The amino acid sensor GCN2 controls red blood cell clearance and iron metabolism through regulation of liver macrophages.

GCN2 (general control nonderepressible 2) is a serine/threonine-protein kinase that controls messenger RNA translation in response to amino acid availability and ribosome stalling. Here, we show that GCN2 controls erythrocyte clearance and iron recycling during stress. Our data highlight the importance of liver macrophages as the primary cell type mediating these effects. During different stress conditions, such as hemolysis, amino acid deficiency or hypoxia, GCN2 knockout (GCN2-/-) mice displayed resistance to anemia compared with wild-type (GCN2+/+) mice. GCN2-/- liver macrophages exhibited defective erythrophagocytosis and lysosome maturation. Molecular analysis of GCN2-/- cells demonstrated that the ATF4-NRF2 pathway is a critical downstream mediator of GCN2 in regulating red blood cell clearance and iron recycling.

Meta-analysis reveals the vaginal microbiome is a better predictor of earlier than later preterm birth.

BACKGROUND: High-throughput sequencing measurements of the vaginal microbiome have yielded intriguing potential relationships between the vaginal microbiome and preterm birth (PTB; live birth prior to 37 weeks of gestation). However, results across studies have been inconsistent. RESULTS: Here, we perform an integrated analysis of previously published datasets from 12 cohorts of pregnant women whose vaginal microbiomes were measured by 16S rRNA gene sequencing. Of 2039 women included in our analysis, 586 went on to deliver prematurely. Substantial variation between these datasets existed in their definition of preterm birth, characteristics of the study populations, and sequencing methodology. Nevertheless, a small group of taxa comprised a vast majority of the measured microbiome in all cohorts. We trained machine learning (ML) models to predict PTB from the composition of the vaginal microbiome, finding low to modest predictive accuracy (0.28-0.79). Predictive accuracy was typically lower when ML models trained in one dataset predicted PTB in another dataset. Earlier preterm birth (< 32 weeks, < 34 weeks) was more predictable from the vaginal microbiome than late preterm birth (34-37 weeks), both within and across datasets. Integrated differential abundance analysis revealed a highly significant negative association between L. crispatus and PTB that was consistent across almost all studies. The presence of the majority (18 out of 25) of genera was associated with a higher risk of PTB, with L. iners, Prevotella, and Gardnerella showing particularly consistent and significant associations. Some example discrepancies between studies could be attributed to specific methodological differences but not most study-to-study variations in the relationship between the vaginal microbiome and preterm birth. CONCLUSIONS: We believe future studies of the vaginal microbiome and PTB will benefit from a focus on earlier preterm births and improved reporting of specific patient metadata shown to influence the vaginal microbiome and/or birth outcomes.

The amino acid sensor GCN2 suppresses terminal oligopyrimidine (TOP) mRNA translation via La-related protein 1 (LARP1).

La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5'TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is largely unexplored. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. Using chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) analysis of activating transcription factor 4 (ATF4), an effector of GCN2 in nutrient stress conditions, in WT and GCN2 KO mouse embryonic fibroblasts, we determined that LARP1 is a GCN2-dependent transcriptional target of ATF4. Moreover, we identified GCN1, a GCN2 activator, participates in a complex with LARP1 on stalled ribosomes, suggesting a role for GCN1 in LARP1-mediated translation inhibition in response to ribosome stalling. Therefore, our data suggest that the GCN2 pathway controls LARP1 activity via two mechanisms: ATF4-dependent transcriptional induction of LARP1 mRNA and GCN1-mediated recruitment of LARP1 to stalled ribosomes.

4E-BP-Dependent Translational Control of Irf8 Mediates Adipose Tissue Macrophage Inflammatory Response.

Deregulation of mRNA translation engenders many human disorders, including obesity, neurodegenerative diseases, and cancer, and is associated with pathogen infections. The role of eIF4E-dependent translational control in macrophage inflammatory responses in vivo is largely unexplored. In this study, we investigated the involvement of the translation inhibitors eIF4E-binding proteins (4E-BPs) in the regulation of macrophage inflammatory responses in vitro and in vivo. We show that the lack of 4E-BPs exacerbates inflammatory polarization of bone marrow-derived macrophages and that 4E-BP-null adipose tissue macrophages display enhanced inflammatory gene expression following exposure to a high-fat diet (HFD). The exaggerated inflammatory response in HFD-fed 4E-BP-null mice coincides with significantly higher weight gain, higher Irf8 mRNA translation, and increased expression of IRF8 in adipose tissue compared with wild-type mice. Thus, 4E-BP-dependent translational control limits, in part, the proinflammatory response during HFD. These data underscore the activity of the 4E-BP-IRF8 axis as a paramount regulatory mechanism of proinflammatory responses in adipose tissue macrophages.

Prenatal vitamin D status and offspring's growth, adiposity and metabolic health: a systematic review and meta-analysis.

In this systematic review and meta-analysis of observational studies, we aimed to estimate the associations between prenatal vitamin D status and offspring growth, adiposity and metabolic health. We searched the literature in human studies on prenatal vitamin D status and offspring growth in PubMed, up to July 2017. Studies were selected according to their methodological quality and outcomes of interest (anthropometry, fat mass and diabetes in offspring). The inverse variance method was used to calculate the pooled mean difference (MD) with 95 % CI for continuous outcomes, and the Mantel-Haenszel method was used to calculate the pooled OR with 95 % CI for dichotomous outcomes. In all, thirty observational studies involving 35 032 mother-offspring pairs were included. Vitamin D status was evaluated by circulating 25-hydroxyvitamin D (25(OH)D) level. Low vitamin D status was based on each study's cut-off for low 25(OH)D levels. Low prenatal vitamin D levels were associated with lower birth weight (g) (MD -100·69; 95 % CI -162·25, -39·13), increased risk of small-for-gestational-age (OR 1·55; 95 % CI 1·16, 2·07) and an elevated weight (g) in infant at the age of 9 months (g) (MD 119·75; 95 % CI 32·97, 206·52). No associations were observed between prenatal vitamin D status and other growth parameters at birth, age 1 year, 4-6 years or 9 years, nor with diabetes type 1. Prenatal vitamin D may play a role in infant adiposity and accelerated postnatal growth. The effects of prenatal vitamin D on long-term metabolic health outcomes in children warrant future studies.

Maternal Vitamin D Insufficiency Early in Pregnancy Is Associated with Increased Risk of Preterm Birth in Ethnic Minority Women in Canada.

Background: Maternal vitamin D insufficiency (plasma 25-hydroxyvitamin D [25(OH)D] <75 nmol/L) may play a role in ethnic disparities in rates of preterm and spontaneous preterm births.Objective: We explored the relation between maternal plasma 25(OH)D concentration in the first trimester (8-14 wk of gestation) and the risk of preterm and spontaneous preterm births (<37 wk of gestation) by ethnicity.Methods: We designed a case-control study that included 120 cases of preterm birth (<37 wk of gestation) and 360 term controls (≥37 wk of gestation) of singleton pregnancies from the 3D cohort, a multicenter study in 2456 pregnant women in Quebec, Canada. Plasma 25(OH)D was measured by LC-mass spectrometry. We compared the distribution of vitamin D status between cases and controls for 8 ethnic minority subgroups. We explored the association between maternal plasma 25(OH)D concentration and preterm and spontaneous preterm births with the use of splines in logistic regression by ethnicity.Results: The distributions of maternal vitamin D status (<50, 50-75, and >75 nmol/L) were different in preterm and spontaneous preterm birth cases compared with controls but only in women of ethnic minority (P-trend = 0.003 and 0.024, respectively). Among ethnic subgroups, sub-Saharan Africans (P-trend = 0.030) and Arab-West Asians (P-trend = 0.045) showed an inverse relation between maternal vitamin D status and the risk of preterm birth. Maternal plasma 25(OH)D concentrations of 30 nmol/L were associated with 4.05 times the risk of preterm birth in the total ethnic minority population (95% CI: 1.16, 14.12; P = 0.028) relative to participants with a concentration of 75 nmol/L. In contrast, there was no such association among nonethnic women (OR: 0.94; 95% CI: 0.48, 1.82; P = 0.85). There was no association when we considered only spontaneous preterm births in the total ethnic minority population (OR: 1.75; 95% CI: 0.39, 7.79; P = 0.46).Conclusion: Vitamin D insufficiency is associated with an increased risk of preterm birth in ethnic minority women in Canada.

Osteocalcin, but not deoxypyridinoline, increases in response to isoflurane-induced anaesthesia in young female guinea pigs.

The effect of the inhaled anaesthetic isoflurane was investigated on bone biomarkers, both during maturation and on minerals and glucose postpartum. Female guinea pigs (n = 10) were anaesthetized during maturation (5 and 9 weeks) and postpartum (26 weeks of age) with isoflurane during dual-energy X-ray absorptiometry scanning. Blood collection was performed at all ages before and after anaesthesia for measurement of plasma osteocalcin (OC), total deoxypyridinoline (tDPD), and cortisol. Postpartum measurements also included: blood ions, acid-base parameters and glucose, plasma minerals, total alkaline phosphatase (tALP), and albumin. Plasma OC concentration almost doubled after exposure to isoflurane at 5 weeks (30.1 ± 5.0-57.9 ± 11.2 nmol/L, p < 0.001) and at 9 weeks (29.1 ± 7.5-62.9 ± 15.9 nmol/L, p < 0.001), but did not change postpartum (3.7 ± 3.3-4.3 ± 3.9 nmol/L, p = 0.88). There was no effect of isoflurane exposure on plasma tDPD at any age. Plasma cortisol increased after exposure to isoflurane at 9 weeks (1859.6 ± 383.2-2748.0 ± 235.3 nmol/L, p < 0.01) and postpartum (3376.7 ± 322.2-4091.6 ± 195.6 nmol/L, p < 0.001) but not at 5 weeks (2088.3 ± 326.4-2464.1 ± 538.0 nmol/L, p > 0.05). Blood ionized Ca(2+), Na(+) and plasma total Ca did not change, whereas plasma albumin decreased, and inorganic phosphate (PO4) and Cl(-) increased upon exposure to isoflurane. Isoflurane decreased tALP (43.2 ± 6.6-40.2 ± 5.9 IU/L, p = 0.01) and increased glucose (7.5 ± 0.6-10.9 ± 1.7 mmol/L, p < 0.0001) postpartum. Isoflurane inflates the assessment of a bone-derived biomarker, OC, during rapid growth, but not following pregnancy when formation is very low. Measurements prior to anaesthesia are recommended to reflect normal metabolism.

High vitamin D status before conception, but not during pregnancy, is inversely associated with maternal gestational diabetes mellitus in guinea pigs.

BACKGROUND: Whether there is a dose-dependent effect of maternal dietary cholecalciferol during pregnancy on maternal glucose tolerance is unknown. In addition, circulating osteocalcin is increased by 1,25-dihydroxyvitamin D [1,25(OH)2D] and may improve glucose homeostasis. OBJECTIVE: This study was designed to test whether dietary cholecalciferol during pregnancy dose-dependently affects maternal glucose tolerance and maternal and neonatal glucose concentrations in relation to plasma osteocalcin and body composition. METHODS: Female guinea pigs (n = 45; 4 mo old) were randomly assigned to 5 doses of cholecalciferol (0, 0.25, 0.5, 1, or 2 IU/g diet) fed from mating to delivery. Plasma vitamin D metabolites, minerals, and osteocalcin, and blood glucose were measured before mating, at midgestation (day 42), and at day 2 postpartum in sows and in 2-d-old pups. At day 50 of pregnancy (early third trimester), a 3-h oral-glucose-tolerance test (OGTT) (2 g/kg) was conducted. Body composition was measured before mating and at day 2 postpartum in sows and in pups. RESULTS: A positive dose-response to dietary cholecalciferol was observed for change in maternal plasma 25-hydroxyvitamin D [25(OH)D] through pregnancy (P < 0.0001), with 1,25(OH)2D increasing by 198% in the 1-IU/g group by midgestation vs. a reduction of 43.6% in the 0-IU/g group (P = 0.05). Twenty-four (54.5%) sows had gestational diabetes mellitus (GDM) on the basis of nonfed glucose and 39 (88.6%) had GDM on the basis of 2-h OGTT glucose concentrations. There were no group differences in maternal OGTT or changes in glucose, minerals, osteocalcin concentrations, and body composition. Pre-mating 25(OH)D was inversely related to 3-h area under the curve for blood glucose from the OGTT (r = -0.31, P = 0.05). In guinea pig pups, although both 25(OH)D (P < 0.0001) and 1,25(OH)2D (P < 0.0001) followed a dose-response to maternal diet, glucose, osteocalcin, minerals, and body composition were not altered. CONCLUSIONS: Dietary vitamin D intake during pregnancy in guinea pigs does not affect the already high rate of GDM, whereas higher prepregnancy vitamin D status appears to be protective.

Dietary vitamin D during pregnancy has dose-dependent effects on long bone density and architecture in guinea pig offspring but not the sows.

BACKGROUND: The effects of vitamin D during pregnancy on maternal and neonatal bone health remain unclear. OBJECTIVE: This study was designed to test whether dietary vitamin D dose-dependently affects maternal and neonatal bone health. METHODS: Female guinea pigs (n = 45; 4 mo old) were randomly assigned at mating to receive 1 of 5 doses of vitamin D3 (cholecalciferol; 0, 0.25, 0.5, 1, or 2 IU/g diet) throughout pregnancy. Plasma vitamin D metabolites, mineral homeostasis, bone biomarkers, and bone mass were tested in sows throughout pregnancy and in 2-d-old pups. Microarchitecture and histology of excised bone were conducted postpartum. RESULTS: By 3 wk of pregnancy, plasma 25-hydroxyvitamin D [25(OH)D] followed a positive dose-response, whereas 1,25-dihydroxyvitamin D [1,25(OH)2D] reached a plateau if vitamin D was ≥0.5 IU/g diet. Weight gain, areal bone mineral density (aBMD), volumetic bone mineral density (vBMD), and bone biomarkers did not differ among maternal groups. A positive dose-response was observed for mean ± SEM pup plasma concentrations of 25(OH)D (10.5 ± 1.50 to 113 ±11.6 nmol/L) and 1,25(OH)2D (123 ± 13.8 to 544 ± 53.3 pmol/L). Pup weight, plasma minerals, and osteocalcin were not different; plasma deoxypyridinoline was lower in the 1- and 0.25-IU/g groups than in all other groups. Pup femur aBMD was higher (9.2-13%; P = 0.04) in the 2-IU/g group than in all other groups except for the 0-IU/g group. Tibia and femur vBMD of pups responded to maternal diet in a U-shaped pattern. The femoral growth plate was 7.9% wider in the 0-IU/g group than in the 1-IU/g group. CONCLUSIONS: Maternal vitamin D supplementation dose-dependently altered pup long bone architecture and mineral density in a manner similar to vitamin D deficient rickets whereas maternal bone was stable. These data reinforce that inadequate maternal vitamin D intake may compromise neonatal bone health and that exceeding recommendations is not advantageous.

Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells.

The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α+/+) and phosphorylation-deficient mutant eIF2α (eIF2αA/A) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/ß) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network.

RNA-based translation activators for targeted gene upregulation.

Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present "translation-activating RNAs" (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research.

Signaling pathways that control mRNA translation initiation in macrophages.

Translational control in mammalian cells plays a critical role in regulating differentiation, cell growth, cell cycle and response to diverse stresses. Macrophages are one of the most versatile cell types in the body. They are professional phagocytic cells that can be found in almost all tissues and adapt tissue-specific functions. Recent studies highlight the importance of translational control in macrophages during invasion of pathogens, exposure to cytokines and in the context of tissue specific functions. In this review, we summarize the current knowledge regarding the role of mRNA translational control in regulation of macrophages.

Effects of dietary selenium supplementation on serum and liver selenium, serum malondialdehyde and liver glutathione peroxidase activity in rats consuming thermally oxidized sunflower oil.

The present study compared the effects of four isocaloric diets containing (1) fresh sunflower oil not supplemented with selenium (Fresh), (2) oxidized sunflower oil not supplemented with selenium (Oxidized), (3) fresh sunflower oil supplemented with 1 ppm selenium as sodium selenite (Fresh+Se), (4) oxidized sunflower oil supplemented with 1 ppm selenium as sodium selenite (Oxidized+Se) on serum MDA concentrations, liver GPx activity and serum and liver selenium contents in growing male Sprague Dawley rats during a period of 43 days. The oxidized oil used was prepared by heating fresh sunflower oil at 180 degrees C for 48 h. Serum and liver selenium contents and liver GPx activity were significantly higher in the selenium supplemented groups compared to the non-selenium supplemented groups, but these parameters did not differ significantly between the oxidized oil fed groups and the fresh oil fed groups. Serum MDA concentrations increased significantly in the Oxidized group compared to the Fresh group. This suggests that the ingestion of oxidized oil resulted in, in vivo lipid peroxidation. Serum MDA concentrations remained significantly higher even in comparison of the Oxidized + Se group with the Oxidized group. Our results emphasize that the consumption of oxidized oil increases in vivo lipid peroxidation and thus can be deleterious to health. However, we did not observe a significant beneficial effect of selenium supplementation upon the ingestion of thermally oxidized oil on lipid peroxidation.

Osteocalcin is higher across pregnancy in Caucasian women with gestational diabetes mellitus.

OBJECTIVE: The purpose of this study was to investigate circulating concentrations of osteocalcin, a bone-derived protein, while accounting for 25-hydroxyvitamin D (25(OH)D) throughout pregnancy, and whether early gestation concentrations and changes in osteocalcin predict the subsequent diagnosis of gestational diabetes mellitus (GDM). METHODS: This was a nested case-control study involving 48 GDM and 48 control pregnant Caucasian women (matched for age, season of conception, pre-pregnancy body mass index and pregnancy length). Maternal serum osteocalcin was measured by enzyme-linked immunosorbent assay and 25(OH)D by chemiluminescence throughout pregnancy (11-13 weeks, 24-28 weeks and predelivery). Differences between groups were compared by mixed model analysis of variance. Predictors of diagnosis of GDM were explored using generalized estimating equation models. Neonatal general health outcomes were also compared between groups. RESULTS: Serum osteocalcin was higher across pregnancy (p=0.006) in women with GDM vs. controls, whereas serum 25(OH)D was not different (p=0.80). Both biomarkers increased with time across pregnancy (p<0.0001). However, serum osteocalcin during early pregnancy and changes in its concentration from early to mid gestation did not predict the development of GDM. There were no significant differences in anthropometry and APGAR (appearance, pulse, grimace, activity, respiration) scores in neonates of controls and cases. CONCLUSIONS: Serum osteocalcin is elevated in Caucasian women with GDM throughout pregnancy, but was not predictive of the onset of GDM. Larger trials evaluating the role of osteocalcin and the development of GDM appear warranted.