Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver.

Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5'-UTR (TISU) motif. The increased translation efficiency of 5'-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation.

Genome-wide analysis of SREBP1 activity around the clock reveals its combined dependency on nutrient and circadian signals.

In mammals, the circadian clock allows them to anticipate and adapt physiology around the 24 hours. Conversely, metabolism and food consumption regulate the internal clock, pointing the existence of an intricate relationship between nutrient state and circadian homeostasis that is far from being understood. The Sterol Regulatory Element Binding Protein 1 (SREBP1) is a key regulator of lipid homeostasis. Hepatic SREBP1 function is influenced by the nutrient-response cycle, but also by the circadian machinery. To systematically understand how the interplay of circadian clock and nutrient-driven rhythm regulates SREBP1 activity, we evaluated the genome-wide binding of SREBP1 to its targets throughout the day in C57BL/6 mice. The recruitment of SREBP1 to the DNA showed a highly circadian behaviour, with a maximum during the fed status. However, the temporal expression of SREBP1 targets was not always synchronized with its binding pattern. In particular, different expression phases were observed for SREBP1 target genes depending on their function, suggesting the involvement of other transcription factors in their regulation. Binding sites for Hepatocyte Nuclear Factor 4 (HNF4) were specifically enriched in the close proximity of SREBP1 peaks of genes, whose expression was shifted by about 8 hours with respect to SREBP1 binding. Thus, the cross-talk between hepatic HNF4 and SREBP1 may underlie the expression timing of this subgroup of SREBP1 targets. Interestingly, the proper temporal expression profile of these genes was dramatically changed in Bmal1-/- mice upon time-restricted feeding, for which a rhythmic, but slightly delayed, binding of SREBP1 was maintained. Collectively, our results show that besides the nutrient-driven regulation of SREBP1 nuclear translocation, a second layer of modulation of SREBP1 transcriptional activity, strongly dependent from the circadian clock, exists. This system allows us to fine tune the expression timing of SREBP1 target genes, thus helping to temporally separate the different physiological processes in which these genes are involved.

Impact of Sleep and Circadian Disruption on Energy Balance and Diabetes: A Summary of Workshop Discussions.

ABSTRACT: A workshop was held at the National Institute for Diabetes and Digestive and Kidney Diseases with a focus on the impact of sleep and circadian disruption on energy balance and diabetes. The workshop identified a number of key principles for research in this area and a number of specific opportunities. Studies in this area would be facilitated by active collaboration between investigators in sleep/circadian research and investigators in metabolism/diabetes. There is a need to translate the elegant findings from basic research into improving the metabolic health of the American public. There is also a need for investigators studying the impact of sleep/circadian disruption in humans to move beyond measurements of insulin and glucose and conduct more in-depth phenotyping. There is also a need for the assessments of sleep and circadian rhythms as well as assessments for sleep-disordered breathing to be incorporated into all ongoing cohort studies related to diabetes risk. Studies in humans need to complement the elegant short-term laboratory-based human studies of simulated short sleep and shift work etc. with studies in subjects in the general population with these disorders. It is conceivable that chronic adaptations occur, and if so, the mechanisms by which they occur needs to be identified and understood. Particular areas of opportunity that are ready for translation are studies to address whether CPAP treatment of patients with pre-diabetes and obstructive sleep apnea (OSA) prevents or delays the onset of diabetes and whether temporal restricted feeding has the same impact on obesity rates in humans as it does in mice.

Strokes restricted to the insular cortex

Résumé: L'objectif de l'étude est de caractériser la manifestation clinique d'une atteinte vasculaire cérébrale ischémique aiguë limitée au cortex insulaire, région intrigante et méconnue du cerveau humain. Dans la pratique clinique, une atteinte vasculaire aiguë limitée à l'insula, sans compromission d'autres régions cérébrales, est exceptionnelle et sa manifestation clinique neurologique est souvent non reconnue. L'étude est focalisée sur quatre patients, inscrits dans le Lausanne Stroke Registry, présentant une nouvelle atteinte vasculaire cérébrale avec une lésion unique purement limitée au cortex insulaire, objectivée à l'aide de la résonance magnétique (IRM). L'étude a mis en évidence cinq manifestations cliniques principales : 1) Troubles de la sensibilité corporelle sont révélé chez trois patients avec une atteinte insulaire postérieure (deux avec un syndrome pseudothalamique, un avec un déficit à distribution partielle). 2) Un patient avec une lésion insulaire postérieure gauche présent des troubles du goût. 3) Un syndrome pseudovestibulaire avec vertiges non rotatoires, instabilité à la marche sans nystagmus, est mis en évidence chez trois patients avec une atteinte ischémique insulaire postérieure. 4) Un patient avec atteinte de l'insula postérieure droite présente des épisodes d'hypertension artérielle d'origine cryptique. 5) Des troubles neuropsychologiques tels qu'aphasie et dysarthrie sont détectés chez les patients avec une atteinte insulaire postérieure gauche, un épisode de somatoparaphrénie est rapporté avec une atteinte insulaire postérieure droite. En conclusion, les atteintes vasculaires cérébrales ischémiques aiguës limitées au cortex insulaire postérieur peuvent se manifester principalement avec un tableau clinique caractérisé par un syndrome pseudothalamique associé à une symptomatologie pseudovertigineuse. Les lésions insulaires postérieures peuvent se manifester avec une dysarthrie et des troubles du goût, une aphasie (gauche), une somatoparaphrénie et une dysfonction hypertensive (droite). L'étude n'a pas mis en évidence de dysphagie, reportée dans les atteintes insulaires antérieures. Abstract: Objective: To characterize clinically acute insular strokes from four patients with, a first ever acute stroke restricted to the insula on MRI. Methods: The authors studied the clinical presentation of four patients with a first ever acute stroke restricted to the insula on MRI. Results: The authors found five main groups of clinical presentations: 1) somatosensory deficits in three patients with posterior insular stroke (two with a transient pseudothalamic sensory syndrome, one with partial distribution); 2) gustatory disorder in a patient with left posterior insular infarct; 3) vestibular-like syndrome, with dizziness, gait instability, and tendency to fall, but no nystagmus, in three patients with posterior insular strokes; 4) cardiovascular disturbances, consisting of hypertensive episodes in a patient with a right posterior insular infarct; and 5) neuropsychological disorders, including aphasia (left posterior insula), dysarthria, and transient somatoparaphrenia (right posterior insula). Conclusion: Strokes restricted to the posterior insula may present with pseudothalamic sensory and vestibular-like syndromes as prominent clinical manifestations, but also dysarthria and aphasia (in left lesions), somatoparaphrenia (right lesions) and gustatory dysfunction and blood pressure with hypertensive episodes in right lesions; we did not find acute dysphagia reported in anterior, insular strokes.

Pédiatrie [Pediatrics].

Several preliminary studies suggest that prophylactic administration of probiotics reduces the incidence of necrotizing enterocolitis (NEC) in preterm infants, and several neonatology units have introduced this treatment under strict surveillance. Nonetheless, breast milk feeding remains the mainstay of NEC prevention. The beta-blocker propranolol, known for its effectiveness on cutaneous hemangiomas, is also proving useful for the treatment of subglottic or visceral hemangiomas. Following the decrease in severe bacterial infections thanks to widespread vaccinations, the McCarthy clinical score has regained importance in the prediction of the risk of bacterial infection in febrile infants. It is easy to use, economical, and has a diagnostic value comparable to laboratory tests. The new WHO growth charts have been introduced in Switzerland in 2011 to take into account the increasing regional and ethnic variations in our country. Any significant change in growth velocity should prompt an evaluation of the need of further investigations.

Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters.

Soil pseudomonads increase their competitiveness by producing toxic secondary metabolites, which inhibit competitors and repel predators. Toxin production is regulated by cell-cell signalling and efficiently protects the bacterial population. However, cell communication is unstable, and natural populations often contain signal blind mutants displaying an altered phenotype defective in exoproduct synthesis. Such mutants are weak competitors, and we hypothesized that their fitness depends on natural communities on the exoproducts of wild-type bacteria, especially defence toxins. We established mixed populations of wild-type and signal blind, non-toxic gacS-deficient mutants of Pseudomonas fluorescens CHA0 in batch and rhizosphere systems. Bacteria were grazed by representatives of the most important bacterial predators in soil, nematodes (Caenorhabditis elegans) and protozoa (Acanthamoeba castellanii). The gacS mutants showed a negative frequency-dependent fitness and could reach up to one-third of the population, suggesting that they rely on the exoproducts of the wild-type bacteria. Both predators preferentially consumed the mutant strain, but populations with a low mutant load were resistant to predation, allowing the mutant to remain competitive at low relative density. The results suggest that signal blind Pseudomonas increase their fitness by exploiting the toxins produced by wild-type bacteria, and that predation promotes the production of bacterial defence compounds by selectively eliminating non-toxic mutants. Therefore, predators not only regulate population dynamics of soil bacteria but also structure the genetic and phenotypic constitution of bacterial communities.

Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.

Detecting epistasis with restricted response patterns in pairs of biallelic loci.

Well-established examples of genetic epistasis between a pair of loci typically show characteristic patterns of phenotypic distributions in joint genotype tables. However, inferring epistasis given such data is difficult due to the lack of power in commonly used approaches, which decompose the epistatic patterns into main plus interaction effects followed by testing the interaction term. Testing additive-only or all terms may have more power, but they are sensitive to nonepistatic patterns. Alternatively, the epistatic patterns of interest can be enumerated and the best matching one is found by searching through the possibilities. Although this approach requires multiple testing correction over possible patterns, each pattern can be fitted with a regression model with just one degree of freedom and thus the overall power can still be high, if the number of possible patterns is limited. Here we compare the power of the linear decomposition and pattern search methods, by applying them to simulated data generated under several patterns of joint genotype effects with simple biological interpretations. Interaction-only tests are the least powerful; while pattern search approach is the most powerful if the range of possibilities is restricted, but still includes the true pattern.

The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines.

The immunogenicity of a novel synthetic peptide consisting of an average of 40 (Asn-Ala-Asn-Pro) repeats of the circumsporozoite protein of Plasmodium falciparum, (NANP)40, was studied in mice without using any carrier proteins. First, high titers of anti-(NANP)40 antibodies could be obtained after immunization of C57BL/6 mice. These antibodies also reacted with an extract of mosquitoes infected with P. falciparum sporozoites. C57BL/6 nu/nu mice did not produce antibodies against (NANP)40. Secondly, when 14 strains of mice with nine different H-2 haplotypes were immunized with (NANP)40 without carrier, only H-2b mice were found to produce anti-(NANP)40 antibodies, whereas all non-H-2b mice were consistently unresponsive. This response was demonstrated to be I-A-linked by using recombinant and mutant mice. I-Ab [B10.A(5R)] mice produced anti-(NANP)40 antibodies as well as H-2b inbred mice. B6CH-2bm12 I-Ab-mutant mice showed only a very low response. Third, the antibody response against (NANP)40 could be induced in nonresponder mice by immunization with the peptide coupled to a carrier protein. In view of the existence of such an exceptional H-2b restriction in the response to sporozoite synthetic peptides in mice, the triggering of peptide-specific T cell responses in humans receiving sporozoite malaria vaccines might be difficult to achieve.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

Attenuated poxviruses expressing a synthetic HIV protein stimulate HLA-A2-restricted cytotoxic T-cell responses.

Efficient HIV vaccines have to trigger cell-mediated immunity directed against various viral antigens. However little is known about the breadth of the response induced by vaccines carrying multiple proteins. Here, we report on the immunogenicity of a construct harbouring a fusion of the HIV-1 IIIB gag, pol and nef genes (gpn) designed for optimal safety and equimolar expression of the HIV proteins. The attenuated poxviruses, MVA and NYVAC, harbouring the gpn construct, induced potent immune responses in conventional mice characterised by stimulation of Gpn-specific IFN-gamma-producing cells and cytotoxic T cells. In HLA-A2 transgenic mice, recombinant MVA elicited cytotoxic responses against epitopes recognised in most HLA-A2+ HIV-1-infected individuals. We also found that the MVA vaccine triggered the in vitro expansion of peripheral blood cells isolated from a HIV-1-seropositive patient and with similar specificity as found in immunised HLA-A2 transgenic mice. In conclusion, the synthetic HIV polyantigen Gpn delivered by MVA is immunogenic, efficiently processed and presented by human MHC class I molecules.

Fas ligand expression is restricted to nonlymphoid thymic components in situ.

The cell surface receptor Fas (Apo-1/CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in activation-induced death of mature T cells and in killing mediated by cytolytic T cells. The role of the Fas pathway in apoptosis associated with thymic selection events is, however, controversial. Although Fas and FasL are known to be expressed in the thymus, the nature and in vivo localization of FasL-expressing cells have not been determined. Using recently developed anti-FasL Abs in combination with in situ hybridization on tissue sections, we show in this work that FasL-expressing cells are present in the thymus, particularly within the medulla. FasL mRNA was detected readily in thymic stromal cell extracts, but not in isolated thymocytes. Moreover, immunohistochemical analysis of serial tissue sections stained with Abs against FasL in conjunction with epithelial and dendritic cell markers indicated that both thymic epithelial and dendritic cells express FasL in situ. The coexistence of FasL-expressing stromal cells and Fas-expressing thymocytes may have important implications for the role of the Fas pathway in apoptosis associated with thymic selection events.

Continuous monitoring of liver oxygenation with near infrared spectroscopy during naso-gastric tube feeding in neonates.

Near infrared spectroscopy (NIRS) is a non-invasive method of estimating the haemoglobin concentration changes in certain tissues. It is frequently used to monitor oxygenation of the brain in neonates. At present it is not clear whether near infrared spectroscopy of other organs (e.g. the liver as a corresponding site in the splanchnic region, which reacts very sensitively to haemodynamic instability) provides reliable values on their tissue oxygenation. The aim of the study was to test near infrared spectroscopy by measuring known physiologic changes in tissue oxygenation of the liver in newborn infants during and after feeding via a naso-gastric tube. The test-retest variability of such measurements was also determined. On 28 occasions in 25 infants we measured the tissue oxygenation index (TOI) of the liver and the brain continuously before, during and 30 minutes after feeding via a gastric tube. Simultaneously we measured arterial oxygen saturation (SaO2), heart rate (HR) and mean arterial blood pressure (MAP). In 10 other newborn infants we performed a test-retest analysis of the liver tissue oxygenation index to estimate the variability in repeated intra-individual measurements. The tissue oxygenation index of the liver increased significantly from 56.7 +/- 7.5% before to 60.3 +/- 5.6% after feeding (p < 0.005), and remained unchanged for the next 30 minutes. The tissue oxygenation index of the brain (62.1 +/- 9.7%), SaO2 (94.4 +/- 7.1%), heart rate (145 +/- 17.3 min-1) and mean arterial blood pressure (52.8 +/- 10.2 mm Hg) did not change significantly. The test-retest variability for intra-individual measurements was 2.7 +/- 2.1%. After bolus feeding the tissue oxygenation index of the liver increased as expected. This indicates that near infrared spectroscopy is suitable for monitoring changes in tissue oxygenation of the liver in newborn infants.

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.