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Culture of Cancer Cells at Physiological Oxygen Levels Affects Gene Expression in a Cell-Type Specific MannerStandard cell culture is routinely performed at supraphysiological oxygen levels (~18% O2). Conversely, O2 levels in most mammalian tissues range from 1–6% (physioxia). Such hyperoxic conditions in cell culture can alter reactive oxygen species (ROS) production, metabolism, mitochondrial networks, and response to drugs and hormones. The aim of this study was to investigate the transcriptional response to different O2 levels and determine whether it is similar across cell lines, or cell line-specific. Using RNA-seq, we performed differential gene expression and functional enrichment analyses in four human cancer cell lines, LNCaP, Huh-7, PC-3, and SH-SY5Y cultured at either 5% or 18% O2 for 14 days. We found that O2 levels affected transcript abundance of thousands of genes, with the affected genes having little overlap between cell lines. Functional enrichment analysis also revealed different processes and pathways being affected by O2 in each cell line. Interestingly, most of the top differentially expressed genes are involved in cancer biology, which highlights the importance of O2 levels in cancer cell research. Further, we observed several hypoxia-inducible factor (HIF) targets, HIF-2α targets particularly, upregulated at 5% O2, consistent with a role for HIFs in physioxia. O2 levels also differentially induced the transcription of mitochondria-encoded genes in most cell lines. Finally, by comparing our transcriptomic data from LNCaP and PC-3 with datasets from the Prostate Cancer Transcriptome Atlas, a correlation between genes upregulated at 5% O2 in LNCaP cells and the in vivo prostate cancer transcriptome was found. We conclude that the transcriptional response to O2 over the range from 5–18% is robust and highly cell-type specific. This latter finding indicates that the effects of O2 levels are difficult to predict and thus highlights the importance of regulating O2 in cell culture.
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Data to accompany manuscript: Thermoconforming rays of the star-nosed moleThe star-nosed mole (Condylura cristata) is well known for its unique star-like rostrum (‘star’) which is formed by 22 nasal appendages highly specialised for tactile sensation. As a northerly distributed insectivorous mammal occupying both aquatic and terrestrial habitats, this sensory appendage is regularly exposed to cold water and thermally conductive soil, leading us to ask whether the surface temperature, a proxy for blood flow to the star, conforms to the local ambient temperature to conserve body heat. Alternatively, given the high functioning and sensory nature of the star, we posited it was possible that the rays may be kept continually warm when foraging, with augmented peripheral blood flow serving metabolic needs of this tactile sensory organ. To test these ideas, we remotely monitored the surface temperatures of the star and other uninsulated appendages in response to changes in local water or ground temperature in wild-caught star-nosed moles briefly studied in captive situation. While the tail responded to increasing heat load through vasodilation, the surface temperature of the star consistently thermoconformed, varying passively in surface temperature, suggesting little evidence for thermoregulatory vasomotion. This thermoconforming response may have evolved as a compensatory response related to the high costs of heat dissipation to water or soil in this actively foraging insectivore.
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Dataset to accompany manuscript "Energetic costs of bill heat exchange demonstrate contributions to thermoregulation at high temperatures in toco toucans (Ramphastos toco)"Body temperature regulation in the face of changes in ambient temperature and/or in metabolic heat production involves adjustments in heat exchange rates between the animal and the environment. One of those mechanisms include the modulation of the surface temperature of specific areas of the body through vasomotor adjustment and blood flow control, to change the thermal conductance of this region, thereby promoting dissipation or conservation of body heat. In homeotherms, this thermoregulatory adjustment is essential for the maintenance of body temperature over a moderate temperature range, known as the thermal neutral zone (TNZ), without increasing metabolic rate (MR). Thermal windows are poorly insulated body regions and highly vascularized that are particularly efficient for heat dissipation through that mechanism. The bill of the toco toucan (Ramphastos toco) has been described as a highly efficient thermal window and hypothesized to assist in the thermal homeostasis of this bird. Herein, we directly evaluated the contribution of heat exchange through the bill of the toco toucan on thermal homeostasis and metabolic rate and also for the delimitation of the TNZ. To do this, we measured metabolic rate, via oxygen consumption, over a range of ambient temperature from 0 to 35°C (every 5°C). MR measurements were made in birds with the bill intact (control group) and also with the bill artificially insulated (experimental group). The limits of the TNZ, 10.9-25.0°C for the control group and 10.8-24.1°C for the experimental group, did not differ between the treatments. MR differed among treatments only at elevated temperatures (30 and 35°C), reaching values of 0.97 ml O2·g-1·h-1·°C-1 (± 0.06) for the control group and 1.20 ml O2·g-1·h-1·°C-1 (± 0.07) for the experimental group at 35°C. These results indicate that while heat dissipation through the bill does not contribute significantly to widening of the TNZ, it may well be critically important in assisting body temperature regulation at higher temperatures extending above the upper limit of the TNZ. We estimate that the contribution of the bill to total heat exchange approaches 31% of basal metabolic heat production, a not insubstantial amount, providing evidence of the role of peripheral heat exchange and linking the role of appendage size as a key factor in the evolution of thermoregulatory responses in endotherms.
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Data from manuscript: "Free-living hummingbirds avoid using torpor while nesting"Reproduction is a trade-off between short-term energetic costs and long-term fitness benefits. This is especially apparent in small endotherms that exhibit high mass-specific metabolic rates and live in unpredictable environments. Many of these animals use torpor, substantially reducing their metabolic rate and body temperature to cope with high energetic demands during non-foraging periods. In birds, when the incubating parent uses torpor, the lowered temperatures that thermally sensitive offspring experience could delay development or increase mortality risk. We used thermal imaging to non-invasively explore how nesting female hummingbirds sustain their own energy balance while effectively incubating their offspring. We located 67 active Allen’s hummingbird (Selasphorus sasin) nests in Los Angeles, California and recorded nightly time-lapse thermal images at 14 of these nests for 108 nights using thermal cameras. We found that nesting females usually avoided entering torpor, with exceptions on two nights. We also modeled the energetic trade-offs of females using vs. avoiding torpor. We confirmed both through the field data and modeling approaches that nest insulation allowed nesting hummingbirds to avoid entering torpor on a regular basis. Nesting female hummingbirds seem to prioritize the nighttime energetic demands of their offspring, within the helpful environment of a warm nest.
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Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future PerspectivesMost conventional incubators used in cell culture do not regulate O2 levels, making the headspace O2 concentration ~18%. In contrast, most human tissues are exposed to 2-6% O2 (physioxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in standard cell culture practices affect a variety of biological processes. In this review, we discuss how supraphysiological O2 levels affect reactive oxygen species (ROS) metabolism and redox homeostasis, gene expression, replicative lifespan, cellular respiration, and mitochondrial dynamics. Furthermore, we present evidence demonstrating how hyperoxic cell culture conditions fail to recapitulate the physiological and pathological behavior of tissues in vivo, including cases of how O2 alters the cellular response to drugs, hormones, and toxicants. We conclude that maintaining physioxia in cell culture is imperative in order to better replicate in vivo-like tissue physiology and pathology, and to avoid artifacts in research involving cell culture.
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Phenological and social characterization of three Lasioglossum (Dialictus) species inferred from long-term trapping collectionsJournal of Hymenoptera Research, 2021-12-30Detailed social and phenological data collected from nesting aggregations exist for relatively few sweat bee species because nesting aggregations are rarely found in large numbers, even when local populations are highly abundant. This limits researchers’ abilities to assess the social status of many species, which in turn, limits our ability to trace the sequence of evolutionary steps between alternative social states. To address this problem, we demonstrate the utility of rehydrated, pinned specimens from pan trap and netting collections for generating inferences about the phenology and social status of a well-studied sweat bee species, Lasioglossum (Dialictus) laevissimum. A detailed comparison of phenology and reproductive traits, between pinned specimens and those in a previous nesting study, produced similar results for bivoltine foraging activity and eusocial colony organization typical in this species. We then used pinned specimens from monitoring studies to describe, for the first time, the foraging phenology and social behaviour of two additional Dialictus species, L. hitchensi and L. ellisiae. Both L. hitchensi and L. ellisiae each exhibited two peaks in abundance during their breeding seasons, indicating two periods of foraging activity, which correspond to provisioning of spring and summer broods. Differences in body size, wear, and ovarian development of spring and summer females indicated that L. hitchensi is most likely eusocial, while L. ellisiae is either solitary or communal. This study demonstrates that analyses of specimens obtained from flower and pan trap collections can be used for assessing the phenology and social organization of temperate sweat bees in the absence of nesting data. The phenological and social lability of many sweat bee species make them ideal for studying geographic and temporal variability in sociality, and analyses of pan trap collections can make these studies possible when direct observations are impossible.
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Foraging gene expression patterns in queens, workers, and males in a eusocial insectReproductive division of labour is based on biased expression of complementary parental behaviours, brood production (egg-laying) by queens and brood care (in particular, brood provisioning) by workers. In many social insect species, queens provision brood when establishing colonies at the beginning of a breeding season and reproductive division of labour begins with the emergence of workers. In many social insect species, the expression of foraging (for) mRNA is associated with the intensity of foraging behaviour, and therefore brood provisioning. However, only two studies have compared queen and worker for expression levels, and neither accounted for transcript splice variation. In this study, we compare the expression level of the for-α transcript variant across four life stages of the queen caste, two behavioural groups of workers, and males of a eusocial sweat bee Lasioglossum laevissimum (Smith, 1853). Foundresses collected prior to the onset of the foraging season and males had the highest for-α expression levels. All active (post-hibernatory) queens and workers had similar for-α expression levels independent of behaviour. These results suggest that the for gene in L. laevissium acts as a primer before foraging activity, and that caste-specific expression patterns correlate with the timing of foraging activity in queens and workers.
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Characterization of neutral sphingomyelinase activity and isoform expression in rodent skeletal muscle mitochondriaSkeletal muscle is composed of fiber types that differ in mitochondrial content, antioxidant capacity, and susceptibility to apoptosis. Ceramides have been linked to oxidative stress-mediated apoptotic intracellular signalling and the enzyme neutral sphingomyelinase (nSMase) is, in part, responsible for generating these ceramides through the hydrolysis of sphingomyelin. Despite the role of ceramides in mediating apoptosis, there is a gap in the literature regarding nSMase in skeletal muscle mitochondria. This study aimed to characterize total nSMase activity and individual isoform expression in isolated subsarcolemmal (SS) mitochondria from soleus, diaphragm, plantaris, and extensor digitorum longus (EDL). Total nSMase activity did not differ between muscle types. nSMase2 content was detectable in all muscles and higher in EDL, soleus, and plantaris compared to diaphragm whereas nSMase3 was undetectable in all muscles. Finally, total nSMase activity positively correlated to nSMase2 protein content in soleus but not the other muscles.These findings suggest that nSMase associated with SS mitochondria may play a role in intracellular signalling processes involving ceramides in skeletal muscle and nSMase2 may be the key isoform, specifically in slow twitch muscle like soleus. Further studies are needed to fully elucidate the specific contribution of nSMase, along with the role of the various isoforms and mitochondrial subpopulation in generating mitochondrial ceramides in skeletal muscle, and its potential effects on mediating apoptosis.
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Imaginer le futur de la mobilisation des connaissancesLa Commission canadienne pour l’UNESCO (CCUNESCO) et le Conseil de recherches en sciences humaines (CRSH) partagent un engagement profond envers le soutien à la production et l’accès à un savoir diversifié et inclusif au profit des générations actuelles et futures. Nous reconnaissons l’importance de lier la recherche aux problèmes mondiaux urgents. Nous avons l’occasion de travailler ensemble pour s’assurer que les résultats de la recherche aient un maximum d’impact, tout en tenant compte des divers systèmes de pensées. Assurer une mobilisation efficace des connaissances est incontournable pour répondre aux défis auxquels notre monde est confronté.
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Is Science a Human Right? Implementing the Principle of Participatory, Equitable, and Universally Accessible ScienceThe right of every human being to have access to scientific knowledge and participate in its development (also called “the right to science”) is enshrined in Article 27.1 of the Universal Declaration of Human Rights, adopted by the United Nations General Assembly on December 10, 1948. This article stipulates that: “Everyone has the right freely to participate in the cultural life of the community, to enjoy the arts and to share in scientific advancement and its benefits.” In 1966, the right to science was included in Article 15 of the International Covenant on Economic, Social and Cultural Rights, which called on the States Parties to “recognize the right of everyone to enjoy the benefits of scientific progress and its applications” and take the necessary steps for “the conservation, the development and the diffusion of science.”
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Pursuing Excellence in Research Reflections from UNESCO Research Chairs in CanadaAssessing or even just defining what excellence in research means can become a monumental task that can lead to frustration. The main reason is that research can take many forms depending on the discipline in which a scientist is working. In this reflection paper, we discuss the potential principles that could be applied when thinking about excellence in research in the context of academic advancement and resourcing. We acknowledge that there are many variants of the term and trying to add a strict framework may lead to discrimination against not only some disciplines but also cultures, as research has a social component that should not be forgotten.
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Vitellogenin expression corresponds with reproductive status and caste in a primitively eusocial bee, Lasioglossum laevissimumVitellogenin (vg) expression is consistently associated with variation in insect phenotypes, particularly egg-laying. Primitively eusocial species, such as eusocial sweat bees, have behaviourally totipotent castes, in which each female is capable of high levels of ovarian development. Few studies have investigated vg expression patterns in primitively eusocial insects, and only one study has focused on a primitively eusocial bee. Here we use a primitively eusocial sweat bee, Lasioglossum laevissimum, and Real Time quantitative PCR (RT-qPCR) to investigate the relationship between vg expression, castes, and variation in phenotypes associated with castes differences. These assays showed that females with high ovarian development had the highest levels of vg expression, and that vg expression levels reflected the reproductive status of females first and caste second. This is in contrast to vg expression patterns observed in advanced eusocial queens and workers, which differ in vg expression based on caste and have caste-specific vg expression patterns. Furthermore, future queens (gynes) do not have ovarian development and had similar vg expression levels to early spring foundresses, which do have ovarian development, supporting Vg’s function as a transporter of lipids and amino acids before diapause.
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Variation among 532 genomes unveils the origin and evolutionary history of a global insect herbivoreThe diamondback moth, Plutella xylostella is a cosmopolitan pest that has evolved resistance to all classes of insecticide, and costs the world economy an estimated US $4-5 billion annually. We analyse patterns of variation among 532 P. xylostella genomes, representing a worldwide sample of 114 populations. We find evidence that suggests South America is the geographical area of origin of this species, challenging earlier hypotheses of an Old-World origin. Our analysis indicates that Plutella xylostella has experienced three major expansions across the world, mainly facilitated by European colonization and global trade. We identify genomic signatures of selection in genes related to metabolic and signaling pathways that could be evidence of environmental adaptation. This evolutionary history of P. xylostella provides insights into transoceanic movements that have enabled it to become a worldwide pest.
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Strengthening our connection to nature and building citizens of the EarthThe authors elaborate on the dangers of rampant consumerism and attempt to explain why most humans are disconnected from the realities of our depleting planet and are not taking action to instigate change to ensure a more sustainable future. They argue that education for sustainable development will play a key role in transforming citizens of this Earth to assume fully their roles as environmental stewards.
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Activity analysis of thermal imaging videos using a difference imaging approachInfrared thermal imaging is a passive imaging technique that captures the emitted radiation from an object to estimate surface temperature, often for inference of heat transfer. Infrared thermal imaging offers the potential to detect movement without the challenges of glare, shadows, or changes in lighting associated with visual digital imaging or active infrared imaging. In this paper, we employ a frame subtraction algorithm for extracting the pixel-by-pixel relative change in signal from a fixed focus video file, tailored for use with thermal imaging videos. By summing the absolute differences across an entire video, we are able to assign quantitative activity assessments to thermal imaging data for comparison with simultaneous recordings of metabolic rates. We tested the accuracy and limits of this approach by analyzing movement of a metronome and provide an example application of the approach to a study of Darwin's finches. In principle, this “Difference Imaging Thermography” (DIT) would allow for activity data to be standardized to energetic measurements and could be applied to any radiometric imaging system.
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Thermal Imaging and Physiological Analysis of Cold-Climate Caribou-Skin ClothingProtective clothing is essential for human existence in the Arctic, and caribou-skin clothing has played a pivotal role for millennia. Although people with northern experience often extol caribou-skin clothing, few scientific studies have investigated its properties. We used infrared thermal imaging in a pilot study to compare authentic caribou-skin clothing sewn by traditional Inuit seamstresses with two other types of cold-weather clothing: a standard-issue, Canadian army, winter uniform and an ensemble of modern retail clothing designed for extreme cold (a down anorak and snowmobile pants). To make the comparison, two subjects sequentially wore the three types of clothing—caribou skin, army uniform, and modern retail—in a still air, uniform thermal environment (where radiant temperatures of all environmental surfaces were equal to air temperature) at −21°C to −23°C (−6°F to −10°F). Thermal imaging quantifies the temperature of the outer surface of clothing, thereby providing key, functionally relevant information on the interface where clothing and environment meet. Under otherwise similar conditions, a low clothing surface temperature indicates superior clothing performance and a reduced rate of heat loss from the body to the environment. Caribou-skin clothing was similar to modern extreme-cold retail clothing: the whole-body composite surface temperature of our subjects wearing caribou-skin clothing was −22.1°C to −22.7°C, compared with −21.6°C in both subjects wearing the modern retail clothing. The army winter uniform (−18.9°C to −20.0°C) was inferior. These quantitative results were mirrored by the subjects’ subjective impressions. A particular advantage of thermal imaging is that it pinpoints locations in clothing where heat leaks occur. Although the two types of modern clothing exhibited heat leaks at zippered structures (even though fully closed), the caribou-skin clothing evaded such heat leaks by lacking such structures, because it is donned over the head. The integral hood characteristic of a caribou-skin parka was also superior in comparison to the detachable hood of the army uniform.
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Development of homeothermic endothermy is delayed in high-altitude native deer mice (Peromyscus maniculatus)Altricial mammals begin to independently thermoregulate during the firstfew weeks of postnatal development. In wild rodent populations, this isalso a time of high mortality (50–95%), making the physiological systemsthat mature during this period potential targets for selection. High altitude(HA) is a particularly challenging environment for small endotherms owingto unremitting low O2and ambient temperatures. While superior thermo-genic capacities have been demonstrated in adults of some HA species, itis unclear if selection has occurred to survive these unique challengesearly in development. We used deer mice (Peromyscus maniculatus) nativeto high and low altitude (LA), and a strictly LA species (Peromyscus leucopus),raised under common garden conditions, to determine if postnatal onset ofendothermy and maturation of brown adipose tissue (BAT) is affected byaltitude ancestry. We found that the onset of endothermy correspondswith the maturation and activation of BAT at an equivalent age in LAnatives, with 10-day-old pups able to thermoregulate in response to acutecold in both species. However, the onset of endothermy in HA pups wassubstantially delayed (by approx. 2 days), possibly driven by delayedsympathetic regulation of BAT. We suggest that this delay may be part ofan evolved cost-saving measure to allow pups to maintain growth ratesunder the O2-limited conditions at HA.
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A Long-Term Study on Massasaugas (Sistrurus catenatus) Inhabiting a Partially Mined Peatland: A Standardized Method to Characterize Snake Overwintering HabitatTemperate snakes occupy overwintering sites for most of their annual life cycle. Microhabitat characteristics of the hibernaculum are largely undescribed, yet are paramount in ensuring snake overwintering survival. We hypothesized that snakes survive hibernation within a vertical subterranean space that we termed a “life zone” (LZ), that is aerobic and flood and frost free throughout winter. We studied an isolated, endangered population of Massasaugas (Sistrurus catenatus) inhabiting an anthropogenically altered peatland and monitored the subterranean habitat during a period of environmental stochasticity. Initial radio telemetry confirmed that snakes moved between altered and natural habitats during the active season and showed hibernation-site fidelity to either habitat. We used a grid of groundwater wells and frost tubes installed in each hibernation area to measure LZ characteristics over 11 consecutive winters. The LZ within the impacted area was periodically reduced to zero during a flood–freeze cycle, but the LZ in the natural area was maintained. Model selection analysis revealed that soil depth and flood status best predicted LZ size. Thermal buffering and groundwater dissolved oxygen increased with LZ size, and annual Massasauga encounters were significantly correlated with LZ size. This analysis suggests a population decline occurred when LZ size was reduced by flooding. Our data give support to the importance and maintenance of an LZ for successful snake hibernation. Our methods apply to subterranean hibernation habitats that are at risk of environmental stochasticity, causing flooding, freezing, or hypoxia.
