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bioRxiv2022

Fatty acids produced by the gut microbiota dampen host inflammatory responses by modulating intestinal SUMOylation

The gut microbiota produces a wide variety of metabolites, which interact with intestinal cells and contribute to host physiology. These metabolites regulate intestinal cell activities by modulating either gene transcription or post-translational modifications of gut proteins. The effect of gut commensal bacteria on SUMOylation, an essential ubiquitin-like modification in intestinal physiology, remains however unknown. Here, we show that short chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) produced by the gut microbiota increase protein SUMOylation in different intestinal cell lines in a pH-dependent manner. We demonstrate that these metabolites induce an oxidative stress which inactivates intestinal deSUMOylases and promotes the hyperSUMOylation of chromatin-bound proteins. In order to determine the impact of these modifications on intestinal physiology, we focused on the NF-{kappa}B signaling pathway, a key player in inflammation known to be regulated by SUMOylation. We demonstrated that the hyperSUMOylation induced by SCFAs/BCFAs inhibits the activation of the NF-{kappa}B pathway in intestinal cells by blocking the degradation of the inhibitory factor I{kappa}B in response to TNF. This results in a decrease in pro-inflammatory cytokines expression, such as IL8 or CCL20, as well as a decrease in intestinal epithelial permeability in response to TNF. Together, our results reveal that fatty acids produced by gut commensal bacteria regulate intestinal physiology by modulating SUMOylation and illustrate a new mechanism of dampening of host inflammatory responses by the gut microbiota.

cell biology↗

bioRxiv2022

Transcriptomic and functional genetic evidence for distinct ecophysiological responses across complex life cycle stages

Organisms with complex life cycles demonstrate a remarkable ability to change their phenotypes across development, presumably as an evolutionary adaptation to developmentally variable environments. Developmental variation in environmentally sensitive performance, and thermal sensitivity in particular, has been well documented in holometabolous insects. For example, thermal performance in adults and juvenile stages exhibit little genetic correlation (genetic decoupling) and can evolve independently, resulting in divergent thermal responses. Yet, we understand very little about how this genetic decoupling occurs. We tested the hypothesis that genetic decoupling of thermal physiology is driven by fundamental differences in physiology between life stages, despite a potentially conserved Cellular Stress Response. We used RNAseq to compare transcript expression in response to a cold stressor in Drosophila melanogaster larvae and adults and used RNAi (RNA interference) to test whether knocking down nine target genes differentially affected larval and adult cold tolerance. Transcriptomic responses of whole larvae and adults during and following exposure to -5{degrees}C were largely unique both in identity of responding transcripts and in temporal dynamics. Further, we found no relationship between stage-specificity and tissue-specificity of transcripts, suggesting that the differences are not simply driven by differences in tissue composition across development. In addition, RNAi of target genes resulted in largely stage-specific and sometimes sex-specific effects on cold tolerance. The combined evidence suggests that thermal physiology is largely stage-specific at the level of gene expression, and thus natural selection may be acting on different loci during the independent thermal adaptation of different life stages. Summary StatementRNAseq and gene knockout via transgenic RNAi lines suggest that physiological responses to low temperatures are largely distinct across life stages of the fly Drosophila melanogaster.

evolutionary biology↗

bioRxiv2022

Gut-liver-axis microphysiological system for studying cellular fluidic shear stress and inter-tissue interaction

Gut-liver-axis (GLA) is a fundamental interaction between the gut and liver for maintaining human health. To clarify the physiological and pathological roles of GLA in the human body, a GLA microphysiological system (GLA-MPS) holds great potential. However, in current GLA-MPS, the importance of a physiologically relevant flow for gut and liver cells cultivation is not fully addressed. In addition, the integration of individual organ perfusion, circulation flow, and organ tissue functions in a single device has not been achieved. Here, we introduce a GLA-MPS by integrating two cell culture chambers with individually applied perfusion flows and a circulation channel with an on-chip pneumatic micropump under cell culture chambers via a porous membrane for interconnecting them. We analyzed the fluid shear stress (FSS) with computational fluid dynamics simulations and confirmed that the physiologically relevant FSS (i.e., 8 x 10-3 and 1.2 x 10-7 dyne cm-2) could be applied for the gut (Caco-2) and liver (HepG2) cells, respectively. Under physiologically relevant flow, the Caco-2 and HepG2 cells in the GLA-MPS maintained a cell survival rate of 95% and 92%, respectively; further, they enhanced the expression of functional proteins such as zonula occludens 1 (ZO-1) and albumin (ALB), respectively. Thus, the presented GLA-MPS can be adapted as an advanced in vitro model in a wide range of applications for disease modeling associated with inter-tissue interactions, such as fatty liver diseases.

bioengineering↗

bioRxiv2022

Impacts and mechanisms of CO2 narcosis in bumble bees: Narcosis depends on dose, caste and mating status and is not induced by anoxia

Carbon dioxide (CO2) is commonly used to immobilize insects and to induce reproduction in bees. However, despite its wide use and potential off-target impacts, its underlying mechanisms are not fully understood. Here we used Bombus impatiens to examine whether CO2 impacts are mediated by anoxia and whether these mechanisms differ between female castes or following mating. We examined the behavior, physiology, and gene expression of workers, mated and virgin queens following exposure to anoxia, hypoxia, full and partial hypercapnia, and control. Hypercapnia and anoxia caused immobilization, but only hypercapnia resulted in behavioral, physiological, and molecular impacts in bees. Recovery from hypercapnia resulted in increased abdominal contractions and took longer in queens. Additionally, hypercapnia activated queens-but inhibited workers ovaries in a dose-dependent manner and caused a depletion of fat-body lipids in both. All responses of hypercapnia were weaker following mating in queens. Analysis of gene expression related to hypoxia and hypercapnia supported the physiological findings in queens, demonstrating that the overall impacts of CO2, excluding virgin queen ovaries, were unique and were not induced by anoxia. This study contributes to our understanding of the impacts and the mechanistic basis of CO2 narcosis in insects and its impacts on bees physiology.

biochemistry↗

bioRxiv2022

On Non-Kolmogorov turbulence in blood flow and its possible role in mechanobiological stimulation

The study of turbulence in physiologic blood flow is important due to its strong relevance to endothelial mechanobiology and vascular disease. Recently, Saqr et al (Sci Rep 10, 15492, 2020) discovered non-Kolmogorov turbulence in physiologic blood flow in vivo, traced its origins to the Navier-Stokes equation and demonstrated some of its properties using chaos and hydrodynamic-stability theories. The present work extends these findings and investigates some inherent characteristics of non-Kolmogorov turbulence in monoharmonic and multiharmonic pulsatile flow under ideal physiologic conditions. The purpose of this work is to propose a conjecture for the origins for picoNewton forces that are known to regulate endothelial cells functions. The new conjecture relates these forces to physiologic momentum-viscous interactions in the near-wall region of the flow. Here, we used high-resolution large eddy simulation (HRLES) to study pulsatile incompressible flow in a straight pipe of L/D = 20. The simulations presented Newtonian and Carreau-Yasuda fluid flows, at Reynolds number of 256 and 228, respectively, each represented by one, two and three boundary harmonics. Comparison was established based on maintaining constant time-averaged mass flow rate in all simulations. First, we report the effect of primary harmonics on the global power budget using primitive variables in phase space. Second, we describe the non-Kolmogorov turbulence in frequency domain. Third, we investigate the near-wall coherent structures in time, space and frequency domains. Finally, we propose a new conjecture for the role of turbulence in endothelial cells mechanobiology. The proposed conjecture correlates near-wall turbulence to a force field of picoNewton scale, suggesting possible relevance to endothelial cells mechanobiology.

cell biology↗

bioRxiv2022

Cervical Spinal Cord Modulation with Repeated Epidural Stimulation in Healthy Adult Rats

The effects of spinal epidural stimulation (ES) in regaining various physiological functions after a spinal cord injury (SCI) are well documented. Spinal evoked motor responses (SEMR) are commonly utilized experimental outcomes in longitudinal pre-clinical and human studies to reflect the in-vivo physiological changes in neural networks secondary to a neurological insult or neuro-rehabilitative treatments utilizing ES. However, it remains unknown if the repeated exposure to ES during SEMRs testing itself modulates the spinal cord physiology and hence the SEMRs characteristics. To address this issue, cervical ES was delivered to the healthy adult rats cervical cord using standard stimulation paradigms during multiple sessions ([~]17 hours that spanned across 100 days). Cervical SEMR and electromyography (EMG) activity from forelimb muscles during a reaching & grasping task were collected before and after 100 days. We noted persistent increase in the cervical SEMR and forelimb muscle activity during reaching & grasping task relative to baseline at the end of the stimulation period indicating increased spinal and cortical excitability. Findings from the present work suggests that cervical SEMRs are amenable to modulation by routine ES testing protocols, with prominent changes in the mono and poly synaptic component of evoked responses. Additionally, since multiple testing sessions of cervical ES alone increases the excitability of the intact spinal cord, we suggest that SEMR data be used with caution to infer the physiological status of the spinal circuitry in longitudinal studies involving multiple SEMR testing sessions. Our findings also recommend involving appropriate control groups, motor behavior correlates, and practicing caution while utilizing normalization methods to allow meaningful functional interpretation of SEMR profiles following a SCI.

neuroscience↗

bioRxiv2022

Generating dynamic carbon-dioxide from the respiratory-volume time series: A feasibility study using neural networks

In the context of fMRI, carbon dioxide (CO2) is a well-known vasodilator that has been widely used to monitor and interrogate vascular physiology. Moreover, spontaneous fluctuations in end-tidal carbon dioxide (PETCO2) reflects changes in arterial CO2 and has been demonstrated as the largest physiological noise source in the low-frequency range of the resting-state fMRI (rs-fMRI) signal. Increasing appreciation for the role of CO2 in fMRI has given rise to methods that use it for physiological denoising or estimating cerebrovascular reactivity. However, the majority of rs-fMRI studies do not involve CO2 recordings, and most often only heart rate and respiration are recorded. While the intrinsic link between these latter metrics and CO2 led to suggested possible analytical models, they have not been widely applied. In this proof-of-concept study, we propose a deep learning approach to reconstruct CO2 and PETCO2 data from respiration waveforms in the resting state. We demonstrate that the one-to-one mapping between respiration and CO2 recordings can be well predicted using fully convolutional networks (FCNs), achieving a Pearson correlation coefficient (r) of 0.946 {+/-} 0.056 with the ground truth CO2. Moreover, dynamic PETCO2 can be successfully derived from the predicted CO2, achieving r of 0.512 {+/-} 0.269 with the ground truth. Importantly, the FCN-based methods outperform previously proposed analytical methods. In addition, we provide guidelines for quality assurance of respiration recordings for the purposes of CO2 prediction. Our results demonstrate that dynamic CO2 can be obtained from respiration-volume using neural networks, complementing the still few reports in deep-learning of physiological fMRI signals, and paving the way for further research in deep-learning based bio-signal processing.

bioinformatics↗

bioRxiv2022

An intrinsic alkalization circuit turns on mntP-riboswitch under manganese stress in Escherichia coli

The trace metal manganese in excess affects iron-sulfur cluster and heme-protein biogenesis eliciting cellular toxicity. The manganese efflux protein MntP is crucial to evading manganese toxicity in bacteria. Recently, two Mn-sensing riboswitches upstream of mntP and alx in Escherichia coli have been reported to mediate the upregulation of their expression under manganese shock. As the alx-riboswitch is also responsive to alkaline shock administered externally, it is intriguing whether mntP-riboswitch is also responsive to alkaline stress. Furthermore, how both manganese and alkaline pH simultaneously regulate these two riboswitches under physiological conditions is a puzzle. Using multiple approaches, we show that manganese shock activated glutamine synthetase (GlnA) and glutaminases (GlsA and GlsB) to spike ammonia production in E. coli. The elevated ammonia intrinsically alkalizes the cytoplasm. We establish that this alkalization under manganese stress is crucial for attaining the highest degree of riboswitch activation. Additional studies showed that alkaline pH promotes a 17 to 22-fold tighter interaction between manganese and the mntP-riboswitch element. Our study uncovers a physiological linkage between manganese efflux and pH homeostasis that mediates enhanced manganese tolerance. Significance statementRiboswitch RNAs are cis-acting elements that can adopt alternative conformations in the presence or absence of a specific ligand(s) to modulate transcription termination or translation initiation processes. In the present work, we show that how manganese and alkaline pH both are necessary for maximal mntP-riboswitch activation to mitigate the manganese toxicity. This study bridges the gap between earlier studies that separately emphasize the importance of alkaline pH and manganese in activating the riboswitches belonging to the yybP-ykoY-family. This study also ascribes a physiological relevance as to how manganese can rewire cellular physiology to render cytoplasmic pH alkaline for its homeostasis.

biochemistry↗

bioRxiv2022

intra-single cell sequencing (iSCseq) spotlights transcriptomic and epigenetic heterogeneity inside multinucleated osteoclast

Single-cell RNA-seq (scRNA-seq) has clarified cellular heterogeneity within cell populations. However, scRNA-seq and spatial transcriptomics cannot capture the dynamic transcriptomic changes inside living cells. To decode subcellular gene expression, we developed intra-single cell sequencing (iSCseq), a novel approach that combines confocal imaging, repeatedly picking up cellular components inside living cells, and next-generation sequencing (intra single-cell RNA-seq; iSCseq). iSCseq illustrated the subcellular heterogeneity of gene expression. iSCseq revealed not only multiple differentiation stages embedded in the same cell, but also physical cytoskeletal connections, physiological activity of mitochondria, and intracellular calcium, as confirmed by transcriptomic evidence. Inclusive iSCseq with in vivo scRNA-seq datasets identified new osteoclast subsets in physiological and pathological bones. Network analysis with centrality provided insights into the connection between subcellular components, and clearly divided differentiation and fusion processes in multinucleation. The iSCseq approach has the potential to enhance cell biology at subcellular resolution and identify new therapeutic targets. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/506360v4_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@115c1d8org.highwire.dtl.DTLVardef@1695ad2org.highwire.dtl.DTLVardef@1c9d257org.highwire.dtl.DTLVardef@1107ddb_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefintra-single cell sequencing (iSCseq) enhances single-cell technology by combining live cell imaging, subcellular sampling from living cells and sequencing, offering deeper insights into cell functions and pathology at subcellular resolution through inclusive analysis with scRNA-seq and advanced centrality-focused network analysis. HighlightsO_LIintra-single cell sequencing (iSCseq) clarifies subcellular heterogeneity C_LIO_LIiSCseq connects morphological and physiological features with transcriptome C_LIO_LIInclusive iSCseq unveils osteoclast subsets in physiological and pathological bones C_LIO_LILinkage at subcellular resolution reveals key players in characteristic fusion C_LI

genomics↗

bioRxiv2022

Adaptive structural and functional evolution of the placenta protects fetal growth in high elevation deer mice

Environmental hypoxia challenges female reproductive physiology in placental mammals, increasing rates of gestational complications. Adaptation to high elevation has limited many of these effects in humans and other mammals, offering potential insight into the developmental processes that lead to and protect against hypoxia-related gestational complications. However, our understanding of these adaptations has been hampered by a lack of experimental work linking the functional, regulatory, and genetic underpinnings of gestational development in locally-adapted populations. Here, we dissect high-elevation adaptation in the reproductive physiology of deer mice, (Peromyscus maniculatus), a rodent species with an exceptionally broad elevational distribution that has emerged as a model for hypoxia adaptation. Using experimental acclimations, we show that lowland mice experience pronounced fetal growth restriction when challenged with gestational hypoxia, while highland mice maintain normal growth by expanding the compartment of the placenta that facilitates nutrient and gas exchange between dam and fetus. We then use compartmentspecific transcriptome analyses to show that adaptive structural remodeling of the placenta is coincident with widespread changes in gene expression within this same compartment. Genes associated with fetal growth in deer mice significantly overlap with genes involved in human placental development, pointing to conserved or convergent pathways underlying these processes. Finally, we overlay our results with genetic data from natural populations to identify can-didate genes and genomic features that contribute to these placental adaptations. Collectively, these experiments advance our understanding of adaptation to hypoxic environments by revealing physiological and genetic mechanisms that shape fetal growth trajectories under maternal hypoxia. Significance StatementResidence at high elevations is associated with higher risk pregnancies and low birth weight, yet the causal mechanisms remain poorly understood. Using a high elevation-adapted rodent model, we investigated the physiological traits that explain fetal growth trajectories in low oxygen environments, and how evolutionary adaptation has modified these traits. We showed that high- and low-elevation populations of deer mice differ in their susceptibility to fetal growth restriction during gestational hypoxia and that these population-level differences are associated with structural and transcriptomic changes in the placenta. We further link placental gene expression to genomic features under selection at high elevation. Our findings identify adaptations that are likely relevant to offsetting the effects of hypoxia on fetal and placental development across mammals.

evolutionary biology↗

bioRxiv2022

Variability and Impact of Musculoskeletal Modeling Parameters for the Human Elbow

Musculoskeletal modeling has significant potential as a translational and clinical research tool for examining neuromuscular injuries and disorders. However its adoption has been limited due, in part, to the difficulty of measuring the subject-specific physiological measures that define model parameters. These measurements may require substantial time and expensive methods, such as MRI, to determine the parameters of a model and thus ensure its accuracy. We used a Monte Carlo simulation to examine the impact of parameter variability on the ill-defined, inverse approximation of muscle activity. We first amalgamated previously published measurements of the physiological characteristics of the upper/lower arm and the biceps/triceps muscles. We then used the observed distributions of these measurements to set physiologically plausible boundaries on uniform distributions and then generated perturbed parameter sets. We computed the root mean squared error (RMSE) between muscle activity patterns generated by the perturbed model parameters to those generated by the original parameters. Regression models were fit to the RMSE of the approximated muscle activity patterns to determine the sensitivity of the simulation results to variation in each parameter. We found that variation in parameters associated with muscle physiology had the most effect on RMSE, suggesting that these parameters may require subject-specific scaling, whereas parameters associated with skeletal bodies had less effect, and might be safely approximated by their population means.

bioengineering↗

bioRxiv2022

Label free autofluorescence imaging permits comprehensive and simultaneous assignment of cell type identity and reveals the existence of airway secretory cell associated antigen passages (SAPs).

The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a properly orchestrated physiologic response. Thus, modern approaches to dissect physiologic mechanism would benefit from an ability to identify specific cell types in live tissues and image them in real time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the simultaneous study of 2 or 3 cell types. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in real time. Furthermore, we find that this methodology for direct cell type specific identification avoid potential pitfalls with the use of ostensibly cell type-specific markers that can be altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology using a clinically relevant model of cholinergic stimulation and identify dynamic secretory cell associated antigen passages (SAPs) that are highly reminiscent of previously reported goblet cell associated antigen passages (GAPs) in the intestine. eLifes Review ProcesseLife works to improve the process of peer review so that it more effectively conveys the assessment of expert reviewers to authors, readers and other interested parties. In the future we envision a system in which research is first published as a preprint and the outputs of peer review are the primary way research is assessed, rather than journal title. Our editorial process produces two outputs: i) an assessment by peers designed to be posted alongside a preprint for the benefit of the readers; i) detailed feedback on the manuscript for the authors, including requests for revisions and suggestions for improvement. Therefore we want to change how we construct and write peer reviews to make themuseful to both authors and readers in a way that better reflects the work you put into reading and thinking about a paper. eLife reviews now have three parts: O_LIAn evaluation summary (in two or three sentences) that captures the major conclusions of the review in a concise manner, accessible to a wide audience. C_LIO_LIA public review that details the strengths and weaknesses of the manuscript before you, and discusses whether the authors claims and conclusions are justified by their data. C_LIO_LIA set of private recommendations for the authors that outline how you think the science and its presentation could be strengthened. C_LI All three sections will be used as the basis for an eLife publishing decision, which will, as always, be made after a consultation among the reviewers and editor. Each of the public reviews will be published (anonymously) alongside the preprint, together with a response from the authors if they choose. In the case of papers we reject after review, the authors can choose to delay posting until their paper has been published elsewhere. If this is your first time going through this new process, we ask that you take some time to read our Reviewer Guide, which discusses how we see each section will be used, what it should contain, and what we hope it accomplishes. And we remind you that, with the shift of reviews from private correspondence to public discourse, it is more important than ever that reviews are written in a clear and constructive manner appropriate for a public audience and mindful of the impact language choices might have on the authors.

cell biology↗

bioRxiv2022

Costs and benefits of maternal nest choice: tradeoffs between brood survival and thermal stress for small carpenter bees

Nest site selection is a crucial decision for bees because where mothers construct their nests influences the developmental environment of their offspring. Small carpenter bees (Ceratina calcarata) nest in sun or shade, suggesting that maternal decisions about nest sites are influenced by thermal conditions that influence juvenile growth and survival. We investigated the costs and benefits to mothers and their offspring of warmer or cooler nest sites using a field experiment in which mothers and newly founded nests were placed in sunny or shady habitats. Maternal costs and benefits in sunny and shady treatments were quantified by comparing brood provisioning behaviour, nest size, number of brood cells, and offspring survival rates. Juvenile costs and benefits were quantified as body size, high temperature tolerance (CTmax), metabolic rate, and pupal duration. The major maternal benefit of nesting in sun was significantly lower rates of total nest failure (caused by predation, parasitism or abandonment), which led to sun mothers producing 3.2 brood on average, while shade mothers produced only 2.9. However, sun nesting entailed costs to brood, which were significantly smaller, less likely to survive to adulthood and had significantly elevated CTmax. This suggests that juvenile bees in sun nests bees experienced thermal stress during development, causing them to shunt resources from growth to thermoprotection, at the cost of smaller size and higher mortality. Pupae raised in a thermal-gradient "BeeCR" machine developed significantly faster at warmer average temperatures, which may be an additional benefit of sun nesting. Overall, our results highlight a tradeoff between maternal benefits and offspring costs when mothers choose nest sites, in which maternal fitness is enhanced by nesting in sun, despite significant physiological costs to offspring, due to the necessity for thermoprotective responses. Thinking through pandemic researchThe first lockdowns of the COVID-19 pandemic began as we prepared to enter the second field season of this study in 2020. Student research halted overnight. Lab access and travel were restricted. With limited access to field sites and no access to lab equipment, we brainstormed alternative approaches that would repeat, if not replicate, our main experiments of 2019 and fulfill degree requirements for JL de Haans MSc in a satisfying way. Our 2019 results had provided convincing evidence developmental temperature has long-term impacts on C. calcarata physiology, so we thought about which physiological measurements would be feasible outside the lab. Authors MH Richards and GJ Tattersall suggested collecting more measurements of CTmax: the Peltier plate device required running water, but a portable water pump and a bucket allowed the apparatus to be set up anywhere. No calibration of instruments was required, and the only maintenance was to change the water in the bucket after a few hours of use. Thus, a students home basement became a laboratory. To investigate how temperatures affect developmental rate, we needed to raise bees in controlled environments, but incubators were not available. Author A Skandalis suggested repurposing a gradient PCR unit as a portable insect incubator ("The BeeCR"). The idea was tested successfully at home in 20202, so a larger study was done by J Maretzki in 2021 when undergraduate lab access was permitted again. Two outcomes of our pandemic pivot produced long-term benefits for our research. The BeeCR is a flexible, inexpensive, easy-to-use incubator perfectly suited for raising small insects at multiple simultaneous sets of variable temperatures. And the ease with which "field" sites could be established in our backyards demonstrates how amenable small carpenter bees are to field manipulations, suggesting this is a model species for addressing a variety of ecological and physiological questions.

ecology↗

bioRxiv2022

Impacts of ocean acidification and warming on post-larval growth and metabolism in two populations of the great scallop (Pecten maximus L.)

Ocean acidification and warming are key stressors for many marine organisms. Some organisms display physiological acclimatisation or plasticity, but this may vary across species ranges, especially if populations are adapted to local climatic conditions. Understanding how acclimatisation potential varies among populations is therefore important in predicting species responses to climate change. We carried out a common garden experiment to investigate how different populations of the economically important great scallop (Pecten maximus) from France and Norway responded to variation in temperature and pCO2 concentration. After acclimation, post-larval scallops (spat) were reared for 31 days at one of two temperatures (13{degrees}C and 19{degrees}C) under either ambient or elevated pCO2 (pH 8.0 and pH 7.7). We combined measures of proteomic, metabolic, and phenotypic traits to produce an integrative picture of how physiological plasticity varies between the populations. The proteome of French spat showed significant sensitivity to environmental variation, with 12 metabolic, structural and stress-response proteins responding to temperature and/or pCO2. Principal component analysis revealed seven energy metabolism proteins in French spat that were consistent with countering ROS stress under elevated temperature. Oxygen uptake in French spat did not change under elevated temperature, but increased under elevated pCO2. In contrast, Norwegian spat reduced oxygen uptake under both elevated temperature and pCO2. Metabolic plasticity seemingly allowed French scallops to maintain greater energy availability for growth than Norwegian spat. However, increased physiological plasticity and growth in French spat may come at a cost, as French (but not Norwegian) spat showed reduced survival under elevated temperature. Summary StatementJuvenile scallops from France and Norway differ in their response to warming and acidification. French scallops show more physiological plasticity, adjusting their proteome and metabolism in order to maintain growth.

evolutionary biology↗

bioRxiv2023

Single Molecule Mechanics and Kinetics of Cardiac Myosin Interacting with Regulated Thin Filaments

The cardiac cycle is a tightly regulated process wherein the heart generates force to pump blood to the body during systole and then relaxes during diastole. Disruption of this finely tuned cycle can lead to a range of diseases including cardiomyopathies and heart failure. Cardiac contraction is driven by the molecular motor myosin, which pulls regulated thin filaments in a calcium-dependent manner. In some muscle and non-muscle myosins, regulatory proteins on actin tune the kinetics, mechanics, and load dependence of the myosin working stroke; however, it is not well understood whether or how thin filament regulatory proteins tune the mechanics of the cardiac myosin motor. To address this critical gap in knowledge, we used single-molecule techniques to measure the kinetics and mechanics of the substeps of the cardiac myosin working stroke in the presence and absence of thin filament regulatory proteins. We found that regulatory proteins gate the calcium-dependent interactions between myosin and the thin filament. At physiologically relevant ATP concentrations, cardiac myosins mechanics and unloaded kinetics are not affected by thin filament regulatory proteins. We also measured the load-dependent kinetics of cardiac myosin at physiologically relevant ATP concentrations using an isometric optical clamp, and we found that thin filament regulatory proteins do not affect either the identity or magnitude of myosins primary load-dependent transition. Interestingly, at low ATP concentrations, thin filament regulatory proteins have a small effect on actomyosin dissociation kinetics, suggesting a mechanism beyond simple steric blocking. These results have important implications for both disease modeling and computational models of muscle contraction. Significance StatementHuman heart contraction is powered by the molecular motor {beta}-cardiac myosin, which pulls on thin filaments consisting of actin and the regulatory proteins troponin and tropomyosin. In some muscle and non-muscle systems, these regulatory proteins tune the kinetics, mechanics, and load dependence of the myosin working stroke. Despite having a central role in health and disease, it is not well understood whether the mechanics or kinetics of {beta}-cardiac myosin are affected by regulatory proteins. We show that regulatory proteins do not affect the mechanics or load-dependent kinetics of the working stroke at physiologically relevant ATP concentrations; however, they can affect the kinetics at low ATP concentrations, suggesting a mechanism beyond simple steric blocking. This has important implications for modeling of cardiac physiology and diseases.

biophysics↗

bioRxiv2023

Extracellular communication between brain cells through functional transfer of Cre mRNA

1In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo overtime. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEVs transfer at physiological levels which will shed light on the role of bdEVs in neural communication within the brain and beyond.

cell biology↗

bioRxiv2023

A Four "Core Genotypes" rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases

BackgroundPrevious research on Four Core Genotypes and XY* mice has been instrumental in establishing important effects of sex-chromosome complement that cause sex differences in physiology and disease. We have generated rat models using similar modifications of the testis-determining gene Sry, to produce XX and XY rats with the same type of gonad, as well as XO, XXY and XYY rats with varying gonads. The models permit discovery of novel sex-chromosome effects (XX vs. XY) that contribute to sex differences in any rat phenotype, and test for effects of different numbers of X or Y chromosomes. MethodsXY rats were created with an autosomal transgene of Sry, producing XX and XY progeny with testes. In other rats, CRISPR-Cas9 technology was used to remove Y chromosome factors that initiate testis differentiation, producing fertile XY gonadal females. Interbreeding of these lines produced rats with interesting combinations of sex chromosomes and gonads: XO, XX, XY, XXY rats with ovaries; and XO, XX, XY, XXY, and XYY rats with testes. These groups can be compared to detect sex differences caused by sex-chromosome complement (XX vs. XY) and/or by gonadal hormones (rats with testes vs. ovaries). Other comparisons detect the effects of X or Y chromosome number (in gonadal females: XO vs. XX, XX vs. XXY, XO vs. XY, XY vs. XXY; in gonadal males: XY vs. XXY, XY vs. XYY; XX vs. XXY, XO vs. XY). ResultsWe measured numerous phenotypes to characterize these models, including gonadal histology, breeding performance, anogenital distance, levels of reproductive hormones, body and organ weights, and central nervous system sexual dimorphisms. Serum testosterone levels were comparable in adult XX and XY gonadal males. Phenotypes previously known to be sexually differentiated by the action of gonadal hormones were found to be similar in XX and XY rats with the same type of gonad, suggesting that XX and XY rats with the same type of gonad have comparable levels of gonadal hormones at various stages of development. ConclusionThe results establish powerful new models to discriminate sex-chromosome and gonadal hormone effects that cause sexual differences in rat physiology and disease. Plain English SummaryThe Four Core Genotypes and XY* mouse models have been broadly useful for determining if sex differences in any mouse phenotype are caused by gonadal hormones, or by sex-chromosome complement (XX vs. XY), and if sex-chromosome effects are caused by X- or Y-linked mechanisms. Using gene knockout and transgenic methods, we have produced laboratory rat models that offer similar capabilities. The new rat models allow investigators to test with relative ease, for the first time, if a sex difference in a rat trait is caused by effects of XX vs. XY sex chromosomes, not mediated by effects of gonadal hormones, and to narrow the search for X or Y genes that have that role. The models produce XO, XX, XY, and XXY rats with ovaries, and XO, XX, XY, XXY, and XYY rats with testes. The four XX and XY groups represent a Four Core Genotypes rat model, comparison of which tests for sex-chromosome and gonadal hormonal effects that cause female and male rats to have different physiological or disease traits. Moreover, comparison of rats with different numbers of X chromosomes, or of Y chromosomes, but with the same type of gonad, provides evidence regarding the effects of X or Y dosage on rat traits. The new models will improve understanding of the impact of sex chromosomes on diseases or traits that are best modeled in rats. They will also improve understanding of the evolution of functional roles of sex chromosomes. HighlightsIt is advantageous to establish the factors that cause sex differences in diseases, because those factors mitigate or exacerbate diseases. We have produced new laboratory rats that have different types and numbers of sex chromosomes but the same type of gonad, allowing investigation of the role of sex chromosomes in causing sex differences in physiology and disease. The new rat lines allow comparison of XX and XY rats with the same type of gonad, to detect sex differences caused in part by the sex chromosomes. Other comparisons of rats with the same gonad but with different numbers of X chromosomes (XO vs. XX, XY vs XXY) or of Y chromosomes (XO vs. XY, XX vs. XXY, XY vs. XYY) detect effects of X or Y chromosome number. These resources can uncover sex-chromosome effects on any rat phenotype.

genetics↗

bioRxiv2023

Escherichia coli aggregates mediated by native or synthetic adhesins exhibit both core and adhesin-specific transcriptional responses

Bacteria can rapidly tune their physiology and metabolism to adapt to environmental fluctuations. In particular, they can adapt their lifestyle to the close proximity of other bacteria or presence of different surfaces. However, whether these interactions trigger transcriptomic responses is poorly understood. We used a specific set up of E. coli strains expressing native or synthetic adhesins mediating bacterial aggregation to study the transcriptomic changes of aggregated compared to non-aggregated bacteria. Our results show that following aggregation, bacteria exhibit a core response independent of the adhesin type, with differential expression of 56.9% of the coding genome, including genes involved in stress response and anaerobic lifestyle. Moreover, when aggregates were formed via a naturally expressed E. coli adhesin (Antigen 43), the transcriptomic response of the bacteria was more exaggerated compared to aggregates formed via a synthetic adhesin. This suggests that the response to aggregation induced by native E. coli adhesins could have been finely tuned during bacterial evolution. Our study therefore provides insights on the effect of self-interaction in bacteria and allows a better understanding of why bacterial aggregates exhibit increased stress tolerance. ImportanceFormation of bacterial aggregates has an important role in both clinical and ecological contexts. Although these structures have been previously shown to be more resistant to stressful conditions, the genetic basis of this stress tolerance associated with the aggregate lifestyle is poorly understood. Surface sensing mediated by different adhesins can result in varying changes on bacterial physiology. However, whether adhesin-adhesin interactions as well as the type of adhesin mediating aggregation affects bacterial cell physiology is unknown. By sequencing the transcriptomes of aggregated and non-aggregated cells expressing native or synthetic adhesins, we characterized the effects of aggregation and adhesin type on E. coli physiology.

microbiology↗