Validation of the ND-PAE Diagnosis in Children with Heavy Prenatal Alcohol Exposure.

This study evaluated criteria for Neurobehavioral Disorder Associated with Prenatal Alcohol Exposure (ND-PAE). Kable et al. (2022) assessed the validity of this diagnosis in a sample with low exposure to alcohol. The current study expanded this assessment to a sample with a wider age range and heavier alcohol exposure. Data were collected from participants (5-17y) with prenatal alcohol exposure (PAE) and typically developing controls at six Collaborative Initiative on Fetal Alcohol Spectrum Disorders sites using neuropsychological assessment and caregiver reports. Impairment was tested at 1SD, 1.5SD, and 2SD below the normative average and a modification of the adaptive functioning requirement was tested. Testing impairment at 1SD resulted in the highest endorsement rates in both groups. Our findings replicated the study by Kable et al. and show that current criteria captured a high rate of those with PAE and that requiring fewer adaptive functioning criteria resulted in higher sensitivity to PAE.

Genomic resources for the Yellowfin tuna Thunnus albacares.

BACKGROUND: The Yellowfin tuna (Thunnus albacares) is a large tuna exploited by major fisheries in tropical and subtropical waters of all oceans except the Mediterranean Sea. Genomic studies of population structure, adaptive variation or of the genetic basis of phenotypic traits are needed to inform fisheries management but are currently limited by the lack of a reference genome for this species. Here we report a draft genome assembly and a linkage map for use in genomic studies of T. albacares. METHODS AND RESULTS: Illumina and PacBio SMRT sequencing were used in combination to generate a hybrid assembly that comprises 743,073,847 base pairs contained in 2,661 scaffolds. The assembly has a N50 of 351,587 and complete and partial BUSCO scores of 86.47% and 3.63%, respectively. Double-digest restriction associated DNA (ddRAD) was used to genotype the 2 parents and 164 of their F1 offspring resulting from a controlled breeding cross, retaining 19,469 biallelic single nucleotide polymorphism (SNP) loci. The SNP loci were used to construct a linkage map that features 24 linkage groups that represent the 24 chromosomes of yellowfin tuna. The male and female maps span 1,243.8 cM and 1,222.9 cM, respectively. The map was used to anchor the assembly in 24 super-scaffolds that contain 79% of the yellowfin tuna genome. Gene prediction identified 46,992 putative genes 20,203 of which could be annotated via gene ontology. CONCLUSIONS: The draft reference will be valuable to interpret studies of genome wide variation in T. albacares and other Scombroid species.

TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway.

Cranial neural crest development is governed by positional gene regulatory networks (GRNs). Fine-tuning of the GRN components underlies facial shape variation, yet how those networks in the midface are connected and activated remain poorly understood. Here, we show that concerted inactivation of Tfap2a and Tfap2b in the murine neural crest, even during the late migratory phase, results in a midfacial cleft and skeletal abnormalities. Bulk and single-cell RNA-seq profiling reveal that loss of both TFAP2 family members dysregulates numerous midface GRN components involved in midface morphogenesis, patterning and differentiation. Notably, Alx1, Alx3 and Alx4 (ALX) transcript levels are reduced, whereas ChIP-seq analyses suggest TFAP2 family members directly and positively regulate ALX gene expression. Tfap2a, Tfap2b and ALX co-expression in midfacial neural crest cells of both mouse and zebrafish implies conservation of this regulatory axis across vertebrates. Consistent with this notion, tfap2a zebrafish mutants present with abnormal alx3 expression patterns, Tfap2a binds ALX loci and tfap2a-alx3 genetic interactions are observed. Together, these data demonstrate TFAP2 paralogs regulate vertebrate midfacial development in part by activating expression of ALX transcription factor genes.

Molecular disparities in colorectal cancers of White Americans, Alabama African Americans, and Oklahoma American Indians.

In the US, the majority of cancer samples analyzed are from white people, leading to biases in racial and ethnic treatment outcomes. Colorectal cancer (CRC) incidence and mortality rates are high in Alabama African Americans (AAs) and Oklahoma American Indians (AIs). We hypothesized that differences between racial groups may partially explain these disparities. Thus, we compared transcriptomic profiles of CRCs of Alabama AAs, Oklahoma AIs, and white people from both states. Compared to CRCs of white people, CRCs of AAs showed (a) higher expression of cytokines and vesicle trafficking toward modulated antitumor-immune activity, and (b) lower expression of the ID1/BMP/SMAD axis, IL22RA1, APOBEC3, and Mucins; and AIs had (c) higher expression of PTGS2/COX2 (an NSAID target/pro-oncogenic inflammation) and splicing regulators, and (d) lower tumor suppressor activities (e.g., TOB2, PCGF2, BAP1). Therefore, targeting strategies designed for white CRC patients may be less effective for AAs/AIs. These findings illustrate needs to develop optimized interventions to overcome racial CRC disparities.

FGF1 supports glycolytic metabolism through the estrogen receptor in endocrine-resistant and obesity-associated breast cancer.

BACKGROUND: Obesity increases breast cancer risk and breast cancer-specific mortality, particularly for people with estrogen receptor (ER)-positive tumors. Body mass index (BMI) is used to define obesity, but it may not be the best predictor of breast cancer risk or prognosis on an individual level. Adult weight gain is an independent indicator of breast cancer risk. Our previous work described a murine model of obesity, ER-positive breast cancer, and weight gain and identified fibroblast growth factor receptor (FGFR) as a potential driver of tumor progression. During adipose tissue expansion, the FGF1 ligand is produced by hypertrophic adipocytes as a stimulus to stromal preadipocytes that proliferate and differentiate to provide additional lipid storage capacity. In breast adipose tissue, FGF1 production may stimulate cancer cell proliferation and tumor progression. METHODS: We explored the effects of FGF1 on ER-positive endocrine-sensitive and resistant breast cancer and compared that to the effects of the canonical ER ligand, estradiol. We used untargeted proteomics, specific immunoblot assays, gene expression profiling, and functional metabolic assessments of breast cancer cells. The results were validated in tumors from obese mice and breast cancer datasets from women with obesity. RESULTS: FGF1 stimulated ER phosphorylation independently of estradiol in cells that grow in obese female mice after estrogen deprivation treatment. Phospho- and total proteomic, genomic, and functional analyses of endocrine-sensitive and resistant breast cancer cells show that FGF1 promoted a cellular phenotype characterized by glycolytic metabolism. In endocrine-sensitive but not endocrine-resistant breast cancer cells, mitochondrial metabolism was also regulated by FGF1. Comparison of gene expression profiles indicated that tumors from women with obesity shared hallmarks with endocrine-resistant breast cancer cells. CONCLUSIONS: Collectively, our data suggest that one mechanism by which obesity and weight gain promote breast cancer progression is through estrogen-independent ER activation and cancer cell metabolic reprogramming, partly driven by FGF/FGFR. The first-line treatment for many patients with ER-positive breast cancer is inhibition of estrogen synthesis using aromatase inhibitors. In women with obesity who are experiencing weight gain, locally produced FGF1 may activate ER to promote cancer cell metabolic reprogramming and tumor progression independently of estrogen.

The Effects of Nitric Oxide on Choroidal Gene Expression.

Purpose: Nitric oxide (NO) is recognized as an important biological mediator that controls several physiological functions, and evidence is now emerging that this molecule may play a significant role in the postnatal control of ocular growth and myopia development. We therefore sought to understand the role that nitric oxide plays in visually-guided ocular growth in order to gain insight into the underlying mechanisms of this process. Methods: Choroids were incubated in organ culture in the presence of the NO donor, PAPA-NONOate (1.5 mM). Following RNA extraction, bulk RNA-seq was used to quantify and compare choroidal gene expression in the presence and absence of PAPA-NONOate. We used bioinformatics to identify enriched canonical pathways, predicted diseases and functions, and regulatory effects of NO in the choroid. Results: Upon treatment of normal chick choroids with the NO donor, PAPA-NONOate, we identified a total of 837 differentially expressed genes (259 upregulated genes, 578 down-regulated genes) compared with untreated controls. Among these, the top five upregulated genes were LSMEM1, STEAP4, HSPB9, and CCL19, and the top five down-regulated genes were CDCA3, SMC2, a novel gene (ENSALGALG00000050836), an uncharacterized gene (LOC107054158), and SPAG5. Bioinformatics predicted that NO treatment will activate pathways involved in cell and organismal death, necrosis, and cardiovascular system development, and inhibit pathways involved in cell proliferation, cell movement, and gene expression. Conclusions: The findings reported herein may provide insight into possible effects of NO in the choroid during visually regulated eye growth, and help to identify targeted therapies for the treatment of myopia and other ocular diseases.

TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway.

Cranial neural crest development is governed by positional gene regulatory networks (GRNs). Fine-tuning of the GRN components underly facial shape variation, yet how those in the midface are connected and activated remain poorly understood. Here, we show that concerted inactivation of Tfap2a and Tfap2b in the murine neural crest even during the late migratory phase results in a midfacial cleft and skeletal abnormalities. Bulk and single-cell RNA-seq profiling reveal that loss of both Tfap2 members dysregulated numerous midface GRN components involved in midface fusion, patterning, and differentiation. Notably, Alx1/3/4 (Alx) transcript levels are reduced, while ChIP-seq analyses suggest TFAP2 directly and positively regulates Alx gene expression. TFAP2 and ALX co-expression in midfacial neural crest cells of both mouse and zebrafish further implies conservation of this regulatory axis across vertebrates. Consistent with this notion, tfap2a mutant zebrafish present abnormal alx3 expression patterns, and the two genes display a genetic interaction in this species. Together, these data demonstrate a critical role for TFAP2 in regulating vertebrate midfacial development in part through ALX transcription factor gene expression.

Single Cell Transcriptomics Identifies Distinct Choroid Cell Populations Involved in Visually Guided Eye Growth.

Postnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. Many studies suggest that the ocular choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refractive development. To elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations during conditions in which the eye is undergoing emmetropization. UMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of significant gene expression changes were relatively small (< 2 fold). The highest changes in gene expression were identified in a rare cell population (0.11% - 0.49% of total choroidal cells). This cell population expressed high levels of neuron-specific genes as well as several opsin genes suggestive of a rare neuronal cell population that is potentially light sensitive. Our results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization as well as insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.

Prenatal Exposure to Cannabis and Risk of Major Structural Birth Defects: A Systematic Review and Meta-analysis.

OBJECTIVE: To review and perform a meta-analysis of observational studies that examined associations between prenatal cannabis exposure and major structural birth defects. DATA SOURCES: Information sources included Google Scholar, BIOSIS, PubMed/MEDLINE, EMBASE CINAHL, and ClinicalTrials.gov. METHODS OF STUDY SELECTION: Study titles and abstracts were reviewed with Abstrackr software. We included observational studies that examined the risk of major structural birth defects in people who used cannabis during pregnancy compared with those who had not used cannabis. We excluded case reports, ecologic studies, conference abstracts, manuscript preprints, studies designed to examine effects of cannabis used concurrently with other drugs, and studies that included synthetic cannabinoids. This process yielded 23 studies that analyzed data from birth years 1968-2021. TABULATION, INTEGRATION, AND RESULTS: We clustered and meta-analyzed measures of association for birth defects by anatomic group. Eleven articles reported an association between cannabis use and the risk of a nonspecific outcome (eg, congenital anomaly). We estimated a pooled odds ratio of 1.33 (95% CI 1.14-1.56) and a pooled adjusted odds ratio (aOR) of 1.22 (95% CI 1.00-1.50). Anatomic groups examined were cardiac (nine studies), oral cleft (three studies), digestive (four studies), genitourinary (three studies), musculoskeletal (seven studies), and nervous system (five studies). Across most outcomes, we reported positive pooled unadjusted associations that were usually attenuated after the inclusion of only adjusted estimates. Two specific anomalies, with limited data, had pooled effect estimates that did not attenuate to the null after adjustment: Ebstein anomaly (two studies, aOR 2.19, 95% CI 1.25-3.82) and gastroschisis (five studies, aOR 2.50, 95% CI 1.09-5.740). CONCLUSION: Studies examining associations between prenatal exposure to cannabis and major structural birth defects were heterogeneous. Most published effect estimates were unadjusted and scored low on our risk-of-bias assessment. Overall, we found inconsistent evidence to suggest that prenatal cannabis exposure is associated with birth defects. However, findings related to specific anomalies should be considered in further research. SYSTEMATIC REVIEW REGISTRATION: PROSPERO, CRD42022319041.

Results of a screening tool for fetal alcohol spectrum disorders are associated with neuropsychological and behavioral measures.

PURPOSE: This study assessed whether the outcome of a screening tool for fetal alcohol spectrum disorders (FASD), the FASD-Tree, was associated with neuropsychological and behavioral outcomes. METHODS: Data for this study were collected as part of the fourth phase of the Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD-4). Participants (N = 175, 5 to 16 years) with or without histories of prenatal alcohol exposure were recruited from San Diego and Minneapolis. Each participant was screened using the FASD-Tree and administered a neuropsychological test battery; parents or guardians completed behavioral questionnaires. The FASD-Tree incorporates physical and behavioral measures and provides an outcome regarding the presence of FASD (FASD-Positive or FASD-Negative). Logistic regression was used to test whether the FASD-Tree outcome was associated with general cognitive ability, executive function, academic achievement, and behavior. Associations were tested in two groups: the whole sample and only correctly classified participants. RESULTS: Results of the FASD-Tree were associated with neuropsychological and behavioral measures. Participants classified as FASD-Positive were more likely than those classified as FASD-Negative to have a lower IQ score and exhibit poorer performance on measures of executive and academic functions. Behaviorally, participants classified as FASD-Positive were rated as having more behavior problems and adaptive difficulties. Similar relationships were found for all measures when including only participants correctly classified by the FASD-Tree screening tool. CONCLUSION: Results from the FASD-Tree screening tool were associated with neuropsychological and behavioral measures. Participants classified as FASD-Positive were more likely to have impairment in all domains tested. The results support the effectiveness of the FASD-Tree as a screening tool for use in clinical settings, providing an efficient and accurate way to identify patients in need of additional evaluation.

Genomic analyses in Cornelia de Lange Syndrome and related diagnoses: Novel candidate genes, genotype-phenotype correlations and common mechanisms.

Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (>60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS-like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or "DTRs"). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype-phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population.

Metformin Disrupts Signaling and Metabolism in Fetal Hepatocytes.

Metformin is used by women during pregnancy to manage diabetes and crosses the placenta, yet its effects on the fetus are unclear. We show that the liver is a site of metformin action in fetal sheep and macaques, given relatively abundant OCT1 transporter expression and hepatic uptake following metformin infusion into fetal sheep. To determine the effects of metformin action, we performed studies in primary hepatocytes from fetal sheep, fetal macaques, and juvenile macaques. Metformin increases AMP-activated protein kinase (AMPK) signaling, decreases mammalian target of rapamycin (mTOR) signaling, and decreases glucose production in fetal and juvenile hepatocytes. Metformin also decreases oxygen consumption in fetal hepatocytes. Unique to fetal hepatocytes, metformin activates stress pathways (e.g., increased PGC1A gene expression, NRF-2 protein abundance, and phosphorylation of eIF2α and CREB proteins) alongside perturbations in hepatokine expression (e.g., increased growth/differentiation factor 15 [GDF15] and fibroblast growth factor 21 [FGF21] expression and decreased insulin-like growth factor 2 [IGF2] expression). Similarly, in liver tissue from sheep fetuses infused with metformin in vivo, AMPK phosphorylation, NRF-2 protein, and PGC1A expression are increased. These results demonstrate disruption of signaling and metabolism, induction of stress, and alterations in hepatokine expression in association with metformin exposure in fetal hepatocytes. ARTICLE HIGHLIGHTS: The major metformin uptake transporter OCT1 is expressed in the fetal liver, and fetal hepatic uptake of metformin is observed in vivo. Metformin activates AMPK, reduces glucose production, and decreases oxygen consumption in fetal hepatocytes, demonstrating similar effects as in juvenile hepatocytes. Unique to fetal hepatocytes, metformin activates metabolic stress pathways and alters the expression of secreted growth factors and hepatokines. Disruption of signaling and metabolism with increased stress pathways and reduced anabolic pathways by metformin in the fetal liver may underlie reduced growth in fetuses exposed to metformin.

Delayed cortical thinning in children and adolescents with prenatal alcohol exposure.

BACKGROUND: Prenatal alcohol exposure (PAE) is associated with abnormalities in cortical structure and maturation, including cortical thickness (CT), cortical volume, and surface area. This study provides a longitudinal context for the developmental trajectory and timing of abnormal cortical maturation in PAE. METHODS: We studied 35 children with PAE and 30 nonexposed typically developing children (Comparisons), aged 8-17 at enrollment, who were recruited from the University of Minnesota FASD Program. Participants were matched on age and sex. They underwent a formal evaluation of growth and dysmorphic facial features associated with PAE and completed cognitive testing. MRI data were collected on a Siemens Prisma 3T scanner. Two sessions, each including MRI scans and cognitive testing, were spaced approximately 15 months apart on average. Change in CT and performance on tests of executive function (EF) were examined. RESULTS: Significant age-by-group (PAE vs. Comparison) linear interaction effects in CT were observed in the parietal, temporal, occipital, and insular cortices suggesting altered developmental trajectories in the PAE vs. Comparison groups. Results suggest a pattern of delayed cortical thinning in PAE, with the Comparison group showing more rapid thinning at younger ages and those with PAE showing accelerated thinning at older ages. Overall, children in the PAE group showed reduced cortical thinning across time relative to the Comparison participants. Symmetrized percent change (SPC) in CT in several regions was significantly correlated with EF performance at 15-month follow-up for the Comparison group but not the group with PAE. CONCLUSIONS: Regional differences were seen longitudinally in the trajectory and timing of CT change in children with PAE, suggesting delayed cortical maturation and an atypical pattern of development compared with typically developing individuals. In addition, exploratory correlation analyses of SPC and EF performance suggest the presence of atypical brain-behavior relationships in PAE. The findings highlight the potential role of altered developmental timing of cortical maturation in contributing to long-term functional impairment in PAE.

Atypical developmental trajectories of white matter microstructure in prenatal alcohol exposure: Preliminary evidence from neurite orientation dispersion and density imaging.

Introduction: Fetal alcohol spectrum disorder (FASD), a life-long condition resulting from prenatal alcohol exposure (PAE), is associated with structural brain anomalies and neurobehavioral differences. Evidence from longitudinal neuroimaging suggest trajectories of white matter microstructure maturation are atypical in PAE. We aimed to further characterize longitudinal trajectories of developmental white matter microstructure change in children and adolescents with PAE compared to typically-developing Controls using diffusion-weighted Neurite Orientation Dispersion and Density Imaging (NODDI). Materials and methods: Participants: Youth with PAE (n = 34) and typically-developing Controls (n = 31) ages 8-17 years at enrollment. Participants underwent formal evaluation of growth and facial dysmorphology. Participants also completed two study visits (17 months apart on average), both of which involved cognitive testing and an MRI scan (data collected on a Siemens Prisma 3 T scanner). Age-related changes in the orientation dispersion index (ODI) and the neurite density index (NDI) were examined across five corpus callosum (CC) regions defined by tractography. Results: While linear trajectories suggested similar overall microstructural integrity in PAE and Controls, analyses of symmetrized percent change (SPC) indicated group differences in the timing and magnitude of age-related increases in ODI (indexing the bending and fanning of axons) in the central region of the CC, with PAE participants demonstrating atypically steep increases in dispersion with age compared to Controls. Participants with PAE also demonstrated greater increases in ODI in the mid posterior CC (trend-level group difference). In addition, SPC in ODI and NDI was differentially correlated with executive function performance for PAE participants and Controls, suggesting an atypical relationship between white matter microstructure maturation and cognitive function in PAE. Discussion: Preliminary findings suggest subtle atypicality in the timing and magnitude of age-related white matter microstructure maturation in PAE compared to typically-developing Controls. These findings add to the existing literature on neurodevelopmental trajectories in PAE and suggest that advanced biophysical diffusion modeling (NODDI) may be sensitive to biologically-meaningful microstructural changes in the CC that are disrupted by PAE. Findings of atypical brain maturation-behavior relationships in PAE highlight the need for further study. Further longitudinal research aimed at characterizing white matter neurodevelopmental trajectories in PAE will be important.

Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

Maternal diet alters long-term innate immune cell memory in fetal and juvenile hematopoietic stem and progenitor cells in nonhuman primate offspring.

Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan.

Cognitive heterogeneity reveals molecular signatures of age-related impairment.

The greatest risk factor for cognitive decline is aging. The biological mechanisms for this decline remain enigmatic due, in part, to the confounding of normal aging mechanisms and those that contribute to cognitive impairment. Importantly, many individuals exhibit impaired cognition in age, while some retain functionality despite their age. Here, we establish a behavioral testing paradigm to characterize age-related cognitive heterogeneity in inbred aged C57BL/6 mice and reliably separate animals into cognitively "intact" (resilient) and "impaired" subgroups using a high-resolution home-cage testing paradigm for spatial discrimination. RNA sequencing and subsequent pathway analyses of cognitively stratified mice revealed molecular signatures unique to cognitively impaired animals, including transcriptional down-regulation of genes involved in mitochondrial oxidative phosphorylation (OXPHOS) and sirtuin (Sirt1 and Sirt3) expression in the hippocampus. Mitochondrial function assessed using high-resolution respirometry indicated a reduced OXPHOS coupling efficiency in cognitively impaired animals with subsequent hippocampal analyses revealing an increase in the oxidative damage marker (3-nitrotyrosine) and an up-regulation of antioxidant enzymes (Sod2, Sod1, Prdx6, etc.). Aged-impaired animals also showed increased levels of IL-6 and TNF-α gene expression in the hippocampus and increased serum levels of proinflammatory cytokines, including IL-6. These results provide critical insight into the diversity of brain aging in inbred animals and reveal the unique mechanisms that separate cognitive resilience from cognitive impairment. Our data indicate the importance of cognitive stratification of aging animals to delineate the mechanisms underlying cognitive impairment and test the efficacy of therapeutic interventions.

Discovery of Hippo signaling as a regulator of CSPG4 expression and as a therapeutic target for Clostridioides difficile disease.

The signaling pathways and networks regulating expression of chondroitin sulfate proteoglycan 4 (CSPG4), a cancer-related protein that serves as a receptor for Clostridiodes difficile TcdB, are poorly defined. In this study, TcdB-resistant/CSPG4-negative HeLa cells were generated by exposure to increasing concentrations of the toxin. The cells that emerged (HeLa R5) lost expression of CSPG4 mRNA and were resistant to binding by TcdB. mRNA expression profiles paired with integrated pathway analysis correlated changes in the Hippo and estrogen signaling pathways with a CSPG4 decrease in HeLa R5 cells. Both signaling pathways altered CSPG4 expression when modulated chemically or through CRISPR-mediated deletion of key transcriptional regulators in the Hippo pathway. Based on the in vitro findings, we predicted and experimentally confirmed that a Hippo pathway inactivating drug (XMU-MP-1) provides protection from C. difficile disease in a mouse model. These results provide insights into key regulators of CSPG4 expression and identify a therapeutic for C. difficile disease.

Maternal Western diet is associated with distinct preclinical pediatric NAFLD phenotypes in juvenile nonhuman primate offspring.

Pediatric NAFLD has distinct and variable pathology, yet causation remains unclear. We have shown that maternal Western-style diet (mWSD) compared with maternal chow diet (CD) consumption in nonhuman primates produces hepatic injury and steatosis in fetal offspring. Here, we define the role of mWSD and postweaning Western-style diet (pwWSD) exposures on molecular mechanisms linked to NAFLD development in a cohort of 3-year-old juvenile nonhuman primates offspring exposed to maternal CD or mWSD followed by CD or Western-style diet after weaning. We used histologic, transcriptomic, and metabolomic analyses to identify hepatic pathways regulating NAFLD. Offspring exposed to mWSD showed increased hepatic periportal collagen deposition but unchanged hepatic triglyceride levels and body weight. mWSD was associated with a downregulation of gene expression pathways underlying HNF4α activity and protein, and downregulation of antioxidant signaling, mitochondrial biogenesis, and PPAR signaling pathways. In offspring exposed to both mWSD and pwWSD, liver RNA profiles showed upregulation of pathways promoting fibrosis and endoplasmic reticulum stress and increased BiP protein expression with pwWSD. pwWSD increased acylcarnitines and decreased anti-inflammatory fatty acids, which was more pronounced when coupled with mWSD exposure. Further, mWSD shifted liver metabolites towards decreased purine catabolism in favor of synthesis, suggesting a mitochondrial DNA repair response. Our findings demonstrate that 3-year-old offspring exposed to mWSD but weaned to a CD have periportal collagen deposition, with transcriptional and metabolic pathways underlying hepatic oxidative stress, compromised mitochondrial lipid sensing, and decreased antioxidant response. Exposure to pwWSD worsens these phenotypes, triggers endoplasmic reticulum stress, and increases fibrosis. Overall, mWSD exposure is associated with altered expression of candidate genes and metabolites related to NAFLD that persist in juvenile offspring preceding clinical presentation of NAFLD.

Neonatal intake of Omega-3 fatty acids enhances lipid oxidation in adipocyte precursors.

Establishing metabolic programming begins during fetal and postnatal development, and early-life lipid exposures play a critical role during neonatal adipogenesis. We define how neonatal consumption of a low omega-6 to -3 fatty acid ratio (n6/n3 FA ratio) establishes FA oxidation in adipocyte precursor cells (APCs) before they become adipocytes. In vivo, APCs isolated from mouse pups exposed to the low n6/n3 FA ratio had superior FA oxidation capacity, elevated beige adipocyte mRNAs Ppargc1α, Ucp2, and Runx1, and increased nuclear receptor NR2F2 protein. In vitro, APC treatment with NR2F2 ligand-induced beige adipocyte mRNAs and increased mitochondrial potential but not mass. Single-cell RNA-sequencing analysis revealed low n6/n3 FA ratio yielded more mitochondrial-high APCs and linked APC NR2F2 levels with beige adipocyte signatures and FA oxidation. Establishing beige adipogenesis is of clinical relevance, because fat depots with energetically active, smaller, and more numerous adipocytes improve metabolism and delay metabolic dysfunction.