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Background:. Geographic information systems are powerful tools for characterizing the geospatial factors influencing access to care. As patients with cleft lip and/or palate (CL/P) require long-term care, with numerous operations and therapies, access to timely, quality care is extremely important. This study uses population level analysis and geographic information systems to identify United States counties with limited access to American Cleft Palate Association–approved cleft teams. Methods:. Natality data were queried from the National Vital Statistics System. Population and geographic data were obtained from the US Census Bureau. The Social Vulnerability Index (SVI) was utilized to account for social inequality. Total births with CL/P, population estimates, SVI, distance to the nearest center, and total centers within 50 km were used to generate the cleft care demand index (CCDI). Results:. Ninety-two counties had CCDIs between 66.7 and 100. The highest scoring county, Hidalgo County, Texas, had 62 births with CL/P, population estimate of 888,367 persons, distance to the nearest cleft center of 368.4 km, and SVI of 0.99. Conclusions:. This study demonstrates the power of geographic information systems for identifying areas with limited access to approved cleft teams. The CCDI measures cleft burden, socioeconomic disadvantage, and geographic barriers to quantify the demand for approved cleft care in each county. Utilizing these scores can help direct future interventions, outreach efforts, and cleft care center planning.

Background. Variation in donation after circulatory death (DCD) organ recovery and liver transplant practices exist among transplant centers. This study aimed to evaluate these practices among centers in the United States. Methods. Scientific Registry of Transplant Recipients data were accessed to identify centers that performed liver transplantation in 2021 and 2022. Surveys were sent to transplant centers that consistently performed ≥5 DCD liver transplants per year. Results. DCD liver transplants were performed by 95 centers (65.1%) of the 146 liver transplant centers in the United States. Survey results were recorded from 42 centers that consistently performed ≥5 DCD liver transplants per year, with a 59.5% response rate. Withdrawal-to-asystole and agonal time were used to define donor warm ischemia time (WIT) in 16% and 84% centers, respectively. Fifty-six percent of the centers did not use oxygen saturation to define donor WIT. Systolic blood pressure cutoffs used to define agonal time varied between 50 and 80 mm Hg, donor age cutoffs ranged between 55 and 75 y, and cold ischemia times varied between 4 and 10 h. Seventy-six percent of centers used normothermic machine perfusion for DCD liver transplantation. Conclusions. This study highlights the wide variation in use, recovery, and definition of donor WIT. Using national data to rigorously define best practices will encourage greater utilization of this important donor resource.

Background Social determinants of health (SDOH) play a significant role in the development of cardiovascular risk factors. We investigated SDOH associations with cardiovascular risk factors among Asian American subgroups. Methods and Results We utilized the National Health Interview Survey, a nationally representative survey of US adults, years 2013 to 2018. SDOH variables were categorized into economic stability, neighborhood and social cohesion, food security, education, and health care utilization. SDOH score was created by categorizing 27 SDOH variables as 0 (favorable) or 1 (unfavorable). Self‐reported cardiovascular risk factors included diabetes, high cholesterol, high blood pressure, obesity, insufficient physical activity, suboptimal sleep, and nicotine exposure. Among 6395 Asian adults aged ≥18 years, 22.1% self‐identified as Filipino, 21.6% as Asian Indian, 21.0% as Chinese, and 35.3% as other Asian. From multivariable‐adjusted logistic regression models, each SD increment of SDOH score was associated with higher odds of diabetes among Chinese (odds ratio [OR], 1.45; 95% CI, 1.04–2.03) and Filipino (OR, 1.24; 95% CI, 1.02–1.51) adults; high blood pressure among Filipino adults (OR, 1.28; 95% CI, 1.03–1.60); insufficient physical activity among Asian Indian (OR, 1.42; 95% CI, 1.22–1.65), Chinese (OR, 1.58; 95% CI, 1.33–1.88), and Filipino (OR, 1.24; 95% CI, 1.06–1.46) adults; suboptimal sleep among Asian Indian adults (OR, 1.20; 95% CI, 1.01–1.42); and nicotine exposure among Chinese (OR, 1.56; 95% CI, 1.15–2.11) and Filipino (OR, 1.50; 95% CI, 1.14–1.97) adults. Conclusions Unfavorable SDOH are associated with higher odds of cardiovascular risk factors in Asian American subgroups. Culturally specific interventions addressing SDOH may help improve cardiovascular health among Asian Americans.

IntroductionVascular and mitochondrial dysfunction are well-established consequences of multiple central nervous system (CNS) disorders, including neurodegenerative diseases and traumatic injuries. We previously reported that 5-hydroxytryptamine 1F receptor (5-HT1FR) agonism induces mitochondrial biogenesis (MB) in multiple organ systems, including the CNS.MethodsLasmiditan is a selective 5-HT1FR agonist that is FDA-approved for the treatment of migraines. We have recently shown that lasmiditan treatment induces MB, promotes vascular recovery and improves locomotor function in a mouse model of spinal cord injury (SCI). To investigate the mechanism of this effect, primary cerebral microvascular endothelial cells from C57bl/6 mice (mBMEC) were used.ResultsLasmiditan treatment increased the maximal oxygen consumption rate, mitochondrial proteins and mitochondrial density in mBMEC, indicative of MB induction. Lasmiditan also enhanced endothelial cell migration and tube formation, key components of angiogenesis. Trans-endothelial electrical resistance (TEER) and tight junction protein expression, including claudin-5, were also increased with lasmiditan, suggesting improved barrier function. Finally, lasmiditan treatment decreased phosphorylated VE-Cadherin and induced activation of the Akt-FoxO1 pathway, which decreases FoxO1-mediated inhibition of claudin-5 transcription.DiscussionThese data demonstrate that lasmiditan induces MB and enhances endothelial cell function, likely via the VE-Cadherin-Akt-FoxO1-claudin-5 signaling axis. Given the importance of mitochondrial and vascular dysfunction in neuropathologies, 5-HT1FR agonism may have broad therapeutic potential to address multiple facets of disease progression by promoting MB and vascular recovery.

Astrocytes are a widely heterogenic cell population that play major roles in central nervous system (CNS) homeostasis and neurotransmission, as well as in various neuropathologies, including spinal cord injury (SCI), traumatic brain injury, and neurodegenerative diseases, such as amyotrophic lateral sclerosis. Spinal cord astrocytes have distinct differences from those in the brain and accurate modeling of disease states is necessary for understanding disease progression and developing therapeutic interventions. Several limitations to modeling spinal cord astrocytes in vitro exist, including lack of commercially available adult-derived cells, lack of purchasable astrocytes with different genotypes, as well as time-consuming and costly in-house primary cell isolations that often result in low yield due to small tissue volume. To address these issues, we developed an efficient adult mouse spinal cord astrocyte isolation method that utilizes enzymatic digestion, debris filtration, and multiple ACSA-2 magnetic microbead purification cycles to achieve an astrocyte monoculture purity of ≅93–98%, based on all markers assessed. Importantly, the isolated cells contain active mitochondria and express key astrocyte markers including ACSA-1, ACSA-2, EAAT2, and GFAP. Furthermore, this isolation method can be applied to the spinal cord of male and female mice, mice subjected to SCI, and genetically modified mice. We present a primary adult mouse spinal cord astrocyte isolation protocol focused on purity, viability, and length of isolation that can be applied to a multitude of models and aid in targeted research on spinal-cord related CNS processes and pathologies.

Introduction. Patients with hormone receptor-positive (HR+), HER2-negative (HER2−) breast cancers have the lowest response to neoadjuvant therapy of all subtypes. The role of neoadjuvant endocrine therapy (NET) in clinically node-positive (cN+), HR+, HER2− patients is evaluated in this meta-analysis. Methods. This study was performed between January 2010 and August 2022. We evaluated the node pathologic complete response (pCR) and axillary lymph node dissection (ALND) rates after neoadjuvant endocrine therapy (NET). Results. 18,037 HR+, HER2−, cN+ stage II and stage III breast cancer patients within eleven studies received neoadjuvant treatments. 3,707 (20.6%) patients received NET and 14,330 (79.4%) received NAC. The average age of the NET patients was higher than that of the neoadjuvant chemotherapy (NAC) patients (64.1 versus 47.6 years old, p

BackgroundThe application of videoconferencing to the assessment and treatment of aphasia has been rapidly increasing; however, there is a need to develop treatments targeting sentence production in persons with aphasia (PWA) that can be delivered through videoconferencing. Structural priming has received recent attention as a potential training method for PWA. We investigated the feasibility and efficacy of a collaborative structural priming task delivered via the internet, TelePriming, in facilitating sentence production in PWA and healthy adults.MethodIn Study 1, young adults (YA), older adults (OA), and PWA participated in a collaborative dialogue-like priming task through videoconferencing, taking turns with an interlocutor (experimenter) to describe transitive action pictures with the goal of finding matching pictures. We measured whether participants produced more passive sentences to describe their picture after hearing their interlocutor produce passive compared to active sentences (primes). In Study 2, we compared the data from the OA and PWA of Study 1 (TelePriming) to different groups of OA and PWA, who completed the same priming task in person.ResultsAll three groups showed robust priming effects in Study 1, producing more passive sentences to describe target pictures after hearing the experimenter produce passive versus active sentences. In Study 2, when controlling for demographic information (age, education) and aphasia severity, TelePriming resulted in larger priming effects for OA and PWA, compared to the in-person priming task. Survey results revealed that both OA and PWA experienced increased comfort and satisfaction with using technology following the task.ConclusionInteractive message-structure alignment processes remain largely intact in PWA, and the positive effects of structural priming in a collaborative communicative task are not diminished by remote delivery. The findings demonstrate the feasibility and validity of TelePriming in OA and PWA, laying experimental groundwork for future use of TelePriming in the assessment and treatment of clinical populations with limited access to face-to-face sessions.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 β-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1’s role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Summary:. Lymphaticovenous anastomosis (LVA) surgery is an effective surgery for the treatment of lymphedema in the extremities. Indocyanine green lymphography is the reference standard for visualizing lymphatics for LVA surgery, but it has several limitations; most notably, superficial dermal congestion can mask deeper lymphatic vessels. To overcome the limitations, we add contrast-enhanced ultrasound (CEUS) lymphography. We have previously reported that CEUS lymphography can identify lymphatic vessels for LVA surgery that indocyanine green lymphography does not. Here, we describe how we perform CEUS lymphography, including workflow, technique, and documentation. Before informed consent, the patient must be screened for possible adverse reactions to microbubbles. The procedure involves multiple intradermal injections of the microbubble agent at various sites along the extremity. After each injection, imaging for microbubble uptake by lymphatic vessels is performed using an ultrasound scanner with contrast-specific software. We use sulfur hexafluoride lipid-type A microspheres (Lumason/SonoVue; Bracco Suisse SA), but we are investigating the performance of other Food & Drug Administration–approved microbubble agents for CEUS lymphography. Having a systematic approach to marking the skin can mitigate the hindrance of marking over ultrasound coupling gel. Another benefit of CEUS lymphography is the rapid identification of neighboring veins compatible in size and location for anastomosis. We hold regular scheduled multidisciplinary meetings for coordination of care, discussion of outcomes, quality assurance, and ongoing innovation.

Background As partial pressure of oxygen (pO2) rises with the first breath, the ductus arteriosus (DA) constricts, diverting blood flow to the pulmonary circulation. The DA's O2 sensor resides within smooth muscle cells. The DA smooth muscle cells’ mitochondrial electron transport chain (ETC) produces reactive oxygen species (ROS) in proportion to oxygen tension, causing vasoconstriction by regulating redox‐sensitive ion channels and enzymes. To identify which ETC complex contributes most to DA O2 sensing and determine whether ROS mediate O2 sensing independent of metabolism, we used electron leak suppressors, S1QEL (suppressor of site IQ electron leak) and S3QEL (suppressor of site IIIQo electron leak), which decrease ROS production by inhibiting electron leak from quinone sites IQ and IIIQo, respectively. Methods and Results The effects of S1QEL, S3QEL, and ETC inhibitors (rotenone and antimycin A) on DA tone, mitochondrial metabolism, O2‐induced changes in intracellular calcium, and ROS were studied in rabbit DA rings, and human and rabbit DA smooth muscle cells. S1QEL's effects on DA patency were assessed in rabbit kits, using micro computed tomography. In DA rings, S1QEL, but not S3QEL, reversed O2‐induced constriction (P=0.0034) without reducing phenylephrine‐induced constriction. S1QEL did not inhibit mitochondrial metabolism or ETC‐I activity. In human DA smooth muscle cells, S1QEL and rotenone inhibited O2‐induced increases in intracellular calcium (P=0.02 and 0.001, respectively), a surrogate for DA constriction. S1QEL inhibited O2‐induced ROS generation (P=0.02). In vivo, S1QEL prevented O2‐induced DA closure (P

This practice report explores a Collaborative Online International Learning (COIL) course focused on awareness and esteem development for women teachers in South Sudan and volunteers associated with a US-based university. Pedagogy of hope was utilized as an educational tool for implementation and evaluation. Assessments of this COIL course focused on data from exit tickets and a final writing assignment. Self-perceptions of the participating teachers were analyzed based on their sense of preparedness, confidence, comfort levels with the class, and any impact the course had on teachers’ sense of teaching as a personal calling. Practitioners and teaching professionals interested in international virtual exchange will find key takeaways related to building partnerships starting with low-stakes initiatives and using COIL to help ignite advances in digital maturity in South Sudan.

Microbial communities contain a broad phylogenetic diversity of organisms; however, the majority of methods center on describing bacteria and archaea. Fungi are important symbionts in many ecosystems and are potentially important members of the human microbiome, beyond those that can cause disease. To expand our analysis of microbial communities to include data from the fungal internal transcribed spacer (ITS) region, five candidate DNA extraction kits were compared against our standardized protocol for describing bacteria and archaea using 16S rRNA gene amplicon- and shotgun metagenomics sequencing. The results are presented considering a diverse panel of host-associated and environmental sample types and comparing the cost, processing time, well-to-well contamination, DNA yield, limit of detection and microbial community composition among protocols. Across all criteria, the MagMAX Microbiome kit was found to perform best. The PowerSoil Pro kit performed comparably but with increased cost per sample and overall processing time. The Zymo MagBead, NucleoMag Food and Norgen Stool kits were included.

Abstract Due to the ubiquity of microplastic contamination in coastal waters, there is potential for adverse impacts to organism development. One organism of interest is the daggerblade grass shrimp, Palaemon pugio, an ecologically important species in estuaries along the east coast of North America. We exposed larval grass shrimp to virgin polyethylene microbeads (35 and 58 µm) at a high (0.375 and 1.95 mg/L), medium (0.0375 and 0.195 mg/L), and a low concentration (0.00375 and 0.0195 mg/L), respectively for 23 days to assess mortality, transformation time from larval to juvenile stage, and weight. Average percent mortality was 3.7 to 4.8 times higher in the experimental treatments compared to controls. The greatest proportion of mortality was observed in the first 11 days. Median time for transformation ranged from 20.2 to 20.8 days. Shrimp exposed to the 35 µm beads in the high treatment (20.2 days) transformed significantly faster than the control shrimp (20.8 days). Although development was not delayed and size of the shrimp did not differ, the acute toxicity of microplastics on grass shrimp is a concern due to their role in energy cycling within tidal-creeks. These findings suggest potential population and community level effects following microplastic exposure.

Abstract Background Depression and obesity are highly prevalent, often co-occurring conditions marked by inflammation. Microbiome perturbations are implicated in obesity-inflammation-depression interrelationships, but how the microbiome mechanistically contributes to pathology remains unclear. Metabolomic investigations into microbial neuroactive metabolites may offer mechanistic insights into host-microbe interactions. Using 16S sequencing and untargeted mass spectrometry of saliva, and blood monocyte inflammation regulation assays, we identified key microbes, metabolites and host inflammation in association with depressive symptomatology, obesity, and depressive symptomatology-obesity comorbidity. Results Gram-negative bacteria with inflammation potential were enriched relative to Gram-positive bacteria in comorbid obesity-depression, supporting the inflammation-oral microbiome link in obesity-depression interrelationships. Oral microbiome was more highly predictive of depressive symptomatology-obesity co-occurrences than of obesity or depressive symptomatology independently, suggesting specific microbial signatures associated with obesity-depression co-occurrences. Mass spectrometry analysis revealed significant changes in levels of signaling molecules of microbiota, microbial or dietary derived signaling peptides and aromatic amino acids among depressive symptomatology, obesity and comorbid obesity-depression. Furthermore, integration of the microbiome and metabolomics data revealed that key oral microbes, many previously shown to have neuroactive potential, co-occurred with potential neuropeptides and biosynthetic precursors of the neurotransmitters dopamine, epinephrine and serotonin. Conclusions Together, our findings offer novel insights into oral microbial-brain connection and potential neuroactive metabolites involved.

Background & Aims: Hyperbaric oxygen therapy (HBOT) is a promising treatment for moderate-to-severe ulcerative colitis. However, our current understanding of the host and microbial response to HBOT remains unclear. This study examined the molecular mechanisms underpinning HBOT using a multi-omic strategy. Methods: Pre- and post-intervention mucosal biopsies, tissue, and fecal samples were collected from HBOT phase 2 clinical trials. Biopsies and fecal samples were subjected to shotgun metaproteomics, metabolomics, 16s rRNA sequencing, and metagenomics. Tissue was subjected to bulk RNA sequencing and digital spatial profiling (DSP) for single-cell RNA and protein analysis, and immunohistochemistry was performed. Fecal samples were also used for colonization experiments in IL10-/- germ-free UC mouse models. Results: Proteomics identified negative associations between HBOT response and neutrophil azurophilic granule abundance. DSP identified an HBOT-specific reduction of neutrophil STAT3, which was confirmed by immunohistochemistry. HBOT decreased microbial diversity with a proportional increase in Firmicutes and a secondary bile acid lithocholic acid. A major source of the reduction in diversity was the loss of mucus-adherent taxa, resulting in increased MUC2 levels post-HBOT. Targeted database searching revealed strain-level associations between Akkermansia muciniphila and HBOT response status. Colonization of IL10-/- with stool obtained from HBOT responders resulted in lower colitis activity compared with non-responders, with no differences in STAT3 expression, suggesting complementary but independent host and microbial responses. Conclusions: HBOT reduces host neutrophil STAT3 and azurophilic granule activity in UC patients and changes in microbial composition and metabolism in ways that improve colitis activity. Intestinal microbiota, especially strain level variations in A muciniphila, may contribute to HBOT non-response.

Background:. The Accreditation Council for Graduate Medical Education (ACGME) implemented duty-hour restrictions limiting residents to 80 hours per week in 2003 and further extended restrictions in 2011 to improve resident and patient well-being. Numerous studies have examined the effects of these restrictions on patient outcomes with inconclusive results. Few efforts have been made to examine the impact of this reform on the safety of common plastic surgery procedures. This study seeks to assess the influence of ACGME duty-hour restrictions on patient outcomes, using bilateral breast reduction mammoplasty as a marker for resident involvement and operative autonomy. Methods:. Bilateral breast reductions performed in the 3 years before and after each reform were collected from the National Inpatient Sample database: pre-duty hours (2000–2002), duty hours (2006–2008), and extended duty hours (2012–2014). Multivariable logistic regression models were constructed to investigate the association between ACGME duty hour restrictions on medical and surgical complications. Results:. Overall, 19,423 bilateral breast reductions were identified. Medical and surgical complication rates in these patients increased with each successive iteration of duty hour restrictions (P < 0.001). The 2003 duty-hour restriction independently associated with increased surgical (OR = 1.51, P < 0.001) and medical complications (OR = 1.85, P < 0.001). The 2011 extended duty-hour restriction was independently associated with increased surgical complications (OR = 1.39, P < 0.001). Conclusions:. ACGME duty-hour restrictions do not seem associated with better patient outcomes for bilateral breast reduction although there are multiple factors involved. These considerations and consequences should be considered in decisions that affect resident quality of life, education, and patient safety.

During an episode of acute kidney injury (AKI), a sudden and rapid decline in renal function is often accompanied by a persistent reduction in mitochondrial function, microvasculature dysfunction/rarefaction, and tubular epithelial injury/necrosis. Additionally, patients who have experienced an AKI are at an elevated risk of developing other progressive renal, cardiovascular, and cardiorenal related diseases. While restoration of the microvasculature is imperative for oxygen and nutrient delivery/transport during proper renal repair processes, the mechanism(s) by which neovascularization and/or inhibition of microvascular dysfunction improves renal recovery remain understudied. Interestingly, pharmacological stimulation of mitochondrial biogenesis (MB) post-AKI has been shown to restore mitochondrial and renal function in mice. Thus, targeting MB pathways in microvasculature endothelial cell (MV-EC) may provide a novel strategy to improve renal vascular function and repair processes post-AKI. However, limitations to studying such mechanisms include a lack of commercially available primary renal peritubular MV-ECs, the variability in both purity and outgrowth of primary renal MV-EC in monoculture, the tendency of primary renal MV-ECs to undergo phenotypic loss in primary monoculture, and a limited quantity of published protocols to obtain primary renal peritubular MV-ECs. Thus, we focused on refining the isolation and phenotypic retention of mouse renal peritubular endothelial cells (MRPEC) for future physiological and pharmacological based studies. Here, we present a refined isolation method that augments the purity, outgrowth, and phenotypic retention of primary MRPEC monocultures by utilizing a collagenase type I enzymatic digestion, CD326+ (EPCAM) magnetic microbead epithelial cell depletion, and two CD146+ (MCAM) magnetic microbead purification cycles to achieve a monoculture MRPEC purity of ≅ 91–99% by all markers evaluated.

Transplant oncology is a relatively new field in which transplantation is used to treat patients who would otherwise be unresectable. New anticancer treatment paradigms using tumor and transplant immunology and cancer immunogenomics are emerging. In turn, liver transplantation (LT) has become a potential therapy for certain patients with colorectal cancer (CRC) with liver metastasis, hepatocellular (HCC), cholangiocarcinoma (CCA), and metastatic neuroendocrine tumor (NET) of the liver. Although there are established criteria for LT in HCC, evidence regarding LT as a treatment modality for certain gastrointestinal malignancies is still debated. The aim of this review is to highlight updates in the role of LT for certain malignancies, including HCC, metastatic CRC, hilar CCA, and neuroendocrine tumor (NET), as well as contextualize LT use and discuss controversies in transplant oncology.

The COVID-19 pandemic poses a significant risk for immunosuppressed groups such as transplant patients. The purpose of this study was to improve our understanding of the impact of the COVID-19 pandemic on kidney transplant recipients, including their views on COVID-19 vaccination. Semi-structured interviews were conducted from December 2021 to August 2022 with 38 kidney transplant recipients who had an appointment with their transplant care team within the previous 6 months. We used qualitative thematic analysis to characterize the perspectives of interviewees. Regardless of COVID-19 vaccination status, most interviewees reported utilizing public health measures such as masking, hand washing, and avoiding crowds to protect themselves against COVID-19. Vaccinated interviewees (n = 31) noted that they chose to receive a COVID-19 vaccine because of their increased risk due to their immunocompromised state. For unvaccinated interviewees (n = 7), reasons for not receiving a COVID-19 vaccine included concerns about the safety and efficacy of the vaccine. Both vaccinated and unvaccinated interviewees expressed concerns about the lack of adequate testing of the vaccine in transplant patients and questioned if the vaccine might have unknown side effects for transplant recipients. Regardless of the vaccination status, most interviewees noted having trust in their healthcare team. Interviewees also described interpersonal tensions that arose during the pandemic, many of which surrounded vaccination and other preventive measures that were important to participants to protect their health. Together, these data demonstrate differing concerns and experiences related to the COVID-19 pandemic for vaccinated and unvaccinated transplant recipients. These findings highlight the unique needs of transplant recipients and reveal opportunities to support this vulnerable patient population in efforts to protect their health as the COVID-19 pandemic evolves.

Nuclear magnetic resonance (NMR) is a non-destructive and atom-specific specific tool that has become a burgeoning analytic method for understanding the detailed molecular interactions in catalysis and energy storage materials. However, the observation of diverse chemical shifts arising from complex molecular interactions makes the interpretation of NMR spectroscopy increasingly challenging, in particular for a novel system without standards for comparison. Density functional theory-NMR (DFT-NMR) is an indispensable tool to mitigate these challenges and provide detailed 3D molecular structures that relate materials and reaction intermediate structures, and information about chemical interactions, dynamics, and reaction mechanisms. This review provides a fundamental background in DFT-NMR relating to theory development, critical parameters for calculating NMR properties, computational accuracy, and the current capabilities. A variety of practical examples from the fields of catalysis and energy storage, including CO2 capture, are summarized to illustrate the capabilities of DFT-NMR application to date. Last but not least, cautionary notes on the application of these strategies are presented for researchers modeling their own systems.

One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols with the authors' previously benchmarked protocol. The authors included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here the authors present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection and well-to-well contamination between these protocols.

Inter-individual differences in the gut microbiome are linked to alterations in inflammation and blood–brain barrier permeability, which may increase the risk of depression in people with HIV (PWH). The microbiome profile of blood, which is considered by many to be typically sterile, remains largely unexplored. We aimed to characterize the blood plasma microbiome composition and assess its association with major depressive disorder (MDD) in PWH and people without HIV (PWoH). In this cross-sectional, observational cohort, we used shallow-shotgun metagenomic sequencing to characterize the plasma microbiome of 151 participants (84 PWH and 67 PWoH), all of whom underwent a comprehensive neuropsychiatric assessment. The microbial composition did not differ between PWH and PWoH or between participants with MDD and those without it. Using the songbird model, we computed the log ratio of the highest and lowest 30% of the ranked classes associated with HIV and MDD. We found that HIV infection and lifetime MDD were enriched in a set of differentially abundant inflammatory classes, such as Flavobacteria and Nitrospira. Our results suggest that the circulating plasma microbiome may increase the risk of MDD related to dysbiosis-induced inflammation in PWH. If confirmed, these findings may indicate new biological mechanisms that could be targeted to improve treatment of MDD in PWH.

Flash nanoprecipitation (FNP) is a turbulent mixing process capable of reproducibly producing polymer nanoparticles loaded with active pharmaceutical ingredients (APIs). The nanoparticles produced with this method consist of a hydrophobic core surrounded by a hydrophilic corona. FNP produces nanoparticles with very high loading levels of nonionic hydrophobic APIs. However, hydrophobic compounds with ionizable groups are not as efficiently incorporated. To overcome this, ion pairing agents (IPs) can be incorporated into the FNP formulation to produce highly hydrophobic drug salts that efficiently precipitate during mixing. We demonstrate the encapsulation of the PI3K inhibitor, LY294002, within poly(ethylene glycol)-b-poly(D,L lactic acid) nanoparticles. We investigated how incorporating two hydrophobic IPs (palmitic acid (PA) and hexadecylphosphonic acid (HDPA)) during the FNP process affected the LY294002 loading and size of the resulting nanoparticles. The effect of organic solvent choice on the synthesis process was also examined. While the presence of either hydrophobic IP effectively increased the encapsulation of LY294002 during FNP, HDPA resulted in well-defined colloidally stable particles, while the PA resulted in ill-defined aggregates. The incorporation of hydrophobic IPs with FNP opens the door for the intravenous administration of APIs that were previously deemed unusable due to their hydrophobic nature.

Abstract The COVID-19 pandemic has claimed the lives of over one million people worldwide, and has affected all aspects of healthcare worldwide, including the delivery of care to patients with fertility-related diagnoses. In the United States, the response of US fertility clinics to the COVID-19 pandemic was coordinated by the American Society for Reproductive Medicine (ASRM). ASRM acted quickly to develop guidelines for limiting fertility treatment and clinic consultations during the early days of the pandemic, and then safely restarting fertility treatment. A survey of patients with fertility-related diagnoses who presented for care during the first months of the pandemic revealed that a majority of patients who presented for care during the early months of the pandemic experienced delayed or cancelled treatment cycles. Patients with infertility subsequently reported a desire to resume fertility care, but emphasized the importance of their clinic having policies and procedures in place to limit the risk of infection.

Abstract Background Determining the role of fomites in the transmission of SARS-CoV-2 is essential in the hospital setting and will likely be important outside of medical facilities as governments around the world make plans to ease COVID-19 public health restrictions and attempt to safely reopen economies. Expanding COVID-19 testing to include environmental surfaces would ideally be performed with inexpensive swabs that could be transported safely without concern of being a source of new infections. However, CDC-approved clinical-grade sampling supplies and techniques using a synthetic swab are expensive, potentially expose laboratory workers to viable virus and prohibit analysis of the microbiome due to the presence of antibiotics in viral transport media (VTM). To this end, we performed a series of experiments comparing the diagnostic yield using five consumer-grade swabs (including plastic and wood shafts and various head materials including cotton, synthetic, and foam) and one clinical-grade swab for inhibition to RNA. For three of these swabs, we evaluated performance to detect SARS-CoV-2 in twenty intensive care unit (ICU) hospital rooms of patients including COVID-19+ patients. All swabs were placed in 95% ethanol and further evaluated in terms of RNase activity. SARS-CoV-2 was measured both directly from the swab and from the swab eluent. Results Compared to samples collected in VTM, 95% ethanol demonstrated significant inhibition properties against RNases. When extracting directly from the swab head as opposed to the eluent, RNA recovery was approximately 2–4× higher from all six swab types tested as compared to the clinical standard of testing the eluent from a CDC-approved synthetic (SYN) swab. The limit of detection (LoD) of SARS-CoV-2 from floor samples collected using the consumer-grade plastic (CGp) or research-grade plastic The Microsetta Initiative (TMI) swabs was similar or better than the SYN swab, further suggesting that swab type does not impact RNA recovery as measured by the abundance of SARS-CoV-2. The LoD for TMI was between 0 and 362.5 viral particles, while SYN and CGp were both between 725 and 1450 particles. Lastly microbiome analyses (16S rRNA gene sequencing) of paired samples (nasal and floor from same patient room) collected using different swab types in triplicate indicated that microbial communities were not impacted by swab type, but instead driven by the patient and sample type. Conclusions Compared to using a clinical-grade synthetic swab, detection of SARS-CoV-2 from environmental samples collected from ICU rooms of patients with COVID was similar using consumer-grade swabs, stored in 95% ethanol. The yield was best from the swab head rather than the eluent and the low level of RNase activity and lack of antibiotics in these samples makes it possible to perform concomitant microbiome analyses. Video abstract

Background. Quality in liver transplantation (LT) is currently measured using 1-y patient and graft survival. Because patient and graft survival rates now exceed 90%, more informative metrics are needed. Textbook outcomes (TOs) describe ideal patient outcomes after surgery. This study critically evaluates TO as a quality metric in LT. Methods. United Network for Organ Sharing data for 25 887 adult LT recipients were used to define TO as patient and graft survival >1 y, length of stay ≤10 d, 0 readmissions within 6 mo, absence of rejection, and bilirubin

ABSTRACT Increasing data volumes on high-throughput sequencing instruments such as the NovaSeq 6000 leads to long computational bottlenecks for common metagenomics data preprocessing tasks such as adaptor and primer trimming and host removal. Here, we test whether faster recently developed computational tools (Fastp and Minimap2) can replace widely used choices (Atropos and Bowtie2), obtaining dramatic accelerations with additional sensitivity and minimal loss of specificity for these tasks. Furthermore, the taxonomic tables resulting from downstream processing provide biologically comparable results. However, we demonstrate that for taxonomic assignment, Bowtie2’s specificity is still required. We suggest that periodic reevaluation of pipeline components, together with improvements to standardized APIs to chain them together, will greatly enhance the efficiency of common bioinformatics tasks while also facilitating incorporation of further optimized steps running on GPUs, FPGAs, or other architectures. We also note that a detailed exploration of available algorithms and pipeline components is an important step that should be taken before optimization of less efficient algorithms on advanced or nonstandard hardware. IMPORTANCE In shotgun metagenomics studies that seek to relate changes in microbial DNA across samples, processing the data on a computer often takes longer than obtaining the data from the sequencing instrument. Recently developed software packages that perform individual steps in the pipeline of data processing in principle offer speed advantages, but in practice they may contain pitfalls that prevent their use, for example, they may make approximations that introduce unacceptable errors in the data. Here, we show that differences in choices of these components can speed up overall data processing by 5-fold or more on the same hardware while maintaining a high degree of correctness, greatly reducing the time taken to interpret results. This is an important step for using the data in clinical settings, where the time taken to obtain the results may be critical for guiding treatment.

ABSTRACT We introduce the operational genomic unit (OGU) method, a metagenome analysis strategy that directly exploits sequence alignment hits to individual reference genomes as the minimum unit for assessing the diversity of microbial communities and their relevance to environmental factors. This approach is independent of taxonomic classification, granting the possibility of maximal resolution of community composition, and organizes features into an accurate hierarchy using a phylogenomic tree. The outputs are suitable for contemporary analytical protocols for community ecology, differential abundance, and supervised learning while supporting phylogenetic methods, such as UniFrac and phylofactorization, that are seldom applied to shotgun metagenomics despite being prevalent in 16S rRNA gene amplicon studies. As demonstrated in two real-world case studies, the OGU method produces biologically meaningful patterns from microbiome data sets. Such patterns further remain detectable at very low metagenomic sequencing depths. Compared with taxonomic unit-based analyses implemented in currently adopted metagenomics tools, and the analysis of 16S rRNA gene amplicon sequence variants, this method shows superiority in informing biologically relevant insights, including stronger correlation with body environment and host sex on the Human Microbiome Project data set and more accurate prediction of human age by the gut microbiomes of Finnish individuals included in the FINRISK 2002 cohort. We provide Woltka, a bioinformatics tool to implement this method, with full integration with the QIIME 2 package and the Qiita web platform, to facilitate adoption of the OGU method in future metagenomics studies. IMPORTANCE Shotgun metagenomics is a powerful, yet computationally challenging, technique compared to 16S rRNA gene amplicon sequencing for decoding the composition and structure of microbial communities. Current analyses of metagenomic data are primarily based on taxonomic classification, which is limited in feature resolution. To solve these challenges, we introduce operational genomic units (OGUs), which are the individual reference genomes derived from sequence alignment results, without further assigning them taxonomy. The OGU method advances current read-based metagenomics in two dimensions: (i) providing maximal resolution of community composition and (ii) permitting use of phylogeny-aware tools. Our analysis of real-world data sets shows that it is advantageous over currently adopted metagenomic analysis methods and the finest-grained 16S rRNA analysis methods in predicting biological traits. We thus propose the adoption of OGUs as an effective practice in metagenomic studies.

A variety of antibiotics are ubiquitous in all freshwater ecosystems that receive wastewater. A wide variety of antibiotics have been developed to kill problematic bacteria and fungi through targeted application, and their use has contributed significantly to public health and livestock management. Unfortunately, a substantial fraction of the antibiotics applied to humans, pets and livestock end up in wastewater, and ultimately many of these chemicals enter freshwater ecosystems. The effect of adding chemicals that are intentionally designed to kill microbes, on freshwater microbial communities remains poorly understood. There are reasons to be concerned, as microbes play an essential role in nutrient uptake, carbon fixation and denitrification in freshwater ecosystems. Chemicals that reduce or alter freshwater microbial communities might reduce their capacity to degrade the excess nutrients and organic matter that characterize wastewater. We performed a laboratory experiment in which we exposed microbial community from unexposed stream sediments to three commonly detected antibiotics found in urban wastewater and urban streams (sulfamethoxazole, danofloxacin, and erythromycin). We assessed how the form and concentration of inorganic nitrogen, microbial carbon, and nitrogen cycling processes changed in response to environmentally relevant doses (10 μg/L) of each of these antibiotics individually and in combination. We expected to find that all antibiotics suppressed rates of microbial mineralization and nitrogen transformations and we anticipated that this suppression of microbial activity would be greatest in the combined treatment. Contrary to our expectations we measured few significant changes in microbially mediated functions in response to our experimental antibiotic dosing. We found no difference in functional gene abundance of key nitrogen cycling genes nosZ, mcrA, nirK, and amoA genes, and we measured no treatment effects on NO3- uptake or N2O, N2, CH4, CO2 production over the course of our seven-day experiment. In the mixture treatment, we measured significant increases in NH4+ concentrations over the first 24 hours of the experiment, which were indistinguishable from controls within six hours. Our results suggest remarkable community resistance to pressure antibiotic exposure poses on naïve stream sediments.

Complications are a part of surgery. Spinal infarctions are a dreaded complication of aortic surgery. We present a patient who developed a spinal infarct after a kidney transplant. We were unable to find a causative factor in our search for etiology. In our review of the literature, we were unable to find a similar report. We present this case report to highlight a rare complication of kidney transplantation and to reinforce that patients requiring kidney transplant are complex patients with multiple comorbidities that can cause a multitude of complications in the periop period.

With the increasing size and complexity of power systems, it is crucial to have an effective protection system in place to ensure its reliability. One of the important components of the protection system are relays. It is important for a relay to operate dependably and securely so that any fault can be cleared in time to minimize damages to the power network. However, it is important to test a relay in a realistic environment before commissioning it to the network. Testing a relay in the actual network can be expensive with limited fault scenarios. Hence, Hardware-in-the-Loop (HIL) testing is an efficient method to perform closed-loop testing of a relay since numerous fault cases can be simulated to provide a realistic operating environment for the relay under test. This paper sheds light on the HIL testing done for protective relays using a sample distribution system using RTDS. Two SEL-351 relays have been used in this experiment, and proper settings for the relays are calculated for coordination. The paper also describes the procedure of configuring the relay and other RTDS components crucial for interfacing of the relay with RTDS. After test setup, a pre-fault, fault, and post-fault analysis was done for the system. The results obtained from these analyses are cross-checked with the event history of the two SEL-351 relays, obtained with AcSELerator Quickset software. This paper provides thorough information for researchers to replicate the presented study or to develop new HIL experiments. It can also help in developing a fundamental understanding of the HIL testing setup that can be further applied to a more complex power system.

Iron-sulfur (Fe–S) clusters are essential cofactors most commonly known for their role mediating electron transfer within the mitochondrial respiratory chain. The Fe–S cluster pathways that function within the respiratory complexes are highly conserved between bacteria and the mitochondria of eukaryotic cells. Within the electron transport chain, Fe–S clusters play a critical role in transporting electrons through Complexes I, II and III to cytochrome c, before subsequent transfer to molecular oxygen. Fe–S clusters are also among the binding sites of classical mitochondrial inhibitors, such as rotenone, and play an important role in the production of mitochondrial reactive oxygen species (ROS). Mitochondrial Fe–S clusters also play a critical role in the pathogenesis of disease. High levels of ROS produced at these sites can cause cell injury or death, however, when produced at low levels can serve as signaling molecules. For example, Ndufs2, a Complex I subunit containing an Fe–S center, N2, has recently been identified as a redox-sensitive oxygen sensor, mediating homeostatic oxygen-sensing in the pulmonary vasculature and carotid body. Fe–S clusters are emerging as transcriptionally-regulated mediators in disease and play a crucial role in normal physiology, offering potential new therapeutic targets for diseases including malaria, diabetes, and cancer.

Hantavirus nucleocapsid protein (NP) inhibits protein kinase R (PKR) dimerization by an unknown mechanism to counteract its antiviral responses during virus infection. Here we demonstrate that NP exploits an endogenous PKR inhibitor P58IPK to inhibit PKR. The activity of P58IPK is normally restricted in cells by the formation of an inactive complex with its negative regulator Hsp40. On the other hand, PKR remains associated with the 40S ribosomal subunit, a unique strategic location that facilitates its free access to the downstream target eIF2α. Although both NP and Hsp40 bind to P58IPK, the binding affinity of NP is much stronger compared to Hsp40. P58IPK harbors an NP binding site, spanning to N-terminal TPR subdomains I and II. The Hsp40 binding site on P58IPK was mapped to the TPR subdomain II. The high affinity binding of NP to P58IPK and the overlap between NP and Hsp40 binding sites releases the P58IPK from its negative regulator by competitive inhibition. The NP-P58IPK complex is selectively recruited to the 40S ribosomal subunit by direct interaction between NP and the ribosomal protein S19 (RPS19), a structural component of the 40S ribosomal subunit. NP has distinct binding sites for P58IPK and RPS19, enabling it to serve as bridge between P58IPK and the 40S ribosomal subunit. NP mutants deficient in binding to either P58IPK or RPS19 fail to inhibit PKR, demonstrating that selective engagement of P58IPK to the 40S ribosomal subunit is required for PKR inhibition. Cells deficient in P58IPK mount a rapid PKR antiviral response and establish an antiviral state, observed by global translational shutdown and rapid decline in viral load. These studies reveal a novel viral strategy in which NP releases P58IPK from its negative regulator and selectively engages it on the 40S ribosomal subunit to promptly combat the PKR antiviral responses.

Cutaneous erythema is used in diagnosis and response assessment of cutaneous chronic graft-versus-host disease (cGVHD). The development of objective erythema evaluation methods remains a challenge. We used a pre-trained neural network to segment cGVHD erythema by detecting changes relative to a patient’s registered baseline photo. We fixed this change detection algorithm on human annotations from a single photo pair, by using either a traditional approach or by marking definitely affected (“Do Not Miss”, DNM) and definitely unaffected skin (“Do Not Include”, DNI). The fixed algorithm was applied to each of the remaining 47 test photo pairs from six follow-up sessions of one patient. We used both the Dice index and the opinion of two board-certified dermatologists to evaluate the algorithm performance. The change detection algorithm correctly assigned 80% of the pixels, regardless of whether it was fixed on traditional (median accuracy: 0.77, interquartile range 0.62–0.87) or DNM/DNI segmentations (0.81, 0.65–0.89). When the algorithm was fixed on markings by different annotators, the DNM/DNI achieved more consistent outputs (median Dice indices: 0.94–0.96) than the traditional method (0.73–0.81). Compared to viewing only rash photos, the addition of baseline photos improved the reliability of dermatologists’ scoring. The inter-rater intraclass correlation coefficient increased from 0.19 (95% confidence interval lower bound: 0.06) to 0.51 (lower bound: 0.35). In conclusion, a change detection algorithm accurately assigned erythema in longitudinal photos of cGVHD. The reliability was significantly improved by exclusively using confident human segmentations to fix the algorithm. Baseline photos improved the agreement among two dermatologists in assessing algorithm performance.

Abstract The last few years have seen tremendous growth in human microbiome research, with a particular focus on the links to both mental and physical health and disease. Medical and experimental settings provide initial sources of information about these links, but individual studies produce disconnected pieces of knowledge bounded in context by the perspective of expert researchers reading full-text publications. Building a knowledge base (KB) consolidating these disconnected pieces is an essential first step to democratize and accelerate the process of accessing the collective discoveries of human disease connections to the human microbiome. In this article, we survey the existing tools and development efforts that have been produced to capture portions of the information needed to construct a KB of all known human microbiome-disease associations and highlight the need for additional innovations in natural language processing (NLP), text mining, taxonomic representations, and field-wide vocabulary standardization in human microbiome research. Addressing these challenges will enable the construction of KBs that help identify new insights amenable to experimental validation and potentially clinical decision support.

Background Sternal rigid plate fixation (RPF) has been adopted in recent years in high-risk cases to reduce complications associated with steel wire cerclage, the traditional approach to sternal closure. While sternal RPF has been associated with lower complication rates than wire cerclage, it has its own complication profile that requires evaluation, necessitating a critical examination from a national perspective. This study will report the outcomes and associated risk factors of sternal RPF using a national database. Methods Patients undergoing sternal RPF from 2005 to 2016 in the American College of Surgeons-National Surgical Quality Improvement Program were identified. Demographics, perioperative information, and complication rates were reviewed. Logistic regression analysis was performed to identify risk factors for postoperative complications. Results There were 381 patient cases of RPF identified. The most common complications included bleeding (28.9%), mechanical ventilation >48 hours (16.5%), and reoperation/readmission (15.2%). Top risk factors for complications included dyspnea (odds ratio [OR], 2.672; P48 hours, and reoperation/readmission, each of which affected over 15% of the study population. Smokers remain at an increased risk for surgical site infection and sternal dehiscence despite RPF’s purported benefit to minimize these outcomes. Complications of primary versus delayed sternal RPF are roughly equivalent, but individual patients may perform better with one versus the other based on identified risk factors.

PurposeTo report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector.DesignSingle-center, retrospective chart review.MethodsIn this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated.ResultsThree out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector.ConclusionsWe observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes.Translational relevanceCaution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies.

ABSTRACT Standard workflows for analyzing microbiomes often include the creation and curation of phylogenetic trees. Here we present EMPress, an interactive web tool for visualizing trees in the context of microbiome, metabolome, and other community data scalable to trees with well over 500,000 nodes. EMPress provides novel functionality—including ordination integration and animations—alongside many standard tree visualization features and thus simplifies exploratory analyses of many forms of ‘omic data. IMPORTANCE Phylogenetic trees are integral data structures for the analysis of microbial communities. Recent work has also shown the utility of trees constructed from certain metabolomic data sets, further highlighting their importance in microbiome research. The ever-growing scale of modern microbiome surveys has led to numerous challenges in visualizing these data. In this paper we used five diverse data sets to showcase the versatility and scalability of EMPress, an interactive web visualization tool. EMPress addresses the growing need for exploratory analysis tools that can accommodate large, complex multi-omic data sets.

High throughput sequencing technologies have revolutionized the identification of mutations responsible for a diverse set of Mendelian disorders, including inherited retinal disorders (IRDs). However, the causal mutations remain elusive for a significant proportion of patients. This may be partially due to pathogenic mutations located in non-coding regions, which are largely missed by capture sequencing targeting the coding regions. The advent of whole-genome sequencing (WGS) allows us to systematically detect non-coding variations. However, the interpretation of these variations remains a significant bottleneck. In this study, we investigated the contribution of deep-intronic splice variants to IRDs. WGS was performed for a cohort of 571 IRD patients who lack a confident molecular diagnosis, and potential deep intronic variants that affect proper splicing were identified using SpliceAI. A total of six deleterious deep intronic variants were identified in eight patients. An in vitro minigene system was applied to further validate the effect of these variants on the splicing pattern of the associated genes. The prediction scores assigned to splice-site disruption positively correlated with the impact of mutations on splicing, as those with lower prediction scores demonstrated partial splicing. Through this study, we estimated the contribution of deep-intronic splice mutations to unassigned IRD patients and leveraged in silico and in vitro methods to establish a framework for prioritizing deep intronic variant candidates for mechanistic and functional analyses.

Pregnant women and developing infants are understudied populations in the opioid crisis, despite the rise in opioid use during pregnancy. Maternal opioid use results in diverse negative outcomes for the fetus/newborn, including death; however, the effects of perinatal (maternal and neonatal) opioids on developing respiratory circuitry are not well understood. Given the profound depressive effects of opioids on central respiratory networks controlling breathing, we tested the hypothesis that perinatal opioid exposure impairs respiratory neural circuitry, creating breathing instability. Our data demonstrate maternal opioids increase apneas and destabilize neonatal breathing. Maternal opioids also blunted opioid-induced respiratory frequency depression acutely in neonates; a unique finding since adult respiratory circuity does not desensitize to opioids. This desensitization normalized rapidly between postnatal days 1 and 2 (P1 and P2), the same age quantal slowing emerged in respiratory rhythm. These data suggest significant reorganization of respiratory rhythm generating circuits at P1–2, the same time as the preBötzinger Complex (key site of respiratory rhythm generation) becomes the dominant respiratory rhythm generator. Thus, these studies provide critical insight relevant to the normal developmental trajectory of respiratory circuits and suggest changes to mutual coupling between respiratory oscillators, while also highlighting how maternal opioids alter these developing circuits. In conclusion, the results presented demonstrate neurorespiratory disruption by maternal opioids and blunted opioid-induced respiratory frequency depression with neonatal opioids, which will be important for understanding and treating the increasing population of neonates exposed to gestational opioids.