Nanoparticle-mediated delivery of placental gene therapy via uterine artery catheterization in a pregnant rhesus macaque.

Nanoparticles offer promise as a mechanism to non-invasively deliver targeted placental therapeutics. Our previous studies utilizing intraplacental administration demonstrate efficient nanoparticle uptake into placental trophoblast cells and overexpression of human IGF1 ( hIGF1 ). Nanoparticle-mediated placental overexpression of hIGF1 in small animal models of placental insufficiency and fetal growth restriction improved nutrient transport and restored fetal growth. The objective of this pilot study was to extend these studies to the pregnant nonhuman primate and develop a method for local delivery of nanoparticles to the placenta via maternal blood flow from the uterine artery. Nanoparticles containing hIGF1 plasmid driven by the placenta-specific PLAC1 promoter were delivered to a mid-gestation pregnant rhesus macaque via a catheterization approach that is clinically used for uterine artery embolization. Maternal-fetal interface, fetal and maternal tissues were collected four days post-treatment to evaluate the efficacy of hIGF1 treatment in the placenta. The uterine artery catheterization procedure and nanoparticle treatment was well tolerated by the dam and fetus through the four-day study period following catheterization. Nanoparticles were taken up by the placenta from maternal blood as plasmid-specific hIGF1 expression was detected in multiple regions of the placenta via in situ hybridization and qPCR. The uterine artery catheterization approach enabled successful delivery of nanoparticles to maternal circulation in close proximity to the placenta with no concerns to maternal or fetal health in this short-term feasibility study. In the future, this delivery approach can be used for preclinical evaluation of the long-term safety and efficacy of nanoparticle-mediated placental therapies in a rhesus macaque model. Highlights: Novel method to deliver therapeutics to maternal-fetal interfaceDelivery of nanoparticles to the placenta via maternal catheterization.

Temporally resolved early BMP-driven transcriptional cascade during human amnion specification.

Amniogenesis, a process critical for continuation of healthy pregnancy, is triggered in a collection of pluripotent epiblast cells as the human embryo implants. Previous studies have established that BMP signaling is a major driver of this lineage specifying process, but the downstream BMP-dependent transcriptional networks that lead to successful amniogenesis remain to be identified. This is, in part, due to the current lack of a robust and reproducible model system that enables mechanistic investigations exclusively into amniogenesis. Here, we developed an improved model of early amnion specification, using a human pluripotent stem cell-based platform in which the activation of BMP signaling is controlled and synchronous. Uniform amniogenesis is seen within 48 hours after BMP activation, and the resulting cells share transcriptomic characteristics with amnion cells of a gastrulating human embryo. Using detailed time-course transcriptomic analyses, we established a previously uncharacterized BMP-dependent amniotic transcriptional cascade, and identified markers that represent five distinct stages of amnion fate specification; the expression of selected markers was validated in early post-implantation macaque embryos. Moreover, a cohort of factors that could potentially control specific stages of amniogenesis was identified, including the transcription factor TFAP2A. Functionally, we determined that, once amniogenesis is triggered by the BMP pathway, TFAP2A controls the progression of amniogenesis. This work presents a temporally resolved transcriptomic resource for several previously uncharacterized amniogenesis states and demonstrates a critical intermediate role for TFAP2A during amnion fate specification.

Cannabis use, decision making, and perceptions of risk among breastfeeding individuals: the Lactation and Cannabis (LAC) Study.

OBJECTIVE: Our primary objective was to understand breastfeeding individuals' decisions to use cannabis. Specifically, we investigated reasons for cannabis use, experiences with healthcare providers regarding use, and potential concerns about cannabis use. METHODS: We collected survey data from twenty breastfeeding participants from Washington and Oregon who used cannabis at least once weekly. We documented individuals' cannabis use and analyzed factors associated with their decisions to use cannabis during lactation. Qualitative description was used to assess responses to an open-ended question about potential concerns. RESULTS: Fifty-five percent of participants (n = 11) reported using cannabis to treat or manage health conditions, mostly related to mental health. Eighty percent of participants (n = 16) reported very few or no concerns about using cannabis while breastfeeding, although participants who used cannabis for medical purposes had significantly more concerns. Most participants (n = 18, 90%) reported receiving either no or unhelpful advice from healthcare providers. Four themes arose through qualitative analysis, indicating that breastfeeding individuals are: 1) identifying research gaps and collecting evidence; 2) monitoring their child's health and development; 3) monitoring and titrating their cannabis use; and 4) comparing risks between cannabis and other controlled substances. CONCLUSIONS: Breastfeeding individuals reported cannabis for medical and non-medical reasons and few had concerns about cannabis use during breastfeeding. Breastfeeding individuals reported using a variety of strategies and resources in their assessment of risk or lack thereof when deciding to use cannabis. Most participants reported receiving no helpful guidance from healthcare providers.

Whole Genome Analysis of SNV and Indel Polymorphism in Common Marmosets (Callithrix jacchus).

The common marmoset (Callithrix jacchus) is one of the most widely used nonhuman primate models of human disease. Owing to limitations in sequencing technology, early genome assemblies of this species using short-read sequencing suffered from gaps. In addition, the genetic diversity of the species has not yet been adequately explored. Using long-read genome sequencing and expert annotation, we generated a high-quality genome resource creating a 2.898 Gb marmoset genome in which most of the euchromatin portion is assembled contiguously (contig N50 = 25.23 Mbp, scaffold N50 = 98.2 Mbp). We then performed whole genome sequencing on 84 marmosets sampling the genetic diversity from several marmoset research centers. We identified a total of 19.1 million single nucleotide variants (SNVs), of which 11.9 million can be reliably mapped to orthologous locations in the human genome. We also observed 2.8 million small insertion/deletion variants. This dataset includes an average of 5.4 million SNVs per marmoset individual and a total of 74,088 missense variants in protein-coding genes. Of the 4956 variants orthologous to human ClinVar SNVs (present in the same annotated gene and with the same functional consequence in marmoset and human), 27 have a clinical significance of pathogenic and/or likely pathogenic. This important marmoset genomic resource will help guide genetic analyses of natural variation, the discovery of spontaneous functional variation relevant to human disease models, and the development of genetically engineered marmoset disease models.

Atypical initial cleavage patterns minimally impact rhesus macaque in vitro embryo morphokinetics and embryo outgrowth development†.

Embryo morphokinetic analysis through time-lapse embryo imaging is envisioned as a method to improve selection of developmentally competent embryos. Morphokinetic analysis could be utilized to evaluate the effects of experimental manipulation on pre-implantation embryo development. The objectives of this study were to establish a normative morphokinetic database for in vitro fertilized rhesus macaque embryos and to assess the impact of atypical initial cleavage patterns on subsequent embryo development and formation of embryo outgrowths. The cleavage pattern and the timing of embryo developmental events were annotated retrospectively for unmanipulated in vitro fertilized rhesus macaque blastocysts produced over four breeding seasons. Approximately 50% of the blastocysts analyzed had an abnormal early cleavage event. The time to the initiation of embryo compaction and the time to completion of hatching was significantly delayed in blastocysts with an abnormal early cleavage event compared to blastocysts that had cleaved normally. Embryo hatching, attachment to an extracellular matrix, and growth during the implantation stage in vitro was not impacted by the initial cleavage pattern. These data establish normative morphokinetic parameters for in vitro fertilized rhesus macaque embryos and suggest that cleavage anomalies may not impact embryo implantation rates following embryo transfer.

Vertical transmission of African-lineage Zika virus through the fetal membranes in a rhesus macaque (Macaca mulatta) model.

Zika virus (ZIKV) can be transmitted vertically from mother to fetus during pregnancy, resulting in a range of outcomes including severe birth defects and fetal/infant death. Potential pathways of vertical transmission in utero have been proposed but remain undefined. Identifying the timing and routes of vertical transmission of ZIKV may help us identify when interventions would be most effective. Furthermore, understanding what barriers ZIKV overcomes to effect vertical transmission may help improve models for evaluating infection by other pathogens during pregnancy. To determine the pathways of vertical transmission, we inoculated 12 pregnant rhesus macaques with an African-lineage ZIKV at gestational day 30 (term is 165 days). Eight pregnancies were surgically terminated at either seven or 14 days post-maternal infection. Maternal-fetal interface and fetal tissues and fluids were collected and evaluated for ZIKV using RT-qPCR, in situ hybridization, immunohistochemistry, and plaque assays. Four additional pregnant macaques were inoculated and terminally perfused with 4% paraformaldehyde at three, six, nine, or ten days post-maternal inoculation. For these four cases, the entire fixed pregnant uterus was evaluated with in situ hybridization for ZIKV RNA. We determined that ZIKV can reach the MFI by six days after infection and infect the fetus by ten days. Infection of the chorionic membrane and the extraembryonic coelomic fluid preceded infection of the fetus and the mesenchymal tissue of the placental villi. We did not find evidence to support a transplacental route of ZIKV vertical transmission via infection of syncytiotrophoblasts or villous cytotrophoblasts. The pattern of infection observed in the maternal-fetal interface provides evidence of paraplacental vertical ZIKV transmission through the chorionic membrane, the outer layer of the fetal membranes.

Rural Communities in the United States Face Persistent Disparities in Access to Orthopaedic Surgical Care.

Background: Access to orthopaedic care across the United States (U.S.) remains an important issue, however, no recent study has examined disparities in rural access to orthopaedic care. The goals of the present study were to (1) investigate trends in the proportion of rural orthopaedic surgeons from 2013 to 2018 as well as the proportion of rural U.S. counties with access to such surgeons and (2) analyze characteristics associated with choice of a rural practice setting. Methods: The study analyzed the Centers for Medicare and Medicaid Services (CMS) Physician Compare National Downloadable File (PC-NDF) for all active orthopaedic surgeons from 2013 to 2018. Rural practice settings were defined using Rural-Urban Commuting Area (RUCA) codes. Linear regression analysis investigated trends in rural orthopaedic surgeon volume. Multivariable logistic regression evaluated the association of surgeon characteristics with rural practice setting. Results: The total number of orthopaedic surgeons increased 1.9%, from 21,045 (2013) to 21,456 (2018). Meanwhile, the proportion of rural orthopaedic surgeons decreased by roughly 0.9%, from 578 (2013) to 559 (2018). From a per capita perspective, the number of orthopaedic surgeons practicing in a rural setting per 100,000 population ranged from 4.55 orthopaedic surgeons per 100,000 in 2013 and 4.47 per 100,000 in 2018. Meanwhile, the number of orthopaedic surgeons practicing in an urban setting ranged from 6.63 per 100,000 in 2013 and 6.35 per 100,000 in 2018. The surgeon characteristics most associated with decreased odds of practicing orthopaedic surgery in a rural setting included earlier career-stage (OR: 0.80, 95% CI: [0.70-0.91]; p < 0.001) and sub-specialization status (OR: 0.40, 95% CI: [0.36-0.45]; p < 0.001). Conclusion: Existing rural-urban disparities in musculoskeletal healthcare access have persisted over the past decade and could worsen. Future research should investigate the effects of orthopaedic workforce shortages on travel times, patient cost burden, and disease specific outcomes. Level of Evidence: IV.

How Female Is the Future of Urology? Projecting Various Trajectories of the United States Urology Workforce.

OBJECTIVE: To project the number and proportion of women in the urology workforce using recent demographic trends and develop an app to explore updated projections using future data. METHODS: Demographic data were obtained from AUA Censuses and ACGME Data Resource Books. The proportion of female graduating urology residents was characterized with a logistic growth model. "Stock and Flow" models were used to project future population numbers and proportions of female practicing urologists, accounting for trainee demographics, retirement trends, and growth in the field. RESULTS: Assuming growth in urology graduate numbers and continued logistic growth in the proportion of women, 10,957 practicing urologists (38%) will be female by 2062. If the rate of women entering urology residency stagnates, 7038 urologists (24%) will be female. If the retirement rates for women in urology change to mirror those of men and the proportion of female residents continues to experience logistic growth, 11,178 urologists (38%) will be female. An interactive app was designed to allow for a range of assumptions and future data: https://stephenrho.shinyapps.io/uro-workforce/. CONCLUSION: Workforce projections should incorporate recent growth in numbers of female residents. If current growth continues, 38% of urologists will be female by 2062. The app allows for exploration of different scenarios and can be updated with new data. The projections demonstrate the need for targeted efforts to recruit women into urology, address disparities within the field, and work toward retaining female urologists. We must continue working toward an equitable future workforce that can address the impending shortage of urologists.

What Factors Are Associated With Early Career Attrition Among Orthopaedic Surgeons in the United States?

BACKGROUND: The attrition of medical personnel in the United States healthcare system has been an ongoing concern among physicians and policymakers alike. Prior studies have shown that reasons for leaving clinical practice vary widely and may range from professional dissatisfaction or disability to the pursuit of alternative career opportunities. Whereas attrition among older personnel has often been understood as a natural phenomenon, attrition among early-career surgeons may pose a host of additional challenges from an individual and societal perspective. QUESTIONS/PURPOSES: (1) What percentage of orthopaedic surgeons experience early-career attrition, defined as leaving active clinical practice within the first 10 years after completion of training? (2) What are the surgeon and practice characteristics associated with early-career attrition? METHODS: In this retrospective analysis drawn from a large database, we used the 2014 Physician Compare National Downloadable File (PC-NDF), a registry of all healthcare professionals in the United States participating in Medicare. A total of 18,107 orthopaedic surgeons were identified, 4853 of whom were within the first 10 years of training completion. The PC-NDF registry was chosen because it has a high degree of granularity, national representativeness, independent validation through the Medicare claims adjudication and enrollment process, and the ability to longitudinally monitor the entry and exit of surgeons from active clinical practice. The primary outcome of early-career attrition was defined by three conditions, all of which had to be simultaneously satisfied ("condition one" AND "condition two" AND "condition three"). The first condition was presence in the Q1 2014 PC-NDF dataset and absence from the same dataset the following year (Q1 2015 PC-NDF). The second condition was consistent absence from the PC-NDF dataset for the following 6 years (Q1 2016, Q1 2017, Q1 2018, Q1 2019, Q1 2020, and Q1 2021), and the third condition was absence from the Centers for Medicare and Medicaid Services Opt-Out registry, which tracks clinicians who have formally discontinued enrollment in the Medicare program. Of the 18,107 orthopaedic surgeons identified in the dataset, 5% (938) were women, 33% (6045) were subspecialty-trained, 77% (13,949) practiced in groups of 10 or more, 24% (4405) practiced in the Midwest, 87% (15,816) practiced in urban areas, and 22% (3887) practiced at academic centers. Surgeons not enrolled in the Medicare program are not represented in this study cohort. A multivariable logistic regression model with adjusted odds ratios and 95% confidence intervals was constructed to investigate characteristics associated with early-career attrition. RESULTS: Among the 4853 early-career orthopaedic surgeons identified in the dataset, 2% (78) were determined to experience attrition between the first quarter 2014 and the same point in 2015. After controlling for potential confounding variables such as years since training completion, practice size, and geographic region, we found that women were more likely than men to experience early-career attrition (adjusted OR 2.8 [95% CI 1.5 to 5.0]; p = 0.006]), as were academic orthopaedic surgeons compared with private practitioners (adjusted OR 1.7 [95% CI 1.02 to 3.0]; p = 0.04), while general orthopaedic surgeons were less likely to experience attrition than subspecialists (adjusted OR 0.5 [95% CI 0.3 to 0.8]; p = 0.01). CONCLUSION: A small but important proportion of orthopaedic surgeons leave the specialty during the first 10 years of practice. Factors most-strongly associated with this attrition were academic affiliation, being a woman, and clinical subspecialization. CLINICAL RELEVANCE: Based on these findings, academic orthopaedic practices might consider expanding the role of routine exit interviews to identify instances in which early-career surgeons face illness, disability, burnout, or any other forms of severe personal hardships. If attrition occurs because of such factors, these individuals could benefit from connection to well-vetted coaching or counseling services. Professional societies might be well positioned to conduct detailed surveys to assess the precise reasons for early attrition and characterize any inequities in workforce retention across a diverse range of demographic subgroups. Future studies should also determine whether orthopaedics is an outlier, or whether 2% attrition is similar to the proportion in the overall medical profession.

Do Patients Reporting Fractures Experience Food Insecurity More Frequently Than the General Population?

BACKGROUND: The economic burden of traumatic injuries forces families into difficult tradeoffs between healthcare and nutrition, particularly among those with a low income. However, the epidemiology of food insecurity among individuals reporting having experienced fractures is not well understood. QUESTIONS/PURPOSES: (1) Do individuals in the National Health Interview Survey reporting having experienced fractures also report food insecurity more frequently than individuals in the general population? (2) Are specific factors associated with a higher risk of food insecurity in patients with fractures? METHODS: This retrospective, cross-sectional analysis of the National Health Interview Survey was conducted to identify patients who reported a fracture within 3 months before survey completion. The National Health Interview Survey is an annual serial, cross-sectional survey administered by the United States Centers for Disease Control, involving approximately 90,000 individuals across 35,000 American households. The survey is designed to be generalizable to the civilian, noninstitutionalized United States population and is therefore well suited to evaluate longitudinal trends in physical, economic, and psychosocial health factors nationwide. We analyzed data from 2011 to 2017 and identified 1399 individuals who reported sustaining a fracture during the 3 months preceding their survey response. Among these patients, 27% (384 of 1399) were older than 65 years, 77% (1074) were White, 57% (796) were women, and 14% (191) were uninsured. A raw score compiled from 10 food security questions developed by the United States Department of Agriculture was used to determine the odds of 30-day food insecurity for each patient. A multivariate logistic regression analysis was performed to determine factors associated with food insecurity among patients reporting fractures . In the overall sample of National Health Interview Survey respondents, approximately 0.6% (1399 of 239,168) reported a fracture. RESULTS: Overall, 17% (241 of 1399) of individuals reporting broken bones or fractures in the National Health Interview Survey also reported food insecurity. Individuals reporting fractures were more likely to report food insecurity if they also were aged between 45 and 64 years (adjusted odds ratio 4.0 [95% confidence interval 2.1 to 7.6]; p < 0.001), had a household income below USD 49,716 (200% of the federal poverty level) per year (adjusted OR 3.1 [95% CI 1.9 to 5.1]; p < 0.001), were current tobacco smokers (adjusted OR 2.8 [95% CI 1.6 to 5.1]; p < 0.001), and were of Black race (adjusted OR 1.9 [95% CI 1.1 to 3.4]; p = 0.02). CONCLUSION: Among patients with fractures, food insecurity screening and routine nutritional assessments may help to direct financially vulnerable patients toward available community resources. Such screening programs may improve adherence to nutritional recommendations in the trauma recovery period and improve the physiologic environment for adequate soft tissue and bone healing. Future research may benefit from the inclusion of clinical nutritional data, a broader representation of high-energy injuries, and a prospective study design to evaluate cost-efficient avenues for food insecurity interventions in the context of locally available social services networks. LEVEL OF EVIDENCE: Level III, prognostic study.

Do Americans Lacking a Local Orthopaedic Surgeon Have Adequate Broadband for Telehealth?

BACKGROUND: Although telehealth holds promise in expanding access to orthopaedic surgical care, high-speed internet connectivity remains a major limiting factor for many communities. Despite persistent federal efforts to study and address the health information technology needs of patients, there is limited information regarding the current high-speed internet landscape as it relates to access to orthopaedic surgical care. QUESTIONS/PURPOSES: (1) What is the distribution of practicing orthopaedic surgeons in the United States relative to the presence of broadband internet access? (2) What geographic, demographic, and socioeconomic factors are associated with the absence of high-speed internet and access to a local orthopaedic surgeon? METHODS: The Federal Communications Commission (FCC) Mapping Broadband in America interactive tool was used to determine the proportion of county residents with access to broadband-speed internet for all 3141 US counties. Data regarding the geographic distribution of orthopaedic surgeons and county-level characteristics were obtained from the 2015 Physician Compare National Downloadable File and the Area Health Resource File, respectively. The FCC mapping broadband public use files are considered the most comprehensive datasets describing high-speed internet infrastructure within the United States. The year 2015 represents the most recently available FCC data for which county-level broadband penetration estimates are available. Third-party audits of the FCC data have shown that broadband expansion has been slow over the past decade and that many large improvements have been driven by changes in the reporting methodology. Therefore, we believe the 2015 FCC data still hold relevance. The primary outcome measure was the simultaneous absence of at least 50% broadband penetration and at least one orthopaedic surgeon practicing in county limits. Statistical analyses using Kruskal-Wallis tests and multivariable logistic regression were conducted to assess for factors associated with inaccessibility to orthopaedic telehealth. All statistical tests were two-sided with a significance threshold of p < 0.05. RESULTS: In 2015, 14% (448 of 3141) of counties were considered "low access" in that they both had no orthopaedic surgeons and possessed less than 50% broadband access. A total of 4,660,559 people lived within these low-access counties, representing approximately 1.4% (4.6 million of 320.7 million) of the US population. After controlling for potential confounding variables, such as the age, sex, income level, and educational attainment, lower population density per square mile (OR 0.92 [95% confidence interval (CI) 0.90 to 0.94]; p < 0.01), a lower number of primary care physicians per 100,000 (OR 0.88 [95% CI 0.81 to 0.97]; p < 0.01), a higher unemployment level (OR 1.3 [95% CI 1.2 to 1.4]; p < 0.01), and greater number preventable hospital stays per 100,000 (OR 1.01 [95% CI 1.01 to 1.02]; p < 0.01) were associated with increased odds of being a low-access county (though the effect size of the finding was small for population density and number of primary care physicians). Stated another way, each additional person per square mile was associated with an 8% (95% CI 6% to 10%; p < 0.01) decrease in the odds of being a low-access county, and each additional percentage point of unemployment was associated with a 30% (95% CI 20% to 40%) increase in the odds of being a low-access county. CONCLUSION: Despite the potential for telehealth programs to improve the delivery of high-quality orthopaedic surgical care, broadband internet access remains a major barrier to implementation. Until targeted investments are made to expand broadband infrastructure across the country, health systems, policymakers, and surgeon leaders must capitalize on existing federal subsidy programs, such as the lifeline or affordability connectivity initiatives, to reach unemployed patients living in economically depressed regions. The incorporation of internet access questions into clinic-based social determinants screening may facilitate the development of alternative follow-up protocols for patients unable to participate in synchronous videoconferencing. CLINICAL RELEVANCE: Some orthopaedic patients lack the broadband capacity necessary for telehealth visits, in which case surgeons may pursue alternative methods of follow-up such as mobile phone-based surveillance of postoperative wounds, surgical sites, and clinical symptoms.

Maternal, placental and fetal response to a non-viral, polymeric nanoparticle gene therapy in nonhuman primates.

Background: Currently, there are no placenta-targeted treatments to alter the in utero environment. Water-soluble polymers have a distinguished record of clinical relevance outside of pregnancy. We have demonstrated the effective delivery of polymer-based nanoparticles containing a non-viral human insulin-like 1 growth factor ( IGF1 ) transgene to correct placental insufficiency in small animal models of fetal growth restriction (FGR). Our goal was to extend these studies to the pregnant nonhuman primate (NHP) and assess maternal, placental and fetal responses to nanoparticle-mediated IGF1 treatment. Methods: Pregnant macaques underwent ultrasound-guided intraplacental injections of nanoparticles ( GFP- or IGF1- expressing plasmid under the control of the trophoblast-specific PLAC1 promoter complexed with a HPMA-DMEAMA co-polymer) at approximately gestational day 100 (term = 165 days). Fetectomy was performed 24 h ( GFP ; n =1), 48 h ( IGF1 ; n = 3) or 10 days ( IGF1 ; n = 3) after nanoparticle delivery. Routine pathological assessment was performed on biopsied maternal tissues, and placental and fetal tissues. Maternal blood was analyzed for complete blood count (CBC), immunomodulatory proteins and growth factors, progesterone (P4) and estradiol (E2). Placental ERK/AKT/mTOR signaling was assessed using western blot and qPCR. Findings: Fluorescent microscopy and in situ hybridization confirmed placental uptake and transgene expression in villous syncytiotrophoblast. No off-target expression was observed in maternal and fetal tissues. Histopathological assessment of the placenta recorded observations not necessarily related to the IGF1 nanoparticle treatment. In maternal blood, CBCs, P4 and E2 remained within the normal range for pregnant macaques across the treatment period. Changes to placental ERK and AKT signaling at 48 h and 10 d after IGF1 nanoparticle treatment indicated an upregulation in placental homeostatic mechanisms to prevent over activity in the normal pregnancy environment. Interpretation: Maternal toxicity profile analysis and lack of adverse reaction to nanoparticle-mediated IGF1 treatment, combined with changes in placental signaling to maintain homeostasis indicates no deleterious impact of treatment. Funding: National Institutes of Health, and Wisconsin National Primate Research Center.

CRISPR/Cas9 genome editing to create nonhuman primate models for studying stem cell therapies for HIV infection.

Nonhuman primates (NHPs) are well-established basic and translational research models for human immunodeficiency virus (HIV) infections and pathophysiology, hematopoietic stem cell (HSC) transplantation, and assisted reproductive technologies. Recent advances in CRISPR/Cas9 gene editing technologies present opportunities to refine NHP HIV models for investigating genetic factors that affect HIV replication and designing cellular therapies that exploit genetic barriers to HIV infections, including engineering mutations into CCR5 and conferring resistance to HIV/simian immunodeficiency virus (SIV) infections. In this report, we provide an overview of recent advances and challenges in gene editing NHP embryos and discuss the value of genetically engineered animal models for developing novel stem cell-based therapies for curing HIV.

Nationwide disparities in transportation related delays to care experienced by children with frequent ear infections.

BACKGROUND: Acute otitis media (AOM), or ear infection, is the most common reason for pediatric medical visits in the United States [1]. Additionally, transportation barriers are a significant driver of missed and delayed care across medical specialties [2,3]. Yet, the role of transportation barriers in impeding access for children with frequent ear infections (FEI) has not been investigated. Assessing the prevalence of transportation barriers across sociodemographic groups may help clinicians improve outcomes for children with FEI. METHODS: A retrospective analysis of the U.S. National Health Interview Survey was completed to examine associations between sociodemographic characteristics among children with FEI and transportations barriers to seeking care between 2011 and 2018. RESULTS: Multivariable logistic regression found that income level, insurance status, and health status were linked to disparities in transportation barriers among children with FEI. Those in the middle (aOR 3.00, 95% CI 1.77-5.08, p < 0.001) and lowest income brackets (aOR 6.33, 95% CI 3.80, p < 0.001), who were publicly insured (aOR 3.24, 95% CI 2.00-5.23, p < 0.001) or uninsured (aOR 3.46, 95% CI 1.84-6.51, p < 0.001), and with Poor to Fair health status were more likely to face transportation delays than patients who were in the highest income bracket, privately insured, or had Good to Excellent health status. CONCLUSION: Children with FEI from families that were lower-income, less insured, and less healthy faced more transportation barriers when accessing care than their counterparts. Future interventions to improve health-related transportation should be targeted toward these patient subgroups to reduce gaps in outcomes.

Zika virus impacts extracellular vesicle composition and cellular gene expression in macaque early gestation trophoblasts.

Zika virus (ZIKV) infection at the maternal-placental interface is associated with adverse pregnancy outcomes including fetal demise and pregnancy loss. To determine how infection impacts placental trophoblasts, we utilized rhesus macaque trophoblast stem cells (TSC) that can be differentiated into early gestation syncytiotrophoblasts (ST) and extravillous trophoblasts (EVT). TSCs and STs, but not EVTs, were highly permissive to productive infection with ZIKV strain DAK AR 41524. The impact of ZIKV on the cellular transcriptome showed that infection of TSCs and STs increased expression of immune related genes, including those involved in type I and type III interferon responses. ZIKV exposure altered extracellular vesicle (EV) mRNA, miRNA and protein cargo, including ZIKV proteins, regardless of productive infection. These findings suggest that early gestation macaque TSCs and STs are permissive to ZIKV infection, and that EV analysis may provide a foundation for identifying non-invasive biomarkers of placental infection in a highly translational model.

Modeling genetic diseases in nonhuman primates through embryonic and germline modification: Considerations and challenges.

Genetic modification of the embryo or germ line of nonhuman primates is envisioned as a method to develop improved models of human disease, yet the promise of such animal models remains unfulfilled. Here, we discuss current methods and their limitations for producing nonhuman primate genetic models that faithfully genocopy and phenocopy human disease. We reflect on how to ethically maximize the translational relevance of such models in the search for new therapeutic strategies to treat human disease.

Robust engraftment of fetal nonhuman primate CD34-positive cells in immune-deficient mice.

Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high-throughput in vivo experimentation leveraged to inform large animal immunology-based studies. We found that adult rhesus macaque mobilized blood (AMb) CD34+-enriched hematopoietic stem and progenitor cells (HSPCs) engrafted at low but persistent levels in immune-deficient mice harboring transgenes for human (NHP cross-reactive) GM-CSF and IL3, but did not in mice with wild-type murine cytokines lacking NHP cross-reactivity. To enhance engraftment, fetal liver-derived HSPCs were selected as the infusion product based on an increased CD34hi fraction compared with AMb and bone marrow. Coupled with cotransplantation of rhesus fetal thymic fragments beneath the mouse kidney capsule, fetal liver-derived HSPC infusion in cytokine-transgenic mice yielded robust multilineage lymphohematopoietic engraftment. The emergent immune system recapitulated that of the fetal monkey, with similar relative frequencies of lymphocyte, granulocyte, and monocyte subsets within the thymic, secondary lymphoid, and peripheral compartments. Importantly, while exhibiting a predominantly naïve phenotype, in vitro functional assays demonstrated robust cellular activation in response to nonspecific and allogenic stimuli. This primatized mouse represents a viable and translatable model for the study of hematopoietic stem cell physiology, immune development, and functional immunology in NHPs. Summary Sentence: Engraftment of rhesus macaque hematopoietic tissues in immune-deficient mice yields a robust BLT/NeoThy-type primatized mouse model for studying nonhuman primate hematopoiesis and immune function in vivo.

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits.

Introduction: Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in CCR5 CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos. Methods: DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis. Results: Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. De novo mutations were also identified at predicted CRISPR-Cas9 off-target sites. Discussion: This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations.

Potential Activities and Long Lifetimes of Organic Carbon-Degrading Extracellular Enzymes in Deep Subsurface Sediments of the Baltic Sea.

Heterotrophic microorganisms in marine sediments produce extracellular enzymes to hydrolyze organic macromolecules, so their products can be transported inside the cell and used for energy and growth. Therefore, extracellular enzymes may mediate the fate of organic carbon in sediments. The Baltic Sea Basin is a primarily depositional environment with high potential for organic matter preservation. The potential activities of multiple organic carbon-degrading enzymes were measured in samples obtained by the International Ocean Discovery Program Expedition 347 from the Little Belt Strait, Denmark, core M0059C. Potential maximum hydrolysis rates (Vmax) were measured at depths down to 77.9mbsf for the following enzymes: alkaline phosphatase, ß-d-xylosidase, ß-d-cellobiohydrolase, N-acetyl-ß-d-glucosaminidase, ß-glucosidase, α-glucosidase, leucyl aminopeptidase, arginyl aminopeptidase, prolyl aminopeptidase, gingipain, and clostripain. Extracellular peptidase activities were detectable at depths shallower than 54.95mbsf, and alkaline phosphatase activity was detectable throughout the core, albeit against a relatively high activity in autoclaved sediments. ß-glucosidase activities were detected above 30mbsf; however, activities of other glycosyl hydrolases (ß-xylosidase, ß-cellobiohydrolase, N-acetyl-ß-glucosaminidase, and α-glucosidase) were generally indistinguishable from zero at all depths. These extracellular enzymes appear to be extremely stable: Among all enzymes, a median of 51.3% of enzyme activity was retained after autoclaving for an hour. We show that enzyme turnover times scale with the inverse of community metabolic rates, such that enzyme lifetimes in subsurface sediments, in which metabolic rates are very slow, are likely to be extraordinarily long. A back-of-the-envelope calculation suggests enzyme lifetimes are, at minimum, on the order of 230days, and may be substantially longer. These results lend empirical support to the hypothesis that a population of subsurface microbes persist by using extracellular enzymes to slowly metabolize old, highly degraded organic carbon.