The Tbx20-TLE interaction is essential for the maintenance of the second heart field.

T-box transcription factor 20 (Tbx20) plays a multifaceted role in cardiac morphogenesis and controls a broad gene regulatory network. However, the mechanism by which Tbx20 activates and represses target genes in a tissue-specific and temporal manner remains unclear. Studies show that Tbx20 directly interacts with the Transducin-like Enhancer of Split (TLE) family of proteins to mediate transcriptional repression. However, a function for the Tbx20-TLE transcriptional repression complex during heart development has yet to be established. We created a mouse model with a two amino acid substitution in the Tbx20 EH1 domain, thereby disrupting the Tbx20-TLE interaction. Disruption of this interaction impaired crucial morphogenic events, including cardiac looping and chamber formation. Transcriptional profiling of Tbx20EH1Mut hearts and analysis of putative direct targets revealed misexpression of the retinoic acid pathway and cardiac progenitor genes. Further, we show that altered cardiac progenitor development and function contribute to the severe cardiac defects in our model. Our studies indicate that TLE-mediated repression is a primary mechanism by which Tbx20 controls gene expression.

PLIN5 interacts with FATP4 at membrane contact sites to promote lipid droplet-to-mitochondria fatty acid transport.

Cells adjust their metabolism by remodeling membrane contact sites that channel metabolites to different fates. Lipid droplet (LD)-mitochondria contacts change in response to fasting, cold exposure, and exercise. However, their function and mechanism of formation have remained controversial. We focused on perilipin 5 (PLIN5), an LD protein that tethers mitochondria, to probe the function and regulation of LD-mitochondria contacts. We demonstrate that efficient LD-to-mitochondria fatty acid (FA) trafficking and ß-oxidation during starvation of myoblasts are promoted by phosphorylation of PLIN5 and require an intact PLIN5 mitochondrial tethering domain. Using human and murine cells, we further identified the acyl-CoA synthetase, FATP4 (ACSVL4), as a mitochondrial interactor of PLIN5. The C-terminal domains of PLIN5 and FATP4 constitute a minimal protein interaction capable of inducing organelle contacts. Our work suggests that starvation leads to phosphorylation of PLIN5, lipolysis, and subsequent channeling of FAs from LDs to FATP4 on mitochondria for conversion to fatty-acyl-CoAs and subsequent oxidation.

Quantitative proteomic profiling identifies global protein network dynamics in murine embryonic heart development.

Defining the mechanisms that govern heart development is essential for identifying the etiology of congenital heart disease. Here, quantitative proteomics was used to measure temporal changes in the proteome at critical stages of murine embryonic heart development. Global temporal profiles of the over 7,300 proteins uncovered signature cardiac protein interaction networks that linked protein dynamics with molecular pathways. Using this integrated dataset, we identified and demonstrated a functional role for the mevalonate pathway in regulating the cell cycle of embryonic cardiomyocytes. Overall, our proteomic datasets are a resource for studying events that regulate embryonic heart development and contribute to congenital heart disease.

Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine.

Coronavirus vaccines that are highly effective against current and anticipated SARS-CoV-2 variants are needed to control COVID-19. We previously reported a receptor-binding domain (RBD)-sortase A-conjugated ferritin nanoparticle (scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected non-human primates (NHPs) from SARS-CoV-2 WA-1 infection. Here, we find the RBD-scNP induced neutralizing antibodies in NHPs against pseudoviruses of SARS-CoV and SARS-CoV-2 variants including 614G, Beta, Delta, Omicron BA.1, BA.2, BA.2.12.1, and BA.4/BA.5, and a designed variant with escape mutations, PMS20. Adjuvant studies demonstrate variant neutralization titers are highest with 3M-052-aqueous formulation (AF). Immunization twice with RBD-scNPs protect NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protect mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect animals from multiple different SARS-related viruses. Such a vaccine could provide broad immunity to SARS-CoV-2 variants.

Breadth of SARS-CoV-2 Neutralization and Protection Induced by a Nanoparticle Vaccine.

Coronavirus vaccines that are highly effective against SARS-CoV-2 variants are needed to control the current pandemic. We previously reported a receptor-binding domain (RBD) sortase A-conjugated ferritin nanoparticle (RBD-scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected monkeys from SARS-CoV-2 WA-1 infection. Here, we demonstrate SARS-CoV-2 RBD-scNP immunization induces potent neutralizing antibodies in non-human primates (NHPs) against all eight SARS-CoV-2 variants tested including the Beta, Delta, and Omicron variants. The Omicron variant was neutralized by RBD-scNP-induced serum antibodies with a mean of 10.6-fold reduction of ID50 titers compared to SARS-CoV-2 D614G. Immunization with RBD-scNPs protected NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protected mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect NHPs and mice from multiple different SARS-related viruses. Such a vaccine could provide the needed immunity to slow the spread of and reduce disease caused by SARS-CoV-2 variants such as Delta and Omicron.

Specificity and effector functions of non-neutralizing gB-specific monoclonal antibodies isolated from healthy individuals with human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common congenital infection. A glycoprotein B (gB) subunit vaccine (gB/MF59) is the most efficacious clinically tested to date, having achieved 50% protection against primary infection of HCMV-seronegative women. We previously identified that gB/MF59 vaccination primarily elicits non-neutralizing antibody responses, with variable binding to gB genotypes, and protection associated with binding to membrane-associated gB. We hypothesized that gB-specific non-neutralizing antibody binding breadth and function are dependent on epitope and genotype specificity, and ability to interact with membrane-associated gB. We mapped twenty-four gB-specific monoclonal antibodies (mAbs) from naturally HCMV-infected individuals for gB domain specificity, genotype preference, and ability to mediate phagocytosis or NK cell activation. gB-specific mAbs were primarily specific for Domain II and demonstrated variable binding to gB genotypes. Two mAbs facilitated phagocytosis with binding specificities of Domain II and AD2. This investigation provides novel understanding on the relationship between gB domain specificity and antigenic variability on gB-specific antibody effector functions.

Human Cytomegalovirus Glycoprotein B Nucleoside-Modified mRNA Vaccine Elicits Antibody Responses with Greater Durability and Breadth than MF59-Adjuvanted gB Protein Immunization.

A vaccine to prevent maternal acquisition of human cytomegalovirus (HCMV) during pregnancy is a primary strategy to reduce the incidence of congenital disease. The MF59-adjuvanted glycoprotein B (gB) protein subunit vaccine (gB/MF59) is the most efficacious vaccine tested to date for this indication. We previously identified that gB/MF59 vaccination elicited poor neutralizing antibody responses and an immunodominant response against gB antigenic domain 3 (AD-3). Thus, we sought to test novel gB vaccines to improve functional antibody responses and reduce AD-3 immunodominance. Groups of juvenile New Zealand White rabbits were administered 3 sequential doses of the full-length gB protein with an MF59-like squalene-based adjuvant, the gB ectodomain protein (lacking AD-3) with squalene adjuvant, or lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA encoding full-length gB. All vaccines were highly immunogenic with similar kinetics and comparable peak gB-binding and functional antibody responses. The AD-3-immunodominant IgG response following human gB/MF59 vaccination was closely mimicked in rabbits. Though gB ectodomain subunit vaccination eliminated targeting of epitopes in AD-3, it did not improve vaccine-elicited neutralizing or nonneutralizing antibody functions. gB nucleoside-modified mRNA-LNP-immunized rabbits exhibited an enhanced durability of vaccine-elicited antibody responses. Furthermore, the gB mRNA-LNP vaccine enhanced the breadth of IgG binding responses against discrete gB peptides. Finally, low-magnitude gB-specific T cell activity was observed in the full-length gB protein and mRNA-LNP groups, though not in ectodomain-vaccinated rabbits. Altogether, these data suggest that the use of gB nucleoside-modified mRNA-LNP vaccines is a viable strategy for improving on the partial efficacy of gB/MF59 vaccination and should be further evaluated in preclinical models.IMPORTANCE Human cytomegalovirus (HCMV) is the most common infectious cause of infant birth defects, resulting in permanent neurological disability for one newborn child every hour in the United States. After more than a half century of research and development, we remain without a clinically licensed vaccine or immunotherapeutic to reduce the burden of HCMV-associated disease. In this study, we sought to improve upon the glycoprotein B protein vaccine (gB/MF59), the most efficacious HCMV vaccine evaluated in a clinical trial, via targeted modifications to either the protein structure or vaccine formulation. Utilization of a novel vaccine platform, nucleoside-modified mRNA formulated in lipid nanoparticles, increased the durability and breadth of vaccine-elicited antibody responses. We propose that an mRNA-based gB vaccine may ultimately prove more efficacious than the gB/MF59 vaccine and should be further evaluated for its ability to elicit antiviral immune factors that can prevent HCMV-associated disease.

Maternal Humoral Immune Responses Do Not Predict Postnatal HIV-1 Transmission Risk in Antiretroviral-Treated Mothers from the IMPAACT PROMISE Study.

To design immune interventions that can synergize with antiretroviral therapy (ART) to reduce the rate of HIV mother-to-child transmission (MTCT), it is essential to characterize maternal immune responses in the setting of ART during pregnancy and breastfeeding and define their effect on MTCT. Prior studies reported an association between breast milk envelope (Env)-specific antibodies and antibody-dependent cell cytotoxicity (ADCC) activity with reduced postnatal transmission. In this study, we investigated whether these immune correlates were similarly associated with protection in a matched case-control study of mother-infant pairs receiving maternal ART or infant nevirapine prophylaxis during breastfeeding in the International Maternal-Pediatric-Adolescent AIDS Clinical Trials Network Promoting Maternal-Infant Survival Everywhere (PROMISE) trial, assessing postnatal transmission risk in 19 transmitting and 57 nontransmitting mothers using conditional logistic regression models adjusted for maternal plasma viral load. The odds ratios of postnatal MTCT for a 1-unit increase in an immune correlate were 3.61 (95% confidence interval [CI], 0.56, 23.14) for breast milk Env-specific secretory IgA (sIgA), 2.32 (95% CI, 0.43, 12.56) for breast milk and 2.16 (95% CI, 0.51, 9.14) for plasma Env-specific IgA, and 4.57 (95% CI, 0.68, 30.48) for breast milk and 0.96 (95% CI, 0.25, 3.67) for plasma ADCC activity, with all CIs spanning 1.0. Interestingly, although mucosal IgA responses are poor in untreated HIV-infected women, there was a strong correlation between the magnitudes of breast milk and plasma Env-specific IgA in this cohort. In this analysis of the small number of postnatal virus transmissions in the landmark PROMISE study, no single antibody response was associated with breast milk transmission risk.IMPORTANCE Each year, >150,000 infants become newly infected with HIV-1 through MTCT despite ART, with up to 42% of infections occurring during breastfeeding. Several factors contribute to continued pediatric infections, including ART nonadherence, the emergence of drug-resistant HIV strains, acute infection during breastfeeding, and poor access to ART in resource-limited areas. A better understanding of the maternal humoral immune responses that provide protection against postnatal transmission in the setting of ART is critical to guide the design of maternal vaccine strategies to further eliminate postnatal HIV transmission. In this study, we found that in women treated with antiretrovirals during pregnancy, there was a positive correlation between plasma viral load and breast milk and plasma IgA responses; however, conclusions regarding odds of MTCT risk were limited by the small sample size. These findings will inform future studies to investigate maternal immune interventions that can synergize with ART to eliminate MTCT during breastfeeding.

Complex integration of intrinsic and peripheral signaling is required for pituitary gland development.

The coordination of pituitary development is complicated and requires input from multiple cellular processes. Recent research has provided insight into key molecular determinants that govern cell fate specification in the pituitary. Moreover, increasing research aimed to identify, characterize, and functionally describe the presumptive pituitary stem cell population has allowed for a better understanding of the processes that govern endocrine cell differentiation in the developing pituitary. The culmination of this research has led to the ability of investigators to recapitulate some of embryonic pituitary development in vitro, the first steps to developing novel regenerative therapies for pituitary diseases. In this current review, we cover the major players in pituitary stem/progenitor cell function and maintenance, and the key molecular determinants of endocrine cell specification. In addition, we discuss the contribution of peripheral hormonal regulation of pituitary gland development, an understudied area of research.

Anterior Pituitary Transcriptome Suggests Differences in ACTH Release in Tame and Aggressive Foxes.

Domesticated species exhibit a suite of behavioral, endocrinological, and morphological changes referred to as "domestication syndrome." These changes may include a reduction in reactivity of the hypothalamic-pituitary-adrenal (HPA) axis and specifically reduced adrenocorticotropic hormone release from the anterior pituitary. To investigate the biological mechanisms targeted during domestication, we investigated gene expression in the pituitaries of experimentally domesticated foxes (Vulpes vulpes). RNA was sequenced from the anterior pituitary of six foxes selectively bred for tameness ("tame foxes") and six foxes selectively bred for aggression ("aggressive foxes"). Expression, splicing, and network differences identified between the two lines indicated the importance of genes related to regulation of exocytosis, specifically mediated by cAMP, organization of pseudopodia, and cell motility. These findings provide new insights into biological mechanisms that may have been targeted when these lines of foxes were selected for behavior and suggest new directions for research into HPA axis regulation and the biological underpinnings of domestication.

Effects of Exposure to the Endocrine-Disrupting Chemical Bisphenol A During Critical Windows of Murine Pituitary Development.

Critical windows of development are often more sensitive to endocrine disruption. The murine pituitary gland has two critical windows of development: embryonic gland establishment and neonatal hormone cell expansion. During embryonic development, one environmentally ubiquitous endocrine-disrupting chemical, bisphenol A (BPA), has been shown to alter pituitary development by increasing proliferation and gonadotrope number in females but not males. However, the effects of exposure during the neonatal period have not been examined. Therefore, we dosed pups from postnatal day (PND)0 to PND7 with 0.05, 0.5, and 50 µg/kg/d BPA, environmentally relevant doses, or 50 µg/kg/d estradiol (E2). Mice were collected after dosing at PND7 and at 5 weeks. Dosing mice neonatally with BPA caused sex-specific gene expression changes distinct from those observed with embryonic exposure. At PND7, pituitary Pit1 messenger RNA (mRNA) expression was decreased with BPA 0.05 and 0.5 µg/kg/d in males only. Expression of Pomc mRNA was decreased at 0.5 µg/kg/d BPA in males and at 0.5 and 50 µg/kg/d BPA in females. Similarly, E2 decreased Pomc mRNA in both males and females. However, no noticeable corresponding changes were found in protein expression. Both E2 and BPA suppressed Pomc mRNA in pituitary organ cultures; this repression appeared to be mediated by estrogen receptor-α and estrogen receptor-ß in females and G protein-coupled estrogen receptor in males, as determined by estrogen receptor subtype-selective agonists. These data demonstrated that BPA exposure during neonatal pituitary development has unique sex-specific effects on gene expression and that Pomc repression in males and females can occur through different mechanisms.

Identification of a novel progenitor cell marker, grainyhead-like 2 in the developing pituitary.

BACKGROUND: Pituitary stem/progenitor cells give rise to all of the endocrine cell types within the pituitary gland and are necessary for both development and gland homeostasis. Recent studies have identified several key factors that characterize the progenitor cell population. However, little is known about the factors that regulate progenitor cell differentiation and maintenance. Therefore, it is crucial to identify novel factors that help elucidate mechanisms of progenitor cell function in the developing pituitary. Our studies are the first to characterize the expression of Grainyhead-like 2 (GRHL2), a transcription factor known to regulate progenitor cell plasticity, in the developing pituitary. RESULTS: Our studies show GRHL2 expression is highest in the embryonic and early postnatal pituitary and is localized in pituitary progenitor cells. We demonstrate GRHL2 expression is changed in Notch2 cKO and Prop1df/df mice, mouse models that display progenitor cell number defects. In addition, our studies indicate a potential relationship between Notch signaling and GRHL2 expression in the developing pituitary. CONCLUSIONS: Taken together, our results indicate GRHL2 as a novel progenitor cell maker in the developing pituitary that may contribute to progenitor cell function and maintenance. Developmental Dynamics 245:1097-1106, 2016. © 2016 Wiley Periodicals, Inc.

Strategies to prevent and reduce diabetes and obesity in Sacramento, California: the African American Leadership Coalition and University of California, Davis.

BACKGROUND: Diabetes is one of the leading causes of illness and death for African Americans and people of African descent throughout the United States and in the city and county of Sacramento, California. The involvement of families and communities in developing prevention strategies can increase the likelihood that behavioral changes will be sustained. CONTEXT: Three member organizations of the African American Leadership Coalition (AALC) entered into a partnership with the University of California, Davis (UC Davis) to engage families in developing a process to identify barriers to diabetes and obesity prevention and reduction, exchange strategies, and create action plans for prevention. METHODS: The intervention comprised 3 phases: 1) coalition formation and training; 2) data collection, analysis, and dissemination of results; and 3) development of family and community action plans. Academic and community partners planned and implemented all project phases together. OUTCOMES: Sources of information about diabetes and obesity were primarily doctors and the Internet; barriers were related to lack of time needed to prepare healthy meals, high food costs, transportation to fresh markets, motivation around healthy habits, and unsafe environments. Action plans addressed behavioral change and family cohesion. The group discussion format encouraged mutual support and suggestions for better eating and physical exercise habits. INTERPRETATION: This collaborative partnership model can strengthen existing group relationships or promote new affiliations that form the basis for future action coalitions. Participants worked both within and across groups to exchange information, stories of success and challenges, and specific health improvement strategies.