👤 Brittni Littlejohn

This study investigated the influence of maternal nutrient restriction and dietary melatonin supplementation on DNA methylation and gene expression in bovine placental cotyledons, with a focus on sex-specific changes. On day 160 of gestation, 29 Brangus heifers (bred to a single sire by AI) were subjected to a 2 × 2 factorial design: adequately fed (ADQ-CON, n = 7), nutrient-restricted (RES-CON, n = 7), and adequately fed or nutrient-restricted supplemented with 20 mg/d of melatonin (ADQ-MEL, n = 7; RES-MEL, n = 8). Cotyledons were collected at day 240 from 12 female and 17 male conceptuses for Methyl MiniSeq-GWBS and RNA-Seq. In RES-CON vs. ADQ-CON, 93 hypomethylated and 143 hypermethylated DMRs were identified, primarily in exonic, intronic, and promoter regions. Melatonin altered the methylation patterns of male and female cotyledons, respectively, with 203 and 460 DMRs associated with axon guidance, RHOC GTPase cycle, and BDNF signaling pathways. RES-MEL showed higher expression of the Show less

The MAST family of microtubule-associated serine-threonine kinases (STKs) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been conclusively associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum. Using exome sequencing, we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells. We identify de novo missense variants in the STK domain in 11 individuals, including 2 recurrent variants p.G510S (n = 5) and p.G515S (n = 3). All 11 individuals had developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at <2 years of age. All patients developed multiple seizure types, 9 of 11 patients had seizures triggered by fever and 9 of 11 patients had drug-resistant seizures. In vitro analysis of HEK293T cells transfected with MAST3 cDNA carrying a subset of these patient-specific missense variants demonstrated variable but generally lower expression, with concomitant increased phosphorylation of the MAST3 target, ARPP-16, compared to wild-type. These findings suggest the patient-specific variants may confer MAST3 gain-of-function. Moreover, single-nuclei RNA sequencing and immunohistochemistry shows that MAST3 expression is restricted to excitatory neurons in the cortex late in prenatal development and postnatally. In summary, we describe MAST3 as a novel epilepsy-associated gene with a potential gain-of-function pathogenic mechanism that may be primarily restricted to excitatory neurons in the cortex. ANN NEUROL 2021;90:274-284. Show less

Changes to milking frequency (MF) affect the metabolic and energetic status of dairy cows. However, the duration of altered MF necessary to modify hepatic transcription during early lactation is less clear. Additionally, long-term responses to short-term alterations in MF have not been established. Holstein-Friesian dairy cows (n = 120) were allocated to 3 or 6 wk of either once-daily (1 ×) or thrice-daily (3 ×) milking, immediately postpartum. Following treatment, cows were switched to twice-daily (2 ×) milking. These 4 treatment groups were compared with cows milked 2 × (n = 30) for the whole lactation. Liver tissue was collected by biopsy at 1, 3, 6, and 9 wk postpartum from 12 cows per treatment, RNA was extracted, and transcript abundance of genes involved in hepatic metabolism was quantified. Milking frequency altered the expression of most of the genes measured; however, we observed no effects caused by the length of time on the alternative milking frequency and no interactions between MF and length. During the MF treatment, mRNA expression of some, but not all, genes involved in gluconeogenesis (G6PC, PCK1), fatty acid β-oxidation (CPT1A, CPT2), ketogenesis (HMGCS2), lipid transport (APOA1), and lipolysis (PNPLA2) were lower for cows milked 1 × and plasma glucose and insulin concentrations were greater. Cows milked 3 × had reduced mRNA expression for some of the genes involved in fatty acid synthesis (ACACA) and lipid transport (APOB) and had greater plasma NEFA concentrations at wk 1. At 9 wk postpartum, expression data indicated that cows previously milked 3 × had a greater capacity for gluconeogenesis (PCK1), ketogenesis (HMGCS2), and urea cycling (ASL, CPS1) and lower glucose concentrations than cows previously milked 1 ×, because some of the genes involved in these processes were still altered. Milking cows 1 × relative to 2 ×, however, did not result in significant carryover effects on the expression of the genes measured in this study, indicating that metabolic changes are not sustained beyond the period of reduced MF. Changes to MF altered the hepatic response during early lactation; however, this was not dependent on the duration of MF change. Although we observed only minimal carryover effects on hepatic metabolism from short periods of reduced MF postpartum, there may be long-term effects on urea cycling (ASL, CPS1) and ketogenesis (HMGCS2) when 3 × milking occurs immediately postpartum. Show less