Sexual Differentiation of Circadian Clock Function in the Adrenal Gland

Sex differences in glucocorticoid production are associated with increased responsiveness of the adrenal gland in females. However, the adrenal-intrinsic mechanisms that establish sexual dimorphic function remain ill defined. Glucocorticoid production is gated at the molecular level by the circadian clock, which may contribute to sexual dimorphic adrenal function. Here we examine sex differences in the adrenal gland using an optical reporter of circadian clock function. Adrenal glands were cultured from male and female Period2::Luciferase (PER2::LUC) mice to assess clock function in vitro in real time. We confirm that there is a pronounced sex difference in the intrinsic capacity to sustain PER2::LUC rhythms in vitro, with higher amplitude rhythms in adrenal glands collected from males than from females. Changes in adrenal PER2::LUC rhythms over the reproductive life span implicate T as an important factor in driving sex differences in adrenal clock function. By directly manipulating hormone levels in adult mice in vivo, we demonstrate that T increases the amplitude of PER2::LUC rhythms in adrenal glands of both male and female mice. In contrast, we find little evidence that ovarian hormones modify adrenal clock function. Lastly, we find that T in vitro can increase the amplitude of PER2::LUC rhythms in male adrenals but not female adrenals, which suggests the existence of sex differences in the mechanisms of T action in vivo. Collectively these results reveal that activational effects of T alter circadian timekeeping in the adrenal gland, which may have implications for sex differences in stress reactivity and stress-related disorders

eIF4E3 forms an active eIF4F complex during stresses (eIF4FS) targeting mTOR and re-programs the translatome.

The eIF4E are a family of initiation factors that bind the mRNA 5' cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active during stress and that it selects specific mRNA populations based on 5' TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have 'moon-lighting' functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote 'stress resistance' and adaptation

Shell Neurons of the Master Circadian Clock Coordinate the Phase of Tissue Clocks Throughout the Brain and Body

Background: Daily rhythms in mammals are programmed by a master clock in the suprachiasmatic nucleus (SCN). The SCN contains two main compartments (shell and core), but the role of each region in system-level coordination remains ill defined. Herein, we use a functional assay to investigate how downstream tissues interpret region-specific outputs by using in vivo exposure to long day photoperiods to temporally dissociate the SCN. We then analyze resulting changes in the rhythms of clocks located throughout the brain and body to examine whether they maintain phase synchrony with the SCN shell or core. Results: Nearly all of the 17 tissues examined in the brain and body maintain phase synchrony with the SCN shell, but not the SCN core, which indicates that downstream oscillators are set by cues controlled specifically by the SCN shell. Interestingly, we also found that SCN dissociation diminished the amplitude of rhythms in core clock gene and protein expression in brain tissues by 50–75 %, which suggests that light-driven changes in the functional organization of the SCN markedly influence the strength of rhythms in downstream tissues. Conclusions: Overall, our results reveal that body clocks receive time-of-day cues specifically from the SCN shell, which may be an adaptive design principle that serves to maintain system-level phase relationships in a changing environment. Further, we demonstrate that lighting conditions alter the amplitude of the molecular clock in downstream tissues, which uncovers a new form of plasticity that may contribute to seasonal changes in physiology and behavior

小学校教科書語彙の研究

<div><p>Abstract</p><p><i>Leishmania</i> parasites replicate within the phagolysosome compartment of mammalian macrophages. Although <i>Leishmania</i> depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient <i>Leishmania major</i> mutant (<i>∆gnat</i>) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in <i>ex vivo</i> infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the <i>L</i>. <i>major ∆gnat</i> mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in <i>ex vivo</i> infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in <i>Leishmania</i>-induced skin lesions and is efficiently internalized into <i>Leishmania</i> containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides <i>Leishmania</i> with essential carbon sources, creating a uniquely favorable niche for these parasites.</p></div

Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal.

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells

A family of dual-activity glycosyltransferasesphosphorylases mediates mannogen turnover and virulence in Leishmania parasites

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches

A patatin-like phospholipase is important for mitochondrial function in malaria parasites.

Plasmodium parasites rely on a functional electron transport chain (ETC) within their mitochondrion for proliferation, and compounds targeting mitochondrial functions are validated antimalarials. Here, we localize Plasmodium falciparum patatin-like phospholipase 2 (PfPNPLA2, PF3D7_1358000) to the mitochondrion and reveal that disruption of the PfPNPLA2 gene impairs asexual replication. PfPNPLA2-null parasites are hypersensitive to proguanil and inhibitors of the mitochondrial ETC, including atovaquone. In addition, PfPNPLA2-deficient parasites show reduced mitochondrial respiration and reduced mitochondrial membrane potential, indicating that disruption of PfPNPLA2 leads to a defect in the parasite ETC. Lipidomic analysis of the mitochondrial phospholipid cardiolipin (CL) reveals that loss of PfPNPLA2 is associated with a moderate shift toward shorter-chained and more saturated CL species, implying a contribution of PfPNPLA2 to CL remodeling. PfPNPLA2-deficient parasites display profound defects in gametocytogenesis, underlining the importance of a functional mitochondrial ETC during both the asexual and sexual development of the parasite. IMPORTANCE For their proliferation within red blood cells, malaria parasites depend on a functional electron transport chain (ETC) within their mitochondrion, which is the target of several antimalarial drugs. Here, we have used gene disruption to identify a patatin-like phospholipase, PfPNPLA2, as important for parasite replication and mitochondrial function in Plasmodium falciparum. Parasites lacking PfPNPLA2 show defects in their ETC and become hypersensitive to mitochondrion-targeting drugs. Furthermore, PfPNPLA2-deficient parasites show differences in the composition of their cardiolipins, a unique class of phospholipids with key roles in mitochondrial functions. Finally, we demonstrate that parasites devoid of PfPNPLA2 have a defect in gametocyte maturation, underlining the importance of a functional ETC for parasite transmission to the mosquito vector

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not infective to mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both

Metabolomic analyses of Leishmania reveal multiple species differences and large differences in amino acid metabolism

Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts