Molecular signatures of longissimus dorsi differ between dairy cattle based on prepartum muscle reserves and branched-chain volatile fatty acid supplementation.

Dairy cattle with high (HM) versus low muscle (LM) reserves exhibit distinct temporal changes in longissimus dorsi muscle depth (LDD) in late gestation. Branched-chain volatile fatty acids (BCVFA) supplementation increased blood glucose levels. We hypothesized that differences in HM and LM reflect distinct muscle metabolism and BCVFA supplementation altered metabolic pathways. At 42 d before expected calving (BEC) Holstein dairy cows were enrolled in a 2 x 2 factorial study of diet and muscle reserves, by assigning to control (CON) or BCVFA supplemented diets and LDD of HM (>4.6 cm) or LM (≤4.6 cm) groups: HM-CON (n=13), HM-BCVFA (n=10), LM-CON (n=9), and LM-BCVFA (n=9). Longisumus dorsi was biopsied at 21 d BEC, total RNA isolated and protein coding gene expression measured with RNA-seq. Between HM and LM 713 genes were differentially expressed and 481 between BCVFA and CON (P<0.05). Transcriptional signatures indicated differential distribution of Type II fibers between groups, with MYH1 greater in LM and MYH2 greater in HM cattle (P<0.05). Signatures of LM cattle relative to HM indicated greater activation of autophagy, ubiquitin-proteasome, and Ca2+-calpain pathways. HM cattle displayed greater expression of genes that encode extracellular matrix proteins and factors that regulate their proteolysis and turnover. BCVFA modified transcriptomes by increasing expression of genes that regulate fatty acid degradation and flux of carbons into the TCA cycle as acetyl CoA. Molecular signatures support distinct metabolic strategies between LM and HM cattle, and that BCVFA supplementation increased substrates for energy generation.

Impact of SARS-CoV-2 Arrival Surveillance Screening by Nucleic Acid Amplification Versus Rapid Antigen Detection on Subsequent COVID-19 Infections in Military Trainees.

BACKGROUND: For persons entering congregate settings, optimal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) arrival surveillance screening method, nucleic acid amplification test (NAAT) versus rapid antigen detection test (RADT), is debated. To aid this, we sought to determine the risk of secondary symptomatic coronavirus disease 19 (COVID-19) among military trainees with negative arrival NAAT or RADT screening. METHODS: Individuals who arrived for US Air Force basic military training from 1 January-31 August 2021 were placed into training groups and screened for SARS-CoV-2 via NAAT or RADT. Secondary symptomatic COVID-19 cases within 2 weeks of training were then measured. A case cluster was defined as ≥5 individual symptomatic COVID-19 cases. RESULTS: 406 (1.6%) of 24 601 trainees screened positive upon arrival. The rate of positive screen was greater for those tested with NAAT versus RADT (2.5% vs 0.4%; RR: 5.4; 95% CI: 4.0-7.3; P < .001). The proportion of training groups with ≥1 positive individual screen was greater in groups screened via NAAT (57.5% vs 10.8%; RR: 5.31; 95% CI: 3.65-7.72; P < .001). However, NAAT versus RADT screening was not associated with a difference in number of training groups to develop a secondary symptomatic case (20.3% vs 22.5%; RR: .9; 95% CI: .66-1.23; P = .53) or case cluster of COVID-19 (4% vs 6.6%; RR: .61; 95% CI: .3-1.22; P = .16). CONCLUSIONS: NAAT versus RADT arrival surveillance screening method impacted individual transmission of COVID-19 but had no effect on number of training groups developing a secondary symptomatic case or case cluster. This study provides consideration for RADT arrival screening in congregate settings.

From Reductionism to Reintegration: Solving society's most pressing problems requires building bridges between data types across the life sciences.

Decades of reductionist approaches in biology have achieved spectacular progress, but the proliferation of subdisciplines, each with its own technical and social practices regarding data, impedes the growth of the multidisciplinary and interdisciplinary approaches now needed to address pressing societal challenges. Data integration is key to a reintegrated biology able to address global issues such as climate change, biodiversity loss, and sustainable ecosystem management. We identify major challenges to data integration and present a vision for a "Data as a Service"-oriented architecture to promote reuse of data for discovery. The proposed architecture includes standards development, new tools and services, and strategies for career-development and sustainability.

Long Chain Fatty Acids Mediate Hepatic Metabolic Flux in Preruminating Dairy Calves Fed Flaxseed Oil, High Oleic Soybean Oil, or Milk Fat.

Nutrition and physiological state affect hepatic metabolism. Our objective was to determine if feeding flaxseed oil (∼50% C18:3n-3 cis), high oleic soybean oil (∼70% C18:1 cis-9), or milk fat (∼50% C16:0) alters hepatic expression of PC, PCK1, and PCK2 and the flow of carbons from propionate and pyruvate into the TCA cycle in preruminating calves. Male Holstein calves (n = 40) were assigned to a diet of skim milk with either: 3% milk fat (MF; n = 8), 3% flaxseed oil (Flax; n = 8), 3% high oleic soybean oil (HOSO; n = 8), 1.5% MF + 1.5% high oleic soybean oil (MF-HOSO; n = 8), or 1.5% MF + 1.5% flaxseed oil (MF-Flax; n = 8) from d 14 to d 21 postnatal. At d 21 postnatal, a liver biopsy was taken for gene expression and metabolic flux analysis. Liver explants were incubated in [U-13C] propionate and [U-13C] pyruvate to trace carbon flux through TCA cycle intermediates or with [U-14C] lactate, [1-14C] palmitic acid, or [2-14C] propionate to quantify substrate oxidation to CO2 and acid soluble products. Compared with other treatments, plasma C18:3n-3 cis was 10 times higher and C18:1 cis-9 was 3 times lower in both flax (Flax and MF-Flax) treatments. PC, PCK1, and PCK2 expression and flux of [U-13C] pyruvate as well as [U-13C] propionate were not different between treatments. PC expression was negatively correlated with the enrichment of citrate M+5 and malate M+3, and PCK2 was negatively correlated with citrate M+5, suggesting that when expression of these enzymes is increased, carbon from pyruvate enters the TCA cycle via PC mediated carboxylation, and then OAA is converted to phosphoenolpyruvate via PCK2. Acid soluble product formation and PC expression were reduced in HOSO (MF-HOSO and HOSO) treatments compared with flax (MF-Flax and Flax), indicating that fatty acids regulate PC expression and carbon flux, but that fatty acid flux control points are not connected to PC, PCK1, or PCK2. In conclusion, fatty acids regulate hepatic expression of PC, PCK1, and PCK2, and carbon flux, but the point of control is distinct.

Follow-up of Military Blood Donors Who Test Positive for Syphilis.

BACKGROUND: Several large studies have demonstrated that syphilis carries a risk of future sexually transmitted infections (STI), such as human immunodeficiency virus. There are limited data on outcomes of syphilis infections that occur in populations that undergo universal syphilis screening, such as blood donors. Military trainees who donate blood can be followed through their military career to determine the future risk of STIs. METHODS: Blood donor data were gathered from the Armed Services Blood Bank Center-San Antonio for those with positive Treponema pallidum antibodies between 2014 and 2021. The medical chart of each case was compared with 6 sex- and military accession date-matched controls with negative T. pallidum antibodies to determine the risk of STI in the 3 years after donation. RESULTS: A total of 63,375 individuals donated blood during the study period. A total of 23 military trainees (0.36 per 1000 donors) had positive T. pallidum antibodies. A minority (n = 7; 30%) of cases were treated for early syphilis. Only 6 cases (26%) received a follow-up nontreponemal test within 1 year. Donors who tested positive had a significantly higher risk of developing an STI within 3 years after blood donation compared with blood donors who tested negative (relative risk, 3.8; 95% confidence interval, 1.3-10.5; P = 0.01) including gonorrhea (9% vs. 0%, P = 0.02) and syphilis (9% vs. 0%, P = 0.02). CONCLUSIONS: This study shows the presence of T. pallidum antibodies in blood donors was associated with an increased risk of future STIs. These cases support the need for close follow-up and broad STI testing in blood donors with positive T. pallidum antibodies.

Infectious etiologies among post-donation deferrals in a military blood donation center.

BACKGROUND: The burden of transfusion-transmitted infections among blood recipients remains low due to extensive pre- and post-donation screening. However, the military has the unique challenge of providing blood in austere environments with limited testing capabilities. This study evaluates the infectious etiologies of deferred blood donors at a large military blood donation center. METHODS: All blood donors at the Armed Service Blood Bank Center, San Antonio, between 2017 and 2022 with positive post-donation screening for hepatitis C (HCV), hepatitis B (HBV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV-I/II), Zika (2018-2021), West Nile virus, Trypanosoma cruzi, Treponema pallidum, or Babesia microti (2020-2022) were evaluated. Donors were deferred based on Food and Drug Administration (FDA) guidance. RESULTS: Two-hundred and thirteen (213) donors met FDA criteria for deferral. T. pallidum (n = 45, 50.3 per 100,000), HCV (n = 34, 38.0 per 100,000), and HBV (n = 19, 21.2 per 100,000) were the most common pathogens among those with both positive screening and confirmatory testing. The majority of HIV (95%), Chagas (78%), HTLV-I/II (50%) deferrals were due to indeterminate confirmatory tests following initial positive screens. The majority of deferrals for HBV were for a second occurrence of a positive screen despite negative confirmatory testing. CONCLUSION: The rates of post-donation deferral for transfusion-transmissible infections were low in this military cohort. Our findings suggest that donor testing in deployed service members should focus on HBV, HCV, and T. pallidum and highlight the need for better diagnostics for HIV, Chagas, and HTLV-I/II.

Effect of circadian system disruption on the concentration and daily oscillations of cortisol, progesterone, melatonin, serotonin, growth hormone, and core body temperature in periparturient dairy cattle.

Metabolic, circadian, sleep, and reproductive systems are integrated and reciprocally regulated, but the understanding of the mechanism is limited. To study this integrated regulation, the circadian timing system was disrupted by exposing late pregnant nonlactating (dry) cows to chronic shifts in the light-dark phase, and rhythms of body temperature and circulating cortisol (CORT), progesterone (P4), serotonin (5HT), melatonin (MEL), and growth hormone (GH) concentrations were measured. Specifically, across 2 identical studies (1 and 2), at 35 d before expected calving (BEC) multiparous cows were assigned to control (CON; n = 24) and exposed to 16 h light and 8 h dark or phase shift (PS; n = 24) treatments and exposed to 6-h light-dark phase shifts every 3 d until parturition. All cows were exposed to control lighting after calving. Blood samples were collected in the first study at 0600 h on d 35 BEC, d 21 BEC, and 2 d before calving, and d 0, 2, 9, 15, and 22 postpartum (PP). A subset of cows (n = 6/group) in study 1 was blood sampled every 4 h over 48 h beginning on d 23 BEC, 9 BEC, and 5 PP. Body temperature was measured every 30 min (n = 8-16/treatment) for 48 h at 23 BEC and 9 BEC in both studies; and at 14 PP and 60 PP only in study 2. Treatment did not affect levels of CORT, GH, or P4 at 0600 h, but overall level of 5HT was lower and MEL higher in PS cows across days sampled. A 2-component versus single-component cosinor model better described [>coefficient of determination (R2);

Proteomic Analysis of the Effect of Salmonella Challenge on Broiler Chicken.

Salmonella enteritidis is a foodborne pathogen that causes high morbidity in poultry. Proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to study the effects of Salmonella infection on spleen proteome in broiler chickens. Day-old broilers were assigned to control (CON; n = 60) or Salmonella challenge (CON−SE; n = 60), and gavaged with Tryptic soy agar broth or SE. A subset of chicks was euthanized on D3 and D7 (n = 4/group/day) and the spleen was removed, and rapidly frozen, subsequently proteome was measured using label-free LC-MS/MS. Protein spectra were mapped to Gallus gallus Uniprot database. Differentially abundant proteins (DAP; FDR < 0.05) between days and treatments were identified using ANOVA. Cecal content of Salmonella in CON−SE was 3.37 log10 CFU/g and CON were negative. Across the 16 samples, 2625 proteins were identified. Proteins that decreased in abundance between days mediated cell cycle progression, while those that increased in abundance function in cytoskeleton and mRNA processing. SE infection caused an increase in proteins that mediated redox homeostasis, lysosomal activities, and energy production, while proteins decreased in abundance-mediated developmental progression. Proteomic signatures of spleen suggest SE infection was metabolically costly, and energy was diverted from normal developmental processes to potentiate disease resistance mechanisms.

Transcriptome analysis reveals disruption of circadian rhythms in late gestation dairy cows may increase risk for fatty liver and reduced mammary remodeling.

Circadian disruption increased insulin resistance and decreased mammary development in late gestation, nonlactating (dry) cows. The objective was to measure the effect of circadian disruption on transcriptomes of the liver and mammary gland. At 35 days before expected calving (BEC), multiparous dry cows were assigned to either control (CON) or phase-shifted treatments (PS). CON was exposed to 16-h light and 8-h dark. PS was exposed to 16-h light to 8-h dark, but phase of the light-dark cycle was shifted 6 h every 3 days. On day 21 BEC, liver and mammary were biopsied. RNA was isolated (n = 6 CON, n = 6 PS per tissue), and libraries were prepared and sequenced using paired-end reads. Reads mapping to bovine genome averaged 27 ± 2 million and aligned to 14,222 protein-coding genes in liver and 15,480 in mammary analysis. In the liver, 834 genes, and in the mammary gland, 862 genes were different (nominal P < 0.05) between PS and CON. In the liver, genes upregulated in PS functioned in cholesterol biosynthesis, endoplasmic reticulum stress, wound healing, and inflammation. Genes downregulated in liver function in cholesterol efflux. In the mammary gland, genes upregulated functioned in mRNA processing and transcription and downregulated genes encoded extracellular matrix proteins and proteases, cathepsins and lysosomal proteases, lipid transporters, and regulated oxidative phosphorylation. Increased cholesterol synthesis and decreased efflux suggest that circadian disruption potentially increases the risk of fatty liver in cows. Decreased remodeling and lipid transport in mammary may decrease milk production capacity during lactation.

One-to-one relationships between milk miRNA content and protein abundance in neonate duodenum support the potential for milk miRNAs regulating neonate development.

Breast milk plays an essential role for offspring development; however, there lacks evidence of how specific milk components like nucleic acids mechanistically function to regulate neonate development. Previously, we found that maternal high-fat diet (HFD) not only significantly affected mRNA and miRNA content of the secreted milk transcriptome in mice but also affected the duodenal proteome of suckling pups. Here, we hypothesized that nucleic acids differentially expressed in milk of HFD fed dams are related to differentially abundant proteins in offspring duodenum nursed by HFD dams. We tested this hypothesis by analyzing one-to-one relationships in RNA-seq data of milk transcriptomes from control (10% kcal fat) and HFD (60% kcal fat) fed mice and liquid chromatography-tandem mass spectrometry (LC-MS/MS) duodenal proteome data from pups exposed to milk. Ten percent of differentially abundant duodenal proteins between controls and HFD-exposed pups had predicted upregulation or downregulation based on differential milk RNA content. Of these, 76% were targets of upregulated miRNA, and linear regression analysis indicated relationships (p < 0.05) between multiple milk miRNA counts and duodenal protein abundance. Duodenal proteins that were potential targets of milk miRNA enriched Gene Ontology (GO) terms and KEGG pathways related to cytoskeletal structure and neural development, suggesting potential regulation of pup enteric nervous system. One-to-one relationships between milk miRNA content and protein abundance in neonate duodenum support the potential for milk miRNAs regulating neonate development. Identification of milk miRNAs that changed in response to maternal diet will enable design of mechanistic studies that test effects on neonate.

High-fat-diet induced obesity increases the proportion of linoleic acyl residues in dam serum and milk and in suckling neonate circulation.

Maternal obesity increases the risk of offspring to become obese and develop related pathologies. Exposure to maternal high-fat diet (HFD) only during lactation increases the risk of obesity-related diseases, suggesting that factors in milk affect long-term health. We hypothesized that prepregnancy obesity induced by HFD alters milk lipidome, and in turn, alterations may affect neonate serum lipidome. The objective of this study was to determine the effect of prepregnancy obesity induced by HFD on circulating lipids in dams and neonates and in milk. Female mice were fed an HFD (60% kcal fat) or control diet (CON, 10% kcal fat) beginning 4 weeks before breeding. On postnatal day 2 (PND2), pups were cross-fostered to create pup groups exposed to HFD during pregnancy, lactation, or both or exposed to CON. On PND12, dams were milked and then euthanized along with pups to collect blood. Serum and milk were processed for multiple reaction monitoring (MRM) lipidomics profiling to quantify the relative expression of lipid classes. Lipidome of HFD dam serum and milk had increased proportion of C18:2 free fatty acid and fatty acyl residues in all lipid classes. Lipidome of serum from pups exposed to maternal HFD during lactation was similarly affected. Thus, maternal HFD induced redistribution of fatty acyl residues in the dam's circulation, which was associated with modification in milk and suckling neonate's lipidome. Further studies are needed to determine if increased circulating levels of C18:2 in neonate affects development and predisposes offspring to obesity and metabolic syndrome.

COVID-19 Monitoring and Response Among U.S. Air Force Basic Military Trainees - Texas, March-April 2020.

The coronavirus disease 2019 (COVID-19) pandemic has resulted in substantial morbidity and mortality since it was first described in December 2019 (1). Based on epidemiologic data showing spread in congregate settings (2-4), national, state, and local governments instituted significant restrictions on large gatherings to prevent transmission of disease in early March 2020. This and other nonpharmaceutical interventions (NPIs) have shown initial success in slowing the pandemic across the country (5). This report examines the first 7 weeks (March 1-April 18) of implementation of NPIs in Basic Military Training (BMT) at a U.S. Air Force base. In a population of 10,579 trainees, COVID-19 incidence was limited to five cases (47 per 100,000 persons), three of which were in persons who were contacts of the first patient. Transmission of symptomatic COVID-19 was successfully limited using strategies of quarantine, social distancing, early screening of trainees, rapid isolation of persons with suspected cases, and monitored reentry into training for trainees with positive test results after resolution of symptoms.

Exposure to chronic light-dark phase shifts during the prepartum nonlactating period attenuates circadian rhythms, decreases blood glucose, and increases milk yield in the subsequent lactation.

Maintaining metabolic balance is a key factor in the health of dairy cattle during the transition from pregnancy to lactation. Little is known regarding the role of the circadian timing system in the regulation of physiological changes during the transition period. We hypothesized that disruption of the cow's circadian timing system by exposure to chronic light-dark phase shifts during the prepartum period would negatively affect the regulation of homeostasis and cause metabolic disturbances, leading to reduced milk production in the subsequent lactation. The objective was to determine the effect of exposure to chronic light-dark phase shift during the last 5 wk prepartum of the nonlactating dry period on core body temperature, melatonin, blood glucose, ß-hydroxybutyric acid (BHB) and nonesterified fatty acid (NEFA) concentrations, and milk production. Multiparous cows were moved to tiestalls at 5 wk before expected calving and assigned to control (CTR; n = 16) or phase-shifted (PS; n = 16) treatments. Control cows were exposed to 16 h of light and 8 h of dark. Phase-shifted cows were exposed to the same photoperiod; however, the light-dark cycle was shifted 6 h every 3 d until parturition. Resting behavior and feed intake were recorded daily. Core body temperature was recorded vaginally for 48 h at 23 and 9 d before expected calving using calibrated data loggers. Blood concentrations of melatonin, glucose, BHB, and NEFA were measured during the pre- and postpartum periods. Milk yield and composition were measured through 60 DIM. Treatment did not affect feed intake or body condition. Cosine fit analysis of 24-h core body temperature and circulating melatonin indicated attenuation of circadian rhythms in the PS treatment compared with the CTR treatment. Phase-shifted cows had lower rest consolidation, as indicated by more total resting time, but shorter resting period durations. Phase-shifted cows had lower blood glucose concentration compared with CTR cows (4 mg/mL decrease), but BHB and NEFA concentrations were similar between PS and CTR cows. Milk yield and milk fat yield were greater in PS compared with CTR cows (2.8 kg/d increase). Thus, exposure to chronic light-dark phase shifts during the prepartum period attenuated circadian rhythms of core body temperature, melatonin, and rest-activity behavior and was associated with increased milk fat and milk yield in the postpartum period despite decreased blood glucose pre- and postpartum. Therefore, less variation in central circadian rhythms may create a more constant milieu that supports the onset of lactogenesis.

Profiling solute-carrier transporters in key metabolic tissues during the postpartum evolution of mammary epithelial cells from nonsecretory to secretory.

Modifications in the abundance of solute-carrier (SLC) transcripts in tandem with adjustments in genes-associated with energy homeostasis during the postpartum transition of the mammary epithelial cells (MEC) from nonsecretory to secretory is pivotal for supporting milk synthesis. The goal of this study was to identify differentially expressed SLC genes across key metabolic tissues between late pregnancy and onset of lactation. Total RNA was isolated from the mammary, liver, and adipose tissues collected from rat dams on day 20 of pregnancy (P20) and day 1 of lactation (L1) and gene expression was measured with Rat 230 2.0 Affymetrix GeneChips. LIMMA was utilized to identify the differential gene expression patterns between P20 and L1 tissues. Transcripts engaged in conveying anions, cations, carboxylates, sugars, amino acids, metals, nucleosides, vitamins, and fatty acids were significantly increased (P < 0.05) in MEC during the P20 to L1 shift. Downregulated (P < 0.05) genes in the mammary during the physiological transition included GLUT8 and SLC45a3. In the liver, SLC genes encoding for anion, carbonyl, and nucleotide sugar transporters were upregulated (P < 0.05) at L1. while genes facilitating transportation of anions and hexose were increased (P < 0.05), from P20 to L1 in the adipose tissue. GLUT1 and GLUT4 in the liver, along with GLUT4 and SGLT2 in the adipose tissue, were repressed (P < 0.05) at L1. Our results illustrate that MEC exhibit dynamic molecular plasticity during the nonsecretory to secretory transition and increase biosynthetic capacity through a coordinated tissue specific SLC transcriptome modification to facilitate substrate transfer.

Diet Impacts Pre-implantation Histotroph Proteomes in Beef Cattle.

In ruminants, the period from fertilization to implantation is relatively prolonged, and the survival of embryos depends on uterine secretions known as histotroph. Our objective was to determine if the pre-breeding diet affected histotroph proteomes in beef cattle. Cows were assigned to one of four diets: a control diet (CON), a high-protein diet (PROT), a high-fat diet (OIL), or a high-protein and high-fat diet (PROT + OIL). After 185 days on these diets, an intravaginal progesterone implant (CIDR) was inserted for 7 days. At 9 days after CIDR removal, animals with a corpus luteum were selected ( n = 16; 4 per treatment). Proteins were isolated from the histotroph collected by uterine lavage and analyzed with liquid chromatography-tandem mass spectrometry. Over 2000 proteins were expressed ( n ≥ 3 cows per treatment), with 1239 proteins being common among all of the groups. There were 20, 37, 85, and 123 proteins unique to CON, PROT + OIL, PROT, and OIL, respectively. Relative to CON, 23, 14, and 51 proteins were differentially expressed in PROT + OIL, PROT, and OIL, respectively. Functional analysis found that 53% of histotroph proteins were categorized as extracellular exosome, 3.28% as cell-cell adhesion, and 17.4% in KEGG metabolic pathways. Differences in proteomes among treatments support the idea that pre-breeding diet affects histotroph. Understanding the impact of diet on histotroph proteins may help improve conception rates.

Novel missense mutation in the bZIP transcription factor, MAF, associated with congenital cataract, developmental delay, seizures and hearing loss (Aymé-Gripp syndrome).

BACKGROUND: Cataract is a major cause of severe visual impairment in childhood. The purpose of this study was to determine the genetic cause of syndromic congenital cataract in an Australian mother and son. METHOD: Fifty-one genes associated with congenital cataract were sequenced in the proband using a custom Ampliseq library on the Ion Torrent Personal Genome Machine (PGM). Reads were aligned against the human genome (hg19) and variants were annotated. Variants were prioritised for validation by Sanger sequencing if they were novel, rare or previously reported to be associated with paediatric cataract and were predicted to be protein changing. Variants were assessed for segregation with the phenotype in the affected mother. RESULT: A novel likely pathogenic variant was identified in the transactivation domain of the MAF gene (c.176C > G, p.(Pro59Arg)) in the proband and his affected mother., but was absent in 326 unrelated controls and absent from public variant databases. CONCLUSION: The MAF variant is the likely cause of the congenital cataract, Asperger syndrome, seizures, hearing loss and facial characteristics in the proband, providinga diagnosis of Aymé-Gripp syndrome for the family.

CLOCK regulates mammary epithelial cell growth and differentiation.

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63 qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.

Transcriptomes reveal alterations in gravity impact circadian clocks and activate mechanotransduction pathways with adaptation through epigenetic change.

Few studies have investigated the impact of alterations in gravity on mammalian transcriptomes. Here, we describe the impact of spaceflight on mammary transcriptome of late pregnant rats and the effect of hypergravity exposure on mammary, liver, and adipose transcriptomes in late pregnancy and at the onset of lactation. RNA was isolated from mammary collected on pregnancy day 20 from rats exposed to spaceflight from days 11 to 20 of gestation. To measure the impact of hypergravity on mammary, liver, and adipose transcriptomes we isolated RNA from tissues collected on P20 and lactation day 1 from rats exposed to hypergravity beginning on pregnancy day 9. Gene expression was measured with Affymetrix GeneChips. Microarray analysis of variance revealed alterations in gravity affected the expression of genes that regulate circadian clocks and activate mechanotransduction pathways. Changes in these systems may explain global gene expression changes in immune response, metabolism, and cell proliferation. Expression of genes that modify chromatin structure and methylation was affected, suggesting adaptation to gravity alterations may proceed through epigenetic change. Altered gravity experiments offer insights into the role of forces omnipresent on Earth that shape genomes in heritable ways. Our study is the first to analyze the impact of alterations in gravity on transcriptomes of pregnant and lactating mammals. Findings provide insight into systems that sense gravity and the way in which they affect phenotype, as well as the possibility of sustaining life beyond Earth's orbit.

Tissue-specific changes in molecular clocks during the transition from pregnancy to lactation in mice.

Circadian clocks regulate homeostasis and mediate responses to stressors. Lactation is one of the most energetically demanding periods of an adult female's life. Peripartum changes occur in almost every organ so the dam can support neonatal growth through milk production while homeostasis is maintained. How circadian clocks are involved in adaptation to lactation is currently unknown. The abundance and temporal pattern of core clock genes' expression were measured in suprachiasmatic nucleus, liver, and mammary from late pregnant and early lactation mice. Tissue-specific changes in molecular clocks occurred between physiological states. Amplitude and robustness of rhythms increased in suprachiasmatic nucleus and liver. Mammary rhythms of core molecular clock genes were suppressed. Attenuated rhythms appeared to be a physiological adaptation of mammary to lactation, because manipulation of timing of suckling resulting in significant differences in plasma prolactin and corticosterone had no effect on amplitude. Analysis of core clock proteins revealed that the stoichiometric relationship between positive (CLOCK) and negative (PER2) components remained 1:1 in liver but was increased to 4:1 in mammary during physiological transition. Induction of differentiation of mammary epithelial cell line HC11 with dexamethasone, insulin, and prolactin resulted in similar stoichiometric changes among positive and negative clock regulators, and prolactin induced phase shifts in HC11 Arntl expression rhythm. Data support that distinct mechanisms drive periparturient changes in mammary clock. Stoichiometric change in clock regulators occurs with gland differentiation. Suppression of mammary clock gene expression rhythms represents a physiological adaptation to suckling cues. Adaptations in mammary clock are likely needed in part to support suckling demands of neonates.

Bacitracin Methylene Disalicylate (BMD) Treatment Affects Spleen Proteome in Broiler Chicks Infected with Salmonella enteritidis.

Bacitracin Methylene Disalicylate (BMD), as a feed additive to poultry diets, enhances digestion, prevents Salmonella enteritidis (SE) colonization, and treats current infections. The objective of this study was to utilize a quantitative proteomic approach to determine the effect of BMD feed additive on broiler chickens challenged with SE in the spleen proteome. At 1 d of age, chicks were randomly allocated into four groups: control with and without SE challenge (CON, n = 60; CON-SE, n = 60), BMD with and without SE challenge (BMD, n = 60; BMD-SE, n = 60). Birds in the CON-SE and BMD-SE treatment were administered SE inoculum by oral gavage. On day three and day seven post-gavage, the spleen was collected aseptically from birds in each treatment group (CON, n = 4/day; CON-SE, n = 4/day; BMD, n = 4/day; BMD-SE, n = 4/day). Proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed an increased abundance of 115 proteins and decreased of 77 due to the BMD. Proteins that decreased in abundance were enriched for fibrinogen complex and extracellular space, whereas proteins that increased in abundance were enriched for proteasome-mediated ubiquitin-dependent protein catabolic process and mitochondrion. Analysis of the interaction between BMD and the Salmonella challenge found 230 differentially abundant proteins including proteins associated with RNA binding, spliceosome, protein transport, and cell adhesion among the upregulated proteins, and those associated with protein folding, carbon metabolism, biosynthesis of nucleotide sugars, response to oxidative stress, positive regulation of NIK/NF-kappaB signaling, and inflammatory response among the downregulated proteins. The impact of BMD treatment on spleen proteome indicates an anti-apoptotic effect. BMD also modified the response of the spleen to the SE challenge with a marked decrease in proteins that prompt cytokine synthesis and an increase in proteins involved in the selective removal of unfolded proteins.