An engineered multicellular stem cell niche for the 3D derivation of human myogenic progenitors from iPSCs.

Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.

Mitochondrial respiratory chain dysfunction alters ER sterol sensing and mevalonate pathway activity.

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.

Longitudinal Human Milk miRNA Composition over the First 3 mo of Lactation in a Cohort of Healthy Mothers Delivering Term Infants.

BACKGROUND: 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. OBJECTIVES: This study aimed to characterize the longitudinal profile of HM miRNA between the second week and third month postpartum. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Sialylated human milk oligosaccharides program cognitive development through a non-genomic transmission mode.

Breastmilk contains bioactive molecules essential for brain and cognitive development. While sialylated human milk oligosaccharides (HMOs) have been implicated in phenotypic programming, their selective role and underlying mechanisms remained elusive. Here, we investigated the long-term consequences of a selective lactational deprivation of a specific sialylated HMO in mice. We capitalized on a knock-out (KO) mouse model (B6.129-St6gal1tm2Jxm/J) lacking the gene responsible for the synthesis of sialyl(alpha2,6)lactose (6'SL), one of the two sources of sialic acid (Neu5Ac) to the lactating offspring. Neu5Ac is involved in the formation of brain structures sustaining cognition. To deprive lactating offspring of 6'SL, we cross-fostered newborn wild-type (WT) pups to KO dams, which provide 6'SL-deficient milk. To test whether lactational 6'SL deprivation affects cognitive capabilities in adulthood, we assessed attention, perseveration, and memory. To detail the associated endophenotypes, we investigated hippocampal electrophysiology, plasma metabolomics, and gut microbiota composition. To investigate the underlying molecular mechanisms, we assessed gene expression (at eye-opening and in adulthood) in two brain regions mediating executive functions and memory (hippocampus and prefrontal cortex, PFC). Compared to control mice, WT offspring deprived of 6'SL during lactation exhibited consistent alterations in all cognitive functions addressed, hippocampal electrophysiology, and in pathways regulating the serotonergic system (identified through gut microbiota and plasma metabolomics). These were associated with a site- (PFC) and time-specific (eye-opening) reduced expression of genes involved in central nervous system development. Our data suggest that 6'SL in maternal milk adjusts cognitive development through a short-term upregulation of genes modulating neuronal patterning in the PFC.

AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.

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.

Development and evaluation of a genome-wide Coffee 8.5K SNP array and its application for high-density genetic mapping and for investigating the origin of Coffea arabica L.

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 (2n = 4x = 44) originating from a hybridization event of the two diploid species C. canephora and C. eugenioides (2n = 2x = 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 SNPs 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 SNPs (~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 SNPs 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.

Protein-leucine ingestion activates a regenerative inflammo-myogenic transcriptome in skeletal muscle following intense endurance exercise.

Protein-leucine supplement ingestion following strenuous endurance exercise accentuates skeletal-muscle protein synthesis and adaptive molecular responses, but the underlying transcriptome is uncharacterized. In a randomized single-blind triple-crossover design, 12 trained men completed 100 min of high-intensity cycling then ingested 70/15/180/30 g protein-leucine-carbohydrate-fat (15LEU), 23/5/180/30 g (5LEU), or 0/0/274/30 g (CON) beverages during the first 90 min of a 240 min recovery period. Vastus lateralis muscle samples (30 and 240 min postexercise) underwent transcriptome analysis by microarray followed by bioinformatic analysis. Gene expression was regulated by protein-leucine in a dose-dependent manner affecting the inflammatory response and muscle growth and development. At 30 min, 15LEU and 5LEU vs. CON activated transcriptome networks with gene-set functions involving cell-cycle arrest (Z-score 2.0-2.7, P < 0.01), leukocyte maturation (1.7, P = 0.007), cell viability (2.4, P = 0.005), promyogenic networks encompassing myocyte differentiation and myogenin (MYOD1, MYOG), and a proteinaceous extracellular matrix, adhesion, and development program correlated with plasma lysine, arginine, tyrosine, taurine, glutamic acid, and asparagine concentrations. High protein-leucine dose (15LEU-5LEU) activated an IL-1I-centered proinflammatory network and leukocyte migration, differentiation, and survival functions (2.0-2.6, <0.001). By 240 min, the protein-leucine transcriptome was anti-inflammatory and promyogenic (IL-6, NF- ß, SMAD, STAT3 network inhibition), with overrepresented functions including decreased leukocyte migration and connective tissue development (-1.8-2.4, P < 0.01), increased apoptosis of myeloid and muscle cells (2.2-3.0, P < 0.002), and cell metabolism (2.0-2.4, P < 0.01). The analysis suggests protein-leucine ingestion modulates inflammatory-myogenic regenerative processes during skeletal muscle recovery from endurance exercise. Further cellular and translational research is warranted to validate amino acid-mediated myeloid and myocellular mechanisms within skeletal-muscle functional plasticity.

Protective effects of maternal nutritional supplementation with lactoferrin on growth and brain metabolism.

BACKGROUND: Intrauterine growth restriction (IUGR) is a major risk factor for both perinatal and long-term morbidity. Bovine lactoferrin (bLf) is a major milk glycoprotein considered as a pleiotropic functional nutrient. The impact of maternal supplementation with bLf on IUGR-induced sequelae, including inadequate growth and altered cerebral development, remains unknown. METHODS: IUGR was induced through maternal dexamethasone infusion (100 µg/kg during last gestational week) in rats. Maternal supplementation with bLf (0.85% in food pellet) was provided during both gestation and lactation. Pup growth was monitored, and Pup brain metabolism and gene expression were studied using in vivo (1)H NMR spectroscopy, quantitative PCR, and microarray in the hippocampus at postnatal day (PND)7. RESULTS: Maternal bLf supplementation did not change gestational weight but increased the birth body weight of control pups (4%) with no effect on the IUGR pups. Maternal bLf supplementation allowed IUGR pups to recover a normalized weight at PND21 (weaning) improving catch-up growth. Significantly altered levels of brain metabolites (γ-aminobutyric acid, glutamate, N-acetylaspartate, and N-acetylaspartylglutamate) and transcripts (brain-derived neurotrophic factor (BDNF), divalent metal transporter 1 (DMT-1), and glutamate receptors) in IUGR pups were normalized with maternal bLf supplementation. CONCLUSION: Our data suggest that maternal bLf supplementation is a beneficial nutritional intervention able to revert some of the IUGR-induced sequelae, including brain hippocampal changes.

A dual-color PAX7 and MYF5 in vivo reporter to investigate muscle stem cell heterogeneity in regeneration and aging.

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.

The genome and population genomics of allopolyploid Coffea arabica reveal the diversification history of modern coffee cultivars.

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.

Effects of increase in fish oil intake on intestinal eicosanoids and inflammation in a mouse model of colitis.

BACKGROUND: Inflammatory bowel diseases (IBD) are chronic intestinal inflammatory diseases affecting about 1% of western populations. New eating behaviors might contribute to the global emergence of IBD. Although the immunoregulatory effects of omega-3 fatty acids have been well characterized in vitro, their role in IBD is controversial. METHODS: The aim of this study was to assess the impact of increased fish oil intake on colonic gene expression, eicosanoid metabolism and development of colitis in a mouse model of IBD. Rag-2 deficient mice were fed fish oil (FO) enriched in omega-3 fatty acids i.e. EPA and DHA or control diet for 4 weeks before colitis induction by adoptive transfer of naïve T cells and maintained in the same diet for 4 additional weeks. Onset of colitis was monitored by colonoscopy and further confirmed by immunological examinations. Whole genome expression profiling was made and eicosanoids were measured by HPLC-MS/MS in colonic samples. RESULTS: A significant reduction of colonic proinflammatory eicosanoids in FO fed mice compared to control was observed. However, neither alteration of colonic gene expression signature nor reduction in IBD scores was observed under FO diet. CONCLUSION: Thus, increased intake of dietary FO did not prevent experimental colitis.

Adipose tissue angiogenesis genes are down-regulated by grape polyphenols supplementation during a human overfeeding trial.

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.

Mice with humanized livers reveal the role of hepatocyte clocks in rhythmic behavior.

The synchronization of circadian clock depends on a central pacemaker located in the suprachiasmatic nuclei. However, the potential feedback of peripheral signals on the central clock remains poorly characterized. To explore whether peripheral organ circadian clocks may affect the central pacemaker, we used a chimeric model in which mouse hepatocytes were replaced by human hepatocytes. Liver humanization led to reprogrammed diurnal gene expression and advanced the phase of the liver circadian clock that extended to muscle and the entire rhythmic physiology. Similar to clock-deficient mice, liver-humanized mice shifted their rhythmic physiology more rapidly to the light phase under day feeding. Our results indicate that hepatocyte clocks can affect the central pacemaker and offer potential perspectives to apprehend pathologies associated with altered circadian physiology.

Network Analyses Reveal Negative Link Between Changes in Adipose Tissue GDF15 and BMI During Dietary-induced Weight Loss.

CONTEXT: Adipose tissue (AT) transcriptome studies provide holistic pictures of adaptation to weight and related bioclinical settings changes. OBJECTIVE: To implement AT gene expression profiling and investigate the link between changes in bioclinical parameters and AT gene expression during 3 steps of a 2-phase dietary intervention (DI). METHODS: AT transcriptome profiling was obtained from sequencing 1051 samples, corresponding to 556 distinct individuals enrolled in a weight loss intervention (8-week low-calorie diet (LCD) at 800 kcal/day) followed with a 6-month ad libitum randomized DI. Transcriptome profiles obtained with QuantSeq sequencing were benchmarked against Illumina RNAseq. Reverse transcription quantitative polymerase chain reaction was used to further confirm associations. Cell specificity was assessed using freshly isolated cells and THP-1 cell line. RESULTS: During LCD, 5 modules were found, of which 3 included at least 1 bioclinical variable. Change in body mass index (BMI) connected with changes in mRNA level of genes with inflammatory response signature. In this module, change in BMI was negatively associated with changes in expression of genes encoding secreted protein (GDF15, CCL3, and SPP1). Through all phases of the DI, change in GDF15 was connected to changes in SPP1, CCL3, LIPA and CD68. Further characterization showed that these genes were specific to macrophages (with LIPA, CD68 and GDF15 expressed in anti-inflammatory macrophages) and GDF15 also expressed in preadipocytes. CONCLUSION: Network analyses identified a novel AT feature with GDF15 upregulated with calorie restriction induced weight loss, concomitantly to macrophage markers. In AT, GDF15 was expressed in preadipocytes and macrophages where it was a hallmark of anti-inflammatory cells.

Nicotinamide riboside kinase 1 protects against diet and age-induced pancreatic ß-cell failure.

OBJECTIVE: Disturbances in NAD+ metabolism have been described as a hallmark for multiple metabolic and age-related diseases, including type 2 diabetes. While alterations in pancreatic ß-cell function are critical determinants of whole-body glucose homeostasis, the role of NAD+ metabolism in the endocrine pancreas remains poorly explored. Here, we aimed to evaluate the role of nicotinamide riboside (NR) metabolism in maintaining NAD+ levels and pancreatic ß-cell function in pathophysiological conditions. METHODS: Whole body and pancreatic ß-cell-specific NRK1 knockout (KO) mice were metabolically phenotyped in situations of high-fat feeding and aging. We also analyzed pancreatic ß-cell function, ß-cell mass and gene expression. RESULTS: We first demonstrate that NRK1, the essential enzyme for the utilization of NR, is abundantly expressed in pancreatic ß-cells. While NR treatment did not alter glucose-stimulated insulin secretion in pancreatic islets from young healthy mice, NRK1 knockout mice displayed glucose intolerance and compromised ß-cells response to a glucose challenge upon high-fat feeding or aging. Interestingly, ß cell dysfunction stemmed from the functional failure of other organs, such as liver and kidney, and the associated changes in circulating peptides and hormones, as mice lacking NRK1 exclusively in ß-cells did not show altered glucose homeostasis. CONCLUSIONS: This work unveils a new physiological role for NR metabolism in the maintenance of glucose tolerance and pancreatic ß-cell function in high-fat feeding or aging conditions.

In vivo transcriptomic profiling using cell encapsulation identifies effector pathways of systemic aging.

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.

Transcriptome and translational signaling following endurance exercise in trained skeletal muscle: impact of dietary protein.

Postexercise protein feeding regulates the skeletal muscle adaptive response to endurance exercise, but the transcriptome guiding these adaptations in well-trained human skeletal muscle is uncharacterized. In a crossover design, eight cyclists ingested beverages containing protein, carbohydrate and fat (PTN: 0.4, 1.2, 0.2 g/kg, respectively) or isocaloric carbohydrate and fat (CON: 1.6, 0.2 g/kg) at 0 and 1 h following 100 min of cycling. Biopsies of the vastus lateralis were collected at 3 and 48 h following to determine the early and late transcriptome and regulatory signaling responses via microarray and immunoblot. The top gene ontology enriched by PTN were: muscle contraction, extracellular matrix--signaling and structure, and nucleoside, nucleotide, and nucleic acid metabolism (3 and 48 h); developmental processes, immunity, and defense (3 h); glycolysis, lipid and fatty acid metabolism (48 h). The transcriptome was also enriched within axonal guidance, actin cytoskeletal, Ca2+, cAMP, MAPK, and PPAR canonical pathways linking protein nutrition to exercise-stimulated signaling regulating extracellular matrix, slow-myofibril, and metabolic gene expression. At 3 h, PTN attenuated AMPKα1Thr172 phosphorylation but increased mTORC1Ser2448, rps6Ser240/244, and 4E-BP1-γ phosphorylation, suggesting increased translation initiation, while at 48 h AMPKα1Thr172 phosphorylation and PPARG and PPARGC1A expression increased, supporting the late metabolic transcriptome, relative to CON. To conclude, protein feeding following endurance exercise affects signaling associated with cell energy status and translation initiation and the transcriptome involved in skeletal muscle development, slow-myofibril remodeling, immunity and defense, and energy metabolism. Further research should determine the time course and posttranscriptional regulation of this transcriptome and the phenotype responding to chronic postexercise protein feeding.

Contribution of genetic ancestry and polygenic risk score in meeting vitamin B12 needs in healthy Brazilian children and adolescents.

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.

The absence of the caffeine synthase gene is involved in the naturally decaffeinated status of Coffea humblotiana, a wild species from Comoro archipelago.

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.

Comparative gene expression profiling between human cultured myotubes and skeletal muscle tissue.

BACKGROUND: A high-sensitivity DNA microarray platform requiring nanograms of RNA input facilitates the application of transcriptome analysis to individual skeletal muscle (SM) tissue samples. Culturing myotubes from SM-biopsies enables investigating transcriptional defects and assaying therapeutic strategies. This study compares the transcriptome of aneurally cultured human SM cells versus that of tissue biopsies. RESULTS: We used the Illumina expression BeadChips to determine the transcriptomic differences between tissue and cultured SM samples from five individuals. Changes in the expression of several genes were confirmed by QuantiGene Plex assay or reverse transcription real-time PCR. In cultured myotubes compared to the tissue, 1216 genes were regulated: 583 down and 633 up. Gene ontology analysis showed that downregulated genes were mainly associated with cytoplasm, particularly mitochondria, and involved in metabolism and the muscle-system/contraction process. Upregulated genes were predominantly related to cytoplasm, endoplasmic reticulum, and extracellular matrix. The most significantly regulated pathway was mitochondrial dysfunction. Apoptosis genes were also modulated. Among the most downregulated genes detected in this study were genes encoding metabolic proteins AMPD1, PYGM, CPT1B and UCP3, muscle-system proteins TMOD4, MYBPC1, MYOZ1 and XIRP2, the proteolytic CAPN3 and the myogenic regulator MYF6. Coordinated reduced expression of five members of the GIMAP gene family, which form a cluster on chromosome 7, was shown, and the GIMAP4-reduction was validated. Within the most upregulated group were genes encoding senescence/apoptosis-related proteins CDKN1A and KIAA1199 and potential regulatory factors HIF1A, TOP2A and CCDC80. CONCLUSIONS: Cultured muscle cells display reductive metabolic and muscle-system transcriptome adaptations as observed in muscle atrophy and they activate tissue-remodeling and senescence/apoptosis processes.