Patrick Descombes

Understanding why some people establish and maintain effective control of HIV-1 and others do not is a priority in the effort to develop new treatments for HIV/AIDS. Using a whole-genome association strategy, we identified polymorphisms that explain nearly 15% of the variation among individuals in viral load during the asymptomatic set-point period of infection. One of these is found within an endogenous retroviral element and is associated with major histocompatibility allele human leukocyte antigen ( HLA )– B*5701 , whereas a second is located near the HLA-C gene. An additional analysis of the time to HIV disease progression implicated two genes, one of which encodes an RNA polymerase I subunit. These findings emphasize the importance of studying human genetic variation as a guide to combating infectious agents.

Background & AimsHepatitis C virus (HCV) induces chronic infection in 50% to 80% of infected persons; approximately 50% of these do not respond to therapy. We performed a genome-wide association study to screen for host genetic determinants of HCV persistence and response to therapy.MethodsThe analysis included 1362 individuals: 1015 with chronic hepatitis C and 347 who spontaneously cleared the virus (448 were coinfected with human immunodeficiency virus [HIV]). Responses to pegylated interferon alfa and ribavirin were assessed in 465 individuals. Associations between more than 500,000 single nucleotide polymorphisms (SNPs) and outcomes were assessed by multivariate logistic regression.ResultsChronic hepatitis C was associated with SNPs in the IL28B locus, which encodes the antiviral cytokine interferon lambda. The rs8099917 minor allele was associated with progression to chronic HCV infection (odds ratio [OR], 2.31; 95% confidence interval [CI], 1.74–3.06; P = 6.07 × 10−9). The association was observed in HCV mono-infected (OR, 2.49; 95% CI, 1.64–3.79; P = 1.96 × 10−5) and HCV/HIV coinfected individuals (OR, 2.16; 95% CI, 1.47–3.18; P = 8.24 × 10−5). rs8099917 was also associated with failure to respond to therapy (OR, 5.19; 95% CI, 2.90–9.30; P = 3.11 × 10−8), with the strongest effects in patients with HCV genotype 1 or 4. This risk allele was identified in 24% of individuals with spontaneous HCV clearance, 32% of chronically infected patients who responded to therapy, and 58% who did not respond (P = 3.2 × 10−10). Resequencing of IL28B identified distinct haplotypes that were associated with the clinical phenotype.ConclusionsThe association of the IL28B locus with natural and treatment-associated control of HCV indicates the importance of innate immunity and interferon lambda in the pathogenesis of HCV infection. Hepatitis C virus (HCV) induces chronic infection in 50% to 80% of infected persons; approximately 50% of these do not respond to therapy. We performed a genome-wide association study to screen for host genetic determinants of HCV persistence and response to therapy. The analysis included 1362 individuals: 1015 with chronic hepatitis C and 347 who spontaneously cleared the virus (448 were coinfected with human immunodeficiency virus [HIV]). Responses to pegylated interferon alfa and ribavirin were assessed in 465 individuals. Associations between more than 500,000 single nucleotide polymorphisms (SNPs) and outcomes were assessed by multivariate logistic regression. Chronic hepatitis C was associated with SNPs in the IL28B locus, which encodes the antiviral cytokine interferon lambda. The rs8099917 minor allele was associated with progression to chronic HCV infection (odds ratio [OR], 2.31; 95% confidence interval [CI], 1.74–3.06; P = 6.07 × 10−9). The association was observed in HCV mono-infected (OR, 2.49; 95% CI, 1.64–3.79; P = 1.96 × 10−5) and HCV/HIV coinfected individuals (OR, 2.16; 95% CI, 1.47–3.18; P = 8.24 × 10−5). rs8099917 was also associated with failure to respond to therapy (OR, 5.19; 95% CI, 2.90–9.30; P = 3.11 × 10−8), with the strongest effects in patients with HCV genotype 1 or 4. This risk allele was identified in 24% of individuals with spontaneous HCV clearance, 32% of chronically infected patients who responded to therapy, and 58% who did not respond (P = 3.2 × 10−10). Resequencing of IL28B identified distinct haplotypes that were associated with the clinical phenotype. The association of the IL28B locus with natural and treatment-associated control of HCV indicates the importance of innate immunity and interferon lambda in the pathogenesis of HCV infection.

LAP, a transcriptional activator, and LIP, a transcriptional repressor, are translated from a single mRNA species by using two AUGs within the same reading frame. These two proteins share the 145 C-terminal amino acids that contain the basic DNA-binding domain and the leucine zipper dimerization helix. Probably owing to its higher affinity for its DNA cognate sequences, LIP can attenuate the transcriptional stimulation by LAP in substoichiometric amounts. As revealed by transient transfection experiments, a moderate increase in the ratio results in a significantly higher transcriptional activation of an appropriate target gene. The ratio increases about 5-fold during terminal rat liver differentiation and is thus likely to modulate the activity of LAP in the intact animal.

A gene, encoding a liver-enriched transcriptional activator protein (LAP) has been isolated. LAP is a 32-kD protein that stimulates the transcription of chimeric genes containing albumin D-promoter elements both in vivo and in vitro. LAP shares extensive sequence homology (71%) in its DNA-binding and leucine zipper domains with C/EBP. As a consequence, these two proteins show an indistinguishable DNA-binding specificity and readily heterodimerize. In addition, both genes, lap and cebp, are devoid of intervening sequences. Although correctly initiated transcripts from the LAP gene accumulate in the six examined tissues--liver, lung, spleen, kidney, brain, and testis--LAP protein is highly enriched in liver nuclei. Thus, the preferential accumulation of LAP protein in liver appears to be regulated post-transcriptionally.

The PAR-domain basic leucine zipper (PAR bZip) transcription factors DBP, TEF, and HLF accumulate in a highly circadian manner in several peripheral tissues, including liver and kidney. Mice devoid of all three of these proteins are born at expected Mendelian ratios, but are epilepsy prone, age at an accelerated rate, and die prematurely. In the hope of identifying PAR bZip target genes whose altered expression might contribute to the high morbidity and mortality of PAR bZip triple knockout mice, we compared the liver and kidney transcriptomes of these animals to those of wild-type or heterozygous mutant mice. These experiments revealed that PAR bZip proteins control the expression of many enzymes and regulators involved in detoxification and drug metabolism, such as cytochrome P450 enzymes, carboxylesterases, and constitutive androstane receptor (CAR). Indeed, PAR bZip triple knockout mice are hypersensitive to xenobiotic compounds, and the deficiency in detoxification may contribute to their early aging.

To extend the understanding of host genetic determinants of HIV-1 control, we performed a genome-wide association study in a cohort of 2,554 infected Caucasian subjects. The study was powered to detect common genetic variants explaining down to 1.3% of the variability in viral load at set point. We provide overwhelming confirmation of three associations previously reported in a genome-wide study and show further independent effects of both common and rare variants in the Major Histocompatibility Complex region (MHC). We also examined the polymorphisms reported in previous candidate gene studies and fail to support a role for any variant outside of the MHC or the chemokine receptor cluster on chromosome 3. In addition, we evaluated functional variants, copy-number polymorphisms, epistatic interactions, and biological pathways. This study thus represents a comprehensive assessment of common human genetic variation in HIV-1 control in Caucasians.

Antibiotic resistance is rising in important bacterial pathogens. Phage therapy (PT), the use of bacterial viruses infecting the pathogen in a species-specific way, is a potential alternative.T4-like coliphages or a commercial Russian coliphage product or placebo was orally given over 4 days to Bangladeshi children hospitalized with acute bacterial diarrhea. Safety of oral phage was assessed clinically and by functional tests; coliphage and Escherichia coli titers and enteropathogens were determined in stool and quantitative diarrhea parameters (stool output, stool frequency) were measured. Stool microbiota was studied by 16S rRNA gene sequencing; the genomes of four fecal Streptococcus isolates were sequenced.No adverse events attributable to oral phage application were observed (primary safety outcome). Fecal coliphage was increased in treated over control children, but the titers did not show substantial intestinal phage replication (secondary microbiology outcome). 60% of the children suffered from a microbiologically proven E. coli diarrhea; the most frequent diagnosis was ETEC infections. Bacterial co-pathogens were also detected. Half of the patients contained phage-susceptible E. coli colonies in the stool. E. coli represented less than 5% of fecal bacteria. Stool ETEC titers showed only a short-lived peak and were otherwise close to the replication threshold determined for T4 phage in vitro. An interim analysis after the enrollment of 120 patients showed no amelioration in quantitative diarrhea parameter by PT over standard care (tertiary clinical outcome). Stool microbiota was characterized by an overgrowth with Streptococcus belonging to the Streptococcus gallolyticus and Streptococcus salivarius species groups, their abundance correlated with quantitative diarrhea outcome, but genome sequencing did not identify virulence genes.Oral coliphages showed a safe gut transit in children, but failed to achieve intestinal amplification and to improve diarrhea outcome, possibly due to insufficient phage coverage and too low E. coli pathogen titers requiring higher oral phage doses. More knowledge is needed on in vivo phage-bacterium interaction and the role of E. coli in childhood diarrhea for successful PT.The study was supported by a grant from Nestlé Nutrition and Nestlé Health Science. The trial was registered with Identifier NCT00937274 at ClinicalTrials.gov.

The CCAAT/enhancer-binding protein (C/EBP) alpha is a leucine zipper protein that is preferentially expressed in certain cell types, such as adipocytes and hepatocytes. Here we show that C/EBP alpha mRNA is translated into two major proteins, C/EBP-42 and C/EBP-30, that differ in their content of N-terminal amino acid sequences. These results are best explained by a ribosome-scanning mechanism in which a fraction of ribosomes ignore the first two AUGs and initiate translation at an AUG located 351 nt downstream of the first one. Because C/EBP-30, the translation product initiated at the third AUG, is devoid of the potent transcription-activation domain contained in C/EBP-42, the former protein stimulates transcription from the mouse albumin promoter much less efficiently than the latter. The gene encoding the liver-enriched transcriptional-activator protein LAP (C/EBP-beta) has also been shown to issue two proteins, LAP and the liver-enriched transcriptional-inhibitory protein LIP, with different transcription-activation potentials. The production of multiple proteins from a single mRNA is not only shared between different C/EBP family members but also appears to be conserved in vertebrate evolution.

Glomalean fungi induce and colonize symbiotic tissue called arbuscular mycorrhiza on the roots of most land plants. Other fungi also colonize plants but cause disease not symbiosis. Whole-transcriptome analysis using a custom-designed Affymetrix Gene-Chip and confirmation with real-time RT-PCR revealed 224 genes affected during arbuscular mycorrhizal symbiosis. We compared these transcription profiles with those from rice roots that were colonized by pathogens (Magnaporthe grisea and Fusarium moniliforme). Over 40% of genes showed differential regulation caused by both the symbiotic and at least one of the pathogenic interactions. A set of genes was similarly expressed in all three associations, revealing a conserved response to fungal colonization. The responses that were shared between pathogen and symbiont infection may play a role in compatibility. Likewise, the responses that are different may cause disease. Some of the genes that respond to mycorrhizal colonization may be involved in the uptake of phosphate. Indeed, phosphate addition mimicked the effect of mycorrhiza on 8% of the tested genes. We found that 34% of the mycorrhiza-associated rice genes were also associated with mycorrhiza in dicots, revealing a conserved pattern of response between the two angiosperm classes.

NMDA receptors promote neuronal survival but also cause cell degeneration and neuron loss. The mechanisms underlying these opposite effects on neuronal fate are unknown. Whole-genome expression profiling revealed that NMDA receptor signaling is decoded at the genomic level through activation of two distinct, largely nonoverlapping gene-expression programs. The location of the NMDA receptor activated specifies the transcriptional response: synaptic NMDA receptors induce a coordinate upregulation of newly identified pro-survival genes and downregulation of pro-death genes. Extrasynaptic NMDA receptors fail to activate this neuroprotective program, but instead induce expression of Clca1, a putative calcium-activated chloride channel that kills neurons. These results help explain the opposing roles of synaptic and extrasynaptic NMDA receptors on neuronal fate. They also demonstrate that the survival function is implemented in neurons through a multicomponent system of functionally related genes, whose coordinate expression is controlled by specific calcium signal initiation sites.

Given the dramatic increase in ageing populations, it is of great importance to understand the genetic and molecular determinants of healthy ageing and longevity. Semi-supercentenarians (subjects who reached an age of 105-109 years) arguably represent the gold standard of successful human ageing because they managed to avoid or postpone the onset of major age-related diseases. Relatively few studies have looked at epigenetic determinants of extreme longevity in humans. Here we test whether families with extreme longevity are epigenetically distinct from controls according to an epigenetic biomarker of ageing which is known as "epigenetic clock". We analyze the DNA methylation levels of peripheral blood mononuclear cells (PBMCs) from Italian families constituted of 82 semi-supercentenarians (mean age: 105.6 ± 1.6 years), 63 semi-supercentenarians' offspring (mean age: 71.8 ± 7.8 years), and 47 age-matched controls (mean age: 69.8 ± 7.2 years). We demonstrate that the offspring of semi-supercentenarians have a lower epigenetic age than age-matched controls (age difference=5.1 years, p=0.00043) and that centenarians are younger (8.6 years) than expected based on their chronological age. By contrast, no significant difference could be observed for estimated blood cell counts (such as naïve or exhausted cytotoxic T cells or helper T cells). Future studies will be needed to replicate these findings in different populations and to extend them to other tissues. Overall, our results suggest that epigenetic processes might play a role in extreme longevity and healthy human ageing.

RT-qPCR is a sensitive and increasingly used method for gene expression quantification. To normalize RT-qPCR measurements between samples, most laboratories use endogenous reference genes as internal controls. There is increasing evidence, however, that the expression of commonly used reference genes can vary significantly in certain contexts.Using the Genevestigator database of normalized and well-annotated microarray experiments, we describe the expression stability characteristics of the transciptomes of several organisms. The results show that a) no genes are universally stable, b) most commonly used reference genes yield very high transcript abundances as compared to the entire transcriptome, and c) for each biological context a subset of stable genes exists that has smaller variance than commonly used reference genes or genes that were selected for their stability across all conditions.We therefore propose the normalization of RT-qPCR data using reference genes that are specifically chosen for the conditions under study. RefGenes is a community tool developed for that purpose. Validation RT-qPCR experiments across several organisms showed that the candidates proposed by RefGenes generally outperformed commonly used reference genes. RefGenes is available within Genevestigator at http://www.genevestigator.com.

Synaptic activity can boost neuroprotection through a mechanism that requires synapse-to-nucleus communication and calcium signals in the cell nucleus. Here we show that in hippocampal neurons nuclear calcium is one of the most potent signals in neuronal gene expression. The induction or repression of 185 neuronal activity-regulated genes is dependent upon nuclear calcium signaling. The nuclear calcium-regulated gene pool contains a genomic program that mediates synaptic activity-induced, acquired neuroprotection. The core set of neuroprotective genes consists of 9 principal components, termed Activity-regulated Inhibitor of Death (AID) genes, and includes Atf3, Btg2, GADD45β, GADD45γ, Inhibin β-A, Interferon activated gene 202B, Npas4, Nr4a1, and Serpinb2, which strongly promote survival of cultured hippocampal neurons. Several AID genes provide neuroprotection through a common process that renders mitochondria more resistant to cellular stress and toxic insults. Stereotaxic delivery of AID gene-expressing recombinant adeno-associated viruses to the hippocampus confers protection in vivo against seizure-induced brain damage. Thus, treatments that enhance nuclear calcium signaling or supplement AID genes represent novel therapies to combat neurodegenerative conditions and neuronal cell loss caused by synaptic dysfunction, which may be accompanied by a deregulation of calcium signal initiation and/or propagation to the cell nucleus.

Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism.

Gut microbes influence their hosts in many ways, in particular by modulating the impact of diet. These effects have been studied most extensively in humans and mice. In this work, we used whole genome metagenomics to investigate the relationship between the gut metagenomes of dogs, humans, mice, and pigs.We present a dog gut microbiome gene catalog containing 1,247,405 genes (based on 129 metagenomes and a total of 1.9 terabasepairs of sequencing data). Based on this catalog and taxonomic abundance profiling, we show that the dog microbiome is closer to the human microbiome than the microbiome of either pigs or mice. To investigate this similarity in terms of response to dietary changes, we report on a randomized intervention with two diets (high-protein/low-carbohydrate vs. lower protein/higher carbohydrate). We show that diet has a large and reproducible effect on the dog microbiome, independent of breed or sex. Moreover, the responses were in agreement with those observed in previous human studies.We conclude that findings in dogs may be predictive of human microbiome results. In particular, a novel finding is that overweight or obese dogs experience larger compositional shifts than lean dogs in response to a high-protein diet.

Significance Rhythmic gene regulation in mouse liver results from an intertwined relationship between feeding cycles and the circadian clock. Significant efforts have been made to understand this interaction but a complete picture of the resulting diurnal transcription–translation processes is still missing. Through the simultaneous quantification of temporal transcription, accumulation, and translation of mRNA in the liver, we investigated the regulatory landscape of mice with intact or deficient circadian clock subjected to different feeding regimens. We showed that circadian clock and feeding rhythms coordinate rhythmic transcription to drive downstream rhythmic mRNA accumulation and translation. However, a subset of genes harboring 5′-Terminal Oligo Pyrimidine tract or Translation Initiator of Short 5′-UTR elements encoding proteins involved in translation and mitochondrial activity, respectively, present a transcription-independent rhythmic translation mainly regulated by feeding.

Research on age-related regenerative failure of skeletal muscle has extensively focused on the phenotypes of muscle stem cells (MuSCs). In contrast, the impact of aging on regulatory cells in the MuSC niche remains largely unexplored. Here, we demonstrate that aging impairs the function of mouse fibro-adipogenic progenitors (FAPs) and thereby indirectly affects the myogenic potential of MuSCs. Using transcriptomic profiling, we identify WNT1 Inducible Signaling Pathway Protein 1 (WISP1) as a FAP-derived matricellular signal that is lost during aging. WISP1 is required for efficient muscle regeneration and controls the expansion and asymmetric commitment of MuSCs through Akt signaling. Transplantation of young FAPs or systemic treatment with WISP1 restores the myogenic capacity of MuSCs in aged mice and rescues skeletal muscle regeneration. Our work establishes that loss of WISP1 from FAPs contributes to MuSC dysfunction in aged skeletal muscles and demonstrates that this mechanism can be targeted to rejuvenate myogenesis.

The causes of impaired skeletal muscle mass and strength during aging are well-studied in healthy populations. Less is known on pathological age-related muscle wasting and weakness termed sarcopenia, which directly impacts physical autonomy and survival. Here, we compare genome-wide transcriptional changes of sarcopenia versus age-matched controls in muscle biopsies from 119 older men from Singapore, Hertfordshire UK and Jamaica. Individuals with sarcopenia reproducibly demonstrate a prominent transcriptional signature of mitochondrial bioenergetic dysfunction in skeletal muscle, with low PGC-1α/ERRα signalling, and downregulation of oxidative phosphorylation and mitochondrial proteostasis genes. These changes translate functionally into fewer mitochondria, reduced mitochondrial respiratory complex expression and activity, and low NAD+ levels through perturbed NAD+ biosynthesis and salvage in sarcopenic muscle. We provide an integrated molecular profile of human sarcopenia across ethnicities, demonstrating a fundamental role of altered mitochondrial metabolism in the pathological loss of skeletal muscle mass and function in older people.

Human milk is the sole and recommended nutrition for the newborn infant and contains one of the largest constituents of diverse oligosaccharides, dubbed human milk oligosaccharides (HMOs). Preclinical and clinical association studies indicate that HMOs have multiple physiological functions largely mediated through the establishment of the gut microbiome. Until recently, HMOs were not available to investigate their role in randomized controlled intervention trials. To our knowledge, this is the first report on the effects of 2 HMOs on establishing microbiota in newborn infants. We provide a detailed description of the microbiota changes observed upon feeding a formula with 2 HMOs in comparison to breastfed reference infants' microbiota. Then, we associate the microbiota to long-term health as assessed by prescribed antibiotic use.

The primary event in mammalian sexual development is the differentiation of the bipotential gonads into either testes or ovaries. Our understanding of the molecular pathways specifying gonadal differentiation is still incomplete. To identify the initial molecular changes accompanying gonadal differentiation in mice, we have performed a large-scale transcriptional analysis of XX and XY Sf1-positive gonadal cells during sex determination. In both male and female genital ridges, a robust genetic program is initiated pre-dating the first morphological changes of the differentiating gonads. Between E10.5 and E13.5, 2306 genes were expressed in a sex-specific manner in the somatic compartment of the gonads; 1223 were overexpressed in XX embryos and 1083 in XY embryos. Although sexually dimorphic genes were scattered throughout the mouse genome, we identified chromosomal regions hosting clusters of genes displaying similar expression profiles. The cyclin-dependent kinase inhibitors Cdkn1a and Cdkn1c are overexpressed in XX gonads at E11.5 and E12.5, suggesting that the increased proliferation of XY gonads relative to XX gonads may result from the overexpression of cell cycle inhibitors in the developing ovaries. These studies define the major characteristics of testicular and ovarian transcriptional programs and reveal the richness of signaling processes in differentiation of the bipotential gonads into testes and ovaries.

Article2 March 1998free access The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts Patrick Descombes Patrick Descombes Department of Molecular Biology, University of Geneva, Science II, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Erich A. Nigg Corresponding Author Erich A. Nigg Department of Molecular Biology, University of Geneva, Science II, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Patrick Descombes Patrick Descombes Department of Molecular Biology, University of Geneva, Science II, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Erich A. Nigg Corresponding Author Erich A. Nigg Department of Molecular Biology, University of Geneva, Science II, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Author Information Patrick Descombes1 and Erich A. Nigg 1 1Department of Molecular Biology, University of Geneva, Science II, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1328-1335https://doi.org/10.1093/emboj/17.5.1328 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Polo-like kinases (Plks), named after the Drosophila gene product polo, have been implicated in the regulation of multiple aspects of mitotic progression, including the activation of the Cdc25 phosphatase, bipolar spindle formation and cytokinesis. Genetic analyses performed in yeast and Drosophila suggest a function for Plks at late stages of mitosis, but biochemical data to support such a function in vertebrate organisms are lacking. Here we have taken advantage of Xenopus egg extracts for exploring the function of Plx1, a Xenopus Plk, during the cell cycle transition from M phase to interphase (I phase). We found that the addition of a catalytically inactive Plx1 mutant to M phase-arrested egg extracts blocked their Ca2+-induced release into interphase. Concomitantly, the proteolytic destruction of several targets of the anaphase-promoting complex and the inactivation of the Cdc2 protein kinase (Cdk1) were prevented. Moreover, the M to I phase transition could be abolished by immunodepletion of Plx1, but was restored upon the addition of recombinant Plx1. These results demonstrate that the exit of egg extracts from M phase arrest requires active Plx1, and they strongly suggest an important role for Plx1 in the activation of the proteolytic machinery that controls the exit from mitosis. Introduction Polo-like kinases (Plks) are emerging as a new family of cell cycle regulators. Prominent members of the Plk family include Drosophila melanogaster polo, Saccharomyces cerevisiae Cdc5p, Schizosaccharomyces pombe plo1+, mammalian Plk1 and Xenopus laevis Plx1 (Glover et al., 1996; Lane and Nigg, 1997). Genetic studies indicate that Plks contribute to promote various aspects of mitotic progression, but biochemical studies on the molecular action of these kinases remain scarce. In Drosophila, mutations in the polo gene result in a high frequency of cells with monopolar spindles and disorganized spindle poles (Sunkel and Glover, 1988). A similar phenotype has also been observed in S.pombe, where disruption of the plo1+ gene results in a defect in bipolar spindle assembly (Ohkura et al., 1995). In mammalian cells, antibody microinjection experiments have revealed a function for Plk1 in centrosome maturation (Lane and Nigg, 1996). Additional support for an important function of Plks at the onset of mitosis has been obtained in Xenopus laevis, where Plx1 has been identified as a protein kinase able to phosphorylate and thereby activate the Cdc25 phosphatase, a positive regulator of Cdk1/cyclin B (M phase-promoting factor, MPF) (Kumagai and Dunphy, 1996). Taken together, these studies indicate that Plks may play important roles at the onset of mitosis. Studies on the CDC5 gene in S.cerevisiae have failed to reveal an essential role for this kinase prior to the onset of anaphase although, interestingly, Cdc5p appears to be required for adaptation to a DNA damage checkpoint at G2/M (Toczyski et al., 1997). Instead, the analysis of cdc5 mutant phenotypes suggests that this kinase is required late during mitosis, as both cdc5 ts and null mutant strains arrest with separated chromosomes and elongated spindles (Hartwell et al., 1973; Kitada et al., 1993). Similarly, plo1+ of S.pombe may be important not only for spindle assembly but also for cytokinesis (Ohkura et al., 1995). Taken together, the available evidence thus suggests that Plks may function during multiple stages of mitosis (Glover et al., 1996; Lane and Nigg, 1997). Progression through mitosis is regulated by two fundamental mechanisms, protein phosphorylation and degradation (Deshaies, 1995; Nigg, 1995; King et al., 1996). In particular, proteolysis has been identified as a key mechanism essential for both sister chromatid separation at the onset of anaphase and the inactivation of Cdk1 upon exit from mitosis. Proteins containing characteristic sequence motifs, termed destruction boxes, are targeted for proteolysis by cell cycle-dependent polyubiquitination (Glotzer et al., 1991). This modification is brought about by the sequential action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3) (reviewed in Hershko and Ciechanover, 1992; Varshavsky, 1997). Then, ubiquitinated proteins are degraded by the 26S proteasome (Jentsch and Schlenker, 1995). A large multiprotein complex with E3 activity specific for mitotic substrates has been purified from several species, including S.cerevisiae (Zachariae et al., 1996), the clam Spisula (Sudakin et al., 1995) and Xenopus (King et al., 1995). This complex consists of some 8–12 subunits and has been termed cyclosome (Sudakin et al., 1995) or APC (anaphase-promoting complex) (King et al., 1995). The first proteins identified as critical targets for degradation during mitosis were the mitotic cyclins (Murray et al., 1989; Glotzer et al., 1991; Lorca et al., 1991; Gallant and Nigg, 1992; Surana et al., 1993). More recently, an important role for proteolysis in the initiation of sister chromatid separation has emerged. Inhibition of APC function either by substrate competition in Xenopus extracts (Holloway et al., 1993) or by mutations in APC subunits in yeast (Irniger et al., 1995) prevents chromosome segregation. These early findings implied that proteins other than mitotic cyclins must be degraded to allow anaphase onset. In support of this view, APC-dependent degradation of the protein Pds1p was shown to be essential for anaphase onset in S.cerevisiae (Cohen-Fix et al., 1996; Shirayama et al., 1998), and similar data have been obtained for Cut2, a probable functional homologue of Pds1p in S.pombe (Funabiki et al., 1996). The fact that different APC substrates are degraded at different times during mitosis (Hunt et al., 1992; Sigrist et al., 1995) implies the existence of regulatory mechanisms that allow the APC to act on different substrates according to a precise temporal (and perhaps spatial) pattern. Several lines of evidence indicate that phosphorylation may represent a major mechanism for regulating APC activity (King et al., 1995; Lahav-Baratz et al., 1995; Sudakin et al., 1995; Grieco et al., 1996; Peters et al., 1996; Yamada et al., 1997), but detailed information on its regulation has not yet been reported. Extrapolating from the phenotype of cdc5 mutants in S.cerevisiae, and, to a lesser extent, data obtained from Drosophila and S.pombe, we expected that vertebrate Plks might play a role during exit from M phase. However, previous attempts at revealing such a role in vertebrate cells had failed to produce conclusive results (Lane and Nigg, 1996; Mundt et al., 1997). In this study, we therefore turned to Xenopus egg extracts, a favourite model system for biochemical studies on the M to I phase transition (Murray, 1991). Using both a dominant-negative approach and immunodepletion–reconstitution experiments, we show that the activity of Plx1 is absolutely required for the Ca2+-induced transition of M phase-arrested extracts to interphase. In the absence of Plx1 activity, the destruction of multiple mitotic substrates is impaired, indicating that Plx1 activity is required for the activation of the degradation machinery in response to a Ca2+ signal. These findings reveal a new role for a vertebrate Plk in the exit from M phase, and they suggest that Plks may be important regulators of APC activity. Results Plx1 activity is cell cycle regulated To study Plx1 in Xenopus egg extracts, we have prepared polyclonal antibodies against recombinant Plx1 produced in baculovirus-infected insect cells. Immunoblotting revealed that both cytostatic factor (CSF)-arrested (M phase) extracts (Murray, 1991) and interphase (I phase) extracts contain similar levels of Plx1 (Figure 1, lanes 1 and 2). This contrasts with the cyclic accumulation of mammalian Plk1 (Golsteyn et al., 1995) and budding yeast Cdc5p (Hardy and Pautz, 1996; Shirayama et al., 1998), but is likely to reflect differences in the regulation of the rapid embryonic cycles as compared with somatic cycles. Similar observations have been made previously for other cell cycle regulators, including cyclin E (Rempel et al., 1995; Hartley et al., 1996). The concentration of Plx1 in the extracts used here was estimated to be ∼10 μg/ml (150 nM) (data not shown). Notably, Plx1 displayed a retarded migration in M phase extracts as compared with I phase extracts (Figure 1, lanes 1 and 2). As suggested by previous studies on Drosophila polo (Tavares et al., 1996) and human Plk1 (Hamanaka et al., 1995), this altered migration most likely reflects phosphorylation of Plx1 in M phase extracts (see also Mundt et al., 1997). Figure 1.Characterization of Plx1 expression and activity in Xenopus egg extracts. Plx1 was detected in M and I phase extracts by immunoblotting with anti-Plx1 antibodies (lanes 1 and 2). The activity of Plx1 was measured in immunoprecipitates prepared from M and I phase extracts (as indicated), using casein as a substrate (lanes 3–6; the arrow marks the migration of casein). Lanes 3 and 5, anti-Plx1 antibodies; lanes 4 and 6, pre-immune IgG. The recovery of Plx1 in the immunoprecipitates was checked by immunoblotting with anti-Plx1 antibodies (lanes 7–9). Lanes 7 and 8, immunoprecipitates prepared with anti-Plx1 antibodies; lanes 9 and 10, immunoprecipitates prepared with pre-immune IgG. Molecular weight markers are indicated in kDa. Download figure Download PowerPoint To compare the activity of Plx1 in M and I phase extracts, immunoprecipitations were performed and kinase activity assayed with casein as a substrate. Plx1 was 5- to 10-fold more active when isolated in its M phase form as compared with the I phase form (Figure 1, lanes 3 and 5). Comparable amounts of Plx1 protein were recovered from both extracts, and the isolated proteins displayed the characteristic mobility difference already observed in whole M and I phase extracts (Figure 1, lanes 7 and 8). No Plx1 protein and no casein kinase activity could be detected in control immunoprecipitates (Figure 1, lanes 4, 6, 9 and 10). These results indicate that in M phase-arrested extracts Plx1 is activated by a post-translational modification (presumably phosphorylation), confirming and extending recent data reported for Drosophila polo (Tavares et al., 1996) and Plk1 in somatic mammalian cells (Hamanaka et al., 1995; Mundt et al., 1997). A dominant-negative approach suggests a role for Plx1 at M phase exit To investigate the function of Plx1 in Xenopus egg extracts, we first chose a dominant-negative approach. Wild-type Plx1 (Plx1wt) and a catalytically inactive mutant [Plx1(N172A)], carrying a substitution of Asn172 by alanine, were purified to near homogeneity from recombinant baculovirus-infected Sf9 cells (Figure 2a). M phase extracts containing sperm nuclei were then supplemented with a 10-fold excess of Plx1wt or Plx1(N172A) (Figure 2b), and Ca2+ was added to trigger the metaphase to anaphase transition (Lohka and Maller, 1985; Lorca et al., 1993). In all extracts, both endogenous and recombinant Plx1 were stable throughout the experiment (Figure 2b). However, in those extracts that were supplemented with control buffer or Plx1wt, both endogenous Plx1 and recombinant Plx1wt displayed increased electrophoretic mobilities after a 90 min incubation (Figure 2b, compare lanes 2 and 4 with lanes 1 and 3, respectively), consistent with dephosphorylation of Plx1 upon exit from M phase (see also Figure 1). Interestingly, no such mobility shift was observed in the extracts containing Plx1(N172A) (Figure 2b, lanes 5 and 6), suggesting that these extracts were blocked in M phase. Figure 2.Plx1(N172A), added in a 10-fold excess to M phase extracts, is not shifted to its interphase position upon Ca2+ addition. (a) Coomassie Blue staining of purified histidine-tagged Plx1wt (lane 1) and Plx1(N172A) (lane 2) resolved by SDS–PAGE (6 μg each). The gel was overloaded to estimate the quality of the purification. (b) Samples of M phase extracts supplemented with control buffer (lanes 1 and 2), Plx1wt (lanes 3 and 4) or Plx1(N172A) (lanes 5 and 6) from time points 0 (lanes 1, 3 and 5) and 90 min after Ca2+ addition (lanes 2, 4 and 6) were analysed for their content in Plx1 by immunoblotting. The slower migration of recombinant Plx1wt and Plx1 N172A, as compared with endogenous Plx1, is due to the presence of the N-terminal His6 tag. Note that exogenous Plx1 was added at ∼10–fold excess over the endogenous kinase. Download figure Download PowerPoint To visualize this putative M phase arrest more directly, cell cycle stages of the various extracts were monitored by microscopic inspection of added sperm chromatin (Figure 3a). After 60 min, control extracts as well as the extracts supplemented with Plx1wt had entered interphase, as indicated by the decondensation of sperm chromatin and the formation of nuclear envelopes (Figure 3a, top and middle rows). In striking contrast, when the Plx1(N172A) mutant was added, sperm chromatin remained condensed even after 90 min of incubation (Figure 3, bottom row), consistent with the view that the catalytically inactive Plx1 had caused a block to the exit from M phase. In parallel, we determined histone H1 kinase activity, a direct measure of MPF (Murray, 1991), and the stability of endogenous cyclin B2 in the very same extracts (Figure 3b). In both control extracts and extracts supplemented with Plx1wt, histone H1 kinase activity dropped drastically within 15 min after Ca2+ addition, but it remained high in the extract containing the catalytically inactive Plx1(N172A) (Figure 3b, top panel). Likewise, endogenous cyclin B2 was degraded rapidly in the former extracts, but was entirely stable in the presence of Plx1(N172A) (Figure 3b, lower panel). Figure 3.Catalytically inactive Plx1 blocks the release of M phase-arrested Xenopus egg extracts into interphase. (a) M phase extracts containing sperm chromatin were supplemented with buffer (control, top panel), Plx1wt (middle panel) or Plx1(N172A) (lower panel). Samples were taken at the indicated times after Ca2+ addition (min), and chromatin was stained with Hoechst 33258. After 90 min, 95% of the input sperm DNA had decondensed in the control sample and 93% in the sample supplemented with Plx1 wt. In contrast, not a single example of decondensed chromatin could be found in the sample supplemented with Plx1(N172A). In each case, at least 300 sperm nuclei were counted. Scale bar denotes 5 μm. (b) Histone H1 kinase activity measurements (top panel), and cyclin B2 levels determined by immunoblotting (lower panel). The immunoreactive band migrating ahead of cyclin B2 represents an unidentified cross-reacting protein and provides an internal control for loading. Download figure Download PowerPoint Taken together, the results described above demonstrate that the presence of catalytically inactive Plx1 blocks the Ca2+-induced release of Xenopus egg extracts from M phase arrest and prevents both cyclin destruction and MPF inactivation. The most likely interpretation of these results is that a 10-fold excess of mutant Plx1(N172A) protein prevents the M to I phase transition by exerting a dominant-negative effect over the endogenous wild-type Plx1, presumably by competing for Plx1 substrates or other interacting proteins. Immunodepletion–reconstitution experiments confirm a requirement for Plx1 activity during M phase exit To corroborate and extend the above findings, immunodepletion and reconstitution experiments were performed. M phase extracts were treated with protein A beads that had been coated with either anti-Plx1 antibodies or pre-immune IgG as a control. More than 95% of Plx1 could be depleted by anti-Plx1 antibody beads (Figure 4a, lane 3), whereas control depletion did not significantly alter the level of Plx1 in the extracts (Figure 4a, lane 2). Also, overall protein profiles were unaffected by both treatments, as determined by Coomassie blue staining (data not shown). The depleted extracts were then tested for their ability to undergo the M to I phase transition in response to Ca2+. Whereas >95% of the sperm nuclei in the control depleted extracts underwent chromatin decondensation and nuclear assembly, the extracts lacking Plx1 could not be released from the metaphase block upon Ca2+ addition and virtually all sperm chromatin remained condensed (Figure 4b, first and second rows). The addition of a roughly physiological amount of recombinant Plx1wt protein (150 nM final concentration; see Figure 4a, lane 4) restored the ability of the Plx1-depleted extracts to exit M phase (Figure 4b, third row), but the addition of the Plx1(N172A) mutant (Figure 4a, lane 5) did not (Figure 4b, fourth row). In a typical rescue experiment, 75–85% of the sperm nuclei decondensed in response to Plx1wt. All extracts remained arrested in M phase in the absence of Ca2+ addition, indicating that immunodepletion had not caused spontaneous release from the metaphase block (data not shown). These results confirm that Plx1 activity is required for the Ca2+-induced release of CSF-arrested Xenopus egg extracts from M phase arrest. Figure 4.A reversible block to M phase exit by immunodepletion–reconstitution of Plx1. Fresh M phase extracts were depleted of Plx1 and assayed for their ability to be released into interphase by Ca2+ addition. Furthermore, the ability of Plx1wt or Plx1(N172A) to rescue the depletion phenotype was tested. (a) Immunoblot showing Plx1 levels in untreated M phase extracts (lane 1), in extracts treated with either control pre-immune beads (lane 2) or anti-Plx1 beads (lane 3), and in Plx1-depleted extracts supplemented with recombinant Plx1wt (lane 4) or Plx1(N172A) (lane 5). Note that recombinant Plx1wt and Plx1(N172A) display a slightly retarded migration, as compared with endogenous Plx1, due to the presence of an N-terminal His6 tag. Molecular weight markers are indicated in kDa. (b) Depleted extracts were supplemented with sperm chromatin and either control buffer, Plx1wt or Plx1(N172A), as indicated. The assembly of nuclei was monitored 120 min after Ca2+ addition by staining chromatin with Hoechst 33258 (left panels, two magnifications are shown) and by phase contrast microscopy (right panels). The scale bars in the left and central panels denote 20 and 5 μm, respectively. Download figure Download PowerPoint Plx1 activity is required for the proteolytic degradation of multiple mitotic regulators Progression through mitosis requires the ubiquitin-dependent proteolytic degradation of multiple proteins (Deshaies, 1995; King et al., 1996). The data shown above clearly demonstrate that a catalytically inactive Plx1 blocks the degradation of a cyclin, but they do not distinguish between a generalized block affecting the entire degradation machinery from a block exerted at the level of a specific substrate. We considered it important, therefore, to determine whether the inability of extracts supplemented with Plx1(N172A) to destroy the mitotic cyclins could be extended to a protein that is expected to be degraded at the metaphase to anaphase transition. Although vertebrate homologues of Pds1p/Cut2 have not yet been identified, the destruction of both Pds1p and Cut2 appears to be faithfully reproduced in Xenopus extracts (Cohen-Fix et al., 1996; Funabiki et al., 1997). We thus performed cell-free destruction assays (Lohka and Maller, 1985; Murray et al., 1989; Stewart et al., 1994), using M phase extracts containing either Plx1wt, Plx1(N172A) or control buffer, supplemented with in vitro translated substrates for APC-dependent proteolytic destruction. When extracts were supplemented with a mixture of in vitro translated full-length cyclin B2 and a non-destructible cyclin B2 mutant (cyclin B2ΔN7–69) as an internal control, the full-length cyclin B2 was destroyed upon Ca2+ addition both in control extracts and in extracts supplemented with Plx1wt (Figure 5a, top and middle panels). However, cyclin B2 was entirely stable in the presence of Plx1(N172A) (Figure 5a, bottom panel), confirming and extending the results shown above (Figure 3b). The non-destructible cyclin B2 mutant was stable in all extracts, as expected. The fate of Cut2 was then investigated following the same procedure. Like cyclin B2, Cut2 was partially destroyed upon Ca2+ addition both in control extracts and in extracts supplemented with Plx1wt (Figure 5b, top and middle panels), but was entirely stable in the presence of Plx1(N172A) (Figure 5b, bottom panel). In agreement with previous results (Funabiki et al., 1996, 1997), only ∼50% of Cut2 was destroyed in these assays (confirmed by densitometric scanning; not shown). Moreover, the Cut2 protein migrated as multiple isoforms, reflecting extensive phosphorylation in M phase extracts (Funabiki et al., 1997). While these isoforms could be seen readily in all extracts at time 0, the non-degraded Cut2 protein remaining in control extracts and in extracts supplemented with Plx1wt (Figure 5b, top and middle panels, time 90 min, plus Ca2+) was clearly shifted to lower migrating forms, probably due to dephosphorylation upon exit of these extracts from M phase. No degradation and no alterations in the migration of Cut2 could be observed in the presence of Plx1(N172A), indicating that the corresponding extracts were unable to degrade a substrate which, at least in yeast, is known to be degraded before the onset of anaphase. Regardless of whether or not S.pombe Cut2 mimics the fate of its (hypothetical) Xenopus homologue, these results clearly indicate that the block to proteolysis imposed by the addition of the Plx1(N172A) mutant to Xenopus egg extracts is not limited to a particular substrate. Figure 5.The destruction of both mitotic cyclins and Cut2 requires Plx1 activity. Destruction assays were performed in M phase extracts supplemented with control buffer (top panels), Plx1wt (middle panels) or Plx1(N172A) (lower panels), and a mixture of in vitro translated full-length cyclin B2 and cyclin B2ΔN7–69 (a) or S.pombe Cut2 (b) as substrates for degradation. The positions of full-length cyclin B2 (B2 wt) and cyclin B2ΔN7–69 (B2 ΔN) (a), and Cut2 (b), as detected by autoradiography, are indicated. Note the complex migration pattern of Cut2 polypeptides, which is due to extensive phosphorylation in M phase extracts. Download figure Download PowerPoint Plx1 activity is required for the degradation of c-Mos In Xenopus egg extracts, the arrest in meiotic metaphase II is imposed by an activity known as cytostatic factor (CSF), which comprises a MAP kinase cascade under the control of the proto-oncogene product c-Mos (Sagata et al., 1989) (reviewed in Sagata, 1997). In response to Ca2+, the proteolytic degradation system is activated in Xenopus egg extracts, before the inhibitory MAP kinase pathway is inactivated through degradation of c-Mos (Ferrell et al., 1991; Lorca et al., 1991; Watanabe et al., 1991). Since c-Mos itself is degraded in a ubiquitin-dependent manner by the 26S proteasome (Ishida et al., 1993), it was of interest to determine the fate of c-Mos in an extract in which the activation of the proteolytic machinery was blocked by Plx1(N172A). As shown by immunoblotting, Ca2+-dependent degradation of c-Mos could be visualized readily in extracts supplemented with control buffer or Plx1wt (Figure 6, left and center panels, lanes 2). In contrast, c-Mos was entirely stable in the extract supplemented with the Plx1(N172A) mutant (Figure 6, right panel, lane 2). Also, c-Mos was stable in all extracts in the absence of Ca2+ (Figure 6, lanes 3 and 4). Together with the results described in the previous sections, these data demonstrate that Plx1(N172A) prevents the degradation of all substrates of ubiquitin-dependent proteolysis tested. The most straightforward interpretation of these results is that Plx1 activity is required for the functional activation of the APC-dependent proteolytic machinery during M phase exit. Figure 6.c-Mos degradation is prevented by Plx1(N172A). Fresh M phase extracts were supplemented with buffer (control, left panel), Plx1wt (middle panel) or Plx1(N172A) (right panel). Samples were taken at the indicated times after Ca2+ addition (min), and their content in c-Mos analysed by immunoblotting. The position of c-Mos (arrow) as well as the molecular weight markers (in kDa) are indicated. Download figure Download PowerPoint Discussion This study reveals a novel role for Plx1 in cell cycle progression. Both a dominant-negative approach and immunodepletion–reconstitution experiments concur to indicate that Plx1 activity is required for the activation of the proteolytic degradation machinery in Xenopus egg extracts. In the absence of Plx1 activity, the Ca2+-dependent activation of the APC-mediated degradation pathway is blocked, with the result that all mitotic targets analysed, i.e. mitotic cyclins, S.pombe Cut2 and c-Mos, remain stable. As a consequence, extracts remain arrested in M phase with high MPF activity. Xenopus eggs are arrested in meiotic metaphase II, because CSF, a c-Mos-dependent MAP kinase cascade (Sagata, 1997), prevents the APC-dependent degradation pathway from being turned on (Abrieu et al., 1996). Upon fertilization, a rise in Ca2+ triggers the release from this block. Our data identify Plx1 as an important new element in the functional activation of the APC-dependent degradation pathway. Formally, active Plx1 counteracts the block imposed by the c-Mos/MAP kinase pathway. However, when interpreting our results, it is important to bear in mind that the functional activation of the proteolytic machinery precedes the inactivation of the c-Mos/MAP kinase pathway (Ferrell et al., 1991; Lorca et al., 1991; Watanabe et al., 1991). In other words, the inactivation of the c-Mos/MAP kinase pathway is the consequence rather than the cause of the Ca2+-induced release from the metaphase II block. Thus, active Plx1 almost certainly acts downstream of the c-Mos/MAP kinase pathway. Figure 7 summarizes, in highly schematic form, a tentative model for how APC-mediated degradation might be regulated in Xenopus egg extracts. This model also illustrates possible sites of action for Plx1. It might seem straightforward to argue that Plx1 could directly phosphorylate subunits of APC and thereby cause the activation of the complex, and/or be important for maintaining its activity. However, the situation may be more complicated, as our data clearly indicate a requirement for Plx1 activity after the Ca2+ signal. In fact, Plx1 is active already during M phase arrest (see Figure 1) and so is APC, at least when assayed in vitro after immunoprecipitation from M phase extracts (King et al., 1995; Peters et al., 1996). To reconcile these findings with the fact that none of the targets of APC are degraded during M phase arrest, it is tempting to postulate the existence of an inhibitor of APC (see Figure 7, and Minshull et al., 1994). This hypothetical APC inhibitor would be maintained in an active state by the c-Mos/MAP kinase pathway (CSF), but would be inactivated in response to a Ca2+ signal. According to this scheme, Plx1 activity might be required for inactivation of the inhibitor or for neutralizing its inhibitory action. Figure 7.Plx1 as a regulator of the M phase degradation machinery in Xenopus egg extracts. M phase extracts are arrested in meiotic metaphase II, due to the action of the c-Mos/MAP kinase pathway (CSF). According to the model presented here, the c-Mos/MAP kinase pathway promotes the activation of an inhibitor of APC/cyclosome function. In response to Ca2+, APC is relieved from the action of this inhibitor, allowing it to degrade its substrates. Subsequently, c-Mos is degraded and the MAP kinase pathway is turned off. As depicted, Plx1 is proposed to (i) mediate the Ca2+-dependent inactivation of the hypothetical APC inhibitor, (ii) counteract the effect of this inhibitor, (iii) maintain the activity of APC, or (iv) contribute to the functional coupling between APC and co-factors of the Fzy family. These mechanisms are not mutually exclusive, and other modes of action cannot be excluded. In particular, it would be premature to exclude regulation of the degradation machinery at the level of substra

Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.

Down syndrome (DS) is characterized by extensive phenotypic variability, with most traits occurring in only a fraction of affected individuals. Substantial gene-expression variation is present among unaffected individuals, and this variation has a strong genetic component. Since DS is caused by genomic-dosage imbalance, we hypothesize that gene-expression variation of human chromosome 21 (HSA21) genes in individuals with DS has an impact on the phenotypic variability among affected individuals. We studied gene-expression variation in 14 lymphoblastoid and 17 fibroblast cell lines from individuals with DS and an equal number of controls. Gene expression was assayed using quantitative real-time polymerase chain reaction on 100 and 106 HSA21 genes and 23 and 26 non-HSA21 genes in lymphoblastoid and fibroblast cell lines, respectively. Surprisingly, only 39% and 62% of HSA21 genes in lymphoblastoid and fibroblast cells, respectively, showed a statistically significant difference between DS and normal samples, although the average up-regulation of HSA21 genes was close to the expected 1.5-fold in both cell types. Gene-expression variation in DS and normal samples was evaluated using the Kolmogorov-Smirnov test. According to the degree of overlap in expression levels, we classified all genes into 3 groups: (A) nonoverlapping, (B) partially overlapping, and (C) extensively overlapping expression distributions between normal and DS samples. We hypothesize that, in each cell type, group A genes are the most dosage sensitive and are most likely involved in the constant DS traits, group B genes might be involved in variable DS traits, and group C genes are not dosage sensitive and are least likely to participate in DS pathological phenotypes. This study provides the first extensive data set on HSA21 gene-expression variation in DS and underscores its role in modulating the outcome of gene-dosage imbalance.

High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4⁺ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1-induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4⁺ and CD8⁺ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4⁺ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.

There is great interindividual variability in HIV-1 viral setpoint after seroconversion, some of which is known to be due to genetic differences among infected individuals. Here, our focus is on determining, genome-wide, the contribution of variable gene expression to viral control, and to relate it to genomic DNA polymorphism. RNA was extracted from purified CD4+ T-cells from 137 HIV-1 seroconverters, 16 elite controllers, and 3 healthy blood donors. Expression levels of more than 48,000 mRNA transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina). Genome-wide SNP data was generated from genomic DNA using the HumanHap550 Genotyping BeadChip (Illumina). We observed two distinct profiles with 260 genes differentially expressed depending on HIV-1 viral load. There was significant upregulation of expression of interferon stimulated genes with increasing viral load, including genes of the intrinsic antiretroviral defense. Upon successful antiretroviral treatment, the transcriptome profile of previously viremic individuals reverted to a pattern comparable to that of elite controllers and of uninfected individuals. Genome-wide evaluation of cis-acting SNPs identified genetic variants modulating expression of 190 genes. Those were compared to the genes whose expression was found associated with viral load: expression of one interferon stimulated gene, OAS1, was found to be regulated by a SNP (rs3177979, p = 4.9E-12); however, we could not detect an independent association of the SNP with viral setpoint. Thus, this study represents an attempt to integrate genome-wide SNP signals with genome-wide expression profiles in the search for biological correlates of HIV-1 control. It underscores the paradox of the association between increasing levels of viral load and greater expression of antiviral defense pathways. It also shows that elite controllers do not have a fully distinctive mRNA expression pattern in CD4+ T cells. Overall, changes in global RNA expression reflect responses to viral replication rather than a mechanism that might explain viral control.

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37-1.73]; combined P=2.58 · 10(-13)). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25-1.44; P=1.032 · 10(-14)). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16-3.66) for systolic and 1.40 (95% CI: 0.25-2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that regulate target mRNAs by binding to their 3′ untranslated regions. There is growing evidence that microRNA-155 (miR155) modulates gene expression in various cell types of the immune system and is a prominent player in the regulation of innate and adaptive immune responses. To define the role of miR155 in dendritic cells (DCs) we performed a detailed analysis of its expression and function in human and mouse DCs. A strong increase in miR155 expression was found to be a general and evolutionarily conserved feature associated with the activation of DCs by diverse maturation stimuli in all DC subtypes tested. Analysis of miR155-deficient DCs demonstrated that miR155 induction is required for efficient DC maturation and is critical for the ability of DCs to promote antigen-specific T-cell activation. Expression-profiling studies performed with miR155−/− DCs and DCs overexpressing miR155, combined with functional assays, revealed that the mRNA encoding the transcription factor c-Fos is a direct target of miR155. Finally, all of the phenotypic and functional defects exhibited by miR155−/− DCs could be reproduced by deregulated c-Fos expression. These results indicate that silencing of c-Fos expression by miR155 is a conserved process that is required for DC maturation and function.

Several million people are exposed to dioxin and dioxin-like compounds, primarily through food consumption. Skin lesions historically called "chloracne" are the most specific sign of abnormal dioxin exposure and classically used as a key marker in humans. We followed for 5 years a man who had been exposed to the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a single oral dose of 5 million-fold more than the accepted daily exposure in the general population. We adopted a molecular medicine approach, aimed at identifying appropriate therapy. Skin lesions, which progressively covered up to 40% of the body surface, were found to be hamartomas, which developed parallel to a complete and sustained involution of sebaceous glands, with concurrent transcriptomic alterations pointing to the inhibition of lipid metabolism and the involvement of bone morphogenetic proteins signaling. Hamartomas created a new compartment that concentrated TCDD up to 10-fold compared with serum and strongly expressed the TCDD-metabolizing enzyme cytochrome P450 1A1, thus representing a potentially significant source of enzymatic activity, which may add to the xenobiotic metabolism potential of the classical organs such as the liver. This historical case provides a unique set of data on the human tissue response to dioxin for the identification of new markers of exposure in human populations. The herein discovered adaptive cutaneous response to TCDD also points to the potential role of the skin in the metabolism of food xenobiotics.

Research in vitro facilitates discovery, screening and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.

Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.—Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems. FASEB J. 30, 1913–1926 (2016). www.fasebj.org

A T4-like coliphage cocktail was given with different oral doses to healthy Bangladeshi children in a placebo-controlled randomized phase I safety trial. Fecal phage detection was oral dose dependent suggesting passive gut transit of coliphages through the gut. No adverse effects of phage application were seen clinically and by clinical chemistry. Similar results were obtained for a commercial phage preparation (Coliproteus from Microgen/Russia). By 16S rRNA gene sequencing, only a low degree of fecal microbiota conservation was seen in healthy children from Bangladesh who were sampled over a time interval of 7 days suggesting a substantial temporal fluctuation of the fecal microbiota composition. Microbiota variability was not associated with the age of the children or the presence of phage in the stool. Stool microbiota composition of Bangladeshi children resembled that found in children of other regions of the world. Marked variability in fecal microbiota composition was also seen in 71 pediatric diarrhea patients receiving only oral rehydration therapy and in 38 patients receiving coliphage preparations or placebo when sampled 1.2 or 4 days apart respectively. Temporal stability of the gut microbiota should be assessed in case-control studies involving children before associating fecal microbiota composition with health or disease phenotypes.

The function of the nuclear receptor Rev-erbα (Nr1d1) in the brain is, apart from its role in the circadian clock mechanism, unknown. Therefore, we compared gene expression profiles in the brain between wild-type and Rev-erbα knock-out (KO) animals. We identified fatty acid binding protein 7 (Fabp7, Blbp) as a direct target of repression by REV-ERBα. Loss of Rev-erbα manifested in memory and mood related behavioral phenotypes and led to overexpression of Fabp7 in various brain areas including the subgranular zone (SGZ) of the hippocampus, where neuronal progenitor cells (NPCs) can initiate adult neurogenesis. We found increased proliferation of hippocampal neurons and loss of its diurnal pattern in Rev-erbα KO mice. In vitro, proliferation and migration of glioblastoma cells were affected by manipulating either Fabp7 expression or REV-ERBα activity. These results suggest an important role of Rev-erbα and Fabp7 in adult neurogenesis, which may open new avenues for treatment of gliomas as well as neurological diseases such as depression and Alzheimer.

The microbiota of breast milk from Chinese lactating mothers at different stages of lactation was examined in the framework of a Maternal Infant Nutrition Growth (MING) study investigating the dietary habits and breast milk composition in Chinese urban mothers. We used microbiota profiling based on the sequencing of fragments of 16S rRNA gene and specific qPCR for bifidobacteria, lactobacilli and total bacteria to study microbiota of the entire breast milk collected using standard protocol without aseptic cleansing (n = 60), and the microbiota of the milk collected aseptically (n = 30). We have also investigated the impact of the delivery mode and the stage of lactation on the microbiota composition. The microbiota of breast milk was dominated by streptococci and staphylococci for both collection protocols and, in the case of standard collection protocol, Acinetobacter sp. While the predominance of streptococci and staphylococci was consistently reported previously for other populations, the abundance of Acinetobacter sp. was reported only once before in a study where milk collection was done without aseptic cleansing of the breast and rejection of foremilk. Higher bacterial counts were found in the milk collected using standard protocol. Bifidobacteria and lactobacilli were present in few samples with low abundance. We observed no effect of the stage of lactation or the delivery mode on microbiota composition. Methodological and geographical differences likely explain the variability in microbiota composition reported to date.

Aging | doi:10.18632/aging.100926. Alice Pannérec, Margherita Springer, Eugenia Migliavacca, Alex Ireland, Mathew Piasecki, Sonia Karaz, Guillaume Jacot, Sylviane Métairon, Esther Danenberg, Frédéric Raymond, Patrick Descombes, Jamie S. McPhee, Jerome N. Feige

Extreme longevity is the paradigm of healthy aging as individuals who reached the extreme decades of human life avoided or largely postponed all major age-related diseases. In this study, we sequenced at high coverage (90X) the whole genome of 81 semi-supercentenarians and supercentenarians [105+/110+] (mean age: 106.6 ± 1.6) and of 36 healthy unrelated geographically matched controls (mean age 68.0 ± 5.9) recruited in Italy. The results showed that 105+/110+ are characterized by a peculiar genetic background associated with efficient DNA repair mechanisms, as evidenced by both germline data (common and rare variants) and somatic mutations patterns (lower mutation load if compared to younger healthy controls). Results were replicated in a second independent cohort of 333 Italian centenarians and 358 geographically matched controls. The genetics of 105+/110+ identified DNA repair and clonal haematopoiesis as crucial players for healthy aging and for the protection from cardiovascular events.

Abstract Coffea arabica , an allotetraploid hybrid of Coffea eugenioides and Coffea canephora , is the source of approximately 60% of coffee products worldwide, and its cultivated accessions have undergone several population bottlenecks. We present chromosome-level assemblies of a di-haploid C. arabica accession and modern representatives of its diploid progenitors, C. eugenioides and C. canephora . The three species exhibit largely conserved genome structures between diploid parents and descendant subgenomes, with no obvious global subgenome dominance. We find evidence for a founding polyploidy event 350,000–610,000 years ago, followed by several pre-domestication bottlenecks, resulting in narrow genetic variation. A split between wild accessions and cultivar progenitors occurred ~30.5 thousand years ago, followed by a period of migration between the two populations. Analysis of modern varieties, including lines historically introgressed with C. canephora , highlights their breeding histories and loci that may contribute to pathogen resistance, laying the groundwork for future genomics-based breeding of C. arabica .

ABSTRACT Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclu-sively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cdc2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

We report a comprehensive large-scale expression profiling analysis of mammalian male germ cells undergoing mitotic growth, meiosis, and gametogenesis by using high-density oligonucleotide microarrays and highly enriched cell populations. Among 11,955 rat loci investigated, 1268 were identified as differentially transcribed in germ cells at subsequent developmental stages compared with total testis, somatic Sertoli cells as well as brain and skeletal muscle controls. The loci were organized into four expression clusters that correspond to somatic, mitotic, meiotic, and postmeiotic cell types. This work provides information about expression patterns of approximately 200 genes known to be important during male germ cell development. Approximately 40 of those are included in a group of 121 transcripts for which we report germ cell expression and lack of transcription in three somatic control cell types. Moreover, we demonstrate the testicular expression and transcriptional induction in mitotic, meiotic, and/or postmeiotic germ cells of 293 as yet uncharacterized transcripts, some of which are likely to encode factors involved in spermatogenesis and fertility. This group also contains potential germ cell-specific targets for innovative contraceptives. A graphical display of the data is conveniently accessible through the GermOnline database at http://www.germonline.org.

Abstract The phenomenon of heterosis describes the increased agronomic performance of heterozygous F1 plants compared to their homozygous parental inbred plants. Heterosis is manifested during the early stages of root development in maize. The goal of this study was to identify nonadditive gene expression in primary roots of maize hybrids compared to the average expression levels of their parental inbred lines. To achieve this goal a two-step strategy was used. First, a microarray preselection of nonadditively expressed candidate genes was performed. Subsequently, gene expression levels in a subset of genes were determined via high-throughput quantitative real-time (qRT)–PCR experiments. Initial microarray experiments identified 1941 distinct microarray features that displayed nonadditive gene expression in at least 1 of the 12 analyzed hybrids compared to the midparent value of their parental inbred lines. Most nonadditively expressed genes were expressed between the parental values (>89%). Comparison of these 1941 genes with nonadditively expressed genes identified in maize shoot apical meristems via the same experimental procedure in the same genotypes revealed significantly less overlap than expected by pure chance. This finding suggests organ-specific patterns of nonadditively expressed genes. qRT–PCR analyses of 64 of the 1941 genes in four different hybrids revealed conserved patterns of nonadditively expressed genes in different hybrids. Subsequently, 22 of the 64 genes that displayed nonadditive expression in all four hybrids were analyzed in 12 hybrids that were generated from four inbred lines. Among those genes a superoxide dismutase 2 was expressed significantly above the midparent value in all 12 hybrids and might thus play a protective role in heterosis-related antioxidative defense in the primary root of maize hybrids. The findings of this study are consistent with the hypothesis that both global expression trends and the consistent differential expression of specific genes contribute to the organ-specific manifestation of heterosis.

An ADME (absorption, distribution, metabolism and excretion)-pharmacogenetics association study may identify functional variants relevant to the pharmacokinetics of lopinavir co-formulated with ritonavir (LPV/r), a first-line anti-HIV agent.An extensive search of literature and web resources helped select ADME genes and single nucleotide polymorphisms (SNPs, functional and HapMap tagging SNPs) with a proven or potentially relevant role in LPV/r pharmacokinetics. The study followed a two-stage design. Stage 1 (discovery) considered a Caucasian population (n=638) receiving LPV/r, where we selected 117 individuals with low LPV clearance (cases) and 90 individuals with high clearance (controls). Genotyping was performed by a 1536-SNP customized GoldenGate Illumina BeadArray. Stage 2 (confirmation) represented a replication study of candidate SNPs from the stage 1 in 148 individuals receiving LPV/r. The analysis led to formal population pharmacokinetic-pharmacogenetic modeling of demographic, environmental and candidate SNP effects.One thousand three hundred and eighty SNPs were successfully genotyped. Nine SNPs prioritized by the stage 1 analysis were brought to replication. Stage 2 confirmed the contribution of two functional SNPs in SLCO1B1, one functional SNP in ABCC2 and a tag SNP of the CYP3A locus in addition to body weight effect and ritonavir coadministration. According to the population pharmacokinetic-pharmacogenetic model, genetic variants explained 5% of LPV variability. Individuals homozygous rs11045819 (SLCO1B1*4) had a clearance of 12.6 l/h, compared with 5.4 l/h in the reference group, and 3.9 l/h in individuals with two or more variant alleles of rs4149056 (SLCO1B1*5), rs717620 (ABCC2) or rs6945984 (CYP3A). A subanalysis confirmed that although a significant part of the variance in LPV clearance was attributed to fluctuation in ritonavir levels, genetic variants had an additional effect on LPV clearance.The two-stage strategy successfully identified genetic variants affecting LPV/r pharmacokinetics. Such a general approach of ADME pharmacogenetics should be generalized to other drugs.

Failure of the testes to descend into the scrotum (cryptorchidism) is one of the most common birth defects in humans. In utero exposure to estrogens, such as 17beta-estradiol (E2) or the synthetic estrogen diethylstilbestrol (DES), down-regulates insulin-like 3 (Insl3) expression in embryonic Leydig cells, which in turn results in cryptorchidism in mice. To identify the molecular mechanism whereby xenoestrogens block Insl3 gene transcription, we performed a microarray analysis of wild-type or estrogen receptor (ER) alpha-mutant testes exposed in utero to pharmacological doses of E2 or DES. Six and 31 genes were respectively down-regulated and up-regulated by estrogen exposure (> or =4-fold). All six genes down-regulated by estrogen exposure, including Insl3 and the steroidogenic genes steroidogenic acute regulatory protein and cytochrome P450 17alpha-hydroxylase/17,20-lyase, were done so by an ERalpha-dependent mechanism. In contrast, up-regulation was mediated either by ERalpha for 12 genes or by an independent mechanism for the 19 remaining genes. Finally, we show that Insl3 gene expression and testicular descent were not affected by in utero exposure to E2 or DES in ERalpha mutant mice, whereas absence of ERbeta did not influence the effect of these estrogens. Collectively, these data demonstrate that xenoestrogens inhibit the endocrine functions of fetal Leydig cells through an ERalpha-dependent mechanism.

Summary The disturbed erythropoiesis in patients with refractory anaemia with ring‐sideroblasts (RARS) is characterized by intramedullary apoptosis of erythroid precursors and increased iron accumulation in mitochondria. To gain insight into these pathophysiological mechanisms we compared the gene expression profile (GEP) of erythroid precursors from RARS patients to the GEP of normal erythroid precursors. Three hundred sixty four probe sets were up‐, and 253 probe sets downregulated in RARS cells. Interestingly, Growth Differentiation factor 15 (GDF15), a cytokine from the TGFβ family, was dramatically upregulated in all RARS patients. Measurement of GDF15 in the sera from twenty RARS patients confirmed this finding by showing significantly, 7·2‐fold, increased protein levels (3254 ± 1400 ng/ml vs. 451 ± 87 ng/ml in normals). In vitro studies demonstrated erythroid‐specific production of GDF15 and dependence on erythropoietin. Induction of apoptosis by arsenic trioxide, a drug which acts via reduction of the mitochondrial membrane potential, also stimulated GDF15 production. Downregulation of endogenous GDF15 production in erythoblasts by specific siRNA led to diminished erythroid differentiation. Taken together, our findings demonstrate a new role for GDF15 in normal erythropoiesis as well as in the ineffective erythropoiesis of RARS patients.

Soy and soy-based products are widely consumed by infants and adult individuals. There has been speculation that the presence of isoflavone phytoestrogens in soybean cause adverse effects on the development and function of the male reproductive system. The purpose of this study was to examine the influence of dietary soy and phytoestrogens on testicular and reproductive functions. Male mice were fed from conception to adulthood with either a high soy-containing diet or a soy-free diet. Although adult mice fed a soy-rich diet exhibited normal male behaviour and were fertile, we observed a reduced proportion of haploid germ cells in testes correlating with a 25% decrease in epididymal sperm counts and a 21% reduction in litter size. LH and androgens levels were not affected but transcripts coding for androgen-response genes in Sertoli cells and Gapd-s, a germ cell-specific gene involved in sperm glycolysis and mobility were significantly reduced. In addition, we found that dietary soy decreased the size of the seminal vesicle but without affecting its proteolytic activity. Taken together, these studies show that long-term exposure to dietary soy and phytoestrogens may affect male reproductive function resulting in a small decrease in sperm count and fertility.

Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3'UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control.

Mitochondrial function can be influenced by mitochondrial shape and connectivity with other cellular organelles through fusion and fission processes. Disturbances in mitochondrial architecture and mitochondrial fusion-related genes are observed in situations of type 2 diabetes and obesity, leading to a highly fissioned mitochondrial network. To directly test the effect of reduced mitochondrial fusion on hepatic metabolism, we generated mice with a liver-specific deletion of the Mfn1 gene (Mfn1LKO) and monitored their energy homeostasis, mitochondrial function, and susceptibility to diet-induced insulin resistance. Livers from Mfn1LKO mice displayed a highly fragmented mitochondrial network. This was coupled to an enhanced mitochondrial respiration capacity and a preference for the use of lipids as the main energy source. Although Mfn1LKO mice are similar to control mice fed a low-fat diet, they are protected against insulin resistance induced by a high-fat diet. Importantly, Mfn1 deficiency increased complex I abundance and sensitized animals to the hypoglycemic effect of metformin. Our results suggest that targeting Mfn1 could provide novel avenues to ameliorate glucose homeostasis in obese patients and improve the effectiveness of metformin.

Abstract Thousands of genetic variants have been associated with complex traits through genome-wide association studies. However, the functional variants or mechanistic consequences remain elusive. Intermediate traits such as gene expression or protein levels are good proxies of the metabolic state of an organism. Proteome analysis especially can provide new insights into the molecular mechanisms of complex traits like obesity. The role of genetic variation in determining protein level variation has not been assessed in obesity. To address this, we design a large-scale protein quantitative trait locus (pQTL) analysis based on a set of 1129 proteins from 494 obese subjects before and after a weight loss intervention. This reveals 55 BMI-associated cis -pQTLs and trans -pQTLs at baseline and 3 trans -pQTLs after the intervention. We provide evidence for distinct genetic mechanisms regulating BMI-associated proteins before and after weight loss. Finally, by functional analysis, we identify and validate FAM46A as a trans regulator for leptin.

AMPK is a central regulator of energy homeostasis. AMPK not only elicits acute metabolic responses but also promotes metabolic reprogramming and adaptations in the long-term through regulation of specific transcription factors and coactivators. We performed a whole-genome transcriptome profiling in wild-type (WT) and AMPK-deficient mouse embryonic fibroblasts (MEFs) and primary hepatocytes that had been treated with 2 distinct classes of small-molecule AMPK activators. We identified unique compound-dependent gene expression signatures and several AMPK-regulated genes, including folliculin (Flcn), which encodes the tumor suppressor FLCN. Bioinformatics analysis highlighted the lysosomal pathway and the associated transcription factor EB (TFEB) as a key transcriptional mediator responsible for AMPK responses. AMPK-induced Flcn expression was abolished in MEFs lacking TFEB and transcription factor E3, 2 transcription factors with partially redundant function; additionally, the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. The AMPK-TFEB-FLCN axis is conserved across species; swimming exercise in WT zebrafish induced Flcn expression in muscle, which was significantly reduced in AMPK-deficient zebrafish. Mechanistically, we have found that AMPK promotes dephosphorylation and nuclear localization of TFEB independently of mammalian target of rapamycin activity. Collectively, we identified the novel AMPK-TFEB-FLCN axis, which may function as a key cascade for cellular and metabolic adaptations.-Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., Lefebvre, G., Raymond, F., Parisi, A., Civiletto, G., Gut, P., Descombes, P., Sakamoto, K. AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.

A low-calorie diet (LCD) reduces fat mass excess, improves insulin sensitivity, and alters adipose tissue (AT) gene expression, yet the relation with clinical outcomes remains unclear. We evaluated AT transcriptome alterations during an LCD and the association with weight and glycemic outcomes both at LCD termination and 6 mo after the LCD. Using RNA sequencing (RNAseq), we analyzed transcriptome changes in AT from 191 obese, nondiabetic patients within a multicenter, controlled dietary intervention. Expression changes were associated with outcomes after an 8-wk LCD (800–1000 kcal/d) and 6 mo after the LCD. Results were validated by using quantitative reverse transcriptase-polymerase chain reaction in 350 subjects from the same cohort. Statistical models were constructed to classify weight maintainers or glycemic improvers. With RNAseq analyses, we identified 1173 genes that were differentially expressed after the LCD, of which 350 and 33 were associated with changes in body mass index (BMI; in kg/m2) and Matsuda index values, respectively, whereas 29 genes were associated with both endpoints. Pathway analyses highlighted enrichment in lipid and glucose metabolism. Classification models were constructed to identify weight maintainers. A model based on clinical baseline variables could not achieve any classification (validation AUC: 0.50; 95% CI: 0.36, 0.64). However, clinical changes during the LCD yielded better performance of the model (AUC: 0.73; 95% CI: 0.60, 0.87]). Adding baseline expression to this model improved the performance significantly (AUC: 0.87; 95% CI: 0.77, 0.96; Delong's P = 0.012). Similar analyses were performed to classify subjects with good glycemic improvements. Baseline- and LCD-based clinical models yielded similar performance (best AUC: 0.73; 95% CI: 0.60, 0.86). The addition of expression changes during the LCD improved the performance substantially (AUC: 0.80; 95% CI: 0.69, 0.92; P = 0.058). This study investigated AT transcriptome alterations after an LCD in a large cohort of obese, nondiabetic patients. Gene expression combined with clinical variables enabled us to distinguish weight and glycemic responders from nonresponders. These potential biomarkers may help clinicians understand intersubject variability and better predict the success of dietary interventions. This trial was registered at clinicaltrials.gov as NCT00390637.

Summary Coffee species such as Coffea canephora P. (Robusta) and C. arabica L. (Arabica) are important cash crops in tropical regions around the world. C. arabica is an allotetraploid (2 n = 4 x = 44) originating from a hybridization event of the two diploid species C. canephora and C. eugenioides (2 n = 2 x = 22). Interestingly, these progenitor species harbour a greater level of genetic variability and are an important source of genes to broaden the narrow Arabica genetic base. Here, we describe the development, evaluation and use of a single‐nucleotide polymorphism ( SNP ) array for coffee trees. A total of 8580 unique and informative SNP s were selected from C. canephora and C. arabica sequencing data, with 40% of the SNP located in annotated genes. In particular, this array contains 227 markers associated to 149 genes and traits of agronomic importance. Among these, 7065 SNP s (~82.3%) were scorable and evenly distributed over the genome with a mean distance of 54.4 Kb between markers. With this array, we improved the Robusta high‐density genetic map by adding 1307 SNP markers, whereas 945 SNP s were found segregating in the Arabica mapping progeny. A panel of C. canephora accessions was successfully discriminated and over 70% of the SNP markers were transferable across the three species. Furthermore, the canephora‐derived subgenome of C. arabica was shown to be more closely related to C. canephora accessions from northern Uganda than to other current populations. These validated SNP markers and high‐density genetic maps will be useful to molecular genetics and for innovative approaches in coffee breeding.

The assessment of population vulnerability under climate change is crucial for planning conservation as well as for ensuring food security. Coffea canephora is, in its native habitat, an understorey tree that is mainly distributed in the lowland rainforests of tropical Africa. Also known as Robusta, its commercial value constitutes a significant revenue for many human populations in tropical countries. Comparing ecological and genomic vulnerabilities within the species' native range can provide valuable insights about habitat loss and the species' adaptive potential, allowing to identify genotypes that may act as a resource for varietal improvement. By applying species distribution models, we assessed ecological vulnerability as the decrease in climatic suitability under future climatic conditions from 492 occurrences. We then quantified genomic vulnerability (or risk of maladaptation) as the allelic composition change required to keep pace with predicted climate change. Genomic vulnerability was estimated from genomic environmental correlations throughout the native range. Suitable habitat was predicted to diminish to half its size by 2050, with populations near coastlines and around the Congo River being the most vulnerable. Whole-genome sequencing revealed 165 candidate SNPs associated with climatic adaptation in C. canephora, which were located in genes involved in plant response to biotic and abiotic stressors. Genomic vulnerability was higher for populations in West Africa and in the region at the border between DRC and Uganda. Despite an overall low correlation between genomic and ecological vulnerability at broad scale, these two components of vulnerability overlap spatially in ways that may become damaging. Genomic vulnerability was estimated to be 23% higher in populations where habitat will be lost in 2050 compared to regions where habitat will remain suitable. These results highlight how ecological and genomic vulnerabilities are relevant when planning on how to cope with climate change regarding an economically important species.

Abstract Background Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial and error approach. We report the first global scale transcriptome profiling performed at all developmental stages of SE in coffee to unravel the mechanisms that regulate cell fate and totipotency. Results RNA-seq of 48 samples (12 developmental stages × 4 biological replicates) generated 90 million high quality reads per sample, approximately 74% of which were uniquely mapped to the Arabica genome. First, the statistical analysis of transcript data clearly grouped SE developmental stages into seven important phases (Leaf, Dedifferentiation, Primary callus, Embryogenic callus, Embryogenic cell clusters, Redifferentiation and Embryo) enabling the identification of six key developmental phase switches, which are strategic for the overall biological efficiency of embryo regeneration. Differential gene expression and functional analysis showed that genes encoding transcription factors, stress-related genes, metabolism-related genes and hormone signaling-related genes were significantly enriched. Second, the standard environmental drivers used to control SE, i.e. light, growth regulators and cell density, were clearly perceived at the molecular level at different developmental stages. Third, expression profiles of auxin-related genes, transcription factor-related genes and secondary metabolism-related genes were analyzed during SE. Gene co-expression networks were also inferred. Auxin-related genes were upregulated during dedifferentiation and redifferentiation while transcription factor-related genes were switched on from the embryogenic callus and onward. Secondary metabolism-related genes were switched off during dedifferentiation and switched back on at the onset of redifferentiation. Secondary metabolites and endogenous IAA content were tightly linked with their respective gene expression. Lastly, comparing Arabica embryogenic and non-embryogenic cell transcriptomes enabled the identification of biological processes involved in the acquisition of embryogenic capacity. Conclusions The present analysis showed that transcript fingerprints are discriminating signatures of cell fate and are under the direct influence of environmental drivers. A total of 23 molecular candidates were successfully identified overall the 12 developmental stages and can be tested in many plant species to optimize SE protocols in a rational way.

Waardenburg anophthalmia syndrome, also known as microphthalmia with limb anomalies, ophthalmoacromelic syndrome, and anophthalmia-syndactyly, is a rare autosomal-recessive developmental disorder that has been mapped to 10p11.23. Here we show that this disease is heterogeneous by reporting on a consanguineous family, not linked to the 10p11.23 locus, whose two affected children have a homozygous mutation in SMOC1. Knockdown experiments of the zebrafish smoc1 revealed that smoc1 is important in eye development and that it is expressed in many organs, including brain and somites.

Methylation of cytosines ((m)C) is essential for epigenetic gene regulation in plants and mammals. Aberrant (m)C patterns are associated with heritable developmental abnormalities in plants and with cancer in mammals. We have developed a genome-wide DNA methylation profiling technology employing a novel amplification step for DNA subjected to bisulfite-mediated cytosine conversion. The methylation patterns detected are not only consistent with previous results obtained with (m)C immunoprecipitation (mCIP) techniques, but also demonstrated improved resolution and sensitivity. The technology, named BiMP (for Bisulfite Methylation Profiling), is more cost-effective than mCIP and requires as little as 100 ng of Arabidopsis DNA.

The DNA-dependent adenosine triphosphatase (ATPase) Plk1-interacting checkpoint helicase (PICH) has recently been implicated in spindle checkpoint (SAC) signaling (Baumann et al., Cell 128(1):101–114, 2007). Depletion of PICH by siRNA abolished the SAC and resulted in an apparently selective loss of Mad2 from kinetochores, suggesting a role for PICH in the regulation of the Mad1–Mad2 interaction. An apparent rescue of SAC functionality by overexpression of PICH in PICH-depleted cells initially seemed to confirm a role for PICH in the SAC. However, we have subsequently discovered that all PICH-directed siRNA oligonucleotides that abolish the SAC also reduce Mad2 mRNA and protein expression. This reduction is functionally significant, as PICH siRNA does not abolish SAC activity in a cell line that harbors a bacterial artificial chromosome driving the expression of murine Mad2. Moreover, we identified several siRNA duplexes that effectively deplete PICH but do not significantly affect SAC functionality or Mad2 abundance or localization. Finally, we discovered that the ability of overexpressed PICH to restore SAC activity in PICH-depleted cells depends on sequestration of the mitotic kinase Plk1 rather than ATPase activity of PICH, pointing to an underlying mechanism of “bypass suppression.” In support of this view, depletion or inhibition of Plk1 also rescued SAC activity in cells harboring low levels of Mad2. This observation suggests that a reduction of Plk1 activity partially compensates for reduced Mad2 levels and argues that Plk1 normally reduces the strength of SAC signaling. Collectively, our results question the role of PICH in the SAC and instead identify Mad2 as a sensitive off target for small RNA duplexes. In support of the latter conclusion, our evidence suggests that an off-target effect on Mad2 may also contribute to explain the apparent role of the Tao1 kinase in SAC signaling (Draviam et al., Nat Cell Biol 9(5):556–564, 2007).

Sinusoidal obstruction syndrome (SOS; formerly veno-occlusive disease) is a well-established complication of hematopoietic stem cell transplantation, pyrrolizidine alkaloid intoxication, and widely used chemotherapeutic agents such as oxaliplatin. It is associated with substantial morbidity and mortality. Pathogenesis of SOS in humans is poorly understood. To explore its molecular mechanisms, we used Affymetrix U133 Plus 2.0 microarrays to investigate the gene expression profile of 11 human livers with oxaliplatin-related SOS and compared it to 12 matched controls. Hierarchical clustering analysis showed that profiles from SOS and controls formed distinct clusters. To identify functional networks and gene ontologies, data were analyzed by the Ingenuity Pathway Analysis Tool. A total of 913 genes were differentially expressed in SOS: 613 being upregulated and 300 downregulated. Reverse transcriptase-PCR results showed excellent concordance with microarray data. Pathway analysis showed major gene upregulation in six pathways in SOS compared with controls: acute phase response (notably interleukin 6), coagulation system (Serpine1, THBD, and VWF), hepatic fibrosis/hepatic stellate cell activation (COL3a1, COL3a2, PDGF-A, TIMP1, and MMP2), and oxidative stress. Angiogenic factors (VEGF-C) and hypoxic factors (HIF1A) were upregulated. The most significant increase was seen in CCL20 mRNA. In conclusion, oxaliplatin-related SOS can be readily distinguished according to morphologic characteristics but also by a molecular signature. Global gene analysis provides new insights into mechanisms underlying chemotherapy-related hepatotoxicity in humans and potential targets relating to its diagnosis, prevention, and treatment. Activation of VEGF and coagulation (vWF) pathways could partially explain at a molecular level the clinical observations that bevacizumab and aspirin have a preventive effect in SOS.

Hyperhomocysteinemia is a risk factor for cognitive decline and dementia, including Alzheimer disease (AD). Homocysteine (Hcy) is a sulfur-containing amino acid and metabolite of the methionine pathway. The interrelated methionine, purine, and thymidylate cycles constitute the one-carbon metabolism that plays a critical role in the synthesis of DNA, neurotransmitters, phospholipids, and myelin. In this study, we tested the hypothesis that one-carbon metabolites beyond Hcy are relevant to cognitive function and cerebrospinal fluid (CSF) measures of AD pathology in older adults.Cross-sectional analysis was performed on matched CSF and plasma collected from 120 older community-dwelling adults with (n = 72) or without (n = 48) cognitive impairment. Liquid chromatography-mass spectrometry was performed to quantify one-carbon metabolites and their cofactors. Least absolute shrinkage and selection operator (LASSO) regression was initially applied to clinical and biomarker measures that generate the highest diagnostic accuracy of a priori-defined cognitive impairment (Clinical Dementia Rating-based) and AD pathology (i.e., CSF tau phosphorylated at threonine 181 [p-tau181]/β-Amyloid 1-42 peptide chain [Aβ1-42] >0.0779) to establish a reference benchmark. Two other LASSO-determined models were generated that included the one-carbon metabolites in CSF and then plasma. Correlations of CSF and plasma one-carbon metabolites with CSF amyloid and tau were explored. LASSO-determined models were stratified by apolipoprotein E (APOE) ε4 carrier status.The diagnostic accuracy of cognitive impairment for the reference model was 80.8% and included age, years of education, Aβ1-42, tau, and p-tau181. A model including CSF cystathionine, methionine, S-adenosyl-L-homocysteine (SAH), S-adenosylmethionine (SAM), serine, cysteine, and 5-methyltetrahydrofolate (5-MTHF) improved the diagnostic accuracy to 87.4%. A second model derived from plasma included cystathionine, glycine, methionine, SAH, SAM, serine, cysteine, and Hcy and reached a diagnostic accuracy of 87.5%. CSF SAH and 5-MTHF were associated with CSF tau and p-tau181. Plasma one-carbon metabolites were able to diagnose subjects with a positive CSF profile of AD pathology in APOE ε4 carriers.We observed significant improvements in the prediction of cognitive impairment by adding one-carbon metabolites. This is partially explained by associations with CSF tau and p-tau181, suggesting a role for one-carbon metabolism in the aggregation of tau and neuronal injury. These metabolites may be particularly critical in APOE ε4 carriers.

Mitochondrial dysfunction is linked to numerous pathological states, in particular related to metabolism, brain health and ageing. Nuclear encoded gene polymorphisms implicated in mitochondrial functions can be analyzed in the context of classical genome wide association studies. By contrast, mitochondrial DNA (mtDNA) variants are more challenging to identify and analyze for several reasons. First, contrary to the diploid nuclear genome, each cell carries several hundred copies of the circular mitochondrial genome. Mutations can therefore be present in only a subset of the mtDNA molecules, resulting in a heterogeneous pool of mtDNA, a situation referred to as heteroplasmy. Consequently, detection and quantification of variants requires extremely accurate tools, especially when this proportion is small. Additionally, the mitochondrial genome has pseudogenized into numerous copies within the nuclear genome over the course of evolution. These nuclear pseudogenes, named NUMTs, must be distinguished from genuine mtDNA sequences and excluded from the analysis.Here we describe a novel method, named MitoRS, in which the entire mitochondrial genome is amplified in a single reaction using rolling circle amplification. This approach is easier to setup and of higher throughput when compared to classical PCR amplification. Sequencing libraries are generated at high throughput exploiting a tagmentation-based method. Fine-tuned parameters are finally applied in the analysis to allow detection of variants even of low frequency heteroplasmy. The method was thoroughly benchmarked in a set of experiments designed to demonstrate its robustness, accuracy and sensitivity. The MitoRS method requires 5 ng total DNA as starting material. More than 96 samples can be processed in less than a day of laboratory work and sequenced in a single lane of an Illumina HiSeq flow cell. The lower limit for accurate quantification of single nucleotide variants has been measured at 1% frequency.The MitoRS method enables the robust, accurate, and sensitive analysis of a large number of samples. Because it is cost effective and simple to setup, we anticipate this method will promote the analysis of mtDNA variants in large cohorts, and may help assessing the impact of mtDNA heteroplasmy on metabolic health, brain function, cancer progression, or ageing.

To identify candidate genes that may be involved in motoneuron degeneration, we combined laser capture microdissection with microarray technology. Gene expression in motoneurons was analyzed during the progression of the disease in transgenic SOD1(G93A) mice that develop motoneuron loss. Three major observations were made: first, there was only a small number of genes that were differentially expressed in motoneurons at a pre-symptomatic age (27 out of 34 000 transcripts). Secondly, there is an early specific up-regulation of the gene coding for the intermediate filament vimentin that is increased even further during disease progression. Using in situ hybridization and immunohistochemical analysis, we show that vimentin expression was not only elevated in motoneurons but that the protein formed inclusions in the motoneuron cytoplasm. Thirdly, a time-course analysis of the motoneurons at a symptomatic age (90 and 120 days) showed a modest de-regulation of only a few genes associated with cell death pathways; however, a massive up-regulation of genes involved in cell growth and/or maintenance was observed. This is the first description of the gene profile of SOD1(G93A) motoneurons during disease progression and unexpectedly, no widespread induction of cell death-associated genes was detected in motoneurons of SOD1(G93A) mice.

Mutations in the transcription factor IPF1/PDX1 have been associated with type 2 diabetes. To elucidate β-cell dysfunction, PDX1 was suppressed by transduction of rat islets with an adenoviral construct encoding a dominant negative form of PDX1. After 2 days, there was a marked inhibition of insulin secretion in response to glucose, leucine, and arginine. Increasing cAMP levels with forskolin and isobutylmethylxanthine restored glucose-stimulated insulin secretion, indicating normal capacity for exocytosis. To identify molecular targets implicated in the altered metabolism secretion coupling, DNA microarray analysis was performed on PDX1-deficient and control islets. Of the 2640 detected transcripts, 70 were up-regulated and 56 were down-regulated. Transcripts were subdivided into 12 clusters; the most prevalent were associated with metabolism. Quantitative reverse transcriptase-PCR confirmed increases in succinate dehydrogenase and ATP synthase mRNAs as well as pyruvate carboxylase and the transcript for the malate shuttle. In parallel there was a 50% reduction in mRNA levels for the mitochondrially encoded <i>nd1</i> gene, a subunit of the NADH dehydrogenase comprising complex I of the mitochondrial respiratory chain. As a consequence, total cellular ATP concentration was drastically decreased by 75%, and glucose failed to augment cytosolic ATP, explaining the blunted glucose-stimulated insulin secretion. Rotenone, an inhibitor of complex I, mimicked this effect. Surprisingly, TFAM, a nuclear-encoded transcription factor important for sustaining expression of mitochondrial genes, was down-regulated in islets expressing DN79PDX1. In conclusion, loss of PDX1 function alters expression of mitochondrially encoded genes through regulation of TFAM leading to impaired insulin secretion.

Bacteria of the genus Gluconacetobacter are usually involved in the industrial production of vinegars with high acetic acid concentrations. We describe here the genome sequence of three Gluconacetobacter europaeus strains, a very common bacterial species from industrial fermentors, as well as of a Gluconacetobacter oboediens strain.

In this review, we address the genetic continuum between aging and age-related diseases, with particular attention to the ecological perspective. We describe the connections between genes that promote longevity and genes associated with age-related diseases considering tradeoff mechanisms in which the same genetic variants could have different effects according to the tissue considered and could be involved in several biological pathways. Then we describe mechanisms of antagonistic pleiotropy, focusing on the complex interplay between genetic variants and environmental changes (internal or external). We sustain the use of centenarians as “super-controls” for the study of the major age-related diseases, starting from the concept that the maximization of the phenotypic differences in the considered cohort, achieved by selecting the most divergent phenotypes, could be useful for increasing the significant differences observed in the genetic association study. We describe the potential impact of the population genetic variability in the study of human longevity and the possible contribution of the past selective pressures in shaping the current genomic background of individuals. In conclusion, we illustrate recent findings emerged from whole-genome sequencing of long-lived individuals and future perspectives for interpreting the huge amount of genetic data that will be generated in the next future.

We report streptococcal dysbiosis in acute diarrhoea irrespective of aetiology. Compared with 20 healthy local controls, 71 Bangladeshi children hospitalized with acute diarrhoea (AD) of viral, mixed viral/bacterial, bacterial and unknown aetiology showed a significantly decreased bacterial diversity with loss of pathways characteristic for the healthy distal colon microbiome (mannan degradation, methylerythritol phosphate and thiamin biosynthesis), an increased proportion of faecal streptococci belonging to the Streptococcus bovis and Streptococcus salivarius species complexes, and an increased level of E. coli-associated virulence genes. No enteropathogens could be attributed to a subgroup of patients. Elevated lytic coliphage DNA was detected in 2 out of 5 investigated enteroaggregative E. coli (EAEC)-infected patients. Streptococcal outgrowth in AD is discussed as a potential nutrient-driven consequence of glucose provided with oral rehydration solution.

Caloric restriction (CR) has been shown to prevent the onset of insulin resistance and to delay age-related physiological decline in mammalian organisms. SIRT1, a NAD(+)-dependent deacetylase enzyme, has been suggested to mediate the adaptive responses to CR, leading to the speculation that SIRT1 activation could be therapeutically used as a CR-mimetic strategy. Here, we used a mouse model of moderate SIRT1 overexpression to test whether SIRT1 gain of function could mimic or boost the metabolic benefits induced by every-other-day feeding (EODF). Our results indicate that SIRT1 transgenesis does not affect the ability of EODF to decrease adiposity and improve insulin sensitivity. Transcriptomic analyses revealed that SIRT1 transgenesis and EODF promote very distinct adaptations in individual tissues, some of which can be even be metabolically opposite, as in brown adipose tissue. Therefore, whereas SIRT1 overexpression and CR both improve glucose metabolism and insulin sensitivity, the etiologies of these benefits are largely different.

In type 1 diabetes, a renewable source of human pancreatic β cells, in particular from human induced pluripotent stem cell (hiPSC) origin, would greatly benefit cell therapy. Earlier work showed that pancreatic progenitors differentiated from human embryonic stem cells in vitro can further mature to become glucose responsive following macroencapsulation and transplantation in mice. Here we took a similar approach optimizing the generation of pancreatic progenitors from hiPSCs. This work demonstrates that hiPSCs differentiated to pancreatic endoderm in vitro can be efficiently and robustly generated under large-scale conditions. The hiPSC-derived pancreatic endoderm cells (HiPECs) can further differentiate into glucose-responsive islet-like cells following macroencapsulation and in vivo implantation. The HiPECs can protect mice from streptozotocin-induced hyperglycemia and maintain normal glucose homeostasis and equilibrated plasma glucose concentrations at levels similar to the human set point. These results further validate the potential use of hiPSC-derived islet cells for application in clinical settings.

Micronutrients are in small amounts in foods, act in concert, and require variable amounts of time to see changes in health and risk for disease. These first principles are incorporated into an intervention study designed to develop new experimental strategies for setting target recommendations for food bioactives for populations and individuals.A 6-week multivitamin/mineral intervention is conducted in 9-13 year olds. Participants (136) are (i) their own control (n-of-1); (ii) monitored for compliance; (iii) measured for 36 circulating vitamin forms, 30 clinical, anthropometric, and food intake parameters at baseline, post intervention, and following a 6-week washout; and (iv) had their ancestry accounted for as modifier of vitamin baseline or response. The same intervention is repeated the following year (135 participants). Most vitamins respond positively and many clinical parameters change in directions consistent with improved metabolic health to the intervention. Baseline levels of any metabolite predict its own response to the intervention. Elastic net penalized regression models are identified, and significantly predict response to intervention on the basis of multiple vitamin/clinical baseline measures.The study design, computational methods, and results are a step toward developing recommendations for optimizing vitamin levels and health parameters for individuals.

Abstract Background The cline of human genetic diversity observable across Europe is recapitulated at a micro-geographic scale by variation within the Italian population. Besides resulting from extensive gene flow, this might be ascribable also to local adaptations to diverse ecological contexts evolved by people who anciently spread along the Italian Peninsula. Dissecting the evolutionary history of the ancestors of present-day Italians may thus improve the understanding of demographic and biological processes that contributed to shape the gene pool of European populations. However, previous SNP array-based studies failed to investigate the full spectrum of Italian variation, generally neglecting low-frequency genetic variants and examining a limited set of small effect size alleles, which may represent important determinants of population structure and complex adaptive traits. To overcome these issues, we analyzed 38 high-coverage whole-genome sequences representative of population clusters at the opposite ends of the cline of Italian variation, along with a large panel of modern and ancient Euro-Mediterranean genomes. Results We provided evidence for the early divergence of Italian groups dating back to the Late Glacial and for Neolithic and distinct Bronze Age migrations having further differentiated their gene pools. We inferred adaptive evolution at insulin-related loci in people from Italian regions with a temperate climate, while possible adaptations to pathogens and ultraviolet radiation were observed in Mediterranean Italians. Some of these adaptive events may also have secondarily modulated population disease or longevity predisposition. Conclusions We disentangled the contribution of multiple migratory and adaptive events in shaping the heterogeneous Italian genomic background, which exemplify population dynamics and gene-environment interactions that played significant roles also in the formation of the Continental and Southern European genomic landscapes.

Abstract Caffeine is the most consumed alkaloid stimulant in the world. It is synthesized through the activity of three known N -methyltransferase proteins. Here we are reporting on the 422-Mb chromosome-level assembly of the Coffea humblotiana genome, a wild and endangered, naturally caffeine-free, species from the Comoro archipelago. We predicted 32,874 genes and anchored 88.7% of the sequence onto the 11 chromosomes. Comparative analyses with the African Robusta coffee genome ( C. canephora ) revealed an extensive genome conservation, despite an estimated 11 million years of divergence and a broad diversity of genome sizes within the Coffea genus. In this genome, the absence of caffeine is likely due to the absence of the caffeine synthase gene which converts theobromine into caffeine through an illegitimate recombination mechanism. These findings pave the way for further characterization of caffeine-free species in the Coffea genus and will guide research towards naturally-decaffeinated coffee drinks for consumers.

MicroRNAs (miRNAs) are small noncoding RNAs involved in posttranscriptional regulation. miRNAs can be secreted and found in many body fluids, and although they are particularly abundant in breastmilk, their functions remain elusive. Human milk (HM) miRNAs start to raise considerable interest, but a comprehensive understanding of the repertoire and expression profiles along lactation has not been well characterized. This study aimed to characterize the longitudinal profile of HM miRNA between the second week and third month postpartum. We used a new sensitive technology to measure HM miRNAs in a cohort of 44 French mothers [mean ± SD age: 31 ± 3.5; BMI (in kg/m2) 21.8 ± 2.3] who delivered at term and provided HM samples at 3 time points (17 ± 3 d, 60 ± 3 d, and 90 ± 3 d) during follow-up visits. We detected 685 miRNAs, of which 35 showed a high and stable expression along the lactation period analyzed. We also described for the first time a set of 11 miRNAs with a dynamic expression profile. To gain insight into the potential functional relevance of this set of miRNAs, we selected miR-3126 and miR-3184 to treat undifferentiated Caco-2 human intestinal cells and then assessed differentially expressed genes and modulation of related biological pathways. Overall, our study provides new insights into HM miRNA composition and, to our knowledge, the first description of its longitudinal dynamics in mothers who delivered at term. Our in vitro results obtained in undifferentiated Caco-2 human intestinal cells transfected with HM miRNAs also provide further support to the hypothesized mother-to-neonate signaling role of HM miRNAs. This trial was registered at clinicaltrials.gov as NCT01894893.

The efficiency and tissue-specificity of transgene expression in animals is usually subject to the position of integration into the host chromatin. We have discovered that a 2.8kbp fragment flanking the rat gene encoding the transcription factor LAP (C/EBP beta) directs position-independent, copy number-dependent expression in transgenic-mouse livers. Concomitantly, the DNAse I hypersensitivity pattern normally observed in the liver is established in the integrated transgene construct demonstrating that this region is capable of creating chromatin structures equivalent to the endogenous situation. These observations are reminiscent of the locus control regions (LCR) described for several genes. Additionally, this LAP element functions with both intron-less and intron-containing genes. The tissue specificity of this element, however, is not restricted to liver. The 2.8kbp region is capable of allowing position-independent, copy number-dependent expression in brain, kidney, heart, spleen, and lung, but in a construct-dependent manner. This is, to our knowledge, the first transcription factor gene with which a cis-linked LCR-like element has been associated.

Cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact.To further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNgamma, CCL2, CCL5, TNF, IL-1beta, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1beta, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E(2)-both involved in inflammatory response-were triggered by T cell contact and inhibited in the presence of HDL.These results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL.

Staphylococcus aureus is a versatile bacterial opportunist responsible for a wide spectrum of infections. Several genomes of this major human pathogen have been publicly available for almost 10 years, but comprehensive links between virulence or epidemicity and genome content of the bacterium are still missing. This project aims at characterizing a set of small transcribed molecules currently ignored by standard automated annotation algorithms. We assessed the NanoString's nCounter Analysis System for its ability to determine the orientation and quantity of the expressed small RNA (sRNA) molecules that we recently detected with RNA-Sequencing (RNA-Seq). The expression of approximately seventy small RNAs, including sRNA localized in pathogenic islands, was assessed at 5 time points during growth of the bacterium in a rich medium. In addition, two extraction strategies were tested: RNA was either purified on columns or simply prepared from crude lysates in the presence of a chaotropic buffer. The nCounter System allowed us to perform these 64 measurements in a single experiment, without any enzymatic reaction, thus avoiding well-known technical biases. We evaluated the reproducibility and reliability of the nCounter compared to quantitative RT-PCR (RT-qPCR). By using two different designs for the two coding strands, we were able to identify the coding strand of 61 small RNA molecules (95%). Overall, the nCounter System provided an identification of the coding strand in perfect concordance with RNA-Seq data. In addition, expression results were also comparable to those obtained with RT-qPCR. The sensitivity and minimal requirements of the nCounter system open new possibilities in the field of gene expression analysis, for assessing bacterial transcript profiles from complex media (i.e. during host–pathogen interactions) or when starting from poorly purified RNA or even directly from lysed infected tissues.

Centromeric regions of plants are generally composed of large array of satellites from a specific lineage of Gypsy LTR-retrotransposons, called Centromeric Retrotransposons. Repeated sequences interact with a specific H3 histone, playing a crucial function on kinetochore formation. To study the structure and composition of centromeric regions in the genus Coffea, we annotated and classified Centromeric Retrotransposons sequences from the allotetraploid C. arabica genome and its two diploid ancestors: Coffea canephora and C. eugenioides. Ten distinct CRC (Centromeric Retrotransposons in Coffea) families were found. The sequence mapping and FISH experiments of CRC Reverse Transcriptase domains in C. canephora, C. eugenioides and C. arabica clearly indicate a strong and specific targeting mainly onto proximal chromosome regions, which can be associated also with heterochromatin. PacBio genome sequence analyses of putative centromeric regions on C. arabica and C. canephora chromosomes showed an exceptional density of one family of CRC elements, and the complete absence of satellite arrays, contrasting with usual structure of plant centromeres. Altogether, our data suggest a specific centromere organization in Coffea, contrasting with other plant genomes.

Hundreds of genetic variants have been associated with Body Mass Index (BMI) through genome-wide association studies (GWAS) using observational cohorts. However, the genetic contribution to efficient weight loss in response to dietary intervention remains unknown. We perform a GWAS in two large low-caloric diet intervention cohorts of obese participants. Two loci close to NKX6.3/MIR486 and RBSG4 are identified in the Canadian discovery cohort (n = 1166) and replicated in the DiOGenes cohort (n = 789). Modulation of HGTX (NKX6.3 ortholog) levels in Drosophila melanogaster leads to significantly altered triglyceride levels. Additional tissue-specific experiments demonstrate an action through the oenocytes, fly hepatocyte-like cells that regulate lipid metabolism. Our results identify genetic variants associated with the efficacy of weight loss in obese subjects and identify a role for NKX6.3 in lipid metabolism, and thereby possibly weight control.

ABSTRACT Nek2 is a mammalian cell cycle-regulated serine/threonine kinase that belongs to the family of proteins related to NIMA of Aspergillus nidulans. Functional studies in diverse species have implicated NIMA-related kinases in G2/M progression, chromatin condensation and centrosome regulation. To directly address the requirements for vertebrate Nek2 kinases in these cell cycle processes, we have turned to the biochemically-tractable system provided by Xenopus laevis egg extracts. Following isolation of a Xenopus homologue of Nek2, called X-Nek2B, we found that X-Nek2B abundance and activity remained constant through the first mitotic cycle implying a fundamental difference in Nek2 regulation between embryonic and somatic cell cycles. Removal of X-Nek2B from extracts did not disturb either entry into mitosis or the accompanying condensation of chromosomes providing no support for a requirement for Nek2 in these processes at least in embryonic cells. In contrast, X-Nek2B localized to centrosomes of adult Xenopus cells and was rapidly recruited to the basal body of Xenopus sperm following incubation in egg extracts. Recruitment led to phosphorylation of the X-Nek2B kinase. Most importantly, depletion of X-Nek2B from extracts significantly delayed both the assembly of microtubule asters and the recruitment of γ-tubulin to the basal body. Hence, these studies demonstrate that X-Nek2B is required for efficient assembly of a functional zygotic centrosome and highlight the possibility of multiple roles for vertebrate Nek2 kinases in the centrosome cycle.

Peroxisome proliferator-activated receptor γ (PPARγ) is an essential regulator of adipocyte differentiation, maintenance, and survival. Deregulations of its functions are associated with metabolic diseases. We show here that deletion of one PPARγ allele not only affected lipid storage but, more surprisingly, also the expression of genes involved in glucose uptake and utilization, the pentose phosphate pathway, fatty acid synthesis, lipolysis, and glycerol export as well as in IR/IGF-1 signaling. These deregulations led to reduced circulating adiponectin levels and an energy crisis in the WAT, reflected in a decrease to nearly half of its intracellular ATP content. In addition, there was a decrease in the metabolic rate and physical activity of the PPARγ+/- mice, which was abolished by thiazolidinedione treatment, thereby linking regulation of the metabolic rate and physical activity to PPARγ. It is likely that the PPARγ+/- phenotype was due to the observed WAT dysfunction, since the gene expression profiles associated with metabolic pathways were not affected either in the liver or the skeletal muscle. These findings highlight novel roles of PPARγ in the adipose tissue and underscore the multifaceted action of this receptor in the functional fine tuning of a tissue that is crucial for maintaining the organism in good health. Peroxisome proliferator-activated receptor γ (PPARγ) is an essential regulator of adipocyte differentiation, maintenance, and survival. Deregulations of its functions are associated with metabolic diseases. We show here that deletion of one PPARγ allele not only affected lipid storage but, more surprisingly, also the expression of genes involved in glucose uptake and utilization, the pentose phosphate pathway, fatty acid synthesis, lipolysis, and glycerol export as well as in IR/IGF-1 signaling. These deregulations led to reduced circulating adiponectin levels and an energy crisis in the WAT, reflected in a decrease to nearly half of its intracellular ATP content. In addition, there was a decrease in the metabolic rate and physical activity of the PPARγ+/- mice, which was abolished by thiazolidinedione treatment, thereby linking regulation of the metabolic rate and physical activity to PPARγ. It is likely that the PPARγ+/- phenotype was due to the observed WAT dysfunction, since the gene expression profiles associated with metabolic pathways were not affected either in the liver or the skeletal muscle. These findings highlight novel roles of PPARγ in the adipose tissue and underscore the multifaceted action of this receptor in the functional fine tuning of a tissue that is crucial for maintaining the organism in good health. White adipose tissue (WAT) 2The abbreviations used are: WAT, white adipose tissue; FFA, free fatty acid; HFD, high fat diet; IGF-1, insulin growth factor 1; WT, wild type; PPARγ, peroxisome proliferator-activated receptor γ; RER, respiratory exchange ratio; SD, standard diet; TG, triglyceride(s); qRT-PCR, quantitative real-time PCR. 2The abbreviations used are: WAT, white adipose tissue; FFA, free fatty acid; HFD, high fat diet; IGF-1, insulin growth factor 1; WT, wild type; PPARγ, peroxisome proliferator-activated receptor γ; RER, respiratory exchange ratio; SD, standard diet; TG, triglyceride(s); qRT-PCR, quantitative real-time PCR. plays a dual role in regulating energy homeostasis (1Cinti S. Prostaglandins Leukot. Essent. Fatty Acids. 2005; 73: 9-15Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). 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In humans, heterozygous PPARγ mutations are responsible for partial lipodystrophy, severe insulin resistance, steatosis, and hypertension (17Agarwal A.K. Garg A. J. Clin. Endocrinol. Metab. 2002; 87: 408-411Crossref PubMed Scopus (245) Google Scholar, 18Agostini M. Gurnell M. Savage D.B. Wood E.M. Smith A.G. Rajanayagam O. Garnes K.T. Levinson S.H. Xu H.E. Schwabe J.W. Willson T.M. O'Rahilly S. Chatterjee V.K. Endocrinology. 2004; 145: 1527-1538Crossref PubMed Scopus (54) Google Scholar, 19Clement K. Hercberg S. Passinge B. Galan P. Varroud-Vial M. Shuldiner A.R. Beamer B.A. Charpentier G. Guy-Grand B. Froguel P. Vaisse C. Int. J. Obes. Relat. Metab. Disord. 2000; 24: 391-393Crossref PubMed Scopus (128) Google Scholar, 20Hegele R.A. Anderson C.M. Wang J. Jones D.C. Cao H. Genome Res. 2000; 10: 652-658Crossref PubMed Scopus (90) Google Scholar, 21Hegele R.A. Cao H. Frankowski C. Mathews S.T. Leff T. Diabetes. 2002; 51: 3586-3590Crossref PubMed Scopus (225) Google Scholar). The activation of PPARγ improves insulin sensitivity in both humans and mice. Agonists of PPARγ, such as the thiazolidinedione Pioglitazone, are used clinically and are effective in reducing hyperglycemia, hyperinsulinemia, and hyperlipidemia in patients suffering from type 2 diabetes (22Miyazaki Y. Mahankali A. Matsuda M. Mahankali S. Hardies J. Cusi K. Mandarino L.J. DeFronzo R.A. J. Clin. Endocrinol. Metab. 2002; 87: 2784-2791Crossref PubMed Scopus (532) Google Scholar, 23Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3443) Google Scholar, 24Boden G. Homko C. Mozzoli M. Showe L.C. Nichols C. Cheung P. Diabetes. 2005; 54: 880-885Crossref PubMed Scopus (97) Google Scholar). Together, these facts underline the functions of PPARγ in adipocyte differentiation and survival and underscore its role in WAT integrity and whole body homeostasis.Although the homozygous deletion of PPARγ in a mouse model was shown to be embryonic lethal, the survival of PPARγ-/- mice by inactivation of PPARγ in all tissues except the trophoblasts was successful. These animals suffered from lipodystrophy, insulin resistance, and hypotension (25Duan S.Z. Ivashchenko C.Y. Whitesall S.E. D'Alecy L.G. Duquaine D.C. Brosius F.C. Gonzalez F.J. Vinson C. Pierre M.A. Milstone D.S. Mortensen R.M. J. Clin. Invest. 2007; 117: 812-822Crossref PubMed Scopus (138) Google Scholar). However, deletion of only one PPARγ allele had some intriguing effects (15Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar, 26Miles P.D. Barak Y. He W. Evans R.M. Olefsky J.M. J. Clin. Invest. 2000; 105: 287-292Crossref PubMed Scopus (373) Google Scholar). In fact, PPARγ+/- mice were resistant to obesity induced by a high fat diet (HFD) and, under these conditions, remained more sensitive to insulin then their WT counterparts (27Yamauchi T. Kamon J. Waki H. Murakami K. Motojima K. Komeda K. Ide T. Kubota N. Terauchi Y. Tobe K. Miki H. Tsuchida A. Akanuma Y. Nagai R. Kimura S. Kadowaki T. J. Biol. Chem. 2001; 276: 41245-41254Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar). Decreased PPARγ activity under HFD conditions had a positive outcome on the development of obesity and diabetes. Based on these observations, a novel approach in type 2 diabetes therapy would include the use of PPARγ antagonists, potentially with fewer side effects compared with the present day synthetic agonists (thiazolidinediones).Taking advantage of our PPARγ+/- mouse model, we aimed at understanding how deletion of one allele of PPARγ, which significantly reduces the activity of the receptor via a gene dosage effect, would affect WAT function and whole body metabolism, when the mice are fed with a standard diet (SD), a condition which does not exacerbate the lipid storage function of the WAT. The results reported herein show that deletion of one PPARγ allele affects specifically the expression of genes associated with metabolic pathways in the WAT. In addition to genes involved in lipid storage, genes involved in glycolysis, de novo fatty acid synthesis, and lipolysis were also down-regulated in the PPARγ heterozygous mice, creating a strong energy deficit in these animals. These defects in WAT functions correlated with a lowering of the metabolic rate of the whole body and were accompanied by a reduction in physical activity. These results cast doubt on a potential long term use of PPARγ antagonists for the treatment of type 2 diabetes.EXPERIMENTAL PROCEDURESIn Vivo Animal Study—WT and PPARγ+/- male mice, of a mixed background Sv129/C56BL/6, were maintained at 23 °C on a 12-h light-dark cycle. The animals studied were between 10 and 12 weeks of age. They had free access to water and to an SD, except during fasting, when they had free access to water only, food being withdrawn for 24 h. In some experiments, 5–6-week-old animals were fed with an SD containing 0.004% of Pioglitazone (w/w) for 5 weeks. The Pioglitazone treatment protocol was adapted from Ref. 15Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar, a study that involved PPARγ+/- animals too. Pioglitazone was kindly provided by Takeda Chemical Industries (Switzerland). The standard food pellets containing the Pioglitazone as well as the control pellets were produced by Provimi-Kliba (Switzerland). For analysis, the animals were killed in the morning between 9 and 11 a.m. by cervical dislocation, and tissues were rapidly frozen in liquid nitrogen. The animal experimentation protocols were approved by the Commission de Surveillance de l'Expérimention Animale of the Canton de Vaud (Switzerland).RNA Preparation—The RNA from epidydymal WAT, gastrocnemius skeletal muscle, and liver was extracted from the frozen tissues using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. The RNA for microarray analyses was further purified using Qiagen RNeasy columns (Qiagen). The RNA quality was assessed by capillary electrophoresis on a 2100 Bioanalyzer (Agilent Technologies).Microarray Experiment and Data Processing—To minimize interindividual variation due to the mixed background of the mouse strain, each PPARγ+/- animal had a WT counterpart coming from the same litter.Three independent sets of total RNA samples (three WT and three PPARγ+/- animals) from epidydymal WAT and gastrocnemius skeletal muscle were isolated. cRNA was synthesized from 5 μg of total RNA, according to Ref. 71. After purification using a Qiagen RNeasy column, aliquots of 20 μg of cRNA were fragmented. Each fragmented cRNA (15 μg) was then hybridized to an Affymetrix “Mouse Genome 430 2.0 Array” Gene-Chip microarray. Hybridization, washing, and scanning were according to Affymetrix instructions.Data from the scanned chips were analyzed using the Affymetrix MAS 5.0 software (28Hubbell E. Liu W.M. Mei R. Bioinformatics. 2002; 18: 1585-1592Crossref PubMed Scopus (494) Google Scholar, 29Liu W.M. Mei R. Di X. Ryder T.B. Hubbell E. Dee S. Webster T.A. Harrington C.A. Ho M.H. Baid J. Smeekens S.P. Bioinformatics. 2002; 18: 1593-1599Crossref PubMed Scopus (358) Google Scholar). To identify differentially expressed transcripts, pairwise comparison analyses were carried out. Each experimental sample was compared with each reference sample, resulting in nine pairwise comparisons. Transcripts were considered to be differentially expressed if their levels changed in the same direction in seven of nine comparisons. Further data filtering and analyses were performed with the Genespring (Agilent) and the Ingenuity Pathway Analysis 4.0 software.Quantitative RT-PCR—Single-stranded cDNA templates for quantitative real time (qRT)-PCR analysis were synthesized using Superscript II reverse transcriptase and random priming, starting from the same RNAs used for the microarray analysis, and from additional independent experiments as described above. Amplicons were designed using the Primer Express software (Applied Biosystems), and their sequences were checked by BLAST against the mouse genome to ensure that they were specific for the gene being assayed. The efficiency of each primer pair was tested in a cDNA dilution series. The list of primers is available on demand.Real time PCR was carried out in optical 384-well plates and labeled by using the SYBR green master mix (Applied Biosystems), and the fluorescence was quantified with a 7900HT SDS system (Applied Biosystems). The relative expression level of target genes was normalized according to geNorm, using β-actin, tubulin α2, and hypoxanthine guanine phosphoribosyl-transferase 1 as references to determine the normalization factor (30Vandesompele J. De Preter K. Pattyn F. Poppe B. Van Roy N. De Paepe A. Speleman F. Genome Biol. 2002; 3research0034.1-research0034.11Crossref Google Scholar). Fold changes were calculated from the ratio of means of the normalized quantities and their statistical significance was determined by a paired Student's t test.ATP Level Measurements—Frozen WAT homogenate was transferred into a plastic tube containing 6% HClO4. Following centrifugation, the supernatant was recovered and neutralized with 5.5 m KOH. The ATP concentration was measured with an ATP determination kit, a time-stable assay from Biaffin GmbH&Co KG (Germany). The kit allows quantitative determination of small amounts of ATP by a bioluminescence assay involving the oxidation of the firefly luciferase depending on the ATP present in the extracts. The ATP concentration was derived according to the manufacturer's instructions.Glycerol Level Measurements—The glycerol content was measured with a glycerol measuring kit (Randox). Briefly, the glycerol present in the samples was converted into a colored product measured at a wavelength of 520 nm. The glycerol concentration was then determined according to the manufacturer's instructions.Metabolic Measurements—Metabolic cage studies were conducted in a comprehensive laboratory animal monitoring System (8-chamber CLAMS system; Columbus Instruments, Columbus, OH). The mice were adapted to powdered food for 24 h before they were introduced into the metabolic cages, where a 48-h acclimation preceded the 24-h recording time. Information was collected on the metabolic activity, food intake, water drinking, and physical activity.Blood was collected from the orbital sinus between 9:00 and 11:00 a.m., using heparinized microcapillary tubes and immediately centrifuged. The serum fraction was frozen immediately. Depending on the experiment, the animals were either normally fed or fasted for 24 h.The plasma concentrations of TG, free fatty acids (FFAs), glycerol, and ketone bodies were measured at the Mouse Clinic Institute (ICS; Strasbourg, France) on a Olympus AU-400 automated laboratory work station (Olympus-SA France) using commercial reagents (Olympus Diagnostica GmbH, Lismeehan, Ireland).The plasma leptin and adiponectin concentrations were measured using the mouse leptin enzyme-linked immunosorbent assay kit and the mouse adiponectin enzyme-linked immunosorbent assay (Linco Reserach).The plasma glucose levels were measured with an Accu-Chek Sensor glucometer (Roche Applied Science), and the plasma insulin concentrations were measured with an Ultra mouse insulin enzyme-linked immunosorbent assay kit (Mercodia SA).RESULTSDecreased Metabolic Rate in PPARγ+/-Mice—In PPARγ+/- animals, PPARγ mRNA and protein (PPARγ1 and PPARγ2) levels were reduced by half compared with those of WT mice (31Rieusset J. Seydoux J. Anghel S.I. Escher P. Michalik L. Soon Tan N. Metzger D. Chambon P. Wahli W. Desvergne B. Mol. Endocrinol. 2004; 18: 2363-2377Crossref PubMed Scopus (30) Google Scholar). This prompted us to explore the impact of this reduced PPARγ expression on whole body metabolism in the absence of any excess energy challenge, as is usually done with HFD feeding in assessing the role of PPARγ in lipid storage. Instead, the PPARγ+/- mice were fed with an SD. Metabolic parameters of the PPARγ+/- mice and their WT littermates were determined using metabolic cages. As expected, both mutated and WT animals consumed more O2 and produced more CO2 during the dark cycle, when they are generally more active (Fig. 1A, left). Although the PPARγ+/- mice had a similar weight (Table S1) and ate an equal amount of food (data not shown), they consumed less oxygen and produced less CO2 during both the light and dark cycles when compared with their WT counterparts, a difference reflected in a decrease of 14% in the metabolic rate (heat production) of PPARγ+/- animals (Fig. 1A). This effect was clearly PPARγ-dependent, since a 5-week treatment with SD containing the PPARγ agonist Pioglitazone, at 0.004%, alleviated the metabolic rate difference between the two genotypes (Fig. 1A, right). Moreover, there was a trend, not statistically significant, for increased O2 consumption, CO2 production, and a higher metabolic rate in Pioglitazone-treated PPARγ+/- mice, whereas such a tendency was not observed in WT animals.FIGURE 1Metabolic rate, fuel consumption, and total physical activity in PPARγ+/- and control mice.A, oxygen (O2) consumption, carbon dioxide (CO2) production, and metabolic rate in the PPARγ+/- and control mice. O2 and CO2 were measured by indirect calorimetry and expressed as average VO2 and VCO2/kg of body weight/h during a 24-h monitoring session (light/dark). Metabolic rate (heat) is calculated from the oxygen production and the respiratory exchange ratio (RER) and is expressed as average kcal/h/kg of body weight during a 24-h monitoring session (light/dark). B, the fuel consumption (RER) is the ratio of CO2 produced to the amount of O2 consumed and serves as a guide of the fuel type consumption (carbohydrate (RER = 1.0) or fat (RER = 0.7)). C, the total physical activity was measured as the horizontal and rearing movements during the 24-h monitoring period (total of light and dark movements). The activity is expressed as the average number of times a mouse crosses both the x and y axes at least twice. n = 12 (CTL experiments); n = 5 (Pioglitazone experiments). Values are expressed as mean ± S.E.; *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Deletion of One PPARγ Allele Does Not Affect the Carbohydrate to Lipid Ratio in Metabolic Fuel Utilization—To determine whether the decrease in the metabolic rate of PPARγ+/- animals was associated with alterations in the use of carbohydrates versus lipids as fuel molecules, we calculated the respiratory exchange ratio (RER). The RER (equal to VO2/VCO2) indicates whether lipids (RER = 0.7) or carbohydrates (RER = 1.0) are being oxidized to produce energy. Both genotypes consumed carbohydrates as the main energy source (Fig. 1B, left). This result disagrees with the notion of a metabolic compensation through increased fat oxidation in PPARγ+/- mice (9Koutnikova H. Cock T.A. Watanabe M. Houten S.M. Champy M.F. Dierich A. Auwerx J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14457-14462Crossref PubMed Scopus (159) Google Scholar). Moreover, the Pioglitazone treatment had no significant effect on the choice of fuel type (Fig. 1B, right).Decreased Physical Activity in PPARγ+/-Mice—Since a decreased metabolic rate in the mutated animals may correlate with a change in behavior, we measured their physical activity (horizontal and rearing movements). Interestingly, the PPARγ+/- mice presented a 23% decrease in total activity (Fig. 1C, left). This phenotype correlated with a strong decrease in the plasma adiponectin concentration in PPARγ+/- mice, whereas the leptin level remained unchanged (Figs. 2A and S1). This observation is in agreement with the reduced spontaneous motor activity of transgenic mice overexpressing an antisense adiponectin RNA, resulting in decreased circulating adiponectin levels (32Saito K. Arata S. Hosono T. Sano Y. Takahashi K. Choi-Miura N.H. Nakano Y. Tobe T. Tomita M. Biochim. Biophys. Acta. 2006; 1761: 709-716Crossref PubMed Scopus (30) Google Scholar). The Pioglitazone treatment corrected this decrease in physical activity, suggesting an implication of PPARγ (Fig. 1C, right). In brief, reduced PPARγ levels decreased the metabolic rate and the physical activity of mice without changing their fuel preference.FIGURE 2Plasma profile of PPARγ+/- and control mice.A, plasma adiponectin concentrations; B, plasma insulin concentrations; C, plasma glucose concentrations; D, FFA plasma levels; E, plasma glycerol concentrations; F, plasma ketone body concentrations; G, plasma TG concentrations. A, fed WT, n = 11; fed PPARγ+/-, n = 12. B–G, fed WT, n = 7; fasted WT, n = 11; fed PPARγ+/-, n = 6; fasted PPARγ+/-, n = 6. Values are expressed as mean ± S.E.; *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PPARγ+/-and WT Mice Have Similar Plasma Insulin and Glucose Profiles—Since a decreased metabolic rate might impact on glucose and lipid homeostasis, we analyzed the plasma profile of the WT and PPARγ+/- mice. The plasma insulin concentration was normal in unchallenged animals and was decreased after a 24-h fast as expected, but no difference was observed between WT and mutant mice (Fig. 2B). Moreover, the glycemia was also normal in PPARγ+/- mice, which however had a significantly attenuated response to fasting (Fig. 2C). In fact, the fasting glycemia was higher in the PPARγ+/- mice compared with that of the WT animals. Fasting for 24 h decreased the glucose level by 45% in WT mice, whereas it was decreased by only 30% in the PPARγ+/- animals. Thus, after fasting, PPARγ+/- mice presented a less pronounced hypoglycemia.The Plasma Lipid Profile of PPARγ+/-Mice Reveals an Alteration in Lipolytic Activity—In fed conditions, the plasma FFA concentrations were normal, and no deregulation was observed in PPARγ+/- mice (Fig. 2D). WT animals responded normally to fasting by liberating FFAs from the WAT into the circulation, thus increasing the plasma FFA concentration. Remarkably, no significant increase was observed in the PPARγ+/- mice, suggesting a deregulation of the lipolytic activity of the PPARγ+/- WAT. This defect was confirmed by measuring the circulating glycerol concentration. As for the FFAs, there was no difference in the fed glycerol concentration between WT and PPARγ+/- mice (Fig. 2E). However, the PPARγ+/- mice responded less well to fasting, since they increased their plasma glycerol concentration by only 32%, compared with the 62% monitored in WT mice. Given that the fasting glycerol and FFA concentrations are indicators of the lipolytic activity in the WAT, we concluded that PPARγ+/- mice might have a decreased lipolytic activity. This alteration should also be detectable in the WAT itself, in which the total glycerol (glycerol + glycerol-3-P) originates from glycolysis, glyceroneogenesis, and lipolysis. There was a 23% decrease in total glycerol content of the PPARγ+/- WAT, suggesting that at least one of the three above functions or all of them were impaired (Fig. 3).FIGURE 3Glycerol content in white adipose tissue of fed PPARγ+/- and WT mice. WT, n = 11; PPARγ+/-, n = 12. Values are expressed as mean ± S.E.; *, p ≤ 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Reduced circulating FFA levels should have consequences for ketone body synthesis in the liver, which depends on FFA availability. We measured the ketone body concentrations after fasting in both WT and PPARγ+/- mice (Fig. 2F). PPARγ+/- mice were less efficient in producing ketone bodies, since their plasma concentration of this peripheral organ fuel was 33% lower than in WT animals. Thus, this decreased supply in ketone bodies might reflect the reduced availability of FFAs in PPARγ+/- mice. The TG concentrations were increased by 38% after fasting in WT mice, which reflects the recycling to TG-very low density lipoprotein by the liver of a portion of the FFA liberated by the WAT during fasting (Fig. 2G). In agreement with the observations reported above, the plasma TG concentration was not increased in PPARγ+/- mice, in contrast to that measured in WT animals. The reason why fed PPARγ+/- animals also presented reduced ketone body levels remains to be elucidated (Fig. 2F).Based on the results described so far, we hypothesized that the PPARγ+/- mice decreased their metabolic rate and their physical activity to adapt to a diminished energy supply. Three organs, the liver, skeletal muscle, and WAT, are primarily involved in energy supply and consumption. Deletion of one PPARγ allele might have affected the expression pattern of PPARγ target genes. This possibility was tested by assessing the expression of genes involved in metabolic pathways of the three key organs mentioned above by microarray analysis (WAT and skeletal muscle) and/or qRT-PCR (WAT and liver). The expression of genes not represented in the microarray was analyzed by qRT-PCR.The Expression of Metabolic Genes Is Not Affected in the Liver of PPARγ+/-Mice—The liver is one of the major organs responsible for whole body energy balance. PPARγ is expressed at low levels in the liver under normal conditions but is increased in steatosis induced by HFD or other pathophysiological conditions (11Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2707) Google Scholar, 12Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3091) Google Scholar, 13Chawla A. Schwarz E.J

Abstract In the natural environment, cadmium is often found as a trace contaminant. Due to the complexity of Cd speciation and the heterogeneity of natural systems and processes, it is often difficult to determine clear relationships between analytical measurements of Cd and its induced biological response. Measurements of gene induction can be used to identify molecular mechanisms underlying toxicity and to quantify sublethal responses to trace contaminants. In the present paper, genes that could be involved in the tolerance of Cd to green algae were examined using two global transcriptome profiling strategies. Microarray and differential display techniques were used for a global transcriptome analysis of Chlamydomonas reinhardtii exposed to micromolar and lower Cd 2+ concentrations for a short period (2 h). Real‐time quantitative polymerase chain reaction analysis confirmed that a small set of 10 genes was differentially expressed in response to trace Cd 2+ exposures ranging from 7.8 nM to 9.0 μM. Since induction was only observed for a few genes, none of which are known to function in a general stress response, it was likely the result of relevant responses to Cd exposure. The identified genes are discussed with respect to their possible involvement in Cd tolerance and to their future use as biomarkers for monitoring Cd bioavailability in natural soils and waters.

Usually the genetics of human longevity is restricted to the nuclear genome (nDNA). However it is well known that the nDNA interacts with a physically and functionally separated genome, the mitochondrial DNA (mtDNA) that, even if limited in length and number of genes encoded, plays a major role in the ageing process. The complex interplay between nDNA/mtDNA and the environment is most likely involved in phenomena such as ageing and longevity. To this scenario we have to add another level of complexity represented by the microbiota, that is, the whole set of bacteria present in the different part of our body with their whole set of genes. In particular, several studies investigated the role of gut microbiota (GM) modifications in ageing and longevity and an age-related GM signature was found. In this view, human being must be considered as "metaorganism" and a more holistic approach is necessary to grasp the complex dynamics of the interaction between the environment and nDNA-mtDNA-GM of the host during ageing. In this review, the relationship between the three genetics and human longevity is addressed to point out that a comprehensive view will allow the researchers to properly address the complex interactions that occur during human lifespan.

Constitutional thinness (CT) is a state of low but stable body weight (BMI ≤18 kg/m2). CT subjects have normal-range hormonal profiles and food intake but exhibit resistance to weight gain despite living in the modern world–s obesogenic environment. The goal of this study is to identify molecular mechanisms underlying this protective phenotype against weight gain. We conducted a clinical overfeeding study on 30 CT subjects and 30 controls (BMI 20–25 kg/m2) matched for age and sex. We performed clinical and integrative molecular and transcriptomic analyses on white adipose and muscle tissues. Our results demonstrate that adipocytes were markedly smaller in CT individuals (mean ± SEM: 2174 ± 142 μm 2) compared with controls (3586 ± 216 μm2) (P < 0.01). The mitochondrial respiratory capacity was higher in CT adipose tissue, particularly at the level of complex II of the electron transport chain (2.2-fold increase; P < 0.01). This higher activity was paralleled by an increase in mitochondrial number (CT compared with control: 784 ± 27 compared with 675 ± 30 mitochondrial DNA molecules per cell; P < 0.05). No evidence for uncoupled respiration or “browning” of the white adipose tissue was found. In accordance with the mitochondrial differences, CT subjects had a distinct adipose transcriptomic profile [62 differentially expressed genes (false discovery rate of 0.1 and log fold change >0.75)], with many differentially expressed genes associating with positive metabolic outcomes. Pathway analyses revealed an increase in fatty acid oxidation ( P = 3 × 10−04) but also triglyceride biosynthesis (P = 3.6 × 10−04). No differential response to the overfeeding was observed in the 2 groups. The distinct molecular signature of the adipose tissue in CT individuals suggests the presence of augm ented futile lipid cycling, rather than mitochondrial uncoupling, as a way to increase energy expenditure in CT individuals. We propose that increased mitochondrial function in adipose tissue is an important mediator in sustaining the low body weight in CT individuals. This knowledge could ultimately allow more targeted approaches for weight management treatment strategies. This trial was registered at clinicaltrials.gov as NCT02004821.

The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning.Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and β subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a β3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function.Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and β2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and β2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice.These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for β3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.

Mitochondrial dysfunction induces a strong adaptive retrograde signaling response; however, many of the downstream effectors of this response remain to be discovered. Here, we studied the shared transcriptional responses to three different mitochondrial respiratory chain inhibitors in human primary skin fibroblasts using QuantSeq 3'-RNA-sequencing. We found that genes involved in the mevalonate pathway were concurrently downregulated, irrespective of the respiratory chain complex affected. Targeted metabolomics demonstrated that impaired mitochondrial respiration at any of the three affected complexes also had functional consequences on the mevalonate pathway, reducing levels of cholesterol precursor metabolites. A deeper study of complex I inhibition showed a reduced activity of endoplasmic reticulum-bound sterol-sensing enzymes through impaired processing of the transcription factor Sterol Regulatory Element-Binding Protein 2 and accelerated degradation of the endoplasmic reticulum cholesterol-sensors squalene epoxidase and HMG-CoA reductase. These adaptations of mevalonate pathway activity affected neither total intracellular cholesterol levels nor the cellular free (nonesterified) cholesterol pool. Finally, measurement of intracellular cholesterol using the fluorescent cholesterol binding dye filipin revealed that complex I inhibition elevated cholesterol on intracellular compartments. Taken together, our study shows that mitochondrial respiratory chain dysfunction elevates intracellular free cholesterol levels and therefore attenuates the expression of mevalonate pathway enzymes, which lowers endogenous cholesterol biosynthesis, disrupting the metabolic output of the mevalonate pathway. We conclude that intracellular disturbances in cholesterol homeostasis may alter systemic cholesterol management in diseases associated with declining mitochondrial function.

The adaptive response to overfeeding is associated with profound modifications of gene expression in adipose tissue to support lipid storage and weight gain. The objective of this study was to assess in healthy lean men whether a supplementation with polyphenols could interact with these molecular adaptations. Abdominal subcutaneous adipose tissue biopsies were sampled from 42 subjects participating to an overfeeding protocol providing an excess of 50% of their total energy expenditure for 31 days, and who were supplemented with 2 g/day of grape polyphenols or a placebo. Gene expression profiling was performed by RNA sequencing. Overfeeding led to a modification of the expression of 163 and 352 genes in the placebo and polyphenol groups, respectively. The GO functions of these genes were mostly involved in lipid metabolism, followed by genes involved in adipose tissue remodeling and expansion. In response to overfeeding, 812 genes were differentially regulated between groups. Among them, a set of 41 genes were related to angiogenesis and were down-regulated in the polyphenol group. Immunohistochemistry targeting PECAM1, as endothelial cell marker, confirmed reduced angiogenesis in this group. Finally, quercetin and isorhamnetin, two polyphenol species enriched in the plasma of the volunteers submitted to the polyphenols, were found to inhibit human umbilical vein endothelial cells migration in vitro. Polyphenol supplementation do not prevent the regulation of genes related to lipid metabolism in human adipose tissue during overfeeding, but impact the angiogenesis pathways. This may potentially contribute to a protection against adipose tissue expansion during dynamic phase of weight gain.

Abstract Neural precursor cells (NPCs) are present in most regions of the adult central nervous system (CNS). Using NPCs in a therapeutical perspective, that is, to regenerate CNS tissue after injury or in neurodegenerative diseases, will require the efficient manipulation of their fate. Proneural gene overexpression in NPCs represents a promising strategy to promote neuronal differentiation. The activity of the proneural proteins is, however, context-dependent and can be inhibited/modulated by binding with other bHLH (basic helix-loop-helix) or HLH transcription factors. In this study, we show that the two proneural proteins, Ngn2 and Mash1, are differentially sensitive to negative regulation by gliogenic factors or a gliogenic substrate (i.e., postnatal spinal cord slices). Coexpressing E-proteins with proneural proteins was efficient to rescue proneural proteins neurogenic activity, suggesting a central role for E-protein sequestration in mediating postnatal CNS gliogenic inhibition. Tethering of proneural proteins with E47 further insulated Mash1 from negative environmental influences whereas this strategy was not successful with Ngn2, suggesting that mechanisms of inhibition differ in between these two proneural proteins. Our results demonstrate that a better understanding of proneural protein modulation by environmental cues is a prerequisite to develop innovative approaches that will permit the manipulation of the fate of NPCs in the adult CNS after trauma or disease. Disclosure of potential conflicts of interest is found at the end of this article.

For decades coffees were associated with the genus Coffea. In 2011, the closely related genus Psilanthus was subsumed into Coffea. However, results obtained in 2017—based on 28,800 nuclear SNPs—indicated that there is not substantial phylogenetic support for this incorporation. In addition, a recent study of 16 plastid full-genome sequences highlighted an incongruous placement of Coffea canephora (Robusta coffee) between maternal and nuclear trees. In this study, similar global features of the plastid genomes of Psilanthus and Coffea are observed. In agreement with morphological and physiological traits, the nuclear phylogenetic tree clearly separates Psilanthus from Coffea (with exception to C. rhamnifolia, closer to Psilanthus than to Coffea). In contrast, the maternal molecular tree was incongruent with both morphological and nuclear differentiation, with four main clades observed, two of which include both Psilanthus and Coffea species, and two with either Psilanthus or Coffea species. Interestingly, Coffea and Psilanthus taxa sampled in West and Central Africa are members of the same group. Several mechanisms such as the retention of ancestral polymorphisms due to incomplete lineage sorting, hybridization leading to homoploidy (without chromosome doubling) and alloploidy (for C. arabica) are involved in the evolutionary history of the coffee species. While sharing similar morphological characteristics, the genetic relationships within C. canephora have shown that some populations are well differentiated and genetically isolated. Given the position of its closely-related species, we may also consider C. canephora to be undergoing a long process of speciation with an intermediate step of (sub-)speciation.

The diagnosis of malignant hematologic diseases has become increasingly complex during the last decade. It is based on the interpretation of results from different laboratory analyses, which range from microscopy to gene expression profiling. Recently, a method for the analysis of RNA phenotypes has been developed, the nCounter technology (Nanostring® Technologies), which allows for simultaneous quantification of hundreds of RNA molecules in biological samples. We evaluated this technique in a Swiss multi-center study on eighty-six samples from acute leukemia patients.mRNA and protein profiles were established for normal peripheral blood and bone marrow samples. Signal intensities of the various tested antigens with surface expression were similar to those found in previously performed Affymetrix microarray analyses. Acute leukemia samples were analyzed for a set of twenty-two validated antigens and the Pearson Correlation Coefficient for nCounter and flow cytometry results was calculated.Highly significant values between 0.40 and 0.97 were found for the twenty-two antigens tested. A second correlation analysis performed on a per sample basis resulted in concordant results between flow cytometry and nCounter in 44-100% of the antigens tested (mean = 76%), depending on the number of blasts present in a sample, the homogeneity of the blast population, and the type of leukemia (AML or ALL).The nCounter technology allows for fast and easy depiction of a mRNA profile from hematologic samples. This technology has the potential to become a valuable tool for the diagnosis of acute leukemias, in addition to multi-color flow cytometry.

Genetic testing, which is now a routine part of clinical practice and disease management protocols, is often based on the assessment of small panels of variants or genes. On the other hand, continuous improvements in the speed and per-base costs of sequencing have now made whole exome sequencing (WES) and whole genome sequencing (WGS) viable strategies for targeted or complete genetic analysis, respectively. Standard WGS/WES data analytical workflows generally rely on calling of sequence variants respect to the reference genome sequence. However, the reference genome sequence contains a large number of sites represented by rare alleles, by known pathogenic alleles and by alleles strongly associated to disease by GWAS. It's thus critical, for clinical applications of WGS and WES, to interpret whether non-variant sites are homozygous for the reference allele or if the corresponding genotype cannot be reliably called. Here we show that an alternative analytical approach based on the analysis of both variant and non-variant sites from WGS data allows to genotype more than 92% of sites corresponding to known SNPs compared to 6% genotyped by standard variant analysis. These include homozygous reference sites of clinical interest, thus leading to a broad and comprehensive characterization of variation necessary to an accurate evaluation of disease risk. Altogether, our findings indicate that characterization of both variant and non-variant clinically informative sites in the genome is necessary to allow an accurate clinical assessment of a personal genome. Finally, we propose a highly efficient extended VCF (eVCF) file format which allows to store genotype calls for sites of clinical interest while remaining compatible with current variant interpretation software.

Malnutrition predisposes to diarrhea and diarrhea adversely affects the nutritional status creating a vicious cycle.1Guerrant R.L. et al.Malnutrition as an enteric infectious disease with long-term effects on child development.Nutr Rev. 2008; 66: 487-505Crossref PubMed Scopus (341) Google Scholar The role of the gut microbiome in malnutrition is an active research area.2Blanton L.V. et al.Childhood undernutrition, the gut microbiota, and microbiota-directed therapeutics.Science. 2016; 352: aad9359Crossref Scopus (137) Google Scholar Parenteral antibiotics are recommended by the World Health Organization in hospitalized pediatric patients with severe acute malnutrition (SAM) presenting signs of infections.3Lazzerini M. et al.Antibiotics in severely malnourished children: systematic review of efficacy, safety and pharmacokinetics.Bull World Health Organ. 2011; 89: 593-606Crossref Scopus (40) Google Scholar Stool microbiota data for such patients are, however, lacking. To fill this gap, we studied the stool microbiota in 19 SAM patients from Bangladesh hospitalized with acute diarrhea (AD) and compared it with that of matched 20 healthy control subjects (HC) (Supplementary Table 1). SAM-AD patients were treated with parenterally administered gentamycin and ampicillin, whereas HC received no antibiotics for at least a month before sample collection. 16S rRNA and metagenome sequencing showed a marked increase of Escherichia and Klebsiella abundances in SAM-AD over HC (Figures 1A, 1C, and 2A), but not of Streptococcus (Figure 1B). Compared with HC, SAM-AD showed a reduced microbiota diversity (Figure 1D) and a decrease in Prevotella, Blautia, Ruminococcus, Faecalibacterium, Megamonas, and Bifidobacterium (Figures 1A and 2A). SAM-AD patients showed a 10-fold-lower 16S copy number of stool bacteria than HC (Figure 1E), which was partially compensated by a 2-fold higher stool frequency. Rotavirus was the dominant pathogen (Supplementary Table 2) in SAM-AD, contradicting reports on protection from rotavirus diarrhea by malnutrition.4Verkerke H. et al.Malnutrition is associated with protection from rotavirus diarrhea: evidence from a longitudinal birth cohort study in Bangladesh.J Clin Microbiol. 2016; 54: 2568-2574Crossref PubMed Scopus (23) Google Scholar All other pathogens (Escherichia coli in 7, Cryptosporidium in 2, Vibrio cholerae in 1, norovirus in 1 patient), except in 1 patient with adenovirus, were associated with copathogens. Salmonella was not detected in any SAM-AD patient. Compared with HC, virulence factor genes were increased in SAM-AD for various pathogenic Enterobacteriaceae (uropathogenic, enterohemorrhagic, and enteroaggregative E coli, Shigella, Salmonella, and Yersinia) (Figure 2B). The top 10 most significant pathway changes in SAM-AD over HC (Supplementary Table 3) included increases in D-glucarate and D-galactarate degradation genes (Figure 2D). In addition, SAM-AD showed more antibiotic resistance genes than HC (Figure 2E), mostly E coli (63%) and Klebsiella (32%) associated. Escherichia phage followed by Vibrio phage DNA was increased in SAM-AD over HC (Figures 2A and 2F). The expansion of coliphages in SAM-AD was likely a consequence of increased abundance of bacterial host cells. Other mechanisms could play a role, such as increased accessibility or modified physiology of the bacterial host cells, for example as a consequence of immune system response to bacteria. Among SAM-AD patients, the abundance of sequences attributed to Escherichia phage phAPEC8 was negatively correlated with the abundance of its host (Figure 2A, SparCC [1000 bootstraps]: -0.52; N = 18; P = .008). It is unclear, however, whether coliphage expansion could lead to a collapse of E coli population because high abundance of phage at enrollment was not associated with a greater decrease of E coli abundance over a period of approximately 1 day (Figure 2G). Longer time series are necessary to determine whether bacteriophages could indeed control the expansion of host bacteria in the gut. Previous attempt to treat E coli–associated AD with a mixture of T4 bacteriophages had failed to demonstrate clinical benefit5Sarker S.A. et al.Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: a randomized trial in children from Bangladesh.EBioMedicine. 2016; 4: 124-137Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar; however, the E coli dominance was much more pronounced in antibiotic-treated SAM-AD patients of the present study than in children with AD. A marked increase of fecal E coli abundance at the expense of bifidobacteria was also described in European newborns not suffering from diarrhea but treated parenterally with ampicillin and gentamicin for suspected sepsis.6Fouhy F. et al.High-throughput sequencing reveals the incomplete, short-term recovery of infant gut microbiota following parenteral antibiotic treatment with ampicillin and gentamicin.Antimicrob Agents Chemother. 2012; 56: 5811-5820Crossref PubMed Scopus (346) Google Scholar Postantibiotics expansion of E coli and Salmonella typhimurium was also observed in mice model7Faber F. et al.Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion.Nature. 2016; 534: 697-699Crossref PubMed Scopus (109) Google Scholar where it was shown to be a consequence of streptomycin-induced production of galactarate and glucarate in host’s cecum. This host-dependent mechanism may have contributed to the observed expansion of E coli, although the main driver was likely the high levels of antibiotic resistance displayed by E coli in Bangladesh.8Rahman M.M. et al.Prevalence of extended spectrum-lactamase-producing Escherichia coli and Klebsiella pneumoniae in an urban hospital in Dhaka, Bangladesh.Int J Antimicrob Agents. 2004; 24: 508-510Crossref PubMed Scopus (35) Google Scholar We think that the treatment with antibiotics rather than malnutrition and diarrhea was the main cause of the observed microbiota alteration, because Bangladeshi children with AD showed an increased abundance of commensal streptococci over control subjects,5Sarker S.A. et al.Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: a randomized trial in children from Bangladesh.EBioMedicine. 2016; 4: 124-137Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar whereas children with SAM displayed a shift to a less mature fecal microbiota composition but not a marked E coli expansion.9Subramanian S. et al.Persistent gut microbiota immaturity in malnourished Bangladeshi children.Nature. 2014; 510: 417-421Crossref PubMed Scopus (752) Google Scholar Antibiotic-induced Enterobacteriaceae expansion studied in mice has been shown to be involved in the disruption of the symbiosis between colonocytes and obligate anaerobic butyrate producers, resulting in a vicious cycle whereby colonocyte metabolism is subverted to permit the outgrowth of oxygen-tolerant, nitrate-dependent Enterobacteriaceae.10Byndloss M.X. et al.Microbiota-activated PPAR-g signaling inhibits dysbiotic Enterobacteriaceae expansion.Science. 2017; 357: 570-575Crossref PubMed Scopus (544) Google Scholar It is known that antibiotic treatment in humans may lead to diarrhea even in a presumed absence of obligate pathogens (antibiotic-associated diarrhea), but the microbiota of pediatric antibiotic-associated diarrhea has not been studied. Currently, there is no evidence from humans that the antibiotic-induced expansion of normally commensal Enterobacteriaceae could be detrimental. However, the observations from animal models suggest that this is a possibility that should be investigated. Silas Kieser: analysis and interpretation of data, and critical revision of the manuscript for important intellectual content statistical analysis. Shafiqul A. Sarker: study concept and design, patient recruitment, and critical revision of the manuscript for important intellectual content. Bernard Berger: analysis and interpretation of data, and critical revision of the manuscript for important intellectual content. Shamima Sultana: patient recruitment, and study supervision. Mohammed J. Chisti: patient recruitment. Shoeb B. Islam: patient recruitment. Francis Foata: acquisition and analysis of data, and technical support. Nadine Porta: acquisition of data, and technical support. Bertrand Betrisey: acquisition of data, and technical support. Coralie Fournier: acquisition of data, and technical support. Patrick Descombes: study supervision, and critical revision of the manuscript for important intellectual content. Annick Mercenier: critical revision of the manuscript for important intellectual content. Olga Sakwinska: analysis and interpretation of data, study supervision, and critical revision of the manuscript for important intellectual content. Harald Brüssow: study concept and design, study supervision, and drafting of the manuscript. The study was approved by the Ethical Review Committee of the International Center for Diarrhoeal Diseases Research in Dhaka, Bangladesh (icddr,b) as protocol #PR-14081. A total of 19 children with severe acute malnutrition (SAM) and acute diarrhea (AD) and 20 matched healthy control (HC) children were enrolled in Dhaka, Bangladesh during the winter season 2014–2015 (Supplementary Table 1). SAM-AD showed z scores <-3 indicative of severe underweight, severe stunting, and severe wasting. Because healthy children do not present to the icddr,b hospital, the control children were recruited at a field clinic maintained by icddr,b (Nandipara), whose population corresponds socioeconomically to the children hospitalized at icddr,b. SAM-AD patients received reduced osmolarity oral rehydration solution supplemented with zinc for diarrhea treatment. SAM-AD is typically associated with a case-fatality rate of 30%–50% because of a high rate of manifest or developing infectious comorbidity (pneumonia, bacteremia, urinary tract infections). Therefore, as recommended by the World Health Organization,1Lazzarini M. et al.Antibiotics in severely malnourished children: systematic review of efficacy, safety and pharmacokinetics.Bull WHO. 2011; 89: 594-607PubMed Google Scholar all SAM-AD were treated on hospitalization with ampicillin (100 mg/kg/day in 4 doses for 48 hours by intravenous injection) and gentamicin (6 mg/kg/d in 2 doses by intramuscular injection) followed with amoxicillin (50 mg/kg/day in 3 divided doses given orally for 5 days). None of the control subjects had received antibiotics for a month before stool sampling. No difference was seen for family income (HC, 7:8:5 and SAM, 6:9:4 with very low:low:moderate income; P = .90), maternal education (illiterate: HC, 2; SAM, 1; P = .96; primary school: HC, 12; SAM, 11; P = .57), or numbers of siblings (for 1, 2, 3, >3 children: HC, 10:4:5:1; SAM, 9:9:1:0; P = .13). Vaccination status was comparable in both groups (HC, 10 and SAM, 13 vaccination completed or running; P = .4) and feeding mode at 6 months of age (HC, 15:1:4; SAM, 10:4:5 exclusive breastfeeding vs formula feeding vs partial breastfeeding; P = .24). However, the 2 groups differed for sex (HC, 12:8 and SAM, 3:16 for female:male; P = .01) (Supplementary Table 4) and previous exposure to cow’s milk (HC, 7 and SAM, 18; P = .003). Stool samples were obtained from the patients at enrollment into the study and the time of transfer to refeeding ward (1.6 + 1.4 days later). Samples were frozen at –80°C immediately after collection. Total stool DNA was extracted using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions, except for addition of a series of mechanical disruption steps using a FastPrep apparatus and Lysing Matrix B tubes (MP Biochemicals, Santa Ana, CA).2Junick J. Blaut M. Quantification of human fecal Bifidobacterium species by use of quantitative real-time PCR analysis targeting the groEL gene.Appl Environ Microbiol. 2012; 78: 2613-2622Crossref PubMed Scopus (83) Google Scholar 16S variable region V3 to V4 were polymerase chain reaction amplified using universal DNA primers with dual indexing3Kozich J.J. et al.Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.Appl Environ Microbiol. 2013; 79: 5112-5120Crossref PubMed Scopus (4133) Google Scholar and sequenced with Miseq reagent kit V3 (Illumina Inc, San Diego, CA) as previously described.4Caporaso J.G. et al.Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms.ISME J. 2012; 6: 1621-1624Crossref PubMed Scopus (5413) Google Scholar Raw sequence data were analyzed using Mothur V.1.33.0 21 and QIIME V.1.8 22 software packages.5Schloss P.D. et al.Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.Appl Environ Microbiol. 2009; 75: 7537-7541Crossref PubMed Scopus (14566) Google Scholar, 6Caporaso J.G. et al.QIIME allows analysis of high-throughput community sequencing data.Nat Methods. 2010; 7: 335-336Crossref PubMed Scopus (24708) Google Scholar Paired-end sequences were demultiplexed and joined as described.5Schloss P.D. et al.Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.Appl Environ Microbiol. 2009; 75: 7537-7541Crossref PubMed Scopus (14566) Google Scholar Chimera were identified and removed. Open reference OTUs picking at 97% identity used pick_otus.py, with options usearch_ref.7Edgar R.C. et al.UCHIME improves sensitivity and speed of chimera detection.Bioinformatics. 2011; 27: 2194-2200Crossref PubMed Scopus (10249) Google Scholar Taxonomy assignment used RDP Classifier8Wang Q. et al.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Appl Environ Microbiol. 2007; 73: 5261-5267Crossref PubMed Scopus (13309) Google Scholar on representative sequences. The resulting multiple alignments were used to build a phylogenetic tree with the FastTree method.9Price M.N. et al.FastTree: computing large minimum evolution trees with profiles instead of a distance matrix.Mol Biol Evol. 2009; 26: 1641-1650Crossref PubMed Scopus (2794) Google Scholar Alpha-diversity was reported as the average of 10 rarefactions. Stool DNA was extracted using MoBio PowerMag Microbiome DNA Isolation Kit (QIAGEN, Hilden, Germany) on an epMotion M5073 (Vaudaux-Eppendorf AG, Basel, Switzerland) followed by Zymo DNA Clean & Concentrator Kit (Zymo Research, Irvine, CA). Library preparation was done according to the Nextera XT protocol from Illumina. The quality and quantity check was based on LabChip GX Touch HT (Perkin Elmer, Waltham, MA) results. Sequencing was performed on HiSeq 2500 using chemistry HighOutput v4 PE125 (Illumina). The paired-end reads were filtered using KneadData v0.5.1 (https://bitbucket.org/biobakery/kneaddata), which included quality filtering based on Trimmomatic and excluded reads mapping to the human genome. A median number of 3.2 × 107 reads and a minimum of 2.2 × 107 reads per sample were evaluated. Taxonomic profiles were generated with MetaPhlAn2 2.5.0.10Truong D.T. et al.MetaPhlAn2 for enhanced metagenomic taxonomic profiling.Nat Methods. 2015; 12: 902-903Crossref PubMed Scopus (1110) Google Scholar Functional annotation was performed with HUMANn2 v0.7.1 and integrated into pathways from the MetaCyc database.11Abubucker S. et al.Metabolic reconstruction for metagenomic data and its application to the human microbiome.PLoS Comput Biol. 2012; 8: e1002358Crossref PubMed Scopus (706) Google Scholar, 12Caspi R. et al.The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases.Nucleic Acids Res. 2014; 42: D459-D471Crossref PubMed Scopus (762) Google Scholar The number of reads are first normalized by the length of the reference genome and then by million reads (counts per million). Functional annotation was used to calculate the abundance of antibiotic resistance genes from CARD database.13McArthur A.G. et al.The comprehensive antibiotic resistance database.Antimicrob Agents Chemother. 2013; 57: 3348-3357Crossref PubMed Scopus (1110) Google Scholar ShortBRED14Kaminski J. et al.High-specificity targeted functional profiling in microbial communities with ShortBRED.PLoS Comput Biol. 2015; 11: e1004557Crossref PubMed Scopus (154) Google Scholar was used to profile the metagenomics samples for virulence factors from the VFDB database.15Chen L. et al.VFDB 2016: hierarchical and refined dataset for big data analysis–10 years on.Nucleic Acids Res. 2016; 44: D694-D697Crossref PubMed Scopus (734) Google Scholar The mapped reads were first normalized by million reads and then by the length of reference sequence (RPKM). The crucial step of bead-beating was included in both protocols of DNA extraction, ensuring an equal efficiency of DNA extraction from Firmicutes, Actinobacteria, and Bacterioides. We found a good correlation between MetaPhlAn and 16S rRNA analysis excluding major differences introduced by the 2 DNA extraction methods. Pathogens were identified by TaqMan Array Card (Thermo Fisher Scientific, Waltham, MA) detecting 19 enteropathogens,16Platts-Mills J.A. et al.Association between stool enteropathogen quantity and disease in Tanzanian children using TaqMan array cards: a nested case-control study.Am J Trop Med Hyg. 2014; 90: 133-138Crossref PubMed Scopus (57) Google Scholar providing semiquantitative cycle threshold values for each target. We normalized the values with respect to the total bacteria by quantitative polymerase chain reaction using universal primers.17Nadkarni M.A. et al.Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.Microbiology. 2002; 148: 257-266Crossref PubMed Scopus (1462) Google Scholar We considered pathogens as detected when the cycle threshold value was lower than in HC children. For the pathogens that were not detected in HC (Salmonella, Vibrio cholera, Ascaris, Cryptosporidium, and Trichuris) a threshold of 35 was imputed. This was complemented by screening of the metagenome sequences for pathogen taxa, defined as taxa targeted by TaqMan Array Card, including pathotypes of Escherichia coli and associated virulence factors. 16S rDNA and metagenome reads are available under the Bio Project accession numbers SRP100410 and SRP100895. Because microbiota abundance have a nonnormal distribution, nonparametric tests were used. The data met all assumptions of nonparametric tests. Nonparametrical tests do not require homoscedasticity for group comparison. If not otherwise mentioned the 2-sided Mann-Whitney test was used for continuous variables and a chi-square test for categorical variables. Data are shown as individual data points or as boxplots. In the boxplots the box represent the quintiles of the dataset, whereas the whisker extend to 1.5 × the interquartile range. Because the HC and SAM-AD differed in female/male ratio, we explored potential impact of sex on the major findings of the study. However, the children of the 2 sexes displayed similar findings (Supplementary Table 4).Supplementary Table 1Baseline Characteristics of SAM-AD Case and Matched HCHCSAM-ADP valueN2019Age child, mo13.0 (10.8 to 16.0)13.0 (9.5 to 18.5).989Age mother, y25.5 (22.0 to 30.5)24.0 (22.0 to 27.5).411Weight, kg8 (8 to 9)5.9 (5.1 to 6.7)2.93e-07Height, cm73.5 (69.8 to 77.4)68.0 (64.5 to 70.7).0064Mid arm circumference, cm13 (13 to 14)12 (11 to 12)3.27e-05Weight for age z score-1 (-1 to -1)-4 (-5 to -4)1.01e-07Height for age z score-1 (-2 to -1)-3 (-4 to -3)4.35e-06Body mass index16 (15 to 16)13 (12 to 13)1.18e-07Weight for height z score-1 (-1 to -0)-3 (-4 to -3)1.01e-07Body mass index z score-0 (-1 to 0)-3.4 (-4.2 to -2.8)1.37e-07Mid arm circumference z score-1 (-1 to -1)-3 (-3 to -3)2.34e-05Rectal temperature, °C36.5 (36.0 to 36.7)37.2 (37.0 to 37.2)1.08e-07Pulse, min-1110.0 (100.0 to 120.0)132.0 (130.0 to 136.0)7.23e-08Respiration rate, min-130.0 (30.0 to 32.0)36.0 (35.0 to 36.0)4.46e-07Vomiting, d-10.0 (0.0 to 0.0)0.0 (0.0 to 2.0).00345Duration of diarrhea, d4 (4 to 4)Stool frequency, d-12.5 (2.5 to 2.5)5.0 (3.5 to 7.0)6.15e-06Systolic blood pressure, mm Hg90.0 (90.0 to 90.0)90.0 (90.0 to 90.0).0166Diastolic blood pressure, mm Hg60.0 (60.0 to 60.0)60.0 (60.0 to 60.0).101Exclusive breastfeeding, mo6.0 (5.8 to 6.0)6.0 (2.5 to 6.0).0578Number of siblings1.5 (1.0 to 3.0)2.0 (1.0 to 2.0).541NOTE. Values are medians (interquartile range: first, third quartile). P values are calculated by a 2-sided Mann-Whitney test. Categorical variables were compared by chi-square test. Open table in a new tab Supplementary Table 2Pathogen Detection in Stools of SAM-AD PatientsPatient IDTaqManPathogen taxaEscherichia coli pathogensVirulence factors506Adenovirus (8)Adenovirus (67)negneg511Cryptosporidium (3)negnegneg503Ascaris (3)Rotavirus (3)negEAEC-aaiC (0.01)EAEC-aaiC (154)516negShigella (1)negneg518Vibrio cholerae (7)EPEC-bfp (5)EPEC-eae (4)EIEC-ipaH (3)V cholerae (3)negEPEC -bfp (36)517Cryptosporidium (15)EPEC -bfp (6)EPEC-eae (6)EIEC-ipaH (6)EAEC-aaiC (5)negnegneg513NAnegEAEC-aaiC (0.01) EAEC-aatA (0.01)EAEC-aaiC (120) EAEC-aatA (79)515Norovirus (5)negEAEC-aaiC (0.01)EAEC-aaiC (123) EAEC-aatA (78)501Rotavirus (10)negnegneg502Rotavirus (5)EAEC-aaiC (3)negEAEC-aaiC (0.02)EAEC-aaiC (275)504Rotavirus (12)negnegneg505Rotavirus (8)EAEC-aatA (3)negEAEC-aaiC (0.01) EAEC-aatA (0.02)EAEC-aaiC (202) EAEC-aatA (62)507Rotavirus (8)Adenovirus (3) Aeromonas (0.2)negneg508Rotavirus (6)negnegneg510Rotavirus (6)negETEC-lt (0.03)ETEC-lt (346)514Rotavirus (11)Adenovirus (0.1)negneg519Rotavirus (10)NANANA509negShigella (0.5)negneg512Cryptosporidium (9)EIEC-ipaH (3)Shigella (2) Cryptosporidium (0.5)negEAEC-aatA (114) EIEC-ipaH (36)NOTE. TaqMan, results of detection of 19 pathogens with TaqMan array card (difference to threshold expressed as cycle threshold), E coli pathotypes and the detected virulence factors are indicated, neg, no pathogen detection; pathogen taxa, percentage of taxons determined in metagenome sequencing of the indicated pathogen, neg, <0.1% of taxa; E coli pathogens, the indicated virulence genes of the specified E coli pathotype with % of identified E coli genes, neg, <0.01 of genes; virulence factors, gene read number corrected per million reads and length of target gene coverage.NA, the corresponding sample was not investigated by metagenome sequencing. Open table in a new tab Supplementary Table 3Top 10 Pathways Significantly Enriched in SAM-AD Over HC in Stool Metagenome DataDescriptionHCSAM-ADQSuperpathway of l-arginine and l-ornithine degradation131050.0002Superpathway of l-arginine, putrescine, and 4-aminobutanoate degradation131050.0002D-glucarate degradation I211610.0004Phytol degradation453910.0004D-galactarate degradation I201200.0007Superpathway of D-glucarate and D-galactarate degradation201200.0007Methylphosphonate degradation I11940.0007Superpathway of fermentation481790.0012Phytate degradation I282180.0012NAD/NADP-NADH/NADPH mitochondrial interconversion321770.0012NOTE. Median counts per million reads as assessed by 1-sided Mann-Whitney U test corrected for multiple testing by the Benjamini-Hochberg procedure.NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate. Open table in a new tab Supplementary Table 4The Main Variables That Were Evaluated in the Study, Stratified By SexHCSAM-ADFemaleMaleFemaleMale16S rRNA sequencingN128216Bifidobacterium (proportion of reads)25.0 (6.62–47.1)24.9 (18.8–55.6)0.024 (0.02–0.02)0.405 (0.05–6.07)Escherichia (proportion of reads)1.46 (0.71–9.76)6.32 1.95–24.1)62.6 (46.6–78.6)57.0 (20.8–73.8)Diversity (Faith index)4.007 (2.86–4.37)3.308 (3.08–3.51)1.360 (1.28–1.44)1.306 (1.06–2.19)Shotgun metagenomicsN128316Antibiotic resistance genes (counts per million reads)955 (767–1213)608 (530–1326)4117 (3063–4140)3978 (2115–6495)D-galactarate degradation pathway (counts per million reads)13.6 (6.94–30.50)20.3 (5.65–25.30)85.7 (78.80–119.01)127 (70.90–186)Phage sequences (percentage of total sequences, normalized for genome size)0.469 (0.00–2.30)0.000 (0.00–0.00)73.0 (49.3–85.4)0.794 (0.04–15.8)NOTE. Median and interquartile range are displayed. Open table in a new tab NOTE. Values are medians (interquartile range: first, third quartile). P values are calculated by a 2-sided Mann-Whitney test. Categorical variables were compared by chi-square test. NOTE. TaqMan, results of detection of 19 pathogens with TaqMan array card (difference to threshold expressed as cycle threshold), E coli pathotypes and the detected virulence factors are indicated, neg, no pathogen detection; pathogen taxa, percentage of taxons determined in metagenome sequencing of the indicated pathogen, neg, <0.1% of taxa; E coli pathogens, the indicated virulence genes of the specified E coli pathotype with % of identified E coli genes, neg, <0.01 of genes; virulence factors, gene read number corrected per million reads and length of target gene coverage. NA, the corresponding sample was not investigated by metagenome sequencing. NOTE. Median counts per million reads as assessed by 1-sided Mann-Whitney U test corrected for multiple testing by the Benjamini-Hochberg procedure. NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate. NOTE. Median and interquartile range are displayed.

Dosage imbalance is responsible for several genetic diseases, among which Down syndrome is caused by the trisomy of human chromosome 21.To elucidate the extent to which the dosage imbalance of specific human chromosome 21 genes perturb distinct molecular pathways, we developed the first mouse embryonic stem (ES) cell bank of human chromosome 21 genes. The human chromosome 21-mouse ES cell bank includes, in triplicate clones, 32 human chromosome 21 genes, which can be overexpressed in an inducible manner. Each clone was transcriptionally profiled in inducing versus non-inducing conditions. Analysis of the transcriptional response yielded results that were consistent with the perturbed gene's known function. Comparison between mouse ES cells containing the whole human chromosome 21 (trisomic mouse ES cells) and mouse ES cells overexpressing single human chromosome 21 genes allowed us to evaluate the contribution of single genes to the trisomic mouse ES cell transcriptome. In addition, for the clones overexpressing the Runx1 gene, we compared the transcriptome changes with the corresponding protein changes by mass spectroscopy analysis.We determined that only a subset of genes produces a strong transcriptional response when overexpressed in mouse ES cells and that this effect can be predicted taking into account the basal gene expression level and the protein secondary structure. We showed that the human chromosome 21-mouse ES cell bank is an important resource, which may be instrumental towards a better understanding of Down syndrome and other human aneuploidy disorders.

Cronobacteris associated with infant infections and the consumption of reconstituted infant formula. Here we sequenced and closed six genomes ofC. condimenti(T),C. muytjensii(T),C. universalis(T),C. malonaticus(T),C. dublinensis(T), andC. sakazakiithat can be used as reference genomes in single nucleotide polymorphism (SNP)-based next-generation sequencing (NGS) analysis for source tracking investigations.

Increased adipogenesis and altered adipocyte function contribute to the development of obesity and associated comorbidities. Fructose modified adipocyte metabolism compared to glucose, but the regulatory mechanisms and consequences for obesity are unknown. Genome-wide methylation and global transcriptomics in SGBS pre-adipocytes exposed to 0, 2.5, 5, and 10 mM fructose, added to a 5-mM glucose-containing medium, were analyzed at 0, 24, 48, 96, 192, and 384 h following the induction of adipogenesis.Time-dependent changes in DNA methylation compared to baseline (0 h) occurred during the final maturation of adipocytes, between 192 and 384 h. Larger percentages (0.1% at 192 h, 3.2% at 384 h) of differentially methylated regions (DMRs) were found in adipocytes differentiated in the glucose-containing control media compared to adipocytes differentiated in fructose-supplemented media (0.0006% for 10 mM, 0.001% for 5 mM, and 0.005% for 2.5 mM at 384 h). A total of 1437 DMRs were identified in 5237 differentially expressed genes at 384 h post-induction in glucose-containing (5 mM) control media. The majority of them inversely correlated with the gene expression, but 666 regions were positively correlated to the gene expression.Our studies demonstrate that DNA methylation regulates or marks the transformation of morphologically differentiating adipocytes (seen at 192 h), to the more mature and metabolically robust adipocytes (as seen at 384 h) in a genome-wide manner. Lower (2.5 mM) concentrations of fructose have the most robust effects on methylation compared to higher concentrations (5 and 10 mM), suggesting that fructose may be playing a signaling/regulatory role at lower concentrations of fructose and as a substrate at higher concentrations.

Abstract Background The high-density oligonucleotide microarray (GeneChip) is an important tool for molecular biological research aiming at large-scale detection of small nucleotide polymorphisms in DNA and genome-wide analysis of mRNA concentrations. Local array data management solutions are instrumental for efficient processing of the results and for subsequent uploading of data and annotations to a global certified data repository at the EBI (ArrayExpress) or the NCBI (GeneOmnibus). Description To facilitate and accelerate annotation of high-throughput expression profiling experiments, the Microarray Information Management and Annotation System (MIMAS) was developed. The system is fully compliant with the Minimal Information About a Microarray Experiment (MIAME) convention. MIMAS provides life scientists with a highly flexible and focused GeneChip data storage and annotation platform essential for subsequent analysis and interpretation of experimental results with clustering and mining tools. The system software can be downloaded for academic use upon request. Conclusion MIMAS implements a novel concept for nation-wide GeneChip data management whereby a network of facilities is centered on one data node directly connected to the European certified public microarray data repository located at the EBI. The solution proposed may serve as a prototype approach to array data management between research institutes organized in a consortium.

Several studies recently reported contradicting results regarding the link between amylase 1 (AMY1) copy numbers (CNs), obesity, and type 2 diabetes. The aim of this study was to assess the impact of AMY1 CN on anthropometrics and glycemic outcomes in obese individuals following a 2-phase dietary weight loss intervention. Using the paralog ratio test, AMY1 CNs were accurately measured in 761 obese individuals from the DiOGenes study. Subjects first underwent an 8-wk low-calorie diet (LCD, at 800 kcal/d) and then were randomly assigned to a 6-mo weight maintenance dietary (WMD) intervention with arms having different glycemic loads. At baseline, a modest association between AMY1 CN and BMI (P = 0.04) was observed. AMY1 CN was not associated with baseline glycemic variables. In addition, AMY1 CN was not associated with anthropometric or glycemic outcomes following either LCD or WMD. Interaction analyses between AMY1 CN and nutrient intake did not reveal any significant association with clinical parameters (at baseline and following LCD or WMD) or when testing gene × WMD interactions during the WMD phase. In the absence of association with weight trajectories or glycemic improvements, the AMY1 CN cannot be considered as an important biomarker for response to a clinical weight loss and weight maintenance programs in overweight/obese subjects. This trial was registered at www.clinicaltrials.gov as NCT00390637.

Abstract Polymorphisms in genes related to the metabolism of vitamin B12 haven’t been examined in a Brazilian population. To (a) determine the correlation between the local genetic ancestry components and vitamin B12 levels using ninety B12-related genes; (b) determine associations between these genes and their SNPs with vitamin B12 levels; (c) determine a polygenic risk score (PRS) using significant variants. This cross-sectional study included 168 children and adolescents, aged 9–13 years old. Total cobalamin was measured in plasma. Genotyping arrays and whole exome data were combined to yield ~ 7000 SNPs in 90 genes related to vitamin B12. The Efficient Local Ancestry Inference was used to estimate local ancestry for African (AFR), Native American, and European (EUR). The association between the genotypes and vitamin B12 levels were determined with generalized estimating equation. Vitamin B12 levels were driven by positive (EUR) and negative (AFR, AMR) correlations with genetic ancestry. A set of 36 variants were used to create a PRS that explained 42% of vitamin level variation. Vitamin B12 levels are influenced by genetic ancestry and a PRS explained almost 50% of the variation in plasma cobalamin in Brazilian children and adolescents.

Abstract Coffea arabica , an allotetraploid hybrid of C. eugenioides and C. canephora , is the source of approximately 60% of coffee products worldwide, and its cultivated accessions have undergone several population bottlenecks. We present chromosome-level assemblies of a di-haploid C. arabica accession and modern representatives of its diploid progenitors, C. eugenioides and C. canephora . The three species exhibit largely conserved genome structures between diploid parents and descendant subgenomes, with no obvious global subgenome dominance. We find evidence for a founding polyploidy event 350,000-610,000 years ago, followed by several pre-domestication bottlenecks, resulting in narrow genetic variation. A split between wild accessions and cultivar progenitors occurred ∼30.5 kya, followed by a period of migration between the two populations. Analysis of modern varieties, including lines historically introgressed with C. canephora , highlights their breeding histories and loci that may contribute to pathogen resistance, laying the groundwork for future genomics-based breeding of C. arabica .

Retinoic acid mediates most of the biological actions of vitamin A. It is oxidized by CYP26A1 to 4-oxoretinoic acid, considered as an inactive catabolite of retinoic acid. However, in the light of studies reporting the presence of 4-oxoretinal or 4-oxoretinol as the predominant retinoids during morphogenesis, we analyzed the retinoid-like biological activity of these oxoretinoids in mouse skin in vivo. Topical 4-oxoretinal and 4-oxoretinol promoted significant epidermal hyperplasia and metaplasia in mouse tail. They induced a moderate response for epidermal inflammation, compared with retinal, whereas neither 4-oxoretinal nor 4-oxoretinol prevented menadione-induced epidermal lipid peroxidation, unlike retinal and retinol. As analyzed by quantitative PCR, 4-oxoretinal and 4-oxoretinol did not reproduce the significant increased expression of genes coding for keratin 4, amphiregulin, heparin-EGF and CYP26A1, that did induce retinal and retinol. However, both retinal and 4-oxoretinal significantly inhibited the lipopolysaccharide-induced maturation of human dendritic cells in vitro. As analyzed in vivo and in vitro, 4-oxoretinal and 4-oxoretinol were not converted into retinoic acid. We conclude that 4-oxoretinal and 4-oxoretinol exert a moderate direct retinoid-like activity in vivo, thus confirming previous in vitro studies in amphibians showing 4-oxometabolites of vitamin A as bioactive agents rather than inactive catabolites.

Sustained exposure to a young systemic environment rejuvenates aged organisms and promotes cellular function. However, due to the intrinsic complexity of tissues it remains challenging to pinpoint niche-independent effects of circulating factors on specific cell populations. Here, we describe a method for the encapsulation of human and mouse skeletal muscle progenitors in diffusible polyethersulfone hollow fiber capsules that can be used to profile systemic aging in vivo independent of heterogeneous short-range tissue interactions. We observed that circulating long-range signaling factors in the old systemic environment lead to an activation of Myc and E2F transcription factors, induce senescence, and suppress myogenic differentiation. Importantly, in vitro profiling using young and old serum in 2D culture does not capture all pathways deregulated in encapsulated cells in aged mice. Thus, in vivo transcriptomic profiling using cell encapsulation allows for the characterization of effector pathways of systemic aging with unparalleled accuracy.

Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H2O2 release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H2O2 release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00×10−8) and 54 suggestive associations (p<1.00×10−5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and ∼2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H2O2 release was observed in Down Syndrome (DS) individuals (p<2.88×10−12). Taken together, our results show strong evidence of genetic control of H2O2 in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders.

Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that ∼50% of the transcriptional signals were located outside of annotated exons and were considered as TARs ( t ranscriptionally a ctive r egions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that ∼40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions.

Supplementary Tables 1-7 from Gene Expression Profiling Provides Insights into Pathways of Oxaliplatin-Related Sinusoidal Obstruction Syndrome in Humans

Supplementary Tables 1-7 from Gene Expression Profiling Provides Insights into Pathways of Oxaliplatin-Related Sinusoidal Obstruction Syndrome in Humans