Identification of novel and conserved microRNA and their expression in the gray mouse lemur, Microcebus murinus, a primate capable of daily torpor.

MicroRNA (miRNA) are endogenous small noncoding RNA gene products, on average 22 nt long, that play important regulatory roles in mediating gene expression by binding to and targeting mRNAs for degradation or translational repression. In this paper we identify both novel and conserved miRNA sequences present in the genome of the gray mouse lemur, Microcebus marinus. In total, 122 conserved and 44 novel miRNA were identified with high confidence from the lemur genome (Mmur_2.0) and were used for expression analysis. All conserved and novel miRNA were subjected to relative quantification by RT-qPCR in liver samples from control and torpid lemurs. A total of 26 miRNA (16 conserved and 10 novel) showed increased levels during primate torpor, whereas 31 (30 conserved and 1 novel) decreased. Additional in silico mapping of the predicted mRNA targets of torpor-responsive mature miRNA suggested that miRNA that increased during torpor were collectively involved in cell development and survival pathways, while miRNA that decreased were enriched in targeting immune function. Overall, the study suggests new regulatory mechanisms of primate torpor via miRNA action.

Caspase-1 activity affects AIM2 speck formation/stability through a negative feedback loop.

The inflammasome is an innate immune signaling platform leading to caspase-1 activation, maturation of pro-inflammatory cytokines and cell death. Recognition of DNA within the host cytosol induces the formation of a large complex composed of the AIM2 receptor, the ASC adaptor and the caspase-1 effector. Francisella tularensis, the agent of tularemia, replicates within the host cytosol. The macrophage cytosolic surveillance system detects Francisella through the AIM2 inflammasome. Upon Francisella novicida infection, we observed a faster kinetics of AIM2 speck formation in ASC(KO) and Casp1(KO) as compared to WT macrophages. This observation was validated by a biochemical approach thus demonstrating for the first time the existence of a negative feedback loop controlled by ASC/caspase-1 that regulates AIM2 complex formation/stability. This regulatory mechanism acted before pyroptosis and required caspase-1 catalytic activity. Our data suggest that sublytic caspase-1 activity could delay the formation of stable AIM2 speck, an inflammasome complex associated with cell death.

AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages.

The inflammasome is a signalling platform leading to caspase-1 activation. Caspase-1 causes pyroptosis, a necrotic-like cell death. AIM2 is an inflammasome sensor for cytosolic DNA. The adaptor molecule ASC mediates AIM2-dependent caspase-1 activation. To date, no function besides caspase-1 activation has been ascribed to the AIM2/ASC complex. Here, by comparing the effect of gene inactivation at different levels of the inflammasome pathway, we uncovered a novel cell death pathway activated in an AIM2/ASC-dependent manner. Francisella tularensis, the agent of tularaemia, triggers AIM2/ASC-dependent caspase-3-mediated apoptosis in caspase-1-deficient macrophages. We further show that AIM2 engagement leads to ASC-dependent, caspase-1-independent activation of caspase-8 and caspase-9 and that caspase-1-independent death is reverted upon caspase-8 inhibition. Caspase-8 interacts with ASC and active caspase-8 specifically colocalizes with the AIM2/ASC speck thus identifying the AIM2/ASC complex as a novel caspase-8 activation platform. Furthermore, we demonstrate that caspase-1-independent apoptosis requires the activation of caspase-9 and of the intrinsic pathway in a typical type II cell manner. Finally, we identify the AIM2/ASC-dependent caspase-1-independent pathway as an innate immune mechanism able to restrict bacterial replication in vitro and control IFN-γ levels in vivo in Casp1(KO) mice. This work underscores the crosstalk between inflammasome components and the apoptotic machinery and highlights the versatility of the pathway, which can switch from pyroptosis to apoptosis.

The grey mouse lemur: a non-human primate model for ageing studies.

The use of non-human primate models is required to understand the ageing process and evaluate new therapies against age-associated pathologies. The present article summarizes all the contributions of the grey mouse lemur Microcebus murinus, a small nocturnal prosimian primate, to the understanding of the mechanisms of ageing. Results from studies of both healthy and pathological ageing research on the grey mouse lemur demonstrated that this animal is a unique model to study age-dependent changes in endocrine systems, biological rhythms, thermoregulation, sensorial, cerebral and cognitive functions.

Gender markedly modulates behavioral thermoregulation in a non-human primate species, the mouse lemur (Microcebus murinus).

Age and gender are known to significantly modulate thermoregulatory capacities in mammals, suggesting strong impacts on behavioral adjustments, which are used to minimize the energy costs of thermoregulation. We tested the effects of sex and age on spontaneous choice of ambient temperature (Ta) in a non-human primate species, the mouse lemur (Microcebus murinus). The animals acclimated to both winter and summer photoperiods, two seasons significantly modifying thermoregulation function, were experimented in a thermal gradient device. During winter, adult males did not show preference for warm Tas whereas old males did. In contrast, female mouse lemurs of both age categories exhibited great preferences for warm Tas. Acclimation to summer revealed that males selected colder Ta for the day than during the night. Such behavior did not exist in females. Old females explored and selected warmer nests than adult ones. This study raised novel issues on the effect of gender on thermoregulatory capacities in the mouse lemur. Females probably use behavioral adjustments to limit energy expenditure and might prefer to preserve energy for maternal investment by anticipation of and during the breeding season. Further experiments focusing on female thermoregulatory capacities are needed to better understand the energy challenge that may occur during winter and summer in female mouse lemurs, and whether this trade-off changes during aging.

[Requests for active euthanasia: which reality in an oncology center.]. / Demandes d'euthanasie : quelle réalité dans un centre anticancéreux.

Euthanasia is a controversial issue in today's society. In countries where euthanasia is legal, it is mainly associated with people with cancer. We retrospectively studied the frequency and basis of patients' requests for active euthanasia in the oncology setting.MethodsRecurrent requests for euthanasia made by the patients of Leon-Berard cancer center (Lyon, France) between 2001 and 2003 were recorded by questioning the physicians and nurse supervisors in charge or by collecting information from the minutes of multidisciplinary palliative care meetings. We also collected information on the general health status of the patients, their motives and their evolution over time, as well as responses from caregivers.ResultsWe identified 16 requests for euthanasia. These involved 8 men, 7 women and 1 child (median age, 56 years), corresponding to 1% of the total deaths recorded during the period. In 2 cases, the request had come from the family only. The most frequent motives were psychological distress (38%), desire for self-autonomy (31%) and pain (31%). Half of the patients, particularly those striving for autonomy, persisted with their request until death, whereas 2 of 3 requests motivated by physical or psychological distress were not maintained. Sedation was administered to 3 patients in response to recurrent requests.ConclusionRequests for euthanasia in cancer patients are rare but may occur. Sometimes suffering is not relieved by palliative care and the request is maintained. Dealing with these patients puts caregivers in a difficult situation.

Effects of age on thermoregulatory responses during cold exposure in a nonhuman primate, Microcebus murinus.

Cold resistance appears altered with aging. Among existing hypotheses, the impaired capacity in response to cold could be related to an altered regulation of plasma IGF-1 concentration. The combined effects of age and cold exposure were studied in a short-living primate, the gray mouse lemur (Microcebus murinus), which adjusts its energy balance using a daily torpor phase, to avoid high energy cost of normothermia maintenance. Changes in body mass, core temperature, locomotor activity, and caloric intake were monitored under 9-day exposures to 25 degrees C and 12 degrees C in captive animals in winter conditions. Short-term (after 2 days) and long-term (after 9 days) cold-induced changes in IGF-1 levels were also evaluated. In thermoneutral conditions (25 degrees C), general characteristics of the daily rhythm of core temperature were preserved with age. At 12 degrees C, age-related changes were mainly characterized by a deeper hypothermia and an increased frequency of torpor phases, associated with a loss of body mass. A short-term cold-induced decrease in plasma IGF-1 levels was observed. IGF-1 levels returned to basal values after 9 days of cold exposure. No significant effect of age could be evidenced on IGF-1 response. However, IGF-1 levels of cold-exposed aged animals were negatively correlated with the frequency of daily torpor. Responses exhibited by aged mouse lemurs exposed to cold revealed difficulties in the maintenance of normothermia and energy balance and might involve modulations of IGF-1 levels.

Guías de práctica clínica: ejemplos en la anestesiología / Clinical practice guidelines: examples in anesthesiology

La medicina basada en las pruebas es una metodología interesante para optimizar lo que leemos, lo que aprendemos y lo que hacemos. Hoy en día, las guías de práctica clínica (GPC) son una de las herramientas más completas para recopilar la mejor evidencia científica disponible. Hay distintos tipos de GPC. Aquellas basadas en la evidencia, halladas en una búsqueda sistemática y exhaustiva de la bibliografía, y las que muestran recomendaciones considerando distintas variables, como efectos, beneficios, costos, inconvenientes y preferencias de los pacientes. Todas las GPC tienen como objetivo ayudarnos a mejorar nuestra práctica clínica y el cuidado de nuestros paciente.

Guías de práctica clínica: ejemplos en la anestesiología / Clinical practice guidelines: examples in anesthesiology

La medicina basada en las pruebas es una metodología interesante para optimizar lo que leemos, lo que aprendemos y lo que hacemos. Hoy en día, las guías de práctica clínica (GPC) son una de las herramientas más completas para recopilar la mejor evidencia científica disponible. Hay distintos tipos de GPC. Aquellas basadas en la evidencia, halladas en una búsqueda sistemática y exhaustiva de la bibliografía, y las que muestran recomendaciones considerando distintas variables, como efectos, beneficios, costos, inconvenientes y preferencias de los pacientes. Todas las GPC tienen como objetivo ayudarnos a mejorar nuestra práctica clínica y el cuidado de nuestros paciente. (AU)

Guías de práctica clínica: ejemplos en la anestesiología / Clinical practice guidelines: examples in anesthesiology

La medicina basada en las pruebas es una metodología interesante para optimizar lo que leemos, lo que aprendemos y lo que hacemos. Hoy en día, las guías de práctica clínica (GPC) son una de las herramientas más completas para recopilar la mejor evidencia científica disponible. Hay distintos tipos de GPC. Aquellas basadas en la evidencia, halladas en una búsqueda sistemática y exhaustiva de la bibliografía, y las que muestran recomendaciones considerando distintas variables, como efectos, beneficios, costos, inconvenientes y preferencias de los pacientes. Todas las GPC tienen como objetivo ayudarnos a mejorar nuestra práctica clínica y el cuidado de nuestros paciente. (AU)

Shortened seasonal photoperiodic cycles accelerate aging of the diurnal and circadian locomotor activity rhythms in a primate.

The gray mouse lemur (Microcebus murinus), a prosimian primate, exhibits seasonal rhythms strictly controlled by photoperiodic variations. Previous studies indicated that longevity can be altered by long-term acceleration of seasonal rhythms, providing a model for assessing various aspects of aging. To assess the effect of aging and accelerated aging on the circadian system of this primate, we compared the circadian rhythm of the locomotor activity in adult mouse lemurs (2-4.5 years, n = 9), aged mouse lemurs (5-9 years, n = 10), and adult mouse lemurs that had been exposed from birth to a shortened seasonal photoperiodic cycle (2-4.5 years, n = 7). Compared to adult animals, aged mouse lemurs showed a significant increase in intradaily variability and an advanced activity onset. Aging was characterized by a decrease in amplitude, with both a decrease in nocturnal activity and an increase in daytime activity. When maintained in constant dim red light, aged animals exhibited a shortening of the free-running period (22.8 +/- 0.1 h) compared to adult animals (23.5 +/- 0.1 h). A 3- to 5-year exposure to an accelerated seasonal photoperiodic rhythm ("annual" duration of 5 months) in accelerated mouse lemurs produced disturbances of the locomotor activity rhythm that resembled those of aged mouse lemurs, whether animals were studied in entrained or in free-running conditions. The present study demonstrated a weakened and fragmented locomotor activity rhythm during normal aging in this primate. Increasing the number of expressed seasonal cycles accelerated aging of parameters related to circadian rhythmicity in adult animals.

Social inhibition of seasonal fattening in wild and captive gray mouse lemurs.

Due to their differential reproductive investment, males and females often exhibit high differences in energy management. The main hypothesis tested in this study is that short photoperiod-induced fattening in the small primate Microcebus murinus is strongly affected by social cues. Short photoperiod-induced changes in body mass, lean mass and thyroid hormone T4 plasma levels measurements were investigated in wild and captive animals. Individual housing showed that both males and females are able to fatten. Fattening was associated with a slight increase of lean mass that reached a plateau after 3 weeks. In contrast, social grouping led to an inhibition of fattening, even when direct interactions were prevented. This social inhibition was mainly observed in males, especially when housed with females, suggesting that their low weight gain in the wild is due to female dominance. Accordingly, wild and captive animals of both sexes exhibited a decrease in T4 plasma levels, revealing a decrease in energy expenditure. Moreover, testis recrudescence observed after 20 weeks of short photoperiod exposure did not lead to any inhibition of fattening, indicating that males take advantage of female hibernation late in the dry season to fatten and use their fat reserves for the mating competition. Therefore, seasonal fattening may be used by females to prepare for hibernation and by males to prepare for the mating competition.

Relationship between urinary estrogen levels before conception and sex ratio at birth in a primate, the gray mouse lemur.

BACKGROUND: In primates, including humans, bias of sex ratio at birth has been reported to depend on maternal condition at conception. In a Malagasy primate, the gray mouse lemur, male-biased sex ratio at birth occurred in captive parous females. The male bias was previously demonstrated to be pre-conceptual and independent of the female's nutritional state. It was hypothesized to be related to changes in hormonal state at ovulation. METHODS AND RESULTS: The relationship between urinary estradiol (E2) levels during the follicular phase until estrus and litter production (number and sex of newborns) was assessed in 91 females mated under controlled conditions. Changes in E2 levels prior to ovulation followed the typical primate pattern characterized by a gradual rise during the 10 days preceding the sharp increase at estrus. A clear decline in E2 levels occurred with ageing. Direction of the sex ratio bias was unrelated to E2 levels at ovulation time but was significantly dependent on E2 levels during the follicular phase. Reduced E2 levels prior to estrus led to male-biased litters. CONCLUSION: This study suggests that hormonal stimulation during the follicular phase plays a role in shifting sex ratio at conception through changes in the local environment of the ova. This hypothesis deserves testing by assessing estrogen levels throughout the follicular phase in other primate species including humans.

Changes in olfactory inputs modify the energy balance response to short days in male gray mouse lemurs.

The role of olfaction/olfactory cues on photoperiodic responses was assessed in Malagasy primate, the gray mouse lemur. When exposed to short photoperiod (SP), this primate demonstrates rapid changes in energy balance as adaptive anticipatory response for winter survival. To follow early changes induced by SP exposure, body mass, food intake, resting metabolism (RMR) and free thyroxin levels in plasma (T4) were measured in males abruptly transferred to SP: six intact males (controls), eight males that underwent bilateral olfactory removal (BOX) and eight males exposed to male urinary cues (U-exposed). To assess the effect of SP exposure, two other groups were maintained for 6 weeks under LP: six controls and six BOX males. Whereas all studied parameters remained constant in controls and BOX males maintained under LP, exposure to SP led to different responses according to groups. In controls, SP exposure led to a regular increase in body mass and after 4 weeks under SP, plasma T4 levels, food consumption and RMR significantly decreased. Even if BOX males demonstrated hyperphagic patterns regardless of the photoperiod, an increase in body mass was also induced by SP exposure but without changes in RMR or food intake that were body mass-dependent. In U-exposed males, body mass gain was significantly reduced while food intake and RMR remained high. In both BOX and U-exposed males, SP exposure led to a transient but high increase in T4 levels compared to controls. These results suggest that olfaction/olfactory cues may delay the SP-mediated changes in energy balance.

Factors affecting the daily rhythm of body temperature of captive mouse lemurs (Microcebus murinus).

Microcebus murinus, a small nocturnal Malagasy primate, exhibits adaptive energy-saving strategies such as daily hypothermia and gregarious patterns during diurnal rest. To determine whether ambient temperature (T(a)), food restriction and nest sharing can modify the daily body temperature (T(b)) rhythm, T(b) was recorded by telemetry during winter in six males exposed to different ambient temperatures (T(a) = 25, 20, 15 degrees C) and/or to a total food restriction for 3 days depending on social condition (isolated versus pair-grouped). At 25 degrees C, the daily rhythm of T(b) was characterized by high T(b) values during the night and lower values during the day. Exposure to cold significantly decreased minimal T(b) values and lengthened the daily hypothermia. Under food restriction, minimal T(b) values were also markedly lowered. The combination of food restriction and cold induced further increases in duration and depth of torpor bouts, minimal T(b) reaching a level just above T(a). Although it influenced daily hypothermia less than environmental factors, nest sharing modified effects of cold and food restriction previously observed by lengthening duration of torpor but without increasing its depth. In response to external conditions, mouse lemurs may thus adjust their energy expenditures through daily modifications of both the duration and the depth of torpor.

[A young woman with severe hyponatremia]. / Hyponatrémie sévère chez une jeune femme.

We report here the case of a young woman, who came by herself at the emergency department, presenting with a severe hyponatremia (106 mmol/L), as shown by her initial biological report. The biological comment leads us to review the hyponatremia considering on one hand osmolarity and on the other hand volemia. Patient's tests results showed hyponatremia with hypoosmolarity and isovolemia, due to her potomania. At last, the authors expose the main guidelines for the correction of hyponatremia.

Daily hypothermia in captive grey mouse lemurs (Microcebus murinus): effects of photoperiod and food restriction.

The grey mouse lemur (Microcebus murinus) is a small nocturnal primate exhibiting daily torpor. In constant ambient temperature (22-24 degrees C), body temperature (Tb) and locomotor activity were monitored by telemetry in animals exposed to short (SP: 10 h light/day) or long (LP: 14 light/day) photoperiods. They were first fed ad libitum for 8 days and then subjected to 80% restricted feeding for 8 more days. During ad libitum feeding, locomotor activity was significantly lower in SP-exposed animals than in LP-exposed animals. Whatever the photoperiod, animals entered daily hypothermia within the first hours following the light onset. Depth of daily hypothermia increased irregularly under SP exposure, whereas minimal daily Tb was constantly above 35 degrees C under LP exposure. After the transfer from long photoperiod to short photoperiod corresponding to the induction of seasonal fattening, locomotor activity and depth of controlled daily hypothermia did not change significantly. In contrast, food restriction led to a significant increase in locomotor activity and in frequency of daily torpor (Tb<33 degrees C) and body temperature reached minimum values averaging 25 degrees C. However, SP-exposed animals exhibited lower minimal daily Tb and higher torpor duration than LP exposed animals. Therefore, daily torpor appears as a rapid response to food restriction occurring whatever the photoperiod, although enhanced by short photoperiod.

Brown fat and nonshivering thermogenesis in the gray mouse lemur (Microcebus murinus).

The gray mouse lemur Microcebus murinus is a rare example of a primate exhibiting daily torpor. In captive animals, we examined the metabolic rate during arousal from torpor and showed that this process involved nonshivering thermogenesis (NST). Under thermoneutrality (28 degrees C), warming-up from daily torpor (body temperature <33 degrees C) involved a rapid (<5 min) increase of O(2) consumption that was proportional to the depth of torpor (n = 8). The injection of a beta-adrenergic agonist (isoproterenol) known to elicit NST induced a dose-dependent increase in metabolic rate (n = 8). Moreover, maximum thermogenesis was increased by cold exposure. For the first time in this species, anatomic and histological examination using an antibody against uncoupling protein (UCP) specifically demonstrated the presence of brown fat. With the use of Western blotting with the same antibody, we showed a likely increase in UCP expression after cold exposure, suggesting that NST is also used to survive low ambient temperatures in this tropical species.

Characterization of low- and very-low-density hepatitis C virus RNA-containing particles.

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.

Quantitative assessment of testicular germ cell production and kinematic and morphometric parameters of ejaculated spermatozoa in the grey mouse lemur, Microcebus murinus.

Germ cell production and organization of the testicular epithelium in a prosimian species, the grey mouse lemur, Microcebus murinus, was investigated to extend knowledge of comparative primate spermatogenesis. In addition, semen samples collected from adult male lemurs (body weight 53-92 g; n = 16) by rectal probe electroejaculation were evaluated using computer-assisted morphometric and kinematic analysis of spermatozoa. Epididymidal spermatozoa were collected from six animals after hemicastration; the testes were weighed and prepared for stereological analysis and flow cytometry. The relative testis mass (as a percentage of body weight) ranged between 1.17 and 5.6%. Twelve stages of testicular seminiferous epithelium as described for macaques were applied and only a single stage was observed in most of the seminiferous tubule cross-sections. On average (mean SD), a single testis contained 1870 +/- 829 x 10(6) germ cells and 35 +/- 12 x 10(6) Sertoli cells. Germ cell ratios (preleptotene:type B spermatogonia = 2, round spermatid:pachytene = 3; elongated spermatid:round spermatids = 1) indicated high spermatogenic efficacy. Sperm head dimensions and tail lengths of the ejaculated and epididymidal spermatozoa were similar. Percentages of defects (neck/mid-piece and tail) were low ( 10%) and similar for ejaculated and epididymidal spermatozoa. Spermatozoa were highly motile, characterized by extensive lateral head displacement, but relatively low progressive motility. In conclusion, the grey mouse lemur has unusually large testes with a highly efficient spermatogenic process and large sperm output. These features, together with the high proportion of morphologically normal and highly motile spermatozoa in the ejaculates, indicate that Microcebus murinus is a species in which sperm competition after ejaculation is likely to occur. The predominantly single spermatogenic stage system seems to be an ancestral feature among primates.