Leveraging African American family connectors for Alzheimer's disease genomic studies.

INTRODUCTION: The underrepresentation of African Americans (AAs) in Alzheimer's disease (AD) research may limit potential benefits from translational applications. This article describes an approach to recruit AA families into an AD genomic study and characteristics of seeds (family connectors) used to overcome recruitment barriers of AA families into AD research. METHODS: A four-step outreach and snowball sampling approach relying on family connectors was used to recruit AA families. Descriptive statistics of a profile survey were gathered to understand the demographic and health characteristics of family connectors. RESULTS: Twenty-five AA families (117 participants) were enrolled in the study via family connectors. Most family connectors self-identified as female (88%), were 60 years of age or older (76%), and attained post-secondary education (77%). DISCUSSION: Community-engaged strategies were essential to recruit AA families. Relationships between study coordinators and family connectors build trust early in the research process among AA families. HIGHLIGHTS: Community events were most effective for recruiting African American families. Family connectors were primarily female, in good health, and highly educated. Systematic efforts by researchers are necessary to "sell" a study to participants.

Parental Guidance Suggested: Engaging Parents as Partners in Research Studies of Genomic Screening for a Pediatric Population.

Recent advances in genomic sequencing and genomic medicine are reshaping the landscape of clinical care. As a screening modality, genetic sequencing has the potential to dramatically expand the clinical utility of newborn screening (NBS), though significant barriers remain regarding ethical, legal, and social implications (ELSI) and technical and evidentiary challenges. Stakeholder-informed implementation research is poised to grapple with many of these barriers, and parents are crucial stakeholders in this process. We describe the formation and activities of a Community Research Board (CRB) composed of parents with diverse backgrounds assembled to participate in an ongoing research partnership with genomic and public health researchers at the University of North Carolina. The mission of the CRB is to provide insight into parental perspectives regarding the prospect of adding genomic sequencing to NBS and collaboratively develop strategies to ensure its equitable uptake. We describe how these contributions can improve the accessibility of research and recruitment methods and promote trust and inclusivity within diverse communities to maximize the societal benefit of population genomic screening in healthy children.

Integration of stakeholder engagement from development to dissemination in genomic medicine research: Approaches and outcomes from the CSER Consortium.

PURPOSE: There is a critical need for genomic medicine research that reflects and benefits socioeconomically and ancestrally diverse populations. However, disparities in research populations persist, highlighting that traditional study designs and materials may be insufficient or inaccessible to all groups. New approaches can be gained through collaborations with patient/community stakeholders. Although some benefits of stakeholder engagement are recognized, routine incorporation into the design and implementation of genomics research has yet to be realized. METHODS: The National Institutes of Health-funded Clinical Sequencing Evidence-Generating Research (CSER) consortium required stakeholder engagement as a dedicated project component. Each CSER project planned and carried out stakeholder engagement activities with differing goals and expected outcomes. Examples were curated from each project to highlight engagement strategies and outcomes throughout the research lifecycle from development through dissemination. RESULTS: Projects tailored strategies to individual study needs, logistical constraints, and other challenges. Lessons learned include starting early with engagement efforts across project stakeholder groups and planned flexibility to enable adaptations throughout the project lifecycle. CONCLUSION: Each CSER project used more than 1 approach to engage with relevant stakeholders, resulting in numerous adaptations and tremendous value added throughout the full research lifecycle. Incorporation of community stakeholder insight improves the outcomes and relevance of genomic medicine research.

Dementia Caregiver Experiences and Recommendations for Using the Behavioral and Environmental Sensing and Intervention System at Home: Usability and Acceptability Study.

BACKGROUND: Caregiver burden associated with dementia-related agitation is one of the most common reasons for a community-dwelling person living with dementia to transition to a care facility. The Behavioral and Environmental Sensing and Intervention (BESI) for the Dementia Caregiver Empowerment system uses sensing technology, smartwatches, tablets, and data analytics to detect and predict agitation in persons living with dementia and to provide just-in-time notifications and dyad-specific intervention recommendations to caregivers. The BESI system has shown that there is a valid relationship between dementia-related agitation and environmental factors and that caregivers prefer a home-based monitoring system. OBJECTIVE: The aim of this study is to obtain input from caregivers of persons living with dementia on the value, usability, and acceptability of the BESI system in the home setting and obtain their insights and recommendations for the next stage of system development. METHODS: A descriptive qualitative design with thematic analysis was used to analyze 10 semistructured interviews with caregivers. The interviews comprised 16 questions, with an 80% (128/160) response rate. RESULTS: Postdeployment caregiver feedback about the BESI system and the overall experience were generally positive. Caregivers acknowledged the acceptability of the system by noting the ease of use and saw the system as a fit for them. Functionality issues such as timeliness in agitation notification and simplicity in the selection of agitation descriptors on the tablet interface were identified, and caregivers indicated a desire for more word options to describe agitation behaviors. Agitation intervention suggestions were well received by the caregivers, and the resulting decrease in the number and severity of agitation events helped confirm that the BESI system has good value and acceptability. Thematic analysis suggested several subjective experiences and yielded the themes of usefulness and helpfulness. CONCLUSIONS: This study determined preferences for assessing caregiver strain and burden, explored caregiver acceptance of the technology system (in-home sensors, actigraph or smart watch technology, and tablet devices), discerned caregiver insights on the burden and stress of caring for persons living with dementia experiencing agitation in dementia, and solicited caregiver input and recommendations for system changes. The themes of usefulness and helpfulness support the use of caregiver knowledge and experience to inform further development of the technology.

Data-Driven Forecasting of Agitation for Persons with Dementia: A Deep Learning-Based Approach.

The World Health Organization estimates that approximately 10 million people are newly diagnosed with dementia each year and a global prevalence of nearly 50 million persons with dementia (PwD). The vast majority of PwD living at home receive the majority of their care from informal familial caregivers. The quality of life (QOL) of familial caregivers may be significantly impacted by their caregiving responsibilities and resultant caregiver burden. A major contributor to caregiver burden is the random occurrence of agitation in PwD and familial caregivers' lack of preparedness to manage these episodes. Caregiver burden may be reduced if it is possible to forecast impending agitation episodes. In this study, we leverage data-driven deep learning models to predict agitation episodes in PwD. We used Long Short-Term Memory (LSTM), a deep learning class of algorithms, to forecast agitations up to 30 min before actual agitation events. In particular, we managed the missing data by estimating the missing values and compensated for the class imbalance challenge by down-sampling the majority class. The simulations were based on real-world data from Alzheimer's disease (AD) caregivers and PwD dyads home environments, including ambient noise level, illumination, room temperature, atmospheric pressure (Pa), and relative humidity. Our results show the efficacy of data-driven deep learning models in predicting agitation episodes in community-dwelling AD dyads with accuracy of 98.6% and recall (sensitivity) of 84.8%.

Ancestral origin of ApoE ε4 Alzheimer disease risk in Puerto Rican and African American populations.

The ApoE ε4 allele is the most significant genetic risk factor for late-onset Alzheimer disease. The risk conferred by ε4, however, differs across populations, with populations of African ancestry showing lower ε4 risk compared to those of European or Asian ancestry. The cause of this heterogeneity in risk effect is currently unknown; it may be due to environmental or cultural factors correlated with ancestry, or it may be due to genetic variation local to the ApoE region that differs among populations. Exploring these hypotheses may lead to novel, population-specific therapeutics and risk predictions. To test these hypotheses, we analyzed ApoE genotypes and genome-wide array data in individuals from African American and Puerto Rican populations. A total of 1,766 African American and 220 Puerto Rican individuals with late-onset Alzheimer disease, and 3,730 African American and 169 Puerto Rican cognitively healthy individuals (> 65 years) participated in the study. We first assessed average ancestry across the genome ("global" ancestry) and then tested it for interaction with ApoE genotypes. Next, we assessed the ancestral background of ApoE alleles ("local" ancestry) and tested if ancestry local to ApoE influenced Alzheimer disease risk while controlling for global ancestry. Measures of global ancestry showed no interaction with ApoE risk (Puerto Rican: p-value = 0.49; African American: p-value = 0.65). Conversely, ancestry local to the ApoE region showed an interaction with the ApoE ε4 allele in both populations (Puerto Rican: p-value = 0.019; African American: p-value = 0.005). ApoE ε4 alleles on an African background conferred a lower risk than those with a European ancestral background, regardless of population (Puerto Rican: OR = 1.26 on African background, OR = 4.49 on European; African American: OR = 2.34 on African background, OR = 3.05 on European background). Factors contributing to the lower risk effect in the ApoE gene ε4 allele are likely due to ancestry-specific genetic factors near ApoE rather than non-genetic ethnic, cultural, and environmental factors.

Determining fungi rRNA copy number by PCR.

The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for ß-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment.

Identification of Bioaerosols Released from an Egg Production Facility in the Southeast United States.

This field study investigated biological characteristics of aerosols emitted from a commercial egg production farm (layer operation). Bioaerosol samples were taken on this farm at five sampling locations covering emission source (inside a layer barn) and four ambient surrounding stations at four wind directions. All-glass impingers (AGI) were used for the field sampling. AGI fluid samples were plated in duplicate on Trypticase Soy Agar for growth of bacteria and Sabouraud Dextrose Agar for growth of fungi. The most prominent bacterial colony types were identified using a combination of methods that include recording characteristics of colony morphology; performing a Gram staining method and metabolic analyses using the Biolog system. Results from thirty-five AGI samples taken at the five stations through seven sampling events over four seasons indicate that there were significantly lower total bacterial concentrations in the samples collected from ambient stations as compared with the samples collected in the layer house; the mean bacterial concentration at the in-house sampling station was 3.86×10(5)±1.74×10(5) cfu/m(3), whereas the mean bacterial concentrations at four ambient stations in the vicinity of the farm ranged from 1.3×10(3) to 6.2×10(3) cfu/m(3) with no significant differences in mean among ambient stations. There were also no significant differences in fungi concentrations among all sampling stations. Mean fungi concentrations at the in-house station was 3.0×10(3)±4.45×10(3) cfu/m(3), whereas the mean concentrations at the ambient stations ranged from 7.4×10(3) to 1.7×10(4) cfu/m(3). The most prominent bacterial species differed among all five stations. Three of the most prominent bacteria from samples taken at all five stations were gram positive. Fungal type also differed from station to station.

VEGF-mediated STAT3 activation inhibits retinal vascularization by down-regulating local erythropoietin expression.

Avascular, hypoxic retina has been postulated to be a source of angiogenic factors that cause aberrant angiogenesis and intravitreal neovascularization (IVNV) in retinopathy of prematurity. Vascular endothelial growth factor (VEGF) is an important factor involved. However, VEGF is also required for normal retinal vascular development, which raises concerns about inhibiting its activity to treat IVNV in retinopathy of prematurity. Therefore, understanding the effects that VEGF has on other factors in the development of avascular retina is important to prevent aberrant angiogenesis and IVNV. Here, we show that STAT3 was activated by increased retinal VEGF in the rat 50/10 oxygen-induced retinopathy model. Phospho-STAT3 colocalized with glutamine synthetase-labeled Müller cells. Inhibition of STAT3 reduced avascular retina and increased retinal erythropoietin (Epo) expression. Epo administered exogenously also reduced avascular retina in the model. In an in vitro study, hypoxia-induced VEGF inhibited Epo gene expression by STAT3 activation in rat Müller cells. The mechanism by which activated STAT3 regulated Epo was by inhibition of Epo promoter activity. Together, these findings show that increased retinal VEGF contributes to avascular retina by regulating retinal Epo expression through Janus kinase/STAT signaling. Our results suggest that rescuing Epo expression in the retina before the development of IVNV may promote normal developmental angiogenesis and, therefore, reduce the stimulus for later pathologic IVNV.

The role of vascular endothelial growth factor-induced activation of NADPH oxidase in choroidal endothelial cells and choroidal neovascularization.

Rac1, a subunit of NADPH oxidase, plays an important role in directed endothelial cell motility. We reported previously that Rac1 activation was necessary for choroidal endothelial cell migration across the retinal pigment epithelium, a critical step in the development of vision-threatening neovascular age-related macular degeneration. Here we explored the roles of Rac1 and NADPH oxidase activation in response to vascular endothelial growth factor treatment in vitro and in a model of laser-induced choroidal neovascularization. We found that vascular endothelial growth factor induced the activation of Rac1 and of NADPH oxidase in cultured human choroidal endothelial cells. Further, vascular endothelial growth factor led to heightened generation of reactive oxygen species from cultured human choroidal endothelial cells, which was prevented by the NADPH oxidase inhibitors, apocynin and diphenyleneiodonium, or the antioxidant, N-acetyl-L-cysteine. In a model of laser-induced injury, inhibition of NADPH oxidase with apocynin significantly reduced reactive oxygen species levels as measured by dihydroethidium fluorescence and the volume of laser-induced choroidal neovascularization. Mice lacking functional p47phox, a subunit of NADPH oxidase, had reduced dihydroethidium fluorescence and choroidal neovascularization compared with wild-type controls. Taken together, these results indicate that vascular endothelial growth factor activates Rac1 upstream from NADPH oxidase in human choroidal endothelial cells and increases generation of reactive oxygen species, contributing to choroidal neovascularization. These steps may contributed to the pathology of neovascular age-related macular degeneration.

Reduction in endothelial tip cell filopodia corresponds to reduced intravitreous but not intraretinal vascularization in a model of ROP.

To determine the effect of a vascular endothelial growth factor receptor 2 tyrosine kinase (VEGFR2) inhibitor on intravitreous neovascularization (IVNV), endothelial tip cell filopodia, and intraretinal vascularization in a rat model of retinopathy of prematurity (ROP). Within 4h of birth, newborn Sprague-Dawley rat pups and their mothers were cycled between 50% and 10% oxygen daily until postnatal day (p)12. Pups were given intravitreous injections of VEGFR2 inhibitor, SU5416, or control (dimethyl sulfoxide, DMSO) and returned to oxygen cycling until p14, then placed into room air. Intravitreous neovascularization (IVNV), avascular/total retinal areas, and endothelial tip cell filopodial number and length were determined in lectin-labeled neurosensory retinal flat mounts. Cryosections or fresh tissue were analyzed for phospho-VEGFR1, phospho-VEGFR2, activated caspase-3, or phospho-beta3 integrin. Human umbilical venous (HUVECs) and human choroidal endothelial cells (ECs) were treated with VEGFR2 inhibitor to determine effect on VEGFR2 phosphorylation and on directed EC migration toward a VEGF gradient. Filopodial length and number of migrated ECs were also measured. Compared to control, the VEGFR2 inhibitor reduced VEGFR2 phosphorylation in HUVECs in vitro and clock hours and areas of IVNV but not percent avascular retina in vivo. Filopodial length and number of filopodia/EC tip cell were reduced in retinal flat mounts at doses that inhibited IVNV, whereas at lower doses, only a reduction in filopodial length/EC tip cell was found. There was no difference in phosphorylated beta3 integrin and cleaved caspase-3 labeling in VEGFR2 inhibitor-treated compared to control in vivo. Doses of the VEGFR2 inhibitor that reduced filopodial length and number of filopodia/migrating EC corresponded to reduced EC migration in in vitro models. VEGFR2 inhibitor reduced IVNV and filopodial number and length/EC tip cell without interfering with intraretinal vascularization. Reducing the number and length of filopodia/endothelial tip cell may reduce guidance cues for endothelial cells to migrate into the vitreous without interfering with migration into the retina toward a VEGF gradient.

The role of supplemental oxygen and JAK/STAT signaling in intravitreous neovascularization in a ROP rat model.

PURPOSE: To investigate whether oxygen stresses experienced in retinopathy of prematurity (ROP) trigger signaling through reactive oxygen species (ROS) and whether the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway lead to intravitreous neovascularization (IVNV) in an oxygen-induced retinopathy (OIR) rat model. METHODS: Newborn rat pups exposed to repeated fluctuations in oxygen and rescued in supplemental oxygen (28% O(2), 50/10 OIR+SO) were treated with apocynin, an NADPH oxidase and ROS inhibitor (10 mg/kg/d), AG490, a JAK2 inhibitor (5 mg/kg/d), or phosphate-buffered saline. Intraperitoneal injections were given from postnatal day (P)12 to P17 (apocynin), or from P3 to P17 (AG490). Outcomes were intravitreous neovascularization and avascular/total retinal areas, vascular endothelial growth factor, phosphorylated JAK2, and phosphorylated STAT3. RESULTS: Apocynin significantly reduced phosphorylated STAT3 in 50/10 OIR+SO (P = 0.04), in association with previously reported inhibition of the IVNV area. Inhibition of JAK with AG490 significantly reduced phosphorylated JAK2 (P < 0.001), phosphorylated STAT3 (P = 0.002), and IVNV area (P = 0.033) in the 50/10 OIR+SO model compared with control. CONCLUSIONS: Activation of NADPH oxidase from supplemental oxygen works through activated STAT3 to lead to IVNV. In addition, inhibition of the JAK/STAT pathway reduces IVNV. Further studies are needed to determine the effects and relationships of oxygen stresses on JAK/STAT and NAPDH oxidase signaling.

Activated NAD(P)H oxidase from supplemental oxygen induces neovascularization independent of VEGF in retinopathy of prematurity model.

PURPOSE: To study NAD(P)H oxidase-dependent outcomes after oxygen stresses that are similar to those experienced by preterm infants today using a rat model of retinopathy of prematurity. METHODS: Within 4 hours of birth, pups and their mothers were cycled between 50% and 10% oxygen daily for 14 days and were returned to room air (21% O2, 50/10 oxygen-induced retinopathy [OIR]) or supplemental oxygen (28% O2, 50/10 OIR+SO) for 4 days. Pups received intraperitoneal injections of the specific NAD(P)H oxidase inhibitor apocynin (10 mg/kg/d) or of PBS from postnatal day (P)12 to P17, and some received intraperitoneal injections of hypoxyprobe before kill. Intravitreous neovascularization (IVNV), avascular/total retinal areas, vascular endothelial growth factor (VEGF), NAD(P)H oxidase activity, or hypoxic retina (conjugated hypoxyprobe) were determined in neurosensory retinas. Human retinal microvascular endothelial cells (RMVECs) treated with apocynin or control were exposed to 1% or 21% O2 and assayed for phosphorylated (p-)Janus kinase (JNK) and NAD(P)H oxidase activity. RESULTS: Retinas from 50/10 OIR+SO had increased NAD(P)H oxidase activity and lower VEGF than did retinas from 50/10 OIR. Apocynin treatment reduced the IVNV area and hypoxic retina in 50/10 OIR+SO. RMVECs treated with 1% O2 had increased p-JNK compared with RMVECs exposed to room air. CONCLUSIONS: Different oxygen stresses activate NAD(P)H oxidase to varying degrees to trigger disparate pathways (angiogenesis or apoptosis). The oxygen stresses and outcomes used in this study are relevant to human ROP and may explain some of the complexity in the pathophysiology of ROP resulting from oxygen exposure.

Neutralizing VEGF decreases tortuosity and alters endothelial cell division orientation in arterioles and veins in a rat model of ROP: relevance to plus disease.

PURPOSE: To study the effects of vascular endothelial growth factor (VEGF) on endothelial nitric oxide synthetase (eNOS) and retinal vascular tortuosity and cleavage planes in a rat model of retinopathy of prematurity (ROP). METHODS: Within 4 hours of birth, pups and mothers were cycled between 50% and 10% oxygen daily. At postnatal day (p)12, pups received either intravitreous anti-rat neutralizing antibody to VEGF or control nonimmune rat IgG in one eye and returned to oxygen cycling until p14 when they were placed in room air (RA) for 4 days (50/10 oxygen-induced retinopathy [50/10 OIR]). Tortuosity indices and endothelial cleavage plane angles relative to the long axes of the major retinal vessels during anaphase were calculated from phosphohistone- and Alexa-isolectin-stained retinal flatmounts. Some retinas were processed for eNOS protein or phosphorylated/total eNOS. RESULTS: Retinas from 50/10 OIR had increased tortuosity over time with peaks at p12 and p14 (P < 0.001 vs. RA) before the development of intravitreous neovascularization, which peaked at p18. Compared with RA, eNOS/actin in 50/10 OIR retinas was increased at p12 (P = 0.0003) and p14 (P = 0.047). Inhibition of VEGF with a neutralizing antibody decreased tortuosity and caused endothelial mitosis cleavage planes to orient in favor of vessel elongation but did not affect eNOS protein or activation. CONCLUSIONS: In the 50/10 OIR model, a model with relevance to ROP, arteriolar tortuosity, and venous dilation are increased through VEGF, which influences the orientation of endothelial cell cleavage in major arterioles and veins, independent of eNOS.