On the Structure, Stability, and Cell Uptake of Nanostructured Lipid Carriers for Drug Delivery.

The efficacy of nanostructured lipid carriers (NLC) for drug delivery strongly depends on their stability and cell uptake. Both properties are governed by their compositions and internal structure. To test the effect of the lipid composition of NLC on cell uptake and stability, three kinds of liquid lipids with different degrees of unsaturation are employed. After ensuring homogeneous size distributions, the thermodynamic characteristics, stability, and mixing properties of NLC are characterized. Then the rates and predominant pathways of cell uptake are determined. Although the same surfactant is used in all cases, different uptake rates are observed. This finding contradicts the view that the surface properties of NLC are dominated by the surfactant. Instead, the uptake rates are explained by the structure of the nanocarrier. Depending on the mixing properties, some liquid lipids remain inside the nanocarrier, while other liquid lipids are present on the surface. Nanocarriers with liquid lipids on the surface are taken up more readily by the cells. This shows that the engineering of efficient lipid nanocarriers requires a delicate balance of interactions between all components of the nanocarrier on the molecular level.

Well-being and burnout: One size does not fit all.

Well-being and burnout are concepts that have become well described throughout emergency medicine. In the past, both well-being and burnout have been defined and addressed as a singular phenomenon, similar for all physicians, regardless of career stage. However, unique stressors may exist for physicians, as a function of their work environment and stage. In this concepts article we present clinician well-being as a dynamic and continuous process, subject to unique factors along the professional lifespan. Specific individual and system-level factors are discussed, ranging from demographic variables, to evolving administrative and professional responsibilities depending on the career stage of a clinician. This detailed description of stressors spanning an emergency physician's professional career may help create more targeted physician well-being and burnout interventions.

Wellness for the Future: Cultural and Systems-based Challenges and Solutions.

The goal of the 2019 Society for Academic Emergency Medicine Consensus Conference was to explore the current cultural and systemic issues in emergency medicine that impact the individual well-being of every emergency physician and to make recommendations for future study. Burnout is epidemic in emergency medicine. Physician wellness is required to enhance patient clinical outcomes as well as to ensure professional satisfaction and longevity. For conference preparation, a consensus steering committee was created, and a decision was made to use the groundbreaking model of the National Academy of Medicine's "Factors Affecting Clinician Well-Being and Resilience" to further identify areas of needed study. On May 14, 2019, the Wellness Consensus Conference was attended by over 50 faculty physicians from across the United States. These attendees discussed key concepts and prior research presented by content experts. Groups of participants engaged in crowdsourcing techniques to consolidate ideas derived from those discussions. These consensus concepts were recorded and are presented within this article. A repetitive theme noted at the conference was the overwhelming effect of the system and organization factors on individual physician well-being. The concept of ongoing assessment of professional fulfillment over the life span of the emergency physician was felt to be crucial in guiding wellness and resilience interventions in a timely manner. Examining ways to enable physicians to flourish rather than experience burnout are strong future directions for study.

Both diet and Helicobacter pylori infection contribute to atherosclerosis in pre- and postmenopausal cynomolgus monkeys.

A number of viruses and bacterial species have been implicated as contributors to atherosclerosis, potentially providing novel pathways for prevention. Epidemiological studies examining the association between Helicobacter pylori and cardiovascular disease have yielded variable results and no studies have been conducted in nonhuman primates. In this investigation, we examined the relationship between H. pylori infection and atherosclerosis development in socially housed, pre- and postmenopausal cynomolgus macaques consuming human-like diets. Ninety-four premenopausal cynomolgus monkeys (Macaca fascicularis) were fed for 36 months an atherogenic diet deriving its protein from either casein lactalbumin(CL) or high isoflavone soy (SOY). Animals were then ovariectomized and fed either the same or the alternate diet for an additional 36 months. Iliac artery biopsies were obtained at the time of ovariectomy and iliac and coronary artery sections were examined at the end of the study. Evidence of H. pylori infection was found in 64% of the monkeys and 46% of animals had live H. pylori within coronary atheromas as determined by mRNA-specific in situ hybridization. There was a significant linear relationship between the densities of gastric and atheroma organisms. Helicobactor pylori infection correlated with increased intimal plaque area and thickness at both the premenopausal and postmenopausal time points and regardless of diet (p< 0.01), although animals consuming the SOY diet throughout had the least amount of atherosclerosis. Additionally, plasma lipid profiles, intimal collagen accumulation, ICAM-1, and plaque macrophage densities were adversely affected by H. pylori infection among animals consuming the CL diet, while the SOY diet had the opposite effect. Plaque measurements were more highly associated with the densities of cagA-positive H. pylori within coronary atheromas than with the densities of gastric organisms, whereas plasma lipid changes were associated with H. pylori infection, but not cagA status. This study provides strong evidence that live H. pylori infects atheromas, exacerbates atherosclerotic plaque development, and alters plasma lipid profiles independently of diet or hormonal status. Finally, socially subordinate animals relative to their dominant counterparts had a greater prevalence of H. pylori, suggesting a stress effect. The results indicate that early H. pylori eradication could prevent or delay development of cardiovascular disease.

In vivo PET Imaging of [11C]CIMBI-5, a 5-HT2AR Agonist Radiotracer in Nonhuman Primates.

PURPOSE: 5-HT2AR exists in high and low affinity states. Agonist PET tracers measure binding to the active high affinity site and thus provide a functionally relevant measure of the receptor. Limited in vivo data have been reported so far for a comparison of agonist versus antagonist tracers for 5-HT2AR used as a proof of principle for measurement of high and low affinity states of this receptor. We compared the in vivo binding of [11C]CIMBI-5, a 5-HT2AR agonist, and of the antagonist [11C]M100907, in monkeys and baboons. METHODS: [11C]CIMBI-5 and [11C]M100907 baseline PET scans were performed in anesthetized male baboons (n=2) and male vervet monkeys (n=2) with an ECAT EXACT HR+ and GE 64-slice PET/CT Discovery VCT scanners. Blocking studies were performed in vervet monkeys by pretreatment with MDL100907 (0.5 mg/kg, i.v.) 60 minutes prior to the scan. Regional distribution volumes and binding potentials were calculated for each ROI using the likelihood estimation in graphical analysis and Logan plot, with either plasma input function or reference region as input, and simplified reference tissue model approaches. RESULTS: PET imaging of [11C]CIMBI-5 in baboons and monkeys showed the highest binding in 5-HT2AR-rich cortical regions, while the lowest binding was observed in cerebellum, consistent with the expected distribution of 5-HT2AR. Very low free fractions and rapid metabolism were observed for [11C]CIMBI-5 in baboon plasma. Binding potential values for [11C]CIMBI-5 were 25-33% lower than those for [11C]MDL100907 in the considered brain regions. CONCLUSION: The lower binding potential of [11C]CIMBI-5 in comparison to [11C]MDL100907 is likely due to the preferential binding of the former to the high affinity site in vivo in contrast to the antagonist,  [11C]MDL100907, which binds to both high and low affinity sites.

A nonhuman primate model of early Alzheimer's disease pathologic change: Implications for disease pathogenesis.

INTRODUCTION: Nonhuman primates may serve as excellent models of sporadic age-associated brain ß-amyloid deposition and Alzheimer's disease pathologic changes. We examined whether a vervet nonhuman primate model recapitulated pathologic, physiologic, and behavioral features of early Alzheimer's disease. METHODS: Nine middle-aged (mean = 11.2 years) and nine aged (mean = 21.7 years) female vervet/African green monkeys underwent cerebrospinal fluid collection, gait speed measurement, and neuroimaging before neuropathologic assessment. RESULTS: ß-amyloid plaques were identified in all aged vervets and paired helical filament tau immunoreactivity was observed in all animals. Cerebrospinal fluid ß-amyloid42 and gait speed correlated negatively with age and plaque density. Greater plaque and paired helical filament tau burden predicted reduced volumes and CMRg in several brain regions. DISCUSSION: We observed a coordinated set of relationships among neuropathologic, cerebrospinal fluid, imaging, and behavioral modalities consistent with early Alzheimer's disease. Our results support future use of the vervet model to explore disease mechanisms, biomarkers, and novel therapeutic strategies.

Neurodegenerative disease biomarkers Aß1-40, Aß1-42, tau, and p-tau181 in the vervet monkey cerebrospinal fluid: Relation to normal aging, genetic influences, and cerebral amyloid angiopathy.

Background: The Caribbean vervet monkey (Chlorocebus aethiops sabaeus) is a potentially valuable animal model of neurodegenerative disease. However, the trajectory of aging in vervets and its relationship to human disease is incompletely understood. Methods: To characterize biomarkers associated with neurodegeneration, we measured cerebrospinal fluid (CSF) concentrations of Aß1-40, Aß1-42, total tau, and p-tau181 in 329 members of a multigenerational pedigree. Linkage and genome-wide association were used to elucidate a genetic contribution to these traits. Results: Aß1-40 concentrations were significantly correlated with age, brain total surface area, and gray matter thickness. Levels of p-tau181 were associated with cerebral volume and brain total surface area. Among the measured analytes, only CSF Aß1-40 was heritable. No significant linkage (LOD > 3.3) was found, though suggestive linkage was highlighted on chromosomes 4 and 12. Genome-wide association identified a suggestive locus near the chromosome 4 linkage peak. Conclusions: Overall, these results support the vervet as a non-human primate model of amyloid-related neurodegeneration, such as Alzheimer's disease and cerebral amyloid angiopathy, and highlight Aß1-40 and p-tau181 as potentially valuable biomarkers of these processes.

Genetic variation and gene expression across multiple tissues and developmental stages in a nonhuman primate.

By analyzing multitissue gene expression and genome-wide genetic variation data in samples from a vervet monkey pedigree, we generated a transcriptome resource and produced the first catalog of expression quantitative trait loci (eQTLs) in a nonhuman primate model. This catalog contains more genome-wide significant eQTLs per sample than comparable human resources and identifies sex- and age-related expression patterns. Findings include a master regulatory locus that likely has a role in immune function and a locus regulating hippocampal long noncoding RNAs (lncRNAs), whose expression correlates with hippocampal volume. This resource will facilitate genetic investigation of quantitative traits, including brain and behavioral phenotypes relevant to neuropsychiatric disorders.

Premenopausal Reproductive Health Modulates Future Cardiovascular Risk - Comparative Evidence from Monkeys and Women.

Coronary heart disease (CHD) remains the major cause of mortality among postmenopausal women living in industrialized countries. Several lines of evidence suggest that ovarian hormones (especially estrogen) protect the coronary arteries of premenopausal women. However, it is also known that women commonly experience disruptions in cyclic hormonal function during their reproductive years. In this perspective, we hypothesize that if regular, cyclic ovarian function affords protection against CHD, ovulatory abnormalities in young women may conversely promote the development of atherosclerosis (the pathobiological process underlying CHD) in the years prior to menopause and thus substantially increase the risk of subsequent heart disease. This hypothesis is supported by evidence from premenopausal nonhuman primates showing that relatively common, subclinical ovarian disruptions - as may be induced by psychosocial stress - are associated with the initiation and acceleration of coronary artery atherosclerosis. If extending to women, these findings would suggest that ovarian dysfunction is an early biomarker for CHD risk and, further, that primary prevention of CHD should begin during the premenopausal phase of life.

Radiosynthesis and in Vivo Evaluation of [11C]A1070722, a High Affinity GSK-3 PET Tracer in Primate Brain.

Dysfunction of glycogen synthase kinase 3 (GSK-3) is implicated in the etiology of Alzheimer's disease, Parkinson's disease, diabetes, pain, and cancer. A radiotracer for functional positron emission tomography (PET) imaging could be used to study the kinase in brain disorders and to facilitate the development of small molecule inhibitors of GSK-3 for treatment. At present, there is no target-specific or validated PET tracer available for the in vivo monitoring of GSK-3. We radiolabeled the small molecule inhibitor [11C]1-(7-methoxy- quinolin-4-yl)-3-(6-(trifluoromethyl)pyridin-2-yl)urea ([11C]A1070722) with high affinity to GSK-3 (Ki = 0.6 nM) in excellent radiochemical yield. PET imaging experiments in anesthetized vervet/African green monkey exhibited that [11C]A1070722 penetrated the blood-brain barrier (BBB) and accumulated in brain regions, with highest radioactivity binding in frontal cortex followed by parietal cortex and anterior cingulate, and with the lowest bindings found in caudate, putamen, and thalamus, similarly to the known distribution of GSK-3 in human brain. Our studies suggest that [11C]A1070722 can be a potential PET radiotracer for the in vivo quantification of GSK-3 in brain.

Functional Hypothalamic Amenorrhea: An Endocrine Society Clinical Practice Guideline.

Cosponsoring Associations: The American Society for Reproductive Medicine, the European Society of Endocrinology, and the Pediatric Endocrine Society. This guideline was funded by the Endocrine Society. Objective: To formulate clinical practice guidelines for the diagnosis and treatment of functional hypothalamic amenorrhea (FHA). Participants: The participants include an Endocrine Society-appointed task force of eight experts, a methodologist, and a medical writer. Evidence: This evidence-based guideline was developed using the Grading of Recommendations, Assessment, Development, and Evaluation approach to describe the strength of recommendations and the quality of evidence. The task force commissioned two systematic reviews and used the best available evidence from other published systematic reviews and individual studies. Consensus Process: One group meeting, several conference calls, and e-mail communications enabled consensus. Endocrine Society committees and members and cosponsoring organizations reviewed and commented on preliminary drafts of this guideline. Conclusions: FHA is a form of chronic anovulation, not due to identifiable organic causes, but often associated with stress, weight loss, excessive exercise, or a combination thereof. Investigations should include assessment of systemic and endocrinologic etiologies, as FHA is a diagnosis of exclusion. A multidisciplinary treatment approach is necessary, including medical, dietary, and mental health support. Medical complications include, among others, bone loss and infertility, and appropriate therapies are under debate and investigation.

Human Striatal Dopaminergic and Regional Serotonergic Synaptic Degeneration with Lewy Body Disease and Inheritance of APOE ε4.

Cognitive impairment in older individuals is a complex trait that in population-based studies most commonly derives from an individually varying mixture of Alzheimer disease, Lewy body disease, and vascular brain injury. We investigated the molecular composition of synaptic particles from three sources: consecutive rapid autopsy brains from the Adult Changes in Thought Study, a population-based cohort; four aged nonhuman primate brains optimally processed for molecular investigation; and targeted replacement transgenic mice homozygous for APOE ε4. Our major goal was to characterize the molecular composition of human synaptic particles in regions of striatum and prefrontal cortex. We performed flow cytometry to measure six markers of synaptic subtypes, as well as amyloid ß 42 and paired helical filament tau. Our results showed selective degeneration of dopaminergic terminals throughout the striatum in individuals with Lewy body disease, and serotonergic degeneration in human ventromedial caudate nucleus from individuals with an APOE ε4 allele. Similar results were seen in mouse caudate nucleus homozygous for APOE ε4 via targeted replacement. Together, extension of these clinical, pathologic, and genetic associations from tissue to the synaptic compartment of cerebral cortex and striatum strongly supports our approach for accurately observing the molecular composition of human synapses by flow cytometry.

In vivo evaluation of [18F]FECIMBI-36, an agonist 5-HT2A/2C receptor PET radioligand in nonhuman primate.

We recently reported the radiosynthesis and in vitro evaluation of [18F]-2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-(2-fluoroethoxy)benzyl)ethanamine, ([18F]FECIMBI-36) or ([18F]1), an agonist radioligand for 5HT2A/2C receptors in postmortem samples of human brain. Herein we describe the in vivo evaluation of [18F]FECIMBI-36 in vervet/African green monkeys by PET imaging. PET images show that [18F]FECIMBI-36 penetrates the blood-brain barrier and a low retention of radioactivity is observed in monkey brain. Although the time activity curves indicate a somehow heterogeneous distribution of the radioligand in the brain, the low level of [18F]FECIMBI-36 in brain may limit the use of this tracer for quantification of 5-HT2A/2C receptors by PET.

Alterations in levels and ratios of n-3 and n-6 polyunsaturated fatty acids in the temporal cortex and liver of vervet monkeys from birth to early adulthood.

Deficiencies in omega-3 (n-3) long chain polyunsaturated fatty acids (LC-PUFAs) and increases in the ratio of omega-6 (n-6) to n-3 LC-PUFAs in brain tissues and blood components have been associated with psychiatric and developmental disorders. Most studies have focused on n-3 LC-PUFA accumulation in the brain from birth until 2years of age, well before the symptomatic onset of such disorders. The current study addresses changes that occur in childhood and adolescence. Postmortem brain (cortical gray matter, inferior temporal lobe; n=50) and liver (n=60) from vervet monkeys fed a uniform diet from birth through young adulthood were collected from archived tissues. Lipids were extracted and fatty acid levels determined. There was a marked reduction in the ratio of n-6 LC-PUFAs, arachidonic acid (ARA) and adrenic acid (ADR), relative to the n-3 LC-PUFA, docosahexaenoic acid (DHA), in temporal cortex lipids from birth to puberty and then a more gradual decrease though adulthood. This decrease in ratio resulted from a 3-fold accumulation of DHA levels while concentrations of ARA remained constant. Early childhood through adolescence appears to be a critical period for DHA accretion in the cortex of vervet monkeys and may represent a vulnerable stage where lack of dietary n-3 LC-PUFAs impacts development in humans.

Vitamin D heritability and effect of pregnancy status in Vervet monkeys (Chlorocebus aethiops sabaeus) under conditions of modest and high dietary supplementation.

OBJECTIVES: The two objectives of the current study were to: 1) investigate the genetic contributions to variations in serum vitamin D concentrations under two dietary conditions (a standard monkey biscuit diet vs. a diet designed to model typical American consumption); and 2) explore the interaction of vitamin D with pregnancy status using a cohort of pedigreed female vervet/African green monkeys. METHODS: This study includes 185 female (≥3.5 years) vervet/African green monkeys (Chlorocebus aethiops sabaeus) from a multi-generational, pedigreed breeding colony. The 25(OH)D3 concentrations were first measured seven to eight weeks after consuming a "typical American" diet (TAD), deriving 37, 18, and 45% of calories from fat, protein sources, and carbohydrates, and supplemented with vitamin D to a human equivalent of 1,000 IU/day. Vitamin D concentrations were assessed again when animals were switched to a low-fat, standard biscuit diet (LabDiet 5038) for 8 months, which provided a human equivalent of approximately 4,000 IU/day of vitamin D. All statistical analyses were implemented in SOLAR. RESULTS: Pregnancy was associated with reduced 25(OH)D3 concentrations. Heritability analyses indicated a significant genetic contribution to 25(OH)D3 concentrations in the same monkeys consuming the biscuit diet (h(2) =0.66, P=0.0004) and TAD (h(2) =0.67, P=0.0078) diets, with higher 25(OH)D3 concentrations in animals consuming the biscuit diet. Additionally, there was a significant genotype-by-pregnancy status interaction on 25(OH)D3 concentrations (P<0.05) only among animals consuming the TAD diet. DISCUSSION: These results support the existence of a genetic contribution to differences in serum 25(OH)D3 concentrations by pregnancy status and emphasize the role of diet (including vitamin D supplementation) in modifying genetic signals as well as vitamin D concentrations.

The genome of the vervet (Chlorocebus aethiops sabaeus).

We describe a genome reference of the African green monkey or vervet (Chlorocebus aethiops). This member of the Old World monkey (OWM) superfamily is uniquely valuable for genetic investigations of simian immunodeficiency virus (SIV), for which it is the most abundant natural host species, and of a wide range of health-related phenotypes assessed in Caribbean vervets (C. a. sabaeus), whose numbers have expanded dramatically since Europeans introduced small numbers of their ancestors from West Africa during the colonial era. We use the reference to characterize the genomic relationship between vervets and other primates, the intra-generic phylogeny of vervet subspecies, and genome-wide structural variations of a pedigreed C. a. sabaeus population. Through comparative analyses with human and rhesus macaque, we characterize at high resolution the unique chromosomal fission events that differentiate the vervets and their close relatives from most other catarrhine primates, in whom karyotype is highly conserved. We also provide a summary of transposable elements and contrast these with the rhesus macaque and human. Analysis of sequenced genomes representing each of the main vervet subspecies supports previously hypothesized relationships between these populations, which range across most of sub-Saharan Africa, while uncovering high levels of genetic diversity within each. Sequence-based analyses of major histocompatibility complex (MHC) polymorphisms reveal extremely low diversity in Caribbean C. a. sabaeus vervets, compared to vervets from putatively ancestral West African regions. In the C. a. sabaeus research population, we discover the first structural variations that are, in some cases, predicted to have a deleterious effect; future studies will determine the phenotypic impact of these variations.

Hematology and Clinical Chemistry Measures During and After Pregnancy and Age- and Sex-Specific Reference Intervals in African Green Monkeys (Chlorocebus aethiops sabaeus).

Clinical decisions and experimental analyses often involve the assessment of hematology and clinical chemistry. Using clinical pathology to assess the health status of NHP in breeding colonies or data from studies than involve pregnancy can often be complicated by pregnancy status. This study had 2 objectives regarding the hematology and clinical chemistry of African green monkeys (AGM, Chlorocebus aethiops sabaeus): 1) to compare pregnant or recently postpartum animals with nonpregnant, nonlactating animals and 2) to create age- and sex-specific reference intervals. Subjects in this study were 491 AGM from the Vervet Research Colony of the Wake Forest University Primate Center. Results indicated that changes in BUN, serum total protein, albumin, ALP, GGT, calcium, phosphorus, sodium, potassium, cholesterol, total CO2, globulins, lipase, amylase, WBC, neutrophils, lymphocytes, platelets, RBC, Hgb, and Hct occur during pregnancy and the postpartum period. Age- and sex-specific reference intervals consistent with guidelines from the American Society for Veterinary Clinical Pathology were established and further expand the understanding of how to define health in AGM on the basis of clinical pathology. The combination of understanding the changes that occur in pregnancy and postpartum and expansive reference intervals will help guide clinical and experimental decisions.

Sequencing strategies and characterization of 721 vervet monkey genomes for future genetic analyses of medically relevant traits.

BACKGROUND: We report here the first genome-wide high-resolution polymorphism resource for non-human primate (NHP) association and linkage studies, constructed for the Caribbean-origin vervet monkey, or African green monkey (Chlorocebus aethiops sabaeus), one of the most widely used NHPs in biomedical research. We generated this resource by whole genome sequencing (WGS) of monkeys from the Vervet Research Colony (VRC), an NIH-supported research resource for which extensive phenotypic data are available. RESULTS: We identified genome-wide single nucleotide polymorphisms (SNPs) by WGS of 721 members of an extended pedigree from the VRC. From high-depth WGS data we identified more than 4 million polymorphic unequivocal segregating sites; by pruning these SNPs based on heterozygosity, quality control filters, and the degree of linkage disequilibrium (LD) between SNPs, we constructed genome-wide panels suitable for genetic association (about 500,000 SNPs) and linkage analysis (about 150,000 SNPs). To further enhance the utility of these resources for linkage analysis, we used a further pruned subset of the linkage panel to generate multipoint identity by descent matrices. CONCLUSIONS: The genetic and phenotypic resources now available for the VRC and other Caribbean-origin vervets enable their use for genetic investigation of traits relevant to human diseases.