France

Understanding molecular mechanisms underlying physiology benefits from studies of isolated and/or cultured cells. However, virtually all experiments in cell culture are done in the absence of O2 regulation, resulting in incubator headspace O2 levels of 18-19%. In vivo, O2 levels in heart, skeletal muscle, brain, liver, and indeed most of the mammalian body, are typically 2-6%. The supra-physiological O2 levels in cell culture drive increased cellular reactive oxygen species (ROS) production, affecting many aspects of cell physiology. We have shown that supra-physiological O2 targets mitochondria, with important effects on bioenergetic function and network dynamics. Cellular responses to hormones like 17β-estradiol are significantly different in 18%, versus 5%, O2. Our RNAseq investigations show widespread effects of 18%, versus 5% O2 on diverse molecular processes ranging from DNA repair and cell cycle to energy metabolism to the viral response. Since supra-physiological O2 affects both transcriptional and translational processes, there are additional effects on relative protein abundance that are evident in proteomic analyses. The effects of supra-physiological O2 we have characterized are generally cell line-specific, making them difficult to predict. Given these myriad cellular effects of O2, it is important to reference hypoxia experiments in cell culture to physiological normoxia (e.g. 2-6%), but the vast majority of CO2 incubators used by cell culturists are not capable of regulating O2. We have developed a low-cost (&amp;lt;1000 CAD) cell culture incubator capable of regulating O2, CO2, and temperature in their appropriate physiological ranges. Improved control over O2 levels will improve data quality in hypoxia experiments.

SEB Annual Conference 2022

Importance of maintaining physioxia in cell culture

Discover the art of creating extraordinary science at SEB’s Annual Conference at Le Corum Conference Center     in Montpellier, France from 5 to  8 July 2022.

SEB’s Annual Conference is celebrated for creating the perfect composition of research, new discoveries and collaborative connections. Be creative and innovative by exchanging ideas and knowledge with masters of biology from all over the world.

2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years.
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Please register for the conference by clicking on this link:

https://www.sebiology.org/events/seb-annual-conference-2022/registration.html

The site will be open for registered participants closer to the event.

2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years - Montpellier, France

Understanding molecular mechanisms underlying physiology benefits from studies of isolated and/or cultured cells. However, virtually all experiments in cell culture are done in the absence of O2 regulation, resulting in incubator headspace O2 levels of 18-19%. In vivo, O2 levels in heart, skeletal muscle, brain, liver, and indeed most of the mammalian body, are typically 2-6%. The supra-physiological O2 levels in cell culture drive increased cellular reactive oxygen species (ROS) production, affecting many aspects of cell physiology. We have shown that supra-physiological O2 targets mitochondria, with important effects on bioenergetic function and network dynamics. Cellular responses to hormones like 17β-estradiol are significantly different in 18%, versus 5%, O2. Our RNAseq investigations show widespread effects of 18%, versus 5% O2 on diverse molecular processes ranging from DNA repair and cell cycle to energy metabolism to the viral response. Since supra-physiological O2 affects both transcriptional and translational processes, there are additional effects on relative protein abundance that are evident in proteomic analyses. The effects of supra-physiological O2 we have characterized are generally cell line-specific, making them difficult to predict. Given these myriad cellular effects of O2, it is important to reference hypoxia experiments in cell culture to physiological normoxia (e.g. 2-6%), but the vast majority of CO2 incubators used by cell culturists are not capable of regulating O2. We have developed a low-cost (&lt;1000 CAD) cell culture incubator capable of regulating O2, CO2, and temperature in their appropriate physiological ranges. Improved control over O2 levels will improve data quality in hypoxia experiments.

technical paper

Energy metabolism (encompassing energy assimilation, conversion and utilization) plays a central role in all life processes and serves as a link between the organismal physiology, behavior, and ecology. Metabolic rates define the physiological and life-history performance of an organism and have direct implications for Darwinian fitness. Anthropogenic and natural stressors affect energy balance because the stress-induced disruption of homeostasis must be corrected to ensure the organism's survival. Pollutants can negatively affect different aspects of energy metabolism of an organism interfering with energy assimilation and conversion or increasing energy costs for basal maintenance. This can diminish the energy fluxes available for other fitness functions and negatively affect the organism’s growth, reproduction and ability to deal with additional stressors or diseases. In this talk, I will discuss the role of bioenergetics in toxicity of legacy and emerging pollutants (including trace metals, nanoparticles and pharmaceuticals) and the pollutant interactions with other abiotic stressors such as temperature, salinity or oxygen deficiency. I will address the mechanisms underlying the pollutant-induced bioenergetics disturbances and discuss the use of bioenergetics-based approaches in linking the molecular and cellular stress responses to the whole-organism fitness in coastal environments exposed to multiple stressors.

Bioenergetic aspects of pollutant toxicity and their role in determining the tolerance limits to multiple stressors in aquatic organisms

We study the potential of a recent invader, the Asian brush-clawed crab (Hemigrapsus takanoi), to expand its distribution range further into the Baltic Sea. H. takanoi has been documented in the southwestern Baltic Sea since 2014. The ability to persist and further expand into the Baltic Sea will depend on their potential to sustain all stages of their complex life cycle, including pelagic larvae, under the Baltic Sea’s conditions. Range limits may be established by the tolerance to low salinity, which in addition may be affected by temperature. A key question is whether local populations at the distribution limit (within the Baltic) are adapting to low salinities and hence promote further expansion. We quantified the combined effects of salinity (10-32 PSU) and temperature (15-24°C) on four populations of H. takanoi (two from the Baltic and two from the North Sea). Our results show substantial differences in larval performance between the populations from the Baltic and North Sea. Larvae from the North Sea populations show higher survival and faster development compared with those from the Baltic Sea. No evidence of elevated tolerance towards low salinity were found in the larvae from the Baltic Sea. In addition, larvae from the population located at the range limit (Neustadt) show very low survival. We did not find evidence of adaptation to low salinity by any of the local populations of H. takanoi we studied. Thus, in order to the population to persist, larvae must either migrate to waters of increased salinity, and then return to the habitat of origin; or such populations are sustained by source-sink dynamics. 


Living at the frontier - salinity and temperature during larval development determine the potential for invasion of the Baltic Sea

Evolution as a contemporary process that can affect changes from one generation to the next has only recently been considered in toxicology. While rapid evolution has recently been linked with the increase of invasive capacity of Invasive Alien Species (IAS), it is still not taken into consideration for Invasive Species Predictive Schemes and risk assessments. 
The Louisiana crayfish, Procambarus clarkii, has a strong adaptive capacity to a variety of aquatic environments, including highly contaminated water bodies, and different salinities. Here, we studied three invasive populations of P. clarkii that inhabit water bodies at different salinities and pollution levels in the South of France. Our results showed that physiological and molecular responses are population-specific when they are exposed to pollutants and salinity shock. Our results suggest that in P. clarkii, the capacity to inhabit polluted environments and the capacity to inhabit water bodies with different salinity levels are not related. While the capacity to face pollutants seems to share a common ancestor in these three populations, the capacity of dealing with salinity change seems to be a more recently acquired trial.

Physiological and molecular responses of P. clarkii to different levels of pollutants and salinity exposure: an Interpopulation study.

Populations of cells must maintain a fairly consistent size, as excessively large or small cells will be non-viable. However, cell division is not necessarily symmetrical, generally leading to the formation of daughter cells of unequal volume. Cells must therefore possess a mechanism of “size control,” by which variation in cell volume at birth may be evened out across the cell cycle. While the basis of size control has been studied in other organisms, such as yeast, it is not yet clear how this mechanism functions in plants. 

It is our view that, while progress through the cell cycle is regulated by a network of many interacting proteins, there exists a handful of key “sizer” proteins which control cell cycle progression and provide an internal scale against which cell size may be measured. Depending upon their rates of synthesis, these proteins may accumulate over time, or be diluted as the cell grows. Activators of cell cycle progression accumulate, while inhibitors are diluted, until a critical point is reached and there is a change of cell phase.   

This talk will consider the mathematical modelling of the cell cycle in Arabidopsis thaliana. In particular, it will focus on the use of differential equations to study size control as an emergent property of a network of interacting proteins. 





Mathematical modelling of the cell cycle and cell size control in plants

The crucian carp has the rare ability to survive days to months of anoxia, depending on temperature. Transcriptome analyses show that this is accompanied by widespread changes in gene expression. However, the importance of changes on the mRNA level can of course be questioned because it is the proteins that ultimately matter. We have therefore, in addition to global analyses of transcription and translation (ribosomal footprint profiling), measured the levels of proteins and phosphopeptides in tissues of crucian carp exposed to one week of anoxia and subsequent reoxygenation at 8°C. A general conclusion is that the transcriptome is a good indicator for the direction of change but that rather few proteins show a detectable change in their concentration during one week of anoxia and subsequent reoxygenation (e.g. 66 out of 3304 proteins in brain), which probably reflects that protein synthesis is slow and occur on top of a background of existing protein pools. Moreover, most proteins may have structural or housekeeping functions that are not altered during anoxia, and making major changes in protein concentrations would be energetically expensive, and energy use needs to be suppressed in anoxia. Phosphorylation of proteins, on the other hand, is an instant response, but also here relatively few changes were seen. In brain 109 out of 4316 phosphopeptides were regulated, while in liver 395 out of 1293 were regulated. The changes found in the proteome and phosphoproteome are likely to point at important adaptive mechanisms, which include mitochondrial adjustments, AMPK signalling and glucose metabolism.

Responses of the proteome and phosphoproteome to anoxia and reoxygenation in crucian carp (Carassius carassius)

Across diverse taxa, maternal allocation to offspring tends to increase until mothers reach a prime, before declining in later life. Offspring born to very young or old mothers thus are of lower quality, as measured for example in size at birth, and potentially experience reduced lifespan. Here, I describe how we can gain new insights into the evolutionary causes of this age-dependent allocation by studying the tsetse fly: a relatively long-lived fly which gives birth to single offspring the same size as the mother. We experimentally test whether the decline in offspring quality produced by older females in the laboratory is exacerbated by nutritional stress, or mitigated by alleviating costs of reproduction. Using state-dependent models, we explore the evolutionary conditions favouring hump-shaped allocation with age, and how this is affected by physiological constraints and environmental uncertainty. Such models also shed light on why the senescence that we find in laboratory flies seems absent in field conditions, where flies have limited access to food and higher mortality. Finally, I discuss the implications for understanding age-dependent maternal allocation not just for considering offspring longevity, but for population processes and disease transmission.

Age-dependent reproductive allocation in a viviparous fly

Maternal age at birth has been demonstrated to affect offspring performance in aspects associated with fitness, including juvenile survival, lifespan, and fitness. A tendency appears to exist for such maternal age effects to be deleterious (‘maternal senescence’). However, there also appears to be much variation in the degree (and even in the direction) of these effects. Why should maternal senescence evolve and where should we expect it to be strongest (or weakest)? The clearest and most general way to explore this question theoretically is to expand William Hamilton’s models of the evolution of ageing to age-specific maternal genetic effects for vital rates. This approach has been applied specifically only to juvenile survival, but it has yielded results that show: 1) that selection always favours the evolution of this manifestation of maternal senescence; 2) that the evolved rate of senescence will differ from actuarial and reproductive senescence; and 3) exactly how selection drives the age-of-onset of maternal senescence. Here I will discuss how this model of maternal genetic effects can be adapted to all other vital rates, and through these, to explain how natural selection can favour other aspects of maternal senescence, such as the Lansing Effect and reduced offspring fitness caused by advancing maternal age.   

Natural selection and the evolution of maternal age effects

Reproductive senescence is an age related decline in reproductive performance, and is commonly studied in terms of age-related patterns of offspring production. However, parental fitness arises via offspring performance as well as production, so both components require attention to gain a more complete understanding of the trajectory and fitness consequences of reproductive senescence. Here we utilise a longitudinal dataset from a long-term study of European badgers (Meles meles) to investigate maternal and paternal reproductive senescence, and quantify how both offspring production and lifetime performance vary with parental age. First, we show that badgers of both sexes experience a senescent decline in annual offspring production, which occurs with a similar age of onset in the two sexes. Second, we show evidence of deleterious maternal age effects on offspring performance; offspring mortality risk increases and offspring lifespan and life-time reproductive success decreases with within-mother increases in maternal age. Crucially, the maternal ages of onset of these declines in offspring performance are significantly earlier than those for offspring production, such that failure to account for them would lead to underestimation of the severity (in terms of timing and fitness consequences) of maternal reproductive senescence. Finally, we found no evidence of paternal age effects on offspring mortality, lifespan or life-time reproductive success. Overall, our findings illustrate the potential importance of attending to parental age-related changes in offspring performance as well as production, and the potential for sex differences in the relative contributions of these two components to the overall fitness consequences of reproductive senescence.

Parental age effects on offspring performance exacerbate reproductive senescence in female, but not male, European badgers.

Annual populations of animals express all of their reproductive effort in a single year only, with some of them producing offspring across a series of reproductive attempts (e.g. clutches) within the same breeding season. In such organisms, the timing of a reproductive attempt correlates with maternal age (so that offspring produced late in the breeding season are mothered by older females), and thus the phenotype of the offspring may differ between reproductive attempts of the same female. Moreover, environmental conditions experienced by the mother during that breeding season may further modify the phenotypic effects of maternal age. In this study, I investigated the effects of the interaction between maternal age and maternal environment on offspring phenotype using an annual population of three-spined stickleback (Gasterosteus aculeatus). I compared the phenotype of offspring from the early, mid- and late clutches of females, half of which were exposed to environmental stressors across their breeding season. Older females produced heavier eggs and fry that were larger at hatching, regardless of environmental stressors. Offspring of both stressed and non-stressed older mothers showed increased hormonal response to an acute stressor, but their survival patterns showed a complex relationship with both maternal age and stress exposure. Moreover, I observed a positive effect of maternal age on their daughters’ reproductive investment, but only if the mothers experienced stressful conditions during the breeding season. I thus show that annual females nearing the end of their reproductive lifespan increase their reproductive investment, with a potentially intergenerational effect. In addition, maternal age influences offspring phenotype, but this influence is moderated by the environmental conditions the mothers experience during the reproductive period.

Maternal age interacts with environmental conditions in shaping of offspring phenotype in an annual fish species.

The Lansing Effect (LE) is the observation of decreased offspring longevity with increasing maternal age at birth. Whilst there appears to be much awareness of this phenomenon, actual support for a LE is mixed, with various studies reporting positive, negative, or no effects of maternal age on lifespan. We performed a meta-analytic review of published studies with two goals: 1) to determine if there exists a tendency across all studies species to express a LE, and 2) identify factors that might related to the severity (or presence) of the effect. We derived 74 relevant estimates from 20 published studies of 14 species. Our analysis revealed a tendency across all studied animal species to exhibit a LE, but this appeared to be driven by estimates from insect and rotifer species, which were the most heavily studied and presented the most consistent LEs. We found no clear tendency from studies of bird or mammal species, but these were severly understudied. These results emphasize both the need to expand the taxonomic breadth of LE studies beyond laboratory model organisms and to develop new evolutionary theory that might help us understand this among-species variation. 









Importance of maintaining physioxia in cell culture

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