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bioRxiv2020

Applying a gene-suite approach to examine the physiological status of wild-caught walleye (Sander vitreus)

Molecular techniques have been increasingly used in a conservation physiology framework to provide valuable information regarding the mechanisms underlying responses of wild organisms to environmental and anthropogenic stressors. In the present study, we developed a reference gill transcriptome for walleye (Sander vitreus) allowing us to pair a gene-suite approach with multivariate statistics to examine the physiological status of wild-caught walleye. For molecular analyses of wild fish, the gill is a useful target for conservation studies, not only because of its importance as an indicator of the physiological status of fish but also because it can be biopsied non-lethally. Walleye were non-lethally sampled following short- ([~]1.5 month) and long-term ([~]3.5 month) holding in the Delta Marsh, that is located south of Lake Manitoba in Manitoba, Canada. Large-bodied walleye are held in the Delta Marsh from late April to early August by exclusion screens used to protect the marsh from invasive common carp (Cyprinus carpio), exposing fish to potentially stressful water quality conditions. Principal components analysis (PCA) revealed patterns of transcript abundance consistent with exposure of fish to increasingly high temperature and low oxygen conditions with longer holding in the marsh. For example, longer-term holding in the marsh was associated with increases in the mRNA levels of heat shock proteins and a shift in the mRNA abundance of aerobic to anaerobic metabolic genes. Overall, the results of the present study suggest that walleye held in the Delta Marsh may be exhibiting sub-lethal responses to high temperature and low oxygen conditions and provides valuable information for managers invested in mediating these impacts to a local species of conservation concern. More broadly, we highlight the usefulness of pairing transcriptomic techniques with multivariate statistics to address potential confounding factors that can affect measured physiological responses of wild-caught fish. Lay summaryNon-lethal molecular techniques were used to assess the physiological status of wild-caught walleye confined in the Delta Marsh, Manitoba, Canada, because of common carp exclusion screens. Walleye sampled during the warmest months of the year exhibited responses to elevated temperature and low oxygen conditions, suggesting sub-lethal effects of local conditions.

physiology↗

bioRxiv2022

Building bridges from genome to physiology using machine learning and Drosophila experimental evolution

Drosophila experimental evolution, with its well-defined selection protocols, has long supplied useful genetic material for the analysis of functional physiology. While there is a long tradition of interpreting the effects of large-effect mutants physiologically, in the genomic era identifying and interpreting gene-to-phenotype relationships has been challenging, with many labs not resolving how physiological traits are affected by multiple genes throughout the genome. Drosophila experimental evolution has demonstrated that multiple phenotypes change due to the evolution of many loci across the genome, creating the scientific challenge of sifting out differentiated but noncausal loci for individual characters. The fused lasso additive model method (FLAM) allows us to infer some of the differentiated loci that have relatively greater causal effects on the differentiation of specific phenotypes. The experimental material used in the present study comes from 50 populations that have been selected for different life-histories and levels of stress resistance. Differentiation of cardiac robustness, starvation resistance, desiccation resistance, lipid content, glycogen content, water content, and body masses was assayed among 40 to 50 of these experimentally-evolved populations. Through FLAM, we combined physiological analysis from eight parameters with whole-body pooled-seq genomic data to identify potentially causally linked genomic regions. We have identified approximately 1,900 significantly differentiated 50 kb genomic windows among our 50 populations, with 161 of those identified genomic regions highly likely to have a causal effect connecting specific genome sites to specific physiological characters.

physiology↗

bioRxiv2022

Mice with humanized livers reveal the involvement of hepatocyte circadian clocks in rhythmic behavior and physiology

The circadian clock is an evolutionarily acquired gene network that synchronizes physiological processes to adapt homeostasis to the succession of day and night. While most mammalian cells have a circadian clock, their synchronization at the body-level depends on a central pacemaker located in the suprachiasmatic nuclei of the hypothalamus that integrates light signals. However, peripheral organs are also synchronized by feeding cues that can uncoupled them from the central pacemaker. Nevertheless, the potential feedback of peripheral signals on the central clock remains poorly characterized. To discover whether peripheral organ circadian clocks may affect the central pacemaker, we used a chimeric model in which mouse hepatocytes were replaced by human hepatocytes. These human hepatocytes showed a specific rhythmic physiology caused by their blunted response to mouse systemic signals. Strikingly, mouse liver humanization reprogrammed the liver diurnal gene expression and modified the phase of the circadian clock. The phase advance was also reflected in the muscle as well as the entire rhythmic physiology of the animals, indicating an impact on the circadian function of the central clock. Like mice with a deficient central clock, the humanized animals shifted their rhythmic physiology more rapidly to the light phase under day feeding. Our results indicate that peripheral clocks may affect the central pacemaker and offer new perspectives to understand the impact of peripheral clocks on the global circadian physiology.

physiology↗

bioRxiv2023

Coral species-specific loss and physiological legacy effects are elicited by extended marine heatwave

Marine heatwaves are increasing in frequency and intensity, with potentially catastrophic consequences for marine ecosystems such as coral reefs. An extended heatwave and recovery time-series that incorporates multiple stressors and is environmentally realistic can provide enhanced predictive capacity for performance under climate change conditions. We exposed common reef-building corals in Hawai i, Montipora capitata and Pocillopora acuta, to a 2-month period of high temperature and high pCO2 conditions or ambient conditions in a factorial design, followed by 2 months of ambient conditions. High temperature, rather than high pCO2, drove multivariate physiology shifts through time in both species, including decreases in respiration rates and endosymbiont densities. Pocillopora acuta exhibited more significantly negatively altered physiology, and substantially higher bleaching and mortality than M. capitata. The sensitivity of P. acuta appears to be driven by higher baseline rates of photosynthesis paired with lower host antioxidant capacity, creating an increased sensitivity to oxidative stress. Thermal tolerance of M. capitata may be partly due to harboring a mixture of Cladocopium and Durusdinium spp., whereas P. acuta was dominated by other distinct Cladocopium spp. Only M. capitata survived the experiment, but physiological state in heatwave exposed M. capitata remained significantly diverged at the end of recovery relative to individuals that experienced ambient conditions. In future climate scenarios, particularly marine heatwaves, our results indicate a species-specific loss of corals that is driven by baseline host and symbiont physiological differences as well as Symbiodiniaceae community compositions, with the surviving species experiencing physiological legacies that are likely to influence future stress responses.

physiology↗

bioRxiv2024

Chronic infection alters pathogen virulence, microbiome composition, and fly physiology across generations.

In many insects, parents and offspring share the same environment. Thus, an infection in the parents has the potential to influence offspring defenses. Moreover, infection can also affect other host aspects, including the microbiome, development, and reproduction. To better understand the intergenerational impacts of infection, we assessed the effects of challenge by the gut pathogen Pseudomonas entomophila (Pe) on Drosophila melanogaster. We found that parental challenge by Pe led to environmental transmission of the pathogen from parents to offspring, resulting in a persistent infection among the population. Pe is a highly virulent pathogen; however, we found that persistent infection was correlated with a loss of pathogen virulence across generations. We explored the impact of chronic pathogen exposure on host physiological traits. Our results showed that pathogen load, virulence, and pathogen-induced microbiome remodeling influence fecundity and starvation resistance. Current research in Drosophila and other insects has shown that immune status can be transmitted to the next generation (transgenerational immunity). Since the offspring were continuously exposed to the pathogen, we explored their response to a new infection. Even though we did not find a protective effect, we observed alterations in gene expression and microbiome remodeling following a new Pe challenge that was dependent on the parental treatment. Altogether, our results provide evidence that the pathogen adapted across generations as part of a tolerance mechanism that allows the pathogen to persist in the environment, which confers a greater probability of survival in subsequent generations. However, chronic exposure to the pathogen resulted in a cost to the host by altering several aspects of host physiology. Author summaryInfection impacts many aspects of animal physiology, including priming host immune responses to repeated pathogen exposure. Whether parental experiences with a pathogen can influence such responses in offspring is less certain. Here, to further our understanding of generational impacts of infection, we studied the interaction between host immunity, the microbiome, and a gut pathogen across generations using the model organism Drosophila melanogaster. Our results showed that parental challenge established a persistent infection in the population, such that offspring were chronically exposed to the pathogen. This chronic pathogen exposure impacted many host physiological traits, but did not confer protection to re-infection with a high-dose of the pathogen. Instead, we found that the transmitted infection led to a loss of pathogen virulence in offspring. At the same time, pathogen density, virulence, and pathogen-induced microbiome remodeling influenced fecundity and starvation resistance. Overall, our results highlight that infection in parents can influence intergenerational responses due to impacts both on the microbiome and on selection on pathogen virulence. Such chronic interactions with the pathogen, even reduced in virulence, alter host physiology.

physiology↗

bioRxiv2024

Ambulatory physiological measures obtained under naturalistic urban mobility conditions have acceptable reliability

Ambulatory assessment methods in psychology and clinical neuroscience are powerful research tools for collecting data outside of the laboratory. These methods encompass physiological, behavioral, and self-report measures obtained while individuals navigate in real-world environments, thereby increasing the ecological validity of experimental approaches. Despite the recent increase in applications of ambulatory physiology, data on the reliability of these measures is still limited. To address this issue, twenty-six healthy participants (N = 15 female, 18-34 years) completed an urban walking route (2.1 km, 30 min walking duration, temperature M = 19.8{degrees} degree Celsius, Range = 12{degrees}-37{degrees} degrees Celsius) on two separate testing days, while GPS-location and ambulatory physiological measures (cardiovascular and electrodermal activity) were continuously recorded. Bootstrapped test-retest reliabilities of single measures and aggregate scores derived via principal component analysis (PCA) were computed. The first principal component (PC#1) accounted for 39% to 45% of variance across measures. PC#1 scores demonstrated an acceptable test-retest reliability (r = .60) across testing days, exceeding the reliabilities of most individual measures (heart rate: r = .53, heart rate variability: r = .50, skin conductance level: r = .53, no. of skin conductance responses: r = .28, skin conductance response amplitude: r = .60). Results confirm that ambulatory physiological measures recorded during naturalistic navigation in urban environments exhibit acceptable test-retest reliability, in particular when compound scores across physiological measures are analyzed, a prerequisite for applications in (clinical) psychology and digital health. Author summaryPsychophysiological assessments have been predominantly limited to controlled laboratory settings, leaving the reliability of field measurements unclear. In this study, we conducted a proof-of-concept investigation in N=26 healthy participants navigating the same urban route on two separate days. Cardiovascular and electrodermal activity were continuously recorded and combined with GPS-based location tracking. Psychophysiological measurements obtained under naturalistic urban mobility conditions showed acceptable test-retest reliability, in particular when multiple measures where combined into a compound score via principal component analysis. Shedding light on the reliability of ambulatory assessments in urban environments emphasizes the potential for psychophysiological measurements to contribute valuable insights beyond the constraints of traditional laboratory settings.

physiology↗

bioRxiv2024

Individual Differences in the Correspondence Between Psychological and Physiological Stress Indicators

Stress threatens physical and mental health. Reactions to acute stress comprise multiple levels, including negative thoughts, bodily symptoms and behaviors. Individuals differ in their reaction to acute stress, and importantly, also in the extent to which these levels align, with a closer correspondence between psychological and physiological stress indicators being beneficial for mental health and well-being. This preregistered study investigates such individual differences systematically by inducing psychological (social-evaluative) and physiological (cold water) stress with the Maastricht Acute Stress Test (MAST) in 149 healthy adults. Participants indicated their perceived stress and four physiological stress indicators (blood pressure, heart rate, salivary cortisol, alpha-amylase) were obtained. Finally, multiple personality traits were assessed as potential moderators, including the Big Five, trait anxiety, and general cognitive ability. In line with previous research, psychological and physiological stress indicators were only weakly correlated and Bayesian analyses provided evidence favoring the absence of close psychophysiological correspondence. Considering individual differences in personality, especially conscientiousness and openness emerged as potential moderators. We propose individual differences in interoceptive abilities as another critical moderator, which deserves further investigation, and discuss how future research on individual differences in psycho-physiological correspondence can contribute to further our understanding of mental and physical diseases.

physiology↗

bioRxiv2019

Quantitative physiology of non-energy-limited retentostat cultures of Saccharomyces cerevisiae at near-zero specific growth rates

So far, the physiology of Saccharomyces cerevisiae at near-zero growth rates has been studied in retentostat cultures with a growth-limiting supply of the carbon and energy source. Despite its relevance in nature and industry, the near-zero growth physiology of S. cerevisiae under conditions where growth is limited by the supply of non-energy substrates remains largely unexplored. This study analyses the physiology of S. cerevisiae in aerobic chemostat and retentostat cultures grown under either ammonium or phosphate limitation. To compensate for loss of extracellular nitrogen- or phosphorus-containing compounds, establishing near-zero growth rates ( < 0.002 h-1) in these retentostats required addition of low concentrations of ammonium or phosphate to reservoir media. In chemostats as well as in retentostats, strongly reduced cellular contents of the growth-limiting element (nitrogen or phosphorus) and high accumulation levels of storage carbohydrates were observed. Even at near-zero growth rates, culture viability in non-energy-limited retentostats remained above 80 % and ATP synthesis was still sufficient to maintain an adequate energy status and keep cells in a metabolic active state. Compared to similar glucose-limited retentostat cultures, the nitrogen- and phosphate-limited cultures showed a partial uncoupling of catabolism and anabolism and aerobic fermentation. The possibility to achieve stable, near-zero growth cultures of S. cerevisiae under nitrogen- or phosphorus-limitation offers interesting prospects for high-yield production of bio-based chemicals.\n\nImportanceThe yeast Saccharomyces cerevisiae is a commonly used microbial host for production of various bio-chemical compounds. From a physiological perspective, biosynthesis of these compounds competes with biomass formation in terms of carbon and/or energy equivalents. Fermentation processes functioning at extremely low or near-zero growth rates would prevent loss of feedstock to biomass production. Establishing S. cerevisiae cultures in which growth is restricted by the limited supply of a non-energy substrate could therefore have a wide range of industrial applications, but remains largely unexplored. In this work we accomplished near-zero growth of S. cerevisiae through limited supply of a non-energy nutrient, namely the nitrogen or phosphorus source and carried out a quantitative physiology study of the cells under these conditions. The possibility to achieve near-zero-growth S. cerevisiae cultures through limited supply of a non-energy nutrient may offer interesting prospects to develop novel fermentation processes for high-yield production of bio-based chemicals.

physiology↗

bioRxiv2026

The physiological dynamic clamp allows insect flight muscle to transition between two actuation modes in virtual reality

In most muscles, contraction is initiated by neural activation. Some groups of insects break this rule, flapping at frequencies far exceeding the neural drive to their flight muscles. These insects muscles (termed asynchronous) produce force in response to stretch, enabling flight at faster frequencies than would be possible through the slow calcium-dependent processes associated with neural activation. The first flapping insects lacked stretch-activated physiology, which then evolved on top of neural activation dynamics before likely being reduced again in some groups including moths. Stretch and neural activation can co-exist, but it remains unclear if stretch-activation alone is sufficient to generate asynchronous flapping in insect flight muscle. Building on prior closed-loop muscle physiology platforms, we develop a new way to perform a gain-of-function muscle physiology experiment called the physiological dynamic clamp. Inspired by dynamic clamp experiments in neuroscience, we couple isolated intact flight muscle from a hawkmoth, Manduca sexta, to simulated stretch-activation in virtual reality. Tuning virtual reality parameters allows us to manipulate the degree of stretch-activation in silico while retaining all other physiological properties of the muscle. With artificially enhanced stretch activation, we find that hawkmoth muscle can support stretch-activated work at typical wingbeat frequencies. When simultaneously stimulated at wingbeat frequency, interference between stretch and neural activation results in variable work production. However, this interference disappears when the two activation timescales are close to each other resulting in entrainment to the neural drive. Matching time scales suggests an evolutionary path for smoothly transitioning to stretch-activated, asynchronous flight and back again.

physiology↗

bioRxiv2016

Inferring physiological energetics of loggerhead turtle (Caretta caretta) from existing data using a general metabolic theory

Loggerhead turtle is an endangered sea turtle species with a migratory lifestyle and worldwide distribution, experiencing markedly different habitats throughout its lifetime. Environmental conditions, especially food availability and temperature, constrain the acquisition and the use of available energy, thus affecting physiological processes such as growth, maturation, and reproduction. These physiological processes at the population level determine survival, fecundity, and ultimately the population growth rate--a key indicator of the success of conservation efforts. As a first step towards the comprehensive understanding of how environment shapes the physiology and the life cycle of a loggerhead turtle, we constructed a full life cycle model based on the principles of energy acquisition and utilization embedded in the Dynamic Energy Budget (DEB) theory. We adapted the standard DEB model using data from published and unpublished sources to obtain parameter estimates and model predictions that could be compared with data. The outcome was a successful mathematical description of ontogeny and life history traits of the loggerhead turtle. Some deviations between the model and the data existed (such as an earlier age at sexual maturity and faster growth of the post-hatchlings), yet probable causes for these deviations were found informative and discussed in great detail. Physiological traits such as the capacity to withstand starvation, trade-offs between reproduction and growth, and changes in the energy budget throughout the ontogeny were inferred from the model. The results offer new insights into physiology and ecology of loggerhead turtle with the potential to lead to novel approaches in conservation of this endangered species.

Zoology↗

bioRxiv2017

The Effects of Sex and Diet on Physiology and Liver Gene Expression in Diversity Outbred Mice

Inter-individual variation in metabolic health and adiposity is driven by many factors. Diet composition and genetic background and the interactions between these two factors affect adiposity and related traits such as circulating cholesterol levels. In this study, we fed 850 Diversity Outbred mice, half females and half males, with either a standard chow diet or a high fat, high sucrose diet beginning at weaning and aged them to 26 weeks. We measured clinical chemistry and body composition at early and late time points during the study, and liver transcription at euthanasia. Males weighed more than females and mice on a high fat diet generally weighed more than those on chow. Many traits showed sex- or diet-specific changes as well as more complex sex by diet interactions. We mapped both the physiological and molecular traits and found that the genetic architecture of the physiological traits is complex, with many single locus associations potentially being driven by more than one polymorphism. For liver transcription, we find that local polymorphisms affect constitutive and sex-specific transcription, but that the response to diet is not affected by local polymorphisms. We identified two loci for circulating cholesterol levels. We performed mediation analysis by mapping the physiological traits, given liver transcript abundance and propose several genes that may be modifiers of the physiological traits. By including both physiological and molecular traits in our analyses, we have created deeper phenotypic profiles to identify additional significant contributors to complex metabolic outcomes such as polygenic obesity. We make the phenotype, liver transcript and genotype data publicly available as a resource for the research community.

genetics↗

bioRxiv2019

Effect of EBV-transformation on Oxidative Phosphorylation Physiology in Human Cell lines

AO_SCPLOWBSTRACTC_SCPLOWDo the immortalized and cryopreserved white blood cells that are part of the 1,000 Human Genomes Project represent a valuable cellular physiological resource to investigate the importance of genome wide sequence variation? While much research exists on the nucleotide variation in the 1,000 Human Genomes, there are few quantitative measures of these humans physiologies. Fortunately, physiological measures can be done on the immortalized and preserved cells from each of the more than 1,000 individuals that are part of Human Genome project. However, these human white blood cells were immortalized by transforming them with the Epstein-Barr virus (EBV-transformed lymphoblastoid cell lines (LCL)). This transformation integrates the viral genome into the human genome and potentially affects important biological differences among individuals. The questions we address here are whether EBV transformations significantly alters the cellular physiology so that 1) replicate transformations within an individual are significantly different, and 2) whether the variance among replicates obscures the variation among individuals. To address these questions, we quantified oxidative phosphorylation (OxPhos) metabolism in LCLs from six individuals with 4 separate and independent EBV-transformations. We examined OxPhos because it is critical for energy production, and mutations in this pathway are responsible for most inborn metabolic diseases. The data presented here demonstrate that there are small but significant effects of EBV-transformations on some OxPhos parameters. In spite of significant variation due to transformations, there is greater and significant variation among individuals in their OxPhos metabolism. Thus, the LCLs from the 1,000 Human Genome project could provide valuable insights into the natural variation of cellular physiology because there is statistically significant variation among individuals when using these EBV-transformed cells.

evolutionary biology↗

bioRxiv2020

A Novel Method for Calculating Beta Band Burst Durations in Parkinson's Disease Using a Physiological Baseline

BackgroundPathological bursts of neural activity in Parkinsons disease present as exaggerated subthalamic neuronal oscillations in the 8-30 Hz frequency range and are related to motor impairment. New MethodThis study introduces a novel method for determining burst dynamics using a baseline that matches physiological 1/f spectrum activity. We used resting state local field potentials from people with Parkinsons disease and a simulated 1/f signal to measure beta burst durations, to demonstrate how tuning parameters (i.e., bandwidth and center frequency) affect burst durations, to compare this with high power threshold methods, and to study the effect of increasing neurostimulation intensities on burst duration. ResultsBurst durations calculated using the Anderson method captured the longest and broadest distribution of burst durations in a pathological beta band compared to previous methods. Mean beta band burst durations were significantly shorter on compared to off neurostimulation (p = 0.011), and their distribution was shifted towards that of the physiological 1/f spectrum during increasing intensities of stimulation. Comparison with Existing MethodExisting methods of measuring local field potential power either lack temporal specificity to detect bursts (power spectral density diagrams and spectrograms) or include only higher power bursts and portions of the neural signal. ConclusionsWe suggest that this novel method is well suited to quantify the full range of fluctuations in beta band neural activity in the Parkinsonian brain. This method may reveal more relevant feedback biomarkers than averaged beta band power for future closed loop algorithms. HighlightsA novel method for measuring variability in subthalamic local field potential oscillations in Parkinsons disease using a physiological baseline of power. Modeling normal brain activity using a physiological 1/f spectrum. Burst durations depend on choice of bandwidth and center frequency. Defining an inert frequency band whose mean burst duration overlap the physiological 1/f spectrum, from which the baseline was determined. Burst durations progressively shortened during increasing intensities of deep brain stimulation.

neuroscience↗

bioRxiv2020

Attention-dependent physiological correlates in sleep deprived healthy humans

Distinct cognitive functions are based on specific brain networks, but they are also affected by workload. The workload is a common factor affecting cognitive functioning that, by activating the Central Autonomic Network, modulates heart rate peripheral correlates of cognitive functioning. Based on these premises, we expected that the peripheral patterns associated with different attentional systems would have common (workload-related) and specific (task-dependent) components. To disentangling the components, a profile of peripheral physiological correlates of cognitive functioning was derived by studying healthy volunteers while performing different cognitive tasks during baseline and post-sleep deprivation conditions. Post-sleep deprivation condition was introduced to increase workload during tasks, allowing the investigation of the same participant at different levels of workload in different conditions. We performed, in each condition, physiological recordings of heart pulse, facial temperature and head movements during tasks assessing attentional networks efficiency (ANT - Attentional Network Task; CCT - Continuous Compensatory Tracker). We assessed perceived workload after the execution of these tasks. Physiological correlates of cognitive performance were identified by associating changes of task indices with the corresponding changes in physiological measures from baseline to post-sleep deprivation condition. Correlation analyses were performed after correction for the between-conditions workload changes: indeed, mental and physical demands of perceived workload increased after sleep deprivation. We found that alerting/vigilance has specific physiological correlates as indicated by the negative correlation between changes in ANT-alerting score and changes in amplitude of head movements and the positive one between changes in CCT-visuomotor speed indexing alertness and changes in facial temperature.

neuroscience↗

bioRxiv2021

The selenophosphate synthetase, selD, is important for Clostridioides difficile physiology

The endospore-forming pathogen, Clostridioides difficile, is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and healthcare. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using RNAseq, we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD+ can be regenerated by other pathways. ImportanceC. difficile is a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop the C. difficile lifecycle, other non-antibiotic treatment options are in urgent need of development. Towards this goal, we find that a metabolic process used by only a small fraction of the microbiota is important for C. difficile physiology - Stickland metabolism. Here, we use our CRISPR-Cas9 system to knock in a copy of the selD gene into the deletion strain to restore selD at its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology - from vegetative growth to spore formation and outgrowth post-germination.

microbiology↗

bioRxiv2021

A high-content platform for physiological profiling and unbiased classification of individual neurons

High-throughput physiological assays often lose single cell resolution, precluding subtype-specific analyses of neuronal activation mechanism and drug effects. Here, we demonstrate APPOINT, Automated Physiological Phenotyping Of Individual Neuronal Types. This physiological assay platform combines calcium imaging, robotic liquid handling, and automated analysis to generate physiological activation profiles of single neurons at a large scale. Using unbiased techniques, we quantify responses to multiple sequential stimuli, enabling subgroup identification by physiology and probing of distinct mechanisms of neuronal activation within subgroups. Using APPOINT, we quantify primary sensory neuron activation by metabotropic receptor agonists and identify potential contributors to pain signaling. Furthermore, we expand the role of neuroimmune interactions by showing that human serum can directly activate sensory neurons, elucidating a new potential pain mechanism. Finally, we apply APPOINT to develop a high-throughput, all-optical approach for quantification of activation threshold and pharmacologically separate the contributions of distinct ion channel subsets to optical activation.

neuroscience↗

bioRxiv2021

Automated, high-dimensional evaluation of physiological aging and resilience in outbred mice

Behavior and physiology are essential readouts in many studies but have not benefited from the high-dimensional data revolution that has transformed molecular and cellular phenotyping. To address this, we developed an approach that combines commercially available automated phenotyping hardware with a systems biology analysis pipeline to generate a high-dimensional readout of mouse behavior/physiology, as well as intuitive and health-relevant summary statistics (resilience and biological age). We used this platform to longitudinally evaluate aging in hundreds of outbred mice across an age range from 6 months to 3.4 years. In contrast to the assumption that aging can only be measured at the limits of animal ability via challenge-based tasks, we observed widespread physiological and behavioral aging starting in early life. Using network connectivity analysis, we found that organism-level resilience exhibited an accelerating decline with age that was distinct from the trajectory of individual phenotypes. We developed a method, Combined Aging and Survival Prediction of Aging Rate (CASPAR), for jointly predicting chronological age and survival time and showed that the resulting model is able to predict both variables simultaneously, a behavior that is not captured by separate age and mortality prediction models. This study provides a uniquely high-resolution view of physiological aging in mice and demonstrates that systems-level analysis of physiology provides insights not captured by individual phenotypes. The approach described here allows aging, and other processes that affect behavior and physiology, to be studied with sophistication and rigor.

systems biology↗

bioRxiv2022

mRNA expression explains metabolic and thermal physiology

Quantifying mRNA expression, which is heritable and physiologically inducible, reveals biologically important networks and pathways underlying complex traits. Here, we quantify mRNA expression in Fundulus heteroclitus, a small teleost fish, among three populations acclimated to 12{degrees}C and 28{degrees}C and relate it to variation in six, complex, physiological traits (whole animal metabolism (WAM), critical thermal maximum (CTmax), and four substrate specific cardiac metabolic rates (CaM)). Although 366 heart mRNAs and 528 brain mRNAs had significant differential expression between the two acclimation temperatures, none of the mRNA acclimation responses were shared across all three populations in any tissue. Yet, within an acclimation temperature across all three populations, weighted gene co-expression network analyses show that mRNA expression patterns explained WAM, CTmax, and CaM trait variation. These analyses revealed 9 significant heart MEs (first principal component of module expression) and 4 significant brain MEs. Heart MEs explained variation in WAM, CTmax, and two of the four substrate specific cardiac metabolic rates at 12{degrees}C, and CTmax at 28C. In contrast, brain MEs explained CTmax and WAM at 28{degrees}C but not at 12{degrees}C. Combining MEs as multiple correlations, 82% of variation in WAM at 12{degrees}C was explained by four heart MEs, 80% of variation in fatty-acid CaM at 12{degrees}C was explained by three heart MEs, and 72% of variation in CTmax at 28{degrees}C was explained by three brain MEs. These MEs were enriched for Kyoto Encyclopedia of Genes and Genomes (KEGG) terms related to specific metabolic pathways, suggesting that they represent biologically relevant pathways. Together these data suggest that mRNA co-expression explains complex traits; moreover, physiological traits are more reliant on heart expression at 12{degrees}C and brain expression at 28{degrees}C. Author SummaryDespite an abundance of genomic data, the molecular and genetic underpinnings of complex traits remain poorly understood. To better understand the molecular basis of complex traits, we used heart and brain mRNA expression to explain complex traits- physiological responses to temperature- in individuals collected from three saltmarsh fish (Fundulus heteroclitus) populations acclimated to 12{degrees}C and 28{degrees}C. We found that while physiological traits did not differ among populations, the mRNAs important for acclimation responses were >88% unique to a single population and differed between heart and brain tissues. We also found tissue specific co-expressed mRNAs that explain up to 82% of complex traits including whole animal metabolism, upper thermal tolerance, and substrate specific cardiac metabolism measured at 12{degrees}C or 28{degrees}C acclimation conditions. Notably, sets of co-expressed mRNAs related to these traits are enriched for molecular pathways affecting metabolism, giving insight into the molecular underpinnings of these traits.

genetics↗