parameters, practices, and preferences for regulatory review of emerging biotechnology products in food and agriculture.

This paper evaluates the U.S. regulatory review of three emerging biotechnology products according to parameters, practices, and endpoints of assessments that are important to stakeholders and publics. First, we present a summary of the literature on variables that are important to non-expert publics in governing biotech products, including ethical, social, policy process, and risk and benefit parameters. Second, we draw from our USDA-funded project results that surveyed stakeholders with subject matter expertise about their attitudes towards important risk, benefit, sustainability, and societal impact parameters for assessing novel agrifood technologies, including biotech. Third, we evaluate the regulatory assessments of three food and agricultural biotechnology case studies that have been reviewed under U.S. regulatory agencies and laws of the Coordinated Framework for the Regulation of Biotechnology, including gene-edited soybeans, beef cattle, and mustard greens. Evaluation of the regulatory review process was based on parameters identified in steps 1 and 2 which were deemed important to both publics and stakeholders. Based on this review, we then propose several policy options for U.S. federal agencies to strengthen their oversight processes to better align with a broader range of parameters to support sustainable agrifood products that rely on novel technologies. These policy options include 1) those that would not require new institutions or legal foundations (such as conducting Environmental Impact Statements and/or requiring a minimal level of safety data), 2) those that would require a novel institutional or cross-institutional framework (such as developing a publicly-available website and/or performing holistic sustainability assessments), and 3) those that would require the agencies to have additional legal authorities (such as requiring agencies to review biotech products according to a minimal set of health, environmental, and socio-economic parameters). Overall, the results of this analysis will be important for guiding policy practice and formulation in the regulatory assessment of emerging biotechnology products that challenge existing legal and institutional frameworks.

Balancing climate resilience and adaptation for Caribbean Small Island Developing States (SIDS): Building institutional capacity.

Although the Caribbean's Small Island Developing States (SIDS) minimally contribute to global greenhouse gas emissions, they face disproportionate climate risks and are particularly susceptible to systemic economic threats posed by climate change and subsequent increases in climate variability. Historically, strategic programs and investments have sought to develop more robust and adaptive engineered systems to absorb climate threats. However, such initiatives are limited and under-resourced in the SIDS' context. This article reviews existing climate strategies in the Caribbean and then critically examines current gaps and barriers relating to climate impact knowledge, needs, and implementation. This examination can assist Caribbean SIDS leadership to identify opportunities to transition from a vulnerability-reducing mindset to one of resilience and transformative adaptation to improve long-term economic outlooks, social welfare, and environmental stewardship despite recurring and escalating climate risks. Integr Environ Assess Manag 2023;00:1-19. © 2023 SETAC.

Informing environmental health and risk priorities through local outreach and extension.

Our society is currently facing an unprecedented number of environmental and societal challenges. Stakeholder and community engagement can help identify priority issues and needs at local levels. One approach to engage stakeholders and communities in the contexts of environmental, health, and societal challenges is to leverage outreach and extension programs. Within this context, and to help identify priority issues to focus subsequent research and extension programs in North Carolina (NC), a survey was conducted with extension agents to identify priority issues as they relate to environmental health and risks and related needs. Based on responses from 66 study participants that represented half of the 100 NC counties, we found that Water pollution, Flooding, Natural resources management, and Engaging stakeholders were top priority issues across all environmental health and risk topics. Participants also identified that practices of Engaging stakeholders as well as Assessing, Managing, and Communicating risks were increasingly important. Participants indicated they needed a moderate-to-significant amount of guidance across a range of areas related to assessing, managing, communicating, and making decisions regarding environmental health and risk topics, as well as engaging with local communities. Outcomes from this work can not only help inform subsequent research and outreach efforts at local scales, but this work demonstrates a simple, low-cost approach to elicit perspectives and priorities can be leveraged in other states and regions with established stakeholder and community outreach programs more broadly. Supplementary Information: The online version contains supplementary material available at 10.1007/s10669-022-09864-0.

Implications of Dosage Deficiencies in CTCF and Cohesin on Genome Organization, Gene Expression, and Human Neurodevelopment.

Properly organizing DNA within the nucleus is critical to ensure normal downstream nuclear functions. CTCF and cohesin act as major architectural proteins, working in concert to generate thousands of high-intensity chromatin loops. Due to their central role in loop formation, a massive research effort has been dedicated to investigating the mechanism by which CTCF and cohesin create these loops. Recent results lead to questioning the direct impact of CTCF loops on gene expression. Additionally, results of controlled depletion experiments in cell lines has indicated that genome architecture may be somewhat resistant to incomplete deficiencies in CTCF or cohesin. However, heterozygous human genetic deficiencies in CTCF and cohesin have illustrated the importance of their dosage in genome architecture, cellular processes, animal behavior, and disease phenotypes. Thus, the importance of considering CTCF or cohesin levels is especially made clear by these heterozygous germline variants that characterize genetic syndromes, which are increasingly recognized in clinical practice. Defined primarily by developmental delay and intellectual disability, the phenotypes of CTCF and cohesin deficiency illustrate the importance of architectural proteins particularly in neurodevelopment. We discuss the distinct roles of CTCF and cohesin in forming chromatin loops, highlight the major role that dosage of each protein plays in the amplitude of observed effects on gene expression, and contrast these results to heterozygous mutation phenotypes in murine models and clinical patients. Insights highlighted by this comparison have implications for future research into these newly emerging genetic syndromes.

Evaluation of Risk Factors and Approach to Screening for Asymptomatic Neonatal Hypoglycemia.

INTRODUCTION: Protocols to identify asymptomatic neonatal hypoglycemia (NH) rely on the presence of established risk factors (late preterm gestation, large or small for gestational age, and infant of a diabetic mother) for inclusion. We analyzed the performance of these risk factors in identifying hypoglycemia in modern practice, and additionally evaluated the optimal duration of screening blood glucose measurements. METHODS: We analyzed a retrospective cohort of 830 infants with 1 or more known risk factor(s) for NH admitted to the mother-baby unit of a single tertiary-care center from May 2017 to April 2018. Manual chart review was performed for data extraction and confirmation of risk factor(s). Infants were excluded if glucose measurements were obtained for any reason other than screening for asymptomatic NH. RESULTS: Of the 830 included infants, 31 (3.7%) ultimately received intravenous dextrose (IVD). Most screened infants (n = 510, 61.4%) did not develop hypoglycemia. None of the established risk factors showed strong association with hypoglycemia. Cesarean delivery was associated with hypoglycemia, although not strongly. All infants who received IVD for feeding-refractory hypoglycemia were identified by the first 2 measurements with nearly all (30/31, 97%) identified at the initial measurement. CONCLUSIONS: Currently accepted risk factors are limited in their ability to identify infants who subsequently develop hypoglycemia, and as a result, most screened infants do not develop hypoglycemia. The majority of infants in our cohort who did develop hypoglycemia achieved normoglycemia with feeding-based interventions and did not require IVD. Those that received IVD were more likely to develop hypoglycemia early and to a more severe degree. Together, our data suggest further refinement of protocol duration and risk factors utilized for screening as potential areas of screening protocol optimization.