Impact of round-the-clock CSF Gram stain on empirical therapy for suspected central nervous system infections.

The impact of round-the-clock cerebrospinal fluid (CSF) Gram stain on overnight empirical therapy for suspected central nervous system (CNS) infections was investigated. All consecutive overnight CSF Gram stains between 2006 and 2011 were included. The impact of a positive or a negative test on empirical therapy was evaluated and compared to other clinical and biological indications based on institutional guidelines. Bacterial CNS infection was documented in 51/241 suspected cases. Overnight CSF Gram stain was positive in 24/51. Upon validation, there were two false-positive and one false-negative results. The sensitivity and specificity were 41 and 99 %, respectively. All patients but one had other indications for empirical therapy than Gram stain alone. Upon obtaining the Gram result, empirical therapy was modified in 7/24, including the addition of an appropriate agent (1), addition of unnecessary agents (3) and simplification of unnecessary combination therapy (3/11). Among 74 cases with a negative CSF Gram stain and without formal indication for empirical therapy, antibiotics were withheld in only 29. Round-the-clock CSF Gram stain had a low impact on overnight empirical therapy for suspected CNS infections and was associated with several misinterpretation errors. Clinicians showed little confidence in CSF direct examination for simplifying or withholding therapy before definite microbiological results.

In vivo imaging of the central and peripheral effects of sleep deprivation and suprachiasmatic nuclei lesion on PERIOD-2 protein in mice.

STUDY OBJECTIVES: That sleep deprivation increases the brain expression of various clock genes has been well documented. Based on these and other findings we hypothesized that clock genes not only underlie circadian rhythm generation but are also implicated in sleep homeostasis. However, long time lags have been reported between the changes in the clock gene messenger RNA levels and their encoded proteins. It is therefore crucial to establish whether also protein levels increase within the time frame known to activate a homeostatic sleep response. We report on the central and peripheral effects of sleep deprivation on PERIOD-2 (PER2) protein both in intact and suprachiasmatic nuclei-lesioned mice. DESIGN: In vivo and in situ PER2 imaging during baseline, sleep deprivation, and recovery. SETTINGS: Mouse sleep-recording facility. PARTICIPANTS: Per2::Luciferase knock-in mice. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Six-hour sleep deprivation increased PER2 not only in the brain but also in liver and kidney. Remarkably, the effects in the liver outlasted those observed in the brain. Within the brain the increase in PER2 concerned the cerebral cortex mainly, while leaving suprachiasmatic nuclei (SCN) levels unaffected. Against expectation, sleep deprivation did not increase PER2 in the brain of arrhythmic SCN-lesioned mice because of higher PER2 levels in baseline. In contrast, liver PER2 levels did increase in these mice similar to the sham and partially lesioned controls. CONCLUSIONS: Our results stress the importance of considering both sleep-wake dependent and circadian processes when quantifying clock-gene levels. Because sleep deprivation alters PERIOD-2 in the brain as well as in the periphery, it is tempting to speculate that clock genes constitute a common pathway mediating the shared and well-known adverse effects of both chronic sleep loss and disrupted circadian rhythmicity on metabolic health.

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.

Epigenetic regulatory elements: Recent advances in understanding their mode of action and use for recombinant protein production in mammalian cells.

Successful generation of high producing cell lines requires the generation of cell clones expressing the recombinant protein at high levels and the characterization of the clones' ability to maintain stable expression levels. The use of cis-acting epigenetic regulatory elements that improve this otherwise long and uncertain process has revolutionized recombinant protein production. Here we review and discuss new insights into the molecular mode of action of the matrix attachment regions (MARs) and ubiquitously-acting chromatin opening elements (UCOEs), i.e. cis-acting elements, and how these elements are being used to improve recombinant protein production. These elements can help maintain the chromatin environment of the transgene genomic integration locus in a transcriptionally favorable state, which increases the numbers of positive clones and the transgene expression levels. Moreover, the high producing clones tend to be more stable in long-term cultures even in the absence of selection pressure. Therefore, by increasing the probability of isolating a high producing clone, as well as by increasing transcription efficiency and stability, these elements can significantly reduce the time and cost required for producing large quantities of recombinant proteins.

Circadian variation of salivary immunoglobin A, alpha-amylase activity and mood in response to repeated double-poling sprints in hypoxia.

PURPOSE: To assess the circadian variations in salivary immunoglobin A (sIgA) and alpha-amylase activity (sAA), biomarkers of mucosal immune function, together with mood during 2 weeks of repeated sprint training in hypoxia (RSH) and normoxia (RSN). METHODS: Over a 2-week period, 17 competitive cross-country skiers performed six training sessions, each consisting of four sets of five 10-s bouts of all-out double-poling under either normobaric hypoxia (FiO2: 13.8 %, 3000 m) or normoxia. The levels of sIgA and sAA activity and mood were determined five times during each of the first (T1) and sixth (T6) days of training, as well as during days preceding (baseline) and after the training intervention (follow-up). RESULTS: With RSH, sIgA was higher on T6 than T1 (P = 0.049), and sAA was increased on days T1, T6, and during the follow-up (P < 0.01). With RSN, sIgA remained unchanged and sAA was elevated on day T1 only (P = 0.04). Similarly, the RSH group demonstrated reduced mood on days T1, T6, and during the follow-up, while mood was lowered only on T1 with RSN (P < 0.01). CONCLUSIONS: The circadian variation of sIgA and sAA activity, biomarkers of mucosal immune function, as well as mood were similar on the first day of training when repeated double-poling sprints were performed with or without hypoxia. Only with RSH did the levels of sIgA and sAA activity rise with time, becoming maximal after six training sessions, when mood was still lowered. Therefore, six sessions of RSH reduced mood, but did not impair mucosal immune function.

Molecular dating of caprines using ancient DNA sequences of Myotragus balearicus, an extinct endemic Balearic mammal

Background: Myotragus balearicus was an endemic bovid from the Balearic Islands (Western Mediterranean) that became extinct around 6,000-4,000 years ago. The Myotragus evolutionary lineage became isolated in the islands most probably at the end of the Messinian crisis, when the desiccation of the Mediterranean ended, in a geological date established at 5.35 Mya. Thus, the sequences of Myotragus could be very valuable for calibrating the mammalian mitochondrial DNA clock and, in particular, the tree of the Caprinae subfamily, to which Myotragus belongs. Results: We have retrieved the complete mitochondrial cytochrome b gene (1,143 base pairs), plus fragments of the mitochondrial 12S gene and the nuclear 28S rDNA multi-copy gene from a well preserved Myotragus subfossil bone. The best resolved phylogenetic trees, obtained with the cytochrome b gene, placed Myotragus in a position basal to the Ovis group. Using the calibration provided by the isolation of Balearic Islands, we calculated that the initial radiation of caprines can be dated at 6.2 ± 0.4 Mya. In addition, alpine and southern chamois, considered until recently the same species, split around 1.6 ± 0.3 Mya, indicating that the two chamois species have been separated much longer than previously thought. Conclusion: Since there are almost no extant endemic mammals in Mediterranean islands, the sequence of the extinct Balearic endemic Myotragus has been crucial for allowing us to use the Messinian crisis calibration point for dating the caprines phylogenetic tree.

Tangential migration of neuronal precursors of glutamatergic neurons in the adult mammalian brain.

In a classic model of mammalian brain formation, precursors of principal glutamatergic neurons migrate radially along radial glia fibers whereas GABAergic interneuron precursors migrate tangentially. These migration modes have significant implications for brain function. Here we used clonal lineage tracing of active radial glia-like neural stem cells in the adult mouse dentate gyrus and made the surprising discovery that proliferating neuronal precursors of glutamatergic granule neurons exhibit significant tangential migration along blood vessels, followed by limited radial migration. Genetic birthdating and morphological and molecular analyses pinpointed the neuroblast stage as the main developmental window when tangential migration occurs. We also developed a partial "whole-mount" dentate gyrus preparation and observed a dense plexus of capillaries, with which only neuroblasts, among the entire population of progenitors, are directly associated. Together, these results provide insight into neuronal migration in the adult mammalian nervous system.

Post-partum persistence of abnormal circadian pattern of blood pressure after preeclampsia [109-POS]

OBJECTIVES: Blunted nocturnal dip of blood pressure (BP) and reversed circadian rhythm have been described in preeclampsia (PE). Non-dipper status and preeclampsia are both associated with an increased risk of cardiovascular disease later in life. Complete recovery of BP in PE is reported to occur over a variable period of time. Twenty-four hours-ambulatory blood pressure measurement (ABPM) in the post-partum follow-up after a PE has not been described. The aim of this study was to assess 24h-ambulatory blood pressure pattern after a PE and to determine the prevalence of non-dipper status, nocturnal hypertension, white coat hypertension and masked hypertension. METHODS: This is an observational, prospective study on women who suffered from a preeclampsia. A 24h-ABPM was done 6 weeks post-partum at the Hypertension Unit of the University Hospitals of Geneva, concomitantly with a clinical and biological evaluation. RESULTS: Forty-five women were included in a preliminary analysis. Mean age was 33±6years, 57.3% were Caucasian, mean BMI before pregnancy was 24±5kg/m(2). Office and ambulatory BP are shown in Table 1. Prevalence of nocturnal hypertension was high and half of the women had no nocturnal dipping. The diagnosis of hypertension based on office BP was discordant with the diagnosis based on ABPM in 25% of women. CONCLUSIONS: The prevalence of increased nighttime BP and abnormal BP pattern is high at 6weeks post-partum in preeclamptic women. Early assessment of BP with ABPM after preeclampsia allows an early identification of women with persistent circadian abnormalities who might be at increased risk. It also provides a more accurate assessment than office BP. DISCLOSURES: A. Ditisheim: None. B. Ponte: None. G. Wuerzner: None. M. Burnier: None. M. Boulvain: None. A. Pechère-Bertschi: None.

Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner.

Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover, mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation, repression of T-bet, enhanced mitochondrial spare respiratory capacity, and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore, mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions.

Altered Sleep Homeostasis in Rev-erbα Knockout Mice.

STUDY OBJECTIVES: The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. METHODS: EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. RESULTS: Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1-4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. CONCLUSIONS: Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context.

The life history of retrocopies illuminates the evolution of new mammalian genes.

New genes contribute substantially to adaptive evolutionary innovation, but the functional evolution of new mammalian genes has been little explored at a broad scale. Previous work established mRNA-derived gene duplicates, known as retrocopies, as models for the study of new gene origination. Here we combine mammalian transcriptomic and epigenomic data to unveil the processes underlying the evolution of stripped-down retrocopies into complex new genes. We show that although some robustly expressed retrocopies are transcribed from preexisting promoters, most evolved new promoters from scratch or recruited proto-promoters in their genomic vicinity. In particular, many retrocopy promoters emerged from ancestral enhancers (or bivalent regulatory elements) or are located in CpG islands not associated with other genes. We detected 88-280 selectively preserved retrocopies per mammalian species, illustrating that these mechanisms facilitated the birth of many functional retrogenes during mammalian evolution. The regulatory evolution of originally monoexonic retrocopies was frequently accompanied by exon gain, which facilitated co-option of distant promoters and allowed expression of alternative isoforms. While young retrogenes are often initially expressed in the testis, increased regulatory and structural complexities allowed retrogenes to functionally diversify and evolve somatic organ functions, sometimes as complex as those of their parents. Thus, some retrogenes evolved the capacity to temporarily substitute for their parents during the process of male meiotic X inactivation, while others rendered parental functions superfluous, allowing for parental gene loss. Overall, our reconstruction of the "life history" of mammalian retrogenes highlights retroposition as a general model for understanding new gene birth and functional evolution.