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1.  Late-born intermittently fasted juvenile garden dormice use torpor to grow and fatten prior to hibernation: consequences for ageing processes 
Torpor is thought to slow age-related processes and to sustain growth and fattening of young individuals. Energy allocation into these processes represents a challenge for juveniles, especially for those born late in the season. We tested the hypothesis that late-born juvenile garden dormice (Eliomys quercinus) fed ad libitum (‘AL’, n = 9) or intermittently fasted (‘IF’, n = 9) use short torpor bouts to enhance growth and fat accumulation to survive winter. IF juveniles displayed more frequent and longer torpor bouts, compared with AL individuals before hibernation. Torpor frequency correlated negatively with energy expenditure and water turnover. Hence, IF juveniles gained mass at the same rate, reached similar pre-hibernation fattening and displayed identical hibernating patterns and mass losses as AL animals. We found no group differences in relative telomere length (RTL), an indicator of ageing, during the period of highest summer mass gain, despite greater torpor use by IF juveniles. Percentage change in RTL was negatively associated with mean and total euthermic durations among all individuals during hibernation. We conclude that torpor use promotes fattening in late-born juvenile dormice prior to hibernation. Furthermore, we provided the first evidence for a functional link between time spent in euthermy and ageing processes over winter.
PMCID: PMC4240977  PMID: 25377448
Eliomys quercinus; intermittent fasting; pre-hibernation fattening; winter survival; doubly labelled water; telomere length
2.  Seasonal variation in haematological and biochemical variables in free-ranging subadult brown bears (Ursus arctos) in Sweden 
BMC Veterinary Research  2015;11:301.
Free-ranging brown bears exhibit highly contrasting physiological states throughout the year. They hibernate 6 months of the year, experiencing a decrease in body temperature, heart rate, respiratory rate and metabolism. An increase in food consumption and the resulting weight gain (mostly through fat storage) prior to hibernation are also part of the brown bear’s annual cycle. Due to these physiological changes, haematological and biochemical variables vary dramatically throughout the year. Seasonal changes in 12 haematological and 34 biochemical variables were evaluated in blood samples collected from 40 free-ranging subadult brown bears (22 females, 18 males) immobilised in Sweden in winter (February-March), spring (April-May), and summer (June).
Higher levels of haemoglobin, haematocrit and red blood cell count, and a lower white blood cell count and mean cell volume was found during hibernation than in spring and summer. Lower values of the enzymes; aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (AP), γ-glutamyl transpeptidase (GGT), glutamate dehydrogenase (GD) and amylase, and increased values of β-hydroxybutyrate (β-HBA) and blood lipids; triglycerides, cholesterol and free fatty acids, were present during hibernation compared to spring and summer.
This study documents significant shifts in haematological and biochemical variables in samples collected from brown bears anaesthetised in winter (February-March) compared to in spring and summer (April-June), reflecting the lowered metabolic, renal and hepatic activity during hibernation. Lower values of enzymes and higher values of blood lipids during hibernation, likely reflect a lipid-based metabolism.
PMCID: PMC4673763  PMID: 26646442
Brown bear; Ursus arctos; Haematology; Biochemistry; Seasonality
3.  Factors Affecting Date of Implantation, Parturition, and Den Entry Estimated from Activity and Body Temperature in Free-Ranging Brown Bears 
PLoS ONE  2014;9(7):e101410.
Knowledge of factors influencing the timing of reproduction is important for animal conservation and management. Brown bears (Ursus arctos) are able to vary the birth date of their cubs in response to their fat stores, but little information is available about the timing of implantation and parturition in free-ranging brown bears. Body temperature and activity of pregnant brown bears is higher during the gestation period than during the rest of hibernation and drops at parturition. We compared mean daily body temperature and activity levels of pregnant and nonpregnant females during preimplantation, gestation, and lactation. Additionally we tested whether age, litter size, primiparity, environmental conditions, and the start of hibernation influence the timing of parturition. The mean date of implantation was 1 December (SD = 12), the mean date of parturition was 26 January (SD = 12), and the mean duration of the gestation period was 56 days (SD = 2). The body temperature of pregnant females was higher during the gestation and lactation periods than that of nonpregnant bears. The body temperature of pregnant females decreased during the gestation period. Activity recordings were also used to determine the date of parturition. The parturition dates calculated with activity and body temperature data did not differ significantly and were the same in 50% of the females. Older females started hibernation earlier. The start of hibernation was earlier during years with favorable environmental conditions. Dates of parturition were later during years with good environmental conditions which was unexpected. We suggest that free-ranging pregnant brown bears in areas with high levels of human activities at the beginning of the denning period, as in our study area, might prioritize investing energy in early denning than in early parturition during years with favorable environmental conditions, as a strategy to prevent disturbances caused by human.
PMCID: PMC4079694  PMID: 24988486
4.  Emperors in Hiding: When Ice-Breakers and Satellites Complement Each Other in Antarctic Exploration 
PLoS ONE  2014;9(6):e100404.
Evaluating the demographic trends of marine top predators is critical to understanding the processes involved in the ongoing rapid changes in Antarctic ecosystems. However, the remoteness and logistical complexity of operating in Antarctica, especially during winter, make such an assessment difficult. Satellite imaging is increasingly recognised as a valuable method for remote animal population monitoring, yet its accuracy and reliability are still to be fully evaluated. We report here the first ground visit of an emperor penguin colony first discovered by satellite, but also the discovery of a second one not indicated by satellite survey at that time. Several successive remote surveys in this coastal region of East Antarctica, both before and after sudden local changes, had indeed only identified one colony. These two colonies (with a total of ca. 7,400 breeding pairs) are located near the Mertz Glacier in an area that underwent tremendous habitat change after the glacier tongue broke off in February 2010. Our findings therefore suggest that a satellite survey, although offering a major advance since it allows a global imaging of emperor penguin colonies, may miss certain colony locations when challenged by certain features of polar ecosystems, such as snow cover, evolving ice topology, and rapidly changing habitat. Moreover our survey shows that this large seabird has considerable potential for rapid adaptation to sudden habitat loss, as the colony detected in 2009 may have moved and settled on new breeding grounds. Overall, the ability of emperor penguin colonies to relocate following habitat modification underlines the continued need for a mix of remote sensing and field surveys (aerial photography and ground counts), especially in the less-frequented parts of Antarctica, to gain reliable knowledge about the population demography and dynamics of this flagship species of the Antarctic ecosystem.
PMCID: PMC4070948  PMID: 24963661
5.  Virus Factories of Cauliflower Mosaic Virus Are Virion Reservoirs That Engage Actively in Vector Transmission 
Journal of Virology  2013;87(22):12207-12215.
Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.
PMCID: PMC3807932  PMID: 24006440
6.  Early interactions during the encounter of plants, aphids and arboviruses 
Plant Signaling & Behavior  2013;8(6):e24225.
Aphids infest many plants and cause damage by depriving them of nutrients and by transmitting many viral diseases. Aphid infestation and arbovirus transmission are controlled by establishment (or not) of a compatible reaction between the insects and the plants. This reaction is the result of defense reactions of the plant and counter-defense reactions of the parasite. Contrarily to plant-bacteria, plant-fungi and plant-herbivorous insects pathosystems, the plant-aphid pathosystem is understudied, although recent advances have begun to uncover some of its details. Especially the very early steps in plant-aphid interactions are hardly known. We here resume the present knowledge of these interactions. We discuss further how an aphid-transmitted plant virus that is transmitted during the first moments of the plant-aphid encounter, might help to study the very early plant aphid interactions.
PMCID: PMC3907434  PMID: 23518584
aphid; plant; virus; arbovirus; transmission; signaling; calcium; ROS; immunity; elicitor; effector
7.  A virus responds instantly to the presence of the vector on the host and forms transmission morphs 
eLife  2013;2:e00183.
Many plant and animal viruses are spread by insect vectors. Cauliflower mosaic virus (CaMV) is aphid-transmitted, with the virus being taken up from specialized transmission bodies (TB) formed within infected plant cells. However, the precise events during TB-mediated virus acquisition by aphids are unknown. Here, we show that TBs react instantly to the presence of the vector by ultra-rapid and reversible redistribution of their key components onto microtubules throughout the cell. Enhancing or inhibiting this TB reaction pharmacologically or by using a mutant virus enhanced or inhibited transmission, respectively, confirming its requirement for efficient virus-acquisition. Our results suggest that CaMV can perceive aphid vectors, either directly or indirectly by sharing the host perception. This novel concept in virology, where viruses respond directly or via the host to the outside world, opens new research horizons, that is, investigating the impact of ‘perceptive behaviors’ on other steps of the infection cycle.
eLife digest
Viruses are infectious agents that can replicate only inside a living host cell. When a virus infects an animal or plant, it introduces its own genetic material and tricks the host cells into producing viral proteins that can be used to assemble new viruses. An essential step in the life cycle of any virus is transmission to a new host: understanding this process can be crucial in the fight against viral epidemics.
Many viruses use living organisms, or vectors, to move between hosts. In the case of plant viruses such as cauliflower mosaic virus, the vectors are often aphids. When an aphid sucks sap out of a leaf, virus particles already present in the leaf become attached to its mouth, and these viruses can be transferred to the next plant that the insect feeds on. However, in order for cauliflower mosaic virus particles to become attached to the aphid, structures called transmission bodies must form beforehand in the infected plant cells. These structures are known to contain helper proteins that bind the viruses to the mouth of the aphid, but the precise role of the transmission body has remained obscure.
Now Martinière et al. show that the transmission body is in fact a dynamic structure that reacts to the presence of aphids and, in so doing, boosts the efficiency of viral transmission. In particular, they show that the action of an aphid feeding on an infected leaf triggers a rapid and massive influx of a protein called tubulin into the transmission body. The transmission body then bursts open, dispersing helper protein-virus particle complexes throughout the cell, where they become more accessible to aphids. This series of events increases viral transmission rates twofold to threefold.
The results show that a virus can detect insect vectors, likely by using the sensory system of its host, and trigger a response that boosts viral uptake and thus transmission. This is a novel concept in virology. It will be important to discover whether similar mechanisms are used by other viruses, including those that infect animals and humans.
PMCID: PMC3552618  PMID: 23358702
aphids; transmission; cell–virus–vector interactions; Arabidopsis; Viruses; Other
8.  Body Temperature during Hibernation Is Highly Correlated with a Decrease in Circulating Innate Immune Cells in the Brown Bear (Ursus arctos): A Common Feature among Hibernators? 
Background: Hibernation involves periods of severely depressed metabolism (torpor) and decreases in body temperature (Tb). Small arctic mammals (<5kg), in which Tb generally drop drastically, display leukopenia during hibernation. This raised the question of whether the decreased leukocyte counts in mammalian hibernators is due to torpor per se or is secondary to low Tb. The present study examined immune cell counts in brown bears (Ursus arctos), where torpor is only associated with shallow decreases in Tb. The results were compared across hibernator species for which immune and Tb data were available.
Methods and Results: The white blood cell counts were determined by flow cytometry in 13 bears captured in the field both during summer and winter over 2 years time. Tb dropped from 39.6±0.8 to 33.5±1.1°C during hibernation. Blood neutrophils and monocytes were lower during hibernation than during the active period (47%, p= 0.001; 43%, p=0.039, respectively), whereas no change in lymphocyte counts was detected (p=0.599). Further, combining our data and those from 10 studies on 9 hibernating species suggested that the decline in Tb explained the decrease in innate immune cells (R2=0.83, p<0.0001).
Conclusions: Bears have fewer innate immune cells in circulation during hibernation, which may represent a suppressed innate immune system. Across species comparison suggests that, both in small and large hibernators, Tb is the main driver of immune function regulation during winter dormancy. The lack of a difference in lymphocyte counts in this context requires further investigations.
PMCID: PMC3607235  PMID: 23532623
Brown bear; Ursus arctos; Hibernation; Innate immunity; Leukocytes; Torpor.
9.  Circulating Virus Load Determines the Size of Bottlenecks in Viral Populations Progressing within a Host 
PLoS Pathogens  2012;8(11):e1003009.
For any organism, population size, and fluctuations thereof, are of primary importance in determining the forces driving its evolution. This is particularly true for viruses—rapidly evolving entities that form populations with transient and explosive expansions alternating with phases of migration, resulting in strong population bottlenecks and associated founder effects that increase genetic drift. A typical illustration of this pattern is the progression of viral disease within a eukaryotic host, where such demographic fluctuations are a key factor in the emergence of new variants with altered virulence. Viruses initiate replication in one or only a few infection foci, then move through the vasculature to seed secondary infection sites and so invade distant organs and tissues. Founder effects during this within-host colonization might depend on the concentration of infectious units accumulating and circulating in the vasculature, as this represents the infection dose reaching new organs or “territories”. Surprisingly, whether or not the easily measurable circulating (plasma) virus load directly drives the size of population bottlenecks during host colonization has not been documented in animal viruses, while in plants the virus load within the sap has never been estimated. Here, we address this important question by monitoring both the virus concentration flowing in host plant sap, and the number of viral genomes founding the population in each successive new leaf. Our results clearly indicate that the concentration of circulating viruses directly determines the size of bottlenecks, which hence controls founder effects and effective population size during disease progression within a host.
Author Summary
Infecting viruses progress within multi-cellular hosts via two distinct mechanisms: cell-to-cell proximal contamination and long-distance migration to remote organs through the vasculature. In distant susceptible organs, it seems logical that the number of initially infected cells, the number of viral genomes entering each of these cells, and thus the number of founders of new viral “colonies”, depends on the concentration of infectious units transported in the vasculature. For any organism, the number of founders colonizing a “virgin territory”, is of prime importance in determining the forces driving its evolution. This is particularly true for viruses where the so-called founder effect is a key factor in the emergence of new variants with altered virulence. It is surprising to note, however, that whether the circulating virus load directly drives the size of viral populations during host colonization remains elusive. By monitoring for the first time the virus concentration flowing in host plant sap, in parallel with the number of viral genome founders in each successive leaf, we provide unequivocal evidence that the concentration of circulating viruses can directly determine the founder effect and effective population size during disease progression in a eucaryotic host.
PMCID: PMC3486874  PMID: 23133389
10.  Effects of Chronic Calorie Restriction or Dietary Resveratrol Supplementation on Insulin Sensitivity Markers in a Primate, Microcebus murinus 
PLoS ONE  2012;7(3):e34289.
The prevalence of diabetes and hyperinsulinemia increases with age, inducing metabolic failure and limiting lifespan. Calorie restriction (CR) without malnutrition delays the aging process, but its long-term application to humans seems difficult. Resveratrol (RSV), a dietary polyphenol, appears to be a promising CR mimetic that can be easily administered in humans. In this work, we hypothesized that both CR and RSV impact insulin sensitivity in a non-human primate compared to standard-fed control (CTL) animals. Four- to five-year-old male grey mouse lemurs (Microcebus murinus) were assigned to three dietary groups: a CTL group, a CR group receiving 30% fewer calories than the CTL and a RSV group receiving the CTL diet supplemented with RSV (200 mg·day−1·kg−1). Insulin sensitivity and glycemia were assessed using an oral glucose tolerance test (OGTT) and the homeostasis model assessment of insulin resistance (HOMA-IR index) evaluation after 21 or 33 months of chronic treatment. Resting metabolic rate was also measured to assess the potential relationships between this energy expenditure parameter and insulin sensitivity markers. No differences were found after a 21-month period of treatment, except for lower glucose levels 30 min after glucose loading in CR animals. After 33 months, CR and RSV decreased glycemia after the oral glucose loading without decreasing fasting blood insulin. A general effect of treatment was observed on the HOMA-IR index, with an 81% reduction in CR animals and 53% in RSV animals after 33 months of treatment compared to CTL. Chronic CR and dietary supplementation with RSV affected insulin sensitivity by improving the glucose tolerance of animals without disturbing their baseline insulin secretion. These results suggest that both CR and RSV have beneficial effects on metabolic alterations, although these effects are different in amplitude between the two anti-aging treatments and potentially rely on different metabolic changes.
PMCID: PMC3316613  PMID: 22479589
11.  Private Heat for Public Warmth: How Huddling Shapes Individual Thermogenic Responses of Rabbit Pups 
PLoS ONE  2012;7(3):e33553.
Within their litter, young altricial mammals compete for energy (constraining growth and survival) but cooperate for warmth. The aim of this study was to examine the mechanisms by which huddling in altricial infants influences individual heat production and loss, while providing public warmth. Although considered as a textbook example, it is surprising to note that physiological mechanisms underlying huddling are still not fully characterised.
Methodology/Principal Findings
The brown adipose tissue (BAT) contribution to energy output was assessed as a function of the ability of rabbit (Oryctolagus cuniculus) pups to huddle (placed in groups of 6 and 2, or isolated) and of their thermoregulatory capacities (non-insulated before 5 days old and insulated at ca. 10 days old). BAT contribution of pups exposed to cold was examined by combining techniques of infrared thermography (surface temperature), indirect calorimetry (total energy expenditure, TEE) and telemetry (body temperature). Through local heating, the huddle provided each pup whatever their age with an ambient “public warmth” in the cold, which particularly benefited non-insulated pups. Huddling allowed pups facing a progressive cold challenge to buffer the decreasing ambient temperature by delaying the activation of their thermogenic response, especially when fur-insulated. In this way, huddling permitted pups to effectively shift from a non-insulated to a pseudo-insulated thermal state while continuously allocating energy to growth. The high correlation between TEE and the difference in surface temperatures between BAT and back areas of the body reveals that energy loss for non-shivering thermogenesis is the major factor constraining the amount of energy allocated to growth in non-insulated altricial pups.
By providing public warmth with minimal individual costs at a stage of life when pups are the most vulnerable, huddling buffers cold challenges and ensures a constant allocation of energy to growth by reducing BAT activation.
PMCID: PMC3306396  PMID: 22438947
12.  Effect of prolonged standardized bed rest on cystatin C and other markers of cardiovascular risk 
BMC Physiology  2011;11:17.
Sedentary lifestyle is associated with coronary artery disease but even shorter periods of physical inactivity may increase cardiovascular risk. Cystatin C is independently associated with cardiovascular disease and our objective was to investigate the relation between this novel biomarker and standardized bed rest. Research of immobilization physiology in humans is challenging because good biological models are in short supply. From the Women International Space simulation for Exploration study (WISE) we studied markers of atherosclerosis and kidney function, including cystatin C, in a standardized bed rest study on healthy volunteers. Fifteen healthy female volunteers participated in a 20-day ambulatory control period followed by 60 days of bed rest in head-down tilt position (-6°) 24 h a day, finalized by 20 days of recovery. The subjects were randomized into two groups during bed rest: a control group (n = 8) that remained physically inactive and an exercise group (n = 7) that participated in both supine resistance and aerobic exercise training.
Compared to baseline values there was a statistically significant increase in cystatin C in both groups after bed rest (P < 0.001). Glomerular filtration rate (GFR), calculated by both cystatin C and Cockcroft-Gault equation, decreased after bed rest while there were no differences in creatinine or creatine kinase levels. CRP did not change during bed rest in the exercise group, but there was an increase of CRP in the control group during recovery compared to both the baseline and the bed rest periods. The apo-B/apo-Ai ratio increased during bed rest and decreased again in the recovery period. Subjects experienced a small but statistically significant reduction in weight during bed rest and compared to baseline weights remained lower at day 8 of recovery.
During and following prolonged standardized bed rest the concentrations of several clinically relevant cardiovascular risk markers change.
PMCID: PMC3298483  PMID: 22152087
13.  VAPA, an Innovative “Virus-Acquisition Phenotyping Assay” Opens New Horizons in Research into the Vector-Transmission of Plant Viruses 
PLoS ONE  2011;6(8):e23241.
Host-to-host transmission—a key step in plant virus infection cycles—is ensured predominantly by vectors, especially aphids and related insects. A deeper understanding of the mechanisms of virus acquisition, which is critical to vector-transmission, might help to design future virus control strategies, because any newly discovered molecular or cellular process is a potential target for hampering viral spread within host populations. With this aim in mind, an aphid membrane-feeding assay was developed where aphids transmitted two non-circulative viruses [cauliflower mosaic virus (CaMV) and turnip mosaic virus] from infected protoplasts. In this assay, virus acquisition occurs exclusively from living cells. Most interestingly, we also show that CaMV is less efficiently transmitted by aphids in the presence of oryzalin—a microtubule-depolymerising drug. The example presented here demonstrates that our technically simple “virus-acquisition phenotyping assay” (VAPA) provides a first opportunity to implement correlative studies relating the physiological state of infected plant cells to vector-transmission efficiency.
PMCID: PMC3154327  PMID: 21853093
14.  Distribution of the Phenotypic Effects of Random Homologous Recombination between Two Virus Species 
PLoS Pathogens  2011;7(5):e1002028.
Recombination has an evident impact on virus evolution and emergence of new pathotypes, and has generated an immense literature. However, the distribution of phenotypic effects caused by genome-wide random homologous recombination has never been formally investigated. Previous data on the subject have promoted the implicit view that most viral recombinant genomes are likely to be deleterious or lethal if the nucleotide identity of parental sequences is below 90%. We decided to challenge this view by creating a bank of near-random recombinants between two viral species of the genus Begomovirus (Family Geminiviridae) exhibiting 82% nucleotide identity, and by testing infectivity and in planta accumulation of recombinant clones randomly extracted from this bank. The bank was created by DNA-shuffling—a technology initially applied to the random shuffling of individual genes, and here implemented for the first time to shuffle full-length viral genomes. Together with our previously described system allowing the direct cloning of full-length infectious geminivirus genomes, it provided a unique opportunity to generate hundreds of “mosaic” virus genomes, directly testable for infectivity. A subset of 47 randomly chosen recombinants was sequenced, individually inoculated into tomato plants, and compared with the parental viruses. Surprisingly, our results showed that all recombinants were infectious and accumulated at levels comparable or intermediate to that of the parental clones. This indicates that, in our experimental system, despite the fact that the parental genomes differ by nearly 20%, lethal and/or large deleterious effects of recombination are very rare, in striking contrast to the common view that has emerged from previous studies published on other viruses.
Author Summary
Recombination creates new genome combinations by joining genome fragments of distinct “parental” origin. This phenomenon, frequent in viral populations, combines mutations originally present on distinct parental genomes, increasing genetic diversity and creating “offspring” with altered biological properties. Consistently, recombination is often associated with the emergence of economically important viruses, with modified host range and virulence. In fact, recombination events can be lethal, deleterious or beneficial, but the respective frequency of these phenotypic effects is unknown and unpredictable. A generally accepted view, which we formally challenge in the present paper, is that most viral recombination events are deleterious or lethal when the parental sequences diverge by more than 10%. However, at present, no dedicated data set supports this supposition. We generated hundreds of “mosaic” genomes randomly from two plant virus species diverging by 18%, and tested a subset of 47 of these recombinants for viability and within-host accumulation. Surprisingly, all were viable, replicated, and accumulated at a pace comparable to that of the parents. Our results are in striking contrast to the current view, and show that viral recombination can have little phenotypic effect, at least in some cases, even when the parental sequences diverge by far more than 10%.
PMCID: PMC3088723  PMID: 21573141
15.  Structural Insights into the Molecular Mechanisms of Cauliflower Mosaic Virus Transmission by Its Insect Vector▿ †  
Journal of Virology  2010;84(9):4706-4713.
Cauliflower mosaic virus (CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex. Here, we describe the conformations of both unliganded CaMV P3 protein and its virion-associated form. X-ray crystallography revealed that the N-terminal domain of unliganded P3 is a tetrameric parallel coiled coil with a unique organization showing two successive four-stranded subdomains with opposite supercoiling handedness stabilized by a ring of interchain disulfide bridges. A structural model of virus-liganded P3 proteins, folding as an antiparallel coiled-coil network coating the virus surface, was derived from molecular modeling. Our results highlight the structural and biological versatility of this coiled-coil structure and provide new insights into the molecular mechanisms involved in CaMV acquisition and transmission by the insect vector.
PMCID: PMC2863735  PMID: 20181714
16.  Dynamics of the Multiplicity of Cellular Infection in a Plant Virus 
PLoS Pathogens  2010;6(9):e1001113.
Recombination, complementation and competition profoundly influence virus evolution and epidemiology. Since viruses are intracellular parasites, the basic parameter determining the potential for such interactions is the multiplicity of cellular infection (cellular MOI), i.e. the number of viral genome units that effectively infect a cell. The cellular MOI values that prevail in host organisms have rarely been investigated, and whether they remain constant or change widely during host invasion is totally unknown. Here, we fill this experimental gap by presenting the first detailed analysis of the dynamics of the cellular MOI during colonization of a host plant by a virus. Our results reveal ample variations between different leaf levels during the course of infection, with values starting close to 2 and increasing up to 13 before decreasing to initial levels in the latest infection stages. By revealing wide dynamic changes throughout a single infection, we here illustrate the existence of complex scenarios where the opportunity for recombination, complementation and competition among viral genomes changes greatly at different infection phases and at different locations within a multi-cellular host.
Author Summary
Viruses are fast evolving organisms for which changes in fitness and virulence are driven by interactions between genomes such as recombination, functional complementation, and competition. Viruses being intra-cellular parasites, one basic parameter determines the potential for such interactions: the cellular multiplicity of infection (cellular MOI), defined as the number of genome units actually penetrating and co-replicating within individual cells of the host. Despite its importance for virus evolution, this trait has scarcely been investigated. For example, there are only three point estimates for eukaryote-infecting viruses while the possibility that the cellular MOI may vary during the infection or across organs of a given host individual has never been conclusively addressed. By monitoring the cellular MOI in plants infected by the Cauliflower mosaic virus we found remarkably ample variations during the development of the infection process in successive leaf levels. Our results reveal that the opportunities for recombination, complementation and competition among viral genomes can greatly change at different infection phases and at different locations within a multi-cellular host.
PMCID: PMC2940754  PMID: 20862320
17.  Resveratrol suppresses body mass gain in a seasonal non-human primate model of obesity 
BMC Physiology  2010;10:11.
Resveratrol, a natural polyphenolic compound, was shown to protect rodents against high-fat-diet induced diabesity by boosting energy metabolism. To the best of our knowledge, no data is yet available on the effects of resveratrol in non-human primates. Six non-human heterotherm primates (grey mouse lemurs, Microcebus murinus) were studied during four weeks of dietary supplementation with resveratrol (200 mg/kg/day) during their winter body-mass gain period. Body mass, spontaneous energy intake, resting metabolic rate, spontaneous locomotor activity and daily variations in body temperature were measured. In addition, the plasma levels of several gut hormones involved in satiety control were evaluated.
Resveratrol reduced the seasonal body-mass gain by concomitantly decreasing energy intake by 13% and increasing resting metabolic rate by 29%. Resveratrol supplementation inhibited the depth of daily torpor, an important energy-saving process in this primate. The daily amount of locomotor activity remained unchanged. Except for an increase in the glucose-dependent insulinotropic polypeptide, a gut hormone known to promote mobilization of fat stores, no major change in satiety hormone plasma levels was observed under resveratrol supplementation.
These results suggest that in a non-human primate, resveratrol reduces body-mass gain by increasing satiety and resting metabolic rate, and by inhibiting torpor expression. The measured anorectic gut hormones did not seem to play a major role in these observations.
PMCID: PMC2903570  PMID: 20569453
18.  Caloric restriction or resveratrol supplementation and ageing in a non-human primate: first-year outcome of the RESTRIKAL study in Microcebus murinus 
Age  2010;33(1):15-31.
A life-long follow-up of physiological and behavioural functions was initiated in 38-month-old mouse lemurs (Microcebus murinus) to test whether caloric restriction (CR) or a potential mimetic compound, resveratrol (RSV), can delay the ageing process and the onset of age-related diseases. Based on their potential survival of 12 years, mouse lemurs were assigned to three different groups: a control (CTL) group fed ad libitum, a CR group fed 70% of the CTL caloric intake and a RSV group (200 mg/–1) fed ad libitum. Since this prosimian primate exhibits a marked annual rhythm in body mass gain during winter, animals were tested throughout the year to assess body composition, daily energy expenditure (DEE), resting metabolic rate (RMR), physical activity and hormonal levels. After 1 year, all mouse lemurs seemed in good health. CR animals showed a significantly decreased body mass compared with the other groups during long day period only. CR or RSV treatments did not affect body composition. CR induced a decrease in DEE without changes in RMR, whereas RSV induced a concomitant increase in DEE and RMR without any obvious modification of locomotor activity in both groups. Hormonal levels remained similar in each group. In summary, after 1 year of treatment CR and RSV induced differential metabolic responses but animals successfully acclimated to their imposed diets. The RESTRIKAL study can now be safely undertaken on a long-term basis to determine whether age-associated alterations in mouse lemurs are delayed with CR and if RSV can mimic these effects.
PMCID: PMC3063642  PMID: 20532988
Ageing; Food restriction; Resveratrol; Energy balance; Biomarkers; Doubly labelled water method
19.  Total Energy Expenditure and Body Composition in Two Free-Living Sympatric Lemurs 
PLoS ONE  2010;5(3):e9860.
Evolutionary theories that account for the unusual socio-ecological traits and life history features of group-living prosimians, compared with other primates, predict behavioral and physiological mechanisms to conserve energy. Low energy output and possible fattening mechanisms are expected, as either an adaptive response to drastic seasonal fluctuations of food supplies in Madagascar, or persisting traits from previously nocturnal hypometabolic ancestors. Free ranging ring-tailed lemurs (Lemur catta) and brown lemurs (Eulemur sp.) of southern Madagascar have different socio-ecological characteristics which allow a test of these theories: Both gregarious primates have a phytophagous diet but different circadian activity rhythms, degree of arboreality, social systems, and slightly different body size.
Methodology and Results
Daily total energy expenditure and body composition were measured in the field with the doubly labeled water procedure. High body fat content was observed at the end of the rainy season, which supports the notion that individuals need to attain a sufficient physical condition prior to the long dry season. However, ring-tailed lemurs exhibited lower water flux rates and energy expenditure than brown lemurs after controlling for body mass differences. The difference was interpreted to reflect higher efficiency for coping with seasonally low quality foods and water scarcity. Daily energy expenditure of both species was much less than the field metabolic rates predicted by various scaling relationships found across mammals.
We argue that low energy output in these species is mainly accounted for by low basal metabolic rate and reflects adaptation to harsh, unpredictable environments. The absence of observed sex differences in body weight, fat content, and daily energy expenditure converge with earlier investigations of physical activity levels in ring-tailed lemurs to suggest the absence of a relationship between energy constraints and the evolution of female dominance over males among lemurs. Nevertheless, additional seasonal data are required to provide a definitive conclusion.
PMCID: PMC2845615  PMID: 20360848
20.  Physical Inactivity Differentially Alters Dietary Oleate and Palmitate Trafficking 
Diabetes  2009;58(2):367-376.
OBJECTIVE— Obesity and diabetes are characterized by the incapacity to use fat as fuel. We hypothesized that this reduced fat oxidation is secondary to a sedentary lifestyle.
RESEARCH DESIGN AND METHODS— We investigated the effect of a 2-month bed rest on the dietary oleate and palmitate trafficking in lean women (control group, n = 8) and the effect of concomitant resistance/aerobic exercise training as a countermeasure (exercise group, n = 8). Trafficking of stable isotope–labeled dietary fats was combined with muscle gene expression and magnetic resonance imaging–derived muscle fat content analyses.
RESULTS— In the control group, bed rest increased the cumulative [1-13C]oleate and [d31]palmitate appearance in triglycerides (37%, P = 0.009, and 34%, P = 0.016, respectively) and nonesterified fatty acids (NEFAs) (37%, P = 0.038, and 38%, P = 0.002) and decreased muscle lipoprotein lipase (P = 0.043) and fatty acid translocase CD36 (P = 0.043) mRNA expressions. Plasma NEFA-to-triglyceride ratios for [1-13C]oleate and [d31]palmitate remained unchanged, suggesting that the same proportion of tracers enters the peripheral tissues after bed rest. Bed rest did not affect [1-13C]oleate oxidation but decreased [d31]palmitate oxidation by −8.2 ± 4.9% (P < 0.0001). Despite a decreased spontaneous energy intake and a reduction of 1.9 ± 0.3 kg (P = 0.001) in fat mass, exercise training did not mitigate these alterations but partially maintained fat-free mass, insulin sensitivity, and total lipid oxidation in fasting and fed states. In both groups, muscle fat content increased by 2.7% after bed rest and negatively correlated with the reduction in [d31]palmitate oxidation (r2 = 0.48, P = 0.003).
CONCLUSIONS— While saturated and monounsaturated fats have similar plasma trafficking and clearance, physical inactivity affects the partitioning of saturated fats toward storage, likely leading to an accumulation of palmitate in muscle fat.
PMCID: PMC2628610  PMID: 19017764
21.  The Grey Mouse Lemur Uses Season-Dependent Fat or Protein Sparing Strategies to Face Chronic Food Restriction 
PLoS ONE  2010;5(1):e8823.
During moderate calorie restriction (CR) the heterotherm Microcebus murinus is able to maintain a stable energy balance whatever the season, even if only wintering animals enter into torpor. To understand its energy saving strategies to respond to food shortages, we assessed protein and energy metabolisms associated with wintering torpor expression or summering torpor avoidance. We investigated body composition, whole body protein turnover, and daily energy expenditure (DEE), during a graded (40 and 80%) 35-day CR in short-days (winter; SD40 and SD80, respectively) and long-days (summer; LD40 and LD80, respectively) acclimated animals. LD40 animals showed no change in fat mass (FM) but a 12% fat free mass (FFM) reduction. Protein balance being positive after CR, the FFM loss was early and rapid. The 25% DEE reduction, in LD40 group was mainly explained by FFM changes. LD80 animals showed a steady body mass loss and were excluded from the CR trial at day 22, reaching a survival-threatened body mass. No data were available for this group. SD40 animals significantly decreased their FM level by 21%, but maintained FFM. Protein sparing was achieved through a 35 and 39% decrease in protein synthesis and catabolism (protein turnover), respectively, overall maintaining nitrogen balance. The 21% reduction in energy requirement was explained by the 30% nitrogen flux drop but also by torpor as DEE FFM-adjusted remained 13% lower compared to ad-libitum. SD80 animals were unable to maintain energy and nitrogen balances, losing both FM and FFM. Thus summering mouse lemurs equilibrate energy balance by a rapid loss of active metabolic mass without using torpor, whereas wintering animals spare protein and energy through increased torpor expression. Both strategies have direct fitness implication: 1) to maintain activities at a lower body size during the mating season and 2) to preserve an optimal wintering muscle mass and function.
PMCID: PMC2809095  PMID: 20098678
22.  A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses 
Virology Journal  2008;5:135.
Viruses of the genus Begomovirus (Geminiviridae) are emerging economically important plant viruses with a circular, single-stranded DNA genome. Previous studies have shown that geminiviruses and RNA viruses exhibit similar mutation frequencies, although geminiviruses are replicated by host DNA polymerases and RNA viruses by their own virus-encoded error-prone RNA-dependent RNA-polymerase. However, the phenotypic effects of naturally occurring mutations have never been extensively investigated in geminiviruses, particularly because, to be infectious, cloned viral genomes usually require sub-cloning as complete or partial tandem repeats into a binary vector from Agrobacterium tumefaciens.
Using Tomato yellow leaf curl virus (TYLCV), we show here that infectivity can be obtained when only a 41-nucleotide region containing a highly conserved stem-loop is repeated. A binary vector containing this 41-nt region and a unique restriction site was created, allowing direct cloning of infectious monomeric viral genomes provided that they harbour the same restriction site at the corresponding nucleotide position. This experimental system, which can be transferable to other geminiviruses, was validated by analysis of the phenotypic effect of mutations appearing in TYLCV genomes in a single tomato host plant originally inoculated with a unique viral sequence. Fourteen full-length infectious genomes extracted from this plant were directly cloned and sequenced. The mutation frequency was 1.38 × 10-4 mutation per nucleotide sequenced, similar to that found previously for another begomovirus by sequencing PCR-amplified partial sequences. Interestingly, even in this minimal pool of analysed genomes, mutants with altered properties were readily identified, one of them being fitter and reducing plant biomass more drastically than the parental clone.
The cloning strategy presented here is useful for any extensive phenotyping of geminivirus variants and particularly of artificially generated mutants or recombinants.
PMCID: PMC2585570  PMID: 18976479
23.  Large Bottleneck Size in Cauliflower Mosaic Virus Populations during Host Plant Colonization 
PLoS Pathogens  2008;4(10):e1000174.
The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Despite their high mutation rate and rapid evolution, this parameter is poorly documented experimentally in viruses, particularly plant viruses. All available studies, however, have demonstrated the existence of huge within-host demographic fluctuations, drastically reducing Ne upon systemic invasion of different organs and tissues. Notably, extreme bottlenecks have been detected at the stage of systemic leaf colonization in all plant viral species investigated so far, sustaining the general idea that some unknown obstacle(s) imposes a barrier on the development of all plant viruses. This idea has important implications, as it appoints genetic drift as a constant major force in plant virus evolution. By co-inoculating several genetic variants of Cauliflower mosaic virus into a large number of replicate host plants, and by monitoring their relative frequency within the viral population over the course of the host systemic infection, only minute stochastic variations were detected. This allowed the estimation of the CaMV Ne during colonization of successive leaves at several hundreds of viral genomes, a value about 100-fold higher than that reported for any other plant virus investigated so far, and indicated the very limited role played by genetic drift during plant systemic infection by this virus. These results suggest that the barriers that generate bottlenecks in some plant virus species might well not exist, or can be surmounted by other viruses, implying that severe bottlenecks during host colonization do not necessarily apply to all plant-infecting viruses.
Author Summary
Whether selection or stochastic genetic drift is the major force driving the evolution of a virus depends largely on the size of the viral population, with the former being predominant in large populations and the latter taking over when population sizes are transiently or durably reduced. This question has been intensively debated in both plant and animal viruses, as demographic fluctuations throughout viral life cycles are poorly understood. In plant viruses, an extremely small population size—down to a few founder genome units colonizing each leaf—has been formally estimated in two instances, and all other virus species investigated so far have consistently been shown to undergo extreme demographic bottlenecks during systemic invasion of their host. This situation conveys the general idea that all viruses are confronted with “universal barriers” in plants, imposing repeated transient decreases in their population size, thus making genetic drift a major constant driver of their evolution. Here, using the example of Cauliflower mosaic virus, we mitigate this general idea by showing that at least one virus species can overcome such putative limiting barriers and massively invade leaves with hundreds to thousands of founding genome units.
PMCID: PMC2553192  PMID: 18846207
24.  Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples 
BMC Genomics  2008;9:85.
Pathogens such as fungi, bacteria and especially viruses, are highly variable even within an individual host, intensifying the difficulty of distinguishing and accurately quantifying numerous allelic variants co-existing in a single nucleic acid sample. The majority of currently available techniques are based on real-time PCR or primer extension and often require multiplexing adjustments that impose a practical limitation of the number of alleles that can be monitored simultaneously at a single locus.
Here, we describe a novel method that allows the simultaneous quantification of numerous allelic variants in a single reaction tube and without multiplexing. Quantitative Single-letter Sequencing (QSS) begins with a single PCR amplification step using a pair of primers flanking the polymorphic region of interest. Next, PCR products are submitted to single-letter sequencing with a fluorescently-labelled primer located upstream of the polymorphic region. The resulting monochromatic electropherogram shows numerous specific diagnostic peaks, attributable to specific variants, signifying their presence/absence in the DNA sample. Moreover, peak fluorescence can be quantified and used to estimate the frequency of the corresponding variant in the DNA population.
Using engineered allelic markers in the genome of Cauliflower mosaic virus, we reliably monitored six different viral genotypes in DNA extracted from infected plants. Evaluation of the intrinsic variance of this method, as applied to both artificial plasmid DNA mixes and viral genome populations, demonstrates that QSS is a robust and reliable method of detection and quantification for variants with a relative frequency of between 0.05 and 1.
This simple method is easily transferable to many other biological systems and questions, including those involving high throughput analysis, and can be performed in any laboratory since it does not require specialized equipment.
PMCID: PMC2276495  PMID: 18291029
25.  Effect of Physical Inactivity on the Oxidation of Saturated and Monounsaturated Dietary Fatty Acids: Results of a Randomized Trial  
PLoS Clinical Trials  2006;1(5):e27.
Changes in the way dietary fat is metabolized can be considered causative in obesity. The role of sedentary behavior in this defect has not been determined. We hypothesized that physical inactivity partitions dietary fats toward storage and that a resistance exercise training program mitigates storage.
We used bed rest, with randomization to resistance training, as a model of physical inactivity.
The trial took place at the Space Clinic (Toulouse, France).
A total of 18 healthy male volunteers, of mean age ± standard deviation 32.6 ± 4.0 y and body mass index 23.6 ± 0.7 kg/m2, were enrolled.
An initial 15 d of baseline data collection were followed by 3 mo of strict bed-rest alone (control group, n = 9) or with the addition of supine resistance exercise training every 3 d (exercise group, n = 9).
Outcome measures:
Oxidation of labeled [d31]palmitate (the main saturated fatty acid of human diet) and [1-13C]oleate (the main monounsaturated fatty acid), body composition, net substrate use, and plasma hormones and metabolites were measured.
Between-group comparisons showed that exercise training did not affect oxidation of both oleate (mean difference 5.6%; 95% confidence interval [95% CI], −3.3% to 14.5%; p = 0.20) and palmitate (mean difference −0.2%; 95% CI, −4.1% to 3.6%; p = 0.89). Within-group comparisons, however, showed that inactivity changed oxidation of palmitate in the control group by −11.0% (95% CI, −19.0% to −2.9%; p = 0.01) and in the exercise group by −11.3% (95% CI, −18.4% to −4.2%; p = 0.008). In contrast, bed rest did not significantly affect oleate oxidation within groups. In the control group, the mean difference in oleate oxidation was 3.2% (95% CI, −4.2% to 10.5%; p = 0.34) and 6.8% (95% CI, −1.2% to 14.7%; p = 0.08) in the exercise group.
Independent of changes in energy balance (intake and/or output), physical inactivity decreased the oxidation of saturated but not monounsaturated dietary fat. The effect is apparently not compensated by resistance exercise training. These results suggest that Mediterranean diets should be recommended in sedentary subjects and recumbent patients.
Editorial Commentary
Background: Obesity is an important contributor to the burden of chronic diseases, particularly type II diabetes, cardiovascular disease, hypertension, and stroke. Being inactive is a risk factor for all of these conditions. However, the physiological effects of inactivity are not well understood. In this trial, supported by the European Space Agency, a group of researchers aimed to further understand the effects of physical inactivity on the way that fat from the diet is metabolized (i.e., broken down to generate energy). 18 healthy male volunteers were randomized into two groups, both of whom underwent 90 days of bed rest, aiming to mimic sedentary behavior. One group also received an exercise training program during the 90 days' bed rest. The researchers examined to what extent two different types of fatty acids common in the diet were metabolized over the duration of the trial: oleate (monounsaturated fat) and palmitate (saturated fat). As secondary objectives of the study, body weight, water, fat, and energy expenditure were also examined in the participants.
What this trial shows: The researchers did not see any statistically significant changes between the groups—that is, participants receiving bed rest, and those receiving bed rest plus exercise training—for any of the primary or secondary outcomes, except for resting metabolic rate, which was higher in the exercise group. However, they did see physiologically relevant changes in fat metabolism of one of the fatty acids, palmitate, over the course of the trial within both groups studied. Although metabolism of oleate (monounsaturated fat) did not show significant changes over the course of the trial, metabolism of palmitate (saturated fat) dropped by nearly 10% in both groups (bed rest, and bed rest plus exercise).
Strengths and limitations: The study design was appropriate to the questions being posed, and the techniques for examining fat metabolism were relevant. Although the number of participants was very small, this problem is true of many such studies due to the cost and complexity of the interventions. The model for inactivity used in this trial—90 days' bed rest—is very extreme. Very few studies of this type have been performed, with most of the evidence relating to activity and fat handling coming from training studies in otherwise sedentary people.
Contribution to the evidence: It is already known that physical activity has numerous health benefits, including the prevention of obesity. This trial provides data showing that inactivity lowers the ability to metabolize fat, specifically saturated fat, from the diet, which would therefore be more likely to be stored in the body.
PMCID: PMC1584255  PMID: 17016547

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