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bioRxiv2024

Cell morphology as a quantifier for functional states of resident tissue macrophages

Resident tissue macrophages (RTMs) are essential for tissue homeostasis. Their diverse functions, from monitoring interstitial fluids to clearing cellular debris, are accompanied by characteristic morphological changes that reflect their functional status. While current knowledge of macrophage behaviour comes primarily from in vitro studies, their dynamic behavior in vivo is fundamentally different, necessitating a more physiologically relevant approach to their understanding. In this study, we employed intravital imaging to generate dynamic data from peritoneal RTMs in mice under various conditions and developed a comprehensive image processing pipeline to quantify RTM morphodynamics over time, defining human-interpretable cell size and shape features. These features allowed for the quantitative and qualitative differentiation of cell populations in various functional states, including pro- and anti-inflammatory activation and endosomal dysfunction. The study revealed that under steady-state conditions, RTMs exhibit a wide range of morphodynamical phenotypes, constituting a naive morphospace of behavioral motifs. Upon challenge, morphodynamic patterns changed uniformly at the population level but predominantly within the constraints of this naive morphospace. Notably, aged animals displayed a markedly shifted naive morphospace, indicating drastically different behavioral patterns compared to their young counterparts. The developed method also proved valuable in optimizing explanted tissue setups, bringing RTM behavior closer to the physiological native state. Our versatile approach thus provides novel insights into the dynamic behavior of bona fide macrophages in vivo, enabling the distinction between physiological and pathological cell states and the assessment of functional tissue age on a population level. Author summaryIn this study, we combine state-of-the-art in vivo imaging with advanced computational analysis to reveal the dynamic behavior of peritoneal resident tissue macrophages (RTMs) in their natural environment. These sentinel cells, which are crucial for tissue homeostasis, constantly monitor their environment and, in the process, undergo dynamic morphological changes that have remained largely uninvestigated due to technical limitations. Using two-photon microscopy, we captured time-lapse images of RTMs in the peritoneal serosa under various experimental conditions. Our customized image processing pipeline allowed a comprehensive assessment of cell morphology and dynamics and provided unprecedented insights into the behavior of RTMs in vivo, enabling us to distinguish cell populations in different physiological and pathological states. Our work opens up new avenues for the dynamic in situ phenotyping of macrophage functionality in disease contexts without their extraction from tissues and provides a novel perspective on the behavior of RTMs in their natural microenvironment. This versatile tool promises to advance our understanding of tissue homeostasis and macrophage function in health and disease, with potential applications in both basic research and clinical settings.

immunology↗

bioRxiv2024

Cilia-mediated cerebrospinal fluid flow modulates neuronal and astroglial activity in the zebrafish larval brain.

The brain uses a specialized system to transport cerebrospinal fluid (CSF). This system consists of interconnected ventricles lined by ependymal cells, which generate a directional flow upon beating of their motile cilia. Motile cilia act jointly with other physiological factors, including active CSF secretion and cardiac pressure gradients, to regulate CSF dynamics. The content and movement of CSF are thought to be important for brain physiology. Yet, the link between cilia-mediated CSF flow and brain function is poorly understood. In this study, we addressed the role of motile cilia-mediated CSF flow on brain development and physiology using zebrafish larvae as a model system. By analyzing mutant animals with paralyzed cilia, we identified that loss of ciliary motility did not alter progenitor proliferation, overall brain morphology, or spontaneous neural activity. Instead, we identified that cilia paralysis led to randomization of brain asymmetry. We also observed altered neuronal responses to photic stimulation, especially in the optic tectum and hindbrain. Since astroglia contact CSF at the ventricular walls and are essential for regulating neuronal activity, we next investigated astroglial activity in motile cilia mutants. Our analyses revealed a striking reduction in astroglial calcium signals both during spontaneous and light-evoked activity. Altogether, our findings highlight a novel role of motile cilia-mediated flow in regulating brain physiology through modulation of neural and astroglial networks.

neuroscience↗

bioRxiv2024

Don't Leave the Past Behind: How Larval Experience Shapes Pupal Antipredator Response in Aedes aegypti

Animals use predation encounters or risk experiences to influence their future antipredator responses. Such carryover effects of predation can benefit them by enhancing their antipredator behaviour and thereby decreasing their risk of mortality through predation. Despite these fitness benefits, behavioural carryovers of predation past may not be a common phenomenon in complex life cycles. Complex life cycles pose a unique evolutionary and physiological challenge to behavioural carryovers since every life stage is distinct in morphology, physiology, and function. Each life stage of a complex life cycle is expected to evolve its independent response based on the real-time threat level and manage the trade-offs accordingly. Along with the evolutionary challenge, physiological barriers can hamper behavioural carryovers, especially in holometabolous insects, where we observe extensive tissue remodelling and developmental compartmentalisation. We investigated behavioural carryover in the holometabolous mosquito model system, Aedes aegypti. We asked whether predation risk during a life stage carries over to the subsequent stage, influencing its behaviour, or if the next life stage responds according to its threat environment. Aedes aegypti has four major stages- egg, larva, pupa, and adult. We examined the effect of predation-risk experience across larval and pupal stages. Larval and pupal stages differ in morphology, physiology and function. They share the same habitat and, therefore, similar threats. We manipulated the threat of predation experienced by larvae and investigated its influence on pupal behaviour. We found behavioural carryover in the pupal stage for the first time and discovered exciting interactions between past experiences and the current threat environment. Our study underscores the crucial role of predation pressure in shaping the evolution of complex life cycles, emphasising the significance of early experiences with predators in influencing behavioural traits across distinct life stages.

ecology↗

bioRxiv2024

Variability in drought gene expression datasets highlight the need for community standardization

Physiologically relevant drought stress is difficult to apply consistently, and the heterogeneity in experimental design, growth conditions, and sampling schemes make it challenging to compare water deficit studies in plants. Here, we re-analyzed hundreds of drought gene expression experiments across diverse model and crop species and quantified the variability across studies. We found that drought studies are surprisingly uncomparable, even when accounting for differences in genotype, environment, drought severity, and method of drying. Many studies, including most Arabidopsis work, lack high-quality phenotypic and physiological datasets to accompany gene expression, making it impossible to assess the severity or in some cases the occurrence of water deficit stress events. From these datasets, we developed supervised learning classifiers that can accurately predict if RNA-seq samples have experienced a physiologically relevant drought stress, and suggest this can be used as a quality control for future studies. Together, our analyses highlight the need for more community standardization, and the importance of paired physiology data to quantify stress severity for reproducibility and future data analyses.

plant biology↗

bioRxiv2024

Microbiome-derived metabolite effects on intestinal barrier integrity and immune cell response to infection

The gut microbiota exerts a significant influence on human health and disease. While compositional changes in the gut microbiota in specific diseases can easily be determined, we lack a detailed mechanistic understanding of how these changes exert effects at the cellular level. However, the putative local and systemic effects on human physiology that are attributed to the gut microbiota are clearly being mediated through molecular communication. Here we determined the effects of a number of gut microbiome-derived metabolites on the first line of defence in the gut. Using in vitro models of intestinal barrier integrity, and studying the interaction of macrophages with pathogenic and non-pathogenic bacteria, we could ascertain the influence of these metabolites at the cellular level at physiologically relevant concentrations. Many metabolites exerted competing influences on intestinal epithelial or immune cells, specific metabolite effects were noted on barrier function, polarised cytokine release and the lifespan of metabolite treated cells. Our findings reiterate the complexity of understanding microbiome effects on host physiology with many metabolites having contrasting effects on host cells. However, our results underline that microbiome metabolites are crucial mediators of barrier function and the innate response to infection. Understanding the effects of these metabolites at the cellular level will allow us to move towards a better mechanistic understanding of microbiome influence over host physiology, a crucial step in advancing microbiome research.

microbiology↗

bioRxiv2024

Variability of immune gene expression among different groups within ant colonies shows a multifaceted response to infection by a non-lethal ectoparasitic fungus

Social insect colonies are known to be targeted by a wide variety of different parasites and pathogens because of their high host abundance. However, within a colony, the level of risk to exposure could vary among individuals depending on their role. Unlike many known parasites, which mostly target specific groups of individuals, e.g. foragers, the myrmecoparasitic fungus Rickia wasmannii infects entire ant colonies, being linked to subtle changes in physiology, morphology and behaviour. We investigated how different groups within the colonies respond to being exposed to the fungus by measuring the expression of the genes defensin 1 and prophenoloxidase, both vital components of ant immunity. We found that workers, queens and broods varied in their immune response. Workers displayed diverse profiles, with variable responses to infection: in same-age workers, both prophenoloxidase and defensin 1 levels exhibited increases in correlation with pathogen loads. Queens exhibited a more pronounced immune response. Highly infected queens had a heightened immune response. Larvae did not show a discernible response. Morphological and physiological characteristics had limited effects on gene expression, except in the case of queens, where larger individuals displayed higher defensin 1 expression. Our study shows that these divergent responses likely stem from the differing physiological needs and priorities of various groups within the colony. HighlightsO_LIIn same-age workers, prophenoloxidase and defensin 1 levels increased with pathogen loads. C_LIO_LIBody size affected defensin 1 expression in a caste-specific manner: larger queens displayed higher expression. C_LIO_LIInfection did not elicit any specific response in larvae. C_LIO_LIThe diverse response to infection likely arise from distinct physiological needs and priorities within colony groups. C_LI

zoology↗

bioRxiv2024

Corticothalamic modelling of sleep neurophysiology with applications to mobile EEG

AO_SCPLOWBSTRACTC_SCPLOWRecent developments in mathematical modelling of EEG enable the tracking of otherwise-inaccessible neurophysiological parameters throughout sleep. Likewise, advancements in wearable electronics have enabled easy & affordable collection of sleep EEG at home. The convergence of these two advances, namely neurophysiological modelling for mobile sleep EEG, can boost preclinical and clinical assessments of sleep. However, this subject area has received limited attention in existing literature. To address this, we used an established model of the corticothalamic system to analyze EEG power spectra from 5 datasets, spanning from research-grade systems to at-home mobile EEG. In the present work, we compare the convergent and divergent features of the data and the estimated physiological model parameters. While data quality and characteristics differ considerably, key patterns consistent with previous theoretical and empirical work are observed. During the transition from lighter to deeper NREM, i) exponent of the aperiodic (1/f) spectral component is increased, ii) bottom-up thalamocortical drive is reduced, iii) corticocortical connection strengths are increased. This effect is observed in healthy subjects but is interestingly absent when taking SSRI antidepressants, suggesting possible effects of ascending neuromodulation on corticothalamic oscillations. We further show a month-long increase in REM% in one mobile EEG subject, associated with boosted high-frequency activity in spectra and higher thalamothalamic gains in the model, pointing to possible changes of thalamic inhibition in REM parasomnias. Our results provide a proof-of-principle for the utility and feasibility of this physiological modelling-based approach to analyzing mobile EEG data, providing a mechanistic measure of brain physiology during sleep. Statement of significanceWe employ a physiological model of the corticothalamic circuitry to model the EEG power spectra in sleep. We fit this model to 5 EEG datasets, and demonstrate that while mobile and non-mobile EEG recordings differ in their characteristics and quality, they can both robustly represent the changes along sleep stages using the aperiodic (1/f) component. We observe an increased corticocortical connection strength and decreased corticothalamic connection strength as the subject goes into deeper stages of NREM sleep; an effect that is, importantly, not observed in subjects taking SSRIs. This work provides a proof-of-concept for using mathematical modelling, working well for large mobile and non-mobile datasets providing valuable insight into the mechanisms generating sleep EEG.

neuroscience↗

bioRxiv2024

Nucleolar accumulation of APE1 through condensates is mediated by rRNA forming G-quadruplex structures

APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is the main endonuclease of the base excision repair (BER) pathway acting on abasic (AP)-sites in damaged DNA. APE1 is an abundant nuclear protein with a higher concentration than other BER pathway enzymes, and therefore, improper expression and localization of this factor could lead to the accumulation of toxic DNA intermediates. Altered APE1 sub-cellular localization, expression levels, or hyper-acetylation are associated with cancer development suggesting the importance of a fine-tuning mechanism for APE1 nuclear-associated processes. Recent work highlighted multi-functional roles of APE1, including rRNA quality control. However, how rRNA influences the sub-cellular localization and activity of APE1 remains poorly understood, but previously underappreciated APE1-RNA interactions may influence the ability of this protein to form biomolecular condensates and tune APE1 partitioning into nucleoli. Since nucleolar accumulation of ectopic proteins could be the result of overexpression strategies, it is imperative to have cellular models to study APE1 trafficking under physiological conditions. Here we created the first cell line to express fluorescently tagged APE1 at its endogenous locus, enabling live-cell imaging. Live-cell imaging demonstrates that APE1 nucleolar accumulation requires active rRNA transcription. When modeled in vitro, APE1 condensate formation depends on RNA G-quadruplex (rG4) structures in rRNA and is modulated by critical lysine residues of APE1. This study sheds light on the mechanisms underlying APE1 trafficking to the nucleolus and formation of RNA-dependent APE1 nucleolar condensates that may modulate a switch between the activity of this factor in rRNA processing and DNA damage repair. Significance StatementWe created and characterized the first endogenous, fluorescently tagged cell line to study APE1 subcellular trafficking under physiological and stress conditions. Using this cell line, we show that APE1 nucleolar enrichment occurs under physiological conditions and, performing in vitro droplet assays, we associate APE1 condensates with active transcription of RNA G-quadruplexes, abundantly present in healthy nucleoli. This work deepens our understanding of APE1s role in healthy cells in the absence of DNA damage and provide a novel mechanism for how this protein responds to stress. Our results suggest that phase separation is an important part of how DNA damage repair proteins switch between their normal physiological functions and their ability to correct DNA lesions.

molecular biology↗

bioRxiv2024

Differential seasonal effects of ephemeral legumes in response to moisture-nitrogen coupling in the deserts of northwestern China

Desert ecosystems are ecosystems limited by both nitrogen and moisture, while legumes, as an important source of nitrogen in desert ecosystems, are extremely sensitive to respond to moisture and nitrogen changes. In order to reveal the growth and physiological metabolic processes of desert legumes in response to moisture and nitrogen changes. In this study, two dominant ephemeral legumes, Trigonella arcuata C. A. Mey. and Astragalus arpilobus Kar. & Kir. were selected from the Gurbantunggut Desert through a controlled experiment in greenhouse pots: four moisture treatment levels were established: W1(soil moisture content of 5 %), W2(soil moisture content of 7 %), W3( 9 % soil moisture content), W4(11 % soil moisture content) and three N treatment levels: N1(0 mmol/L 15NH4 15NO3), N2(18 mmol/L 15NH4 15NO3), N3(72 mmol/L 15NH4 15NO3), and we conducted a 15N tracking experiment for the pot treatments throughout the growing season. The results showed that: (1) moisture and nitrogen treatments had similar patterns on above-ground and below-ground as well as total fresh weight, plant height and basal diameter, Chla, Chlb and Chla+b of the two legumes, all of which were greatest in the W3N3 treatment and smallest in the W4N1 treatment, but the growth characteristics and chlorophyll parameters had cumulative differences over the growing season. (2) When subjected to severe moisture and nitrogen stresses, the 2 plants would increase the physiological levels of stress tolerance in vivo to enhance cellular resistance, with the osmoregulatory substance content being the most sensitive in response to moisture, and the cell membrane and antioxidant enzyme systems being the most sensitive in response to nitrogen stress. (3) The adaptive characteristics of physiological metabolic processes in plants to seasonal changes, in which growth characteristics, cell membrane and antioxidant enzyme content accumulation showed: rapid growth period < maximum biomass period, while chlorophyll content and osmoregulatory substance content accumulation content accumulation showed: rapid growth period > maximum biomass period. In general, drought can hinder nitrogen uptake and transport, while sufficient moisture can accelerate the nutrient metabolism process of plants, and there is a nutrient dilution effect once the moisture is in excess, but in the nitrogen starvation state, the moisture factor has almost no effect on plants, therefore, the moisture-nitrogen coupling effect is more reflective of the physiological response characteristics of the plants when they encountered the adversity stress, and the degree of accumulation is different in different growing seasons, and the plants in the The differences in different growing seasons reflect the changes in photosynthetic intensity and energy demand.

ecology↗

bioRxiv2024

Geographic variation in vulnerability to warming temperatures in an intertidal barnacle species

Vulnerability to warming temperatures under climate change arises when there is a gap between local climate and local physiology. Intertidal species are unique because they face two distinct thermal environments, and it is unclear which is the bigger driver of thermal physiology and vulnerability. Here we compare the thermal environments and physiology of three populations of the intertidal barnacle Balanus glandula, spanning 1460 km of its geographic range. We measured energy consumption in the laboratory across a 5-hour emersion and subsequent 6-hour immersion at 7 different emersion temperatures (10-38{degrees}C). We compared these results to one year of emersion and immersion temperature data from each location. Our results suggest that the temperatures experienced during emersion are a bigger driver of each populations emersion thermal physiology than those experienced during immersion. We also estimated vulnerability to future warming in two ways: as the total annual energy demand and as the number of days above each populations thermal peak. These produced conflicting results. The central population spent the most days over its thermal peak, but the northernmost population had the greatest total costs over a year. The higher energetic costs in the northernmost population may be explained in part by a strong latitudinal gradient in primary productivity that is selecting for higher energy demand in higher latitude populations. Thus, accurate predictions of B. glandulas response to warming temperatures will require knowledge of both future temperature and food availability.

ecology↗

bioRxiv2024

Carbonic anhydrase plays multiple roles in acetotrophic growth of a model marine methanogen from the domain Archaea

Carbonic anhydrase (CA) catalyzes the reversible hydration of CO2 to bicarbonate and a proton. The enzyme is universally distributed in all three domains of life and plays diverse physiological roles in the domains Eukarya and Bacteria. Remarkably, a physiological role has not been identified for any CA from the domain Archaea. Herein are described roles for a gamma class CA (Cam) from the methane-producing marine archaeon Methanosarcina acetivorans. Acetate-dependent growth of a {Delta}cam mutant showed an extended lag phase, lower final cell density, and metabolized acetate to a threshold of 20.0 mM compared to 1.0 mM for wild-type. Molar growth yields (Ymethane) were substantially greater for wild-type compared to the mutant. In contrast, growth parameters were identical for the methanol-grown wild-type and mutant. Rates of methane formation in resting cell suspensions containing 20.0 mM acetate were significantly less in the mutant versus wild-type and dependent on the presence of CO2. Rates for the wild-type decreased with increasing pH that was more pronounced for the mutant. CA activity was 100-fold greater in the membrane versus soluble fraction of acetate-grown cells. Addition of a surrogate CA stimulated acetate-dependent methanogenesis in resting cell suspensions of the mutant. The results support a role for Cam to supply protons for symport of acetate by the AceP symporter that also optimizes and facilitates growth at low acetate concentrations and high pH values encountered in the marine environment where M. acetivorans was isolated. Significance StatementAlthough CA plays major physiological roles in the domains Eukarya and Bacteria, a role has not been reported for the domain Archaea in which methanogens comprise the major group with abundant genomic annotations for CAs. Acetotrophic methanogens account for most of the methane produced in Earths biosphere where it is a major greenhouse gas. Although the biochemistry of the conversion of acetate to methane and carbon dioxide is well known, little is understood of acetate transport. The finding that CA has multiple roles facilitating thermodynamically constrained growth of a model marine acetotrophic methanogen has implications for advancing ecological understanding of the methane cycle that impacts global warming and climate change. Finally, the work is an introduction to anticipated physiological roles of CAs in the domain Archaea for which genomic annotations are abundant.

microbiology↗

bioRxiv2024

Cell-Driven Fluid Dynamics: A Physical Model of Active Systemic Circulation

Active fluid circulation and transport are key functions of living organisms, which drive efficient delivery of oxygen and nutrients to various physiological compartments. Because fluid circulation occurs in a network, the systemic flux and pressure are not simple outcomes of any given component. Rather, they are emergent properties of network elements and network topology. Moreover, consistent pressure and osmolarity gradients across compartments such as the kidney, interstitium, and vessels are known. How these gradients and network properties are established and maintained is an unanswered question in systems physiology. Previous studies have shown that epithelial cells are fluid pumps that actively generate pressure and osmolarity gradients. Polarization and activity of ion exchangers that drive fluid flux in epithelial cells are affected by pressure and osmolarity gradients. Therefore, there is an unexplored coupling between the pressure and osmolarity in the circulating network. Here we develop a mathematical theory that integrates the influence of pressure and osmolarity on solute transport and explores both cell fluid transport and systemic circulation. This model naturally generates pressure and osmolarity gradients across physiological compartments, and demonstrates how systemic transport properties can depend on cell properties, and how the cell state can depend on systemic properties. When epithelial and en-dothelial pumps are considered together, we predict how pressures at various points in the network depend on the overall osmolarity of the system. The model can be improved by including physiological geometries and expanding solute species, and highlights the interplay of fluid properties with cell function in living organisms.

biophysics↗

bioRxiv2024

The fingerprints of pupillary dynamics

The size of the pupils reflects directly the balance of different branches of the autonomic nervous system. This measure is inexpensive, non-invasive, and has provided invaluable insights on a wide range of mental processes, from attention to emotion and executive functions. Two outstanding limitations of current pupillometry research are the lack of consensus in the analytical approaches, which vary wildly across research groups and disciplines, and the fact that, unlike other neuroimaging techniques, pupillometry lacks the dimensionality to shed light on the different sources of the observed effects. In other words, pupillometry provides an integrated readout of several distinct networks, but it is unclear whether each has a specific fingerprint, stemming from its function or physiological substrate. Here we show that phasic changes in pupil size are inherently low-dimensional, with modes that are highly consistent across behavioral tasks of very different nature, suggesting that these changes occur along pupillary manifolds that are highly constrained by the underlying physiological structures rather than functions. These results provide not only a unified approach to analyze pupillary data, but also the opportunity for physiology and psychology to refer to the same processes by tracing the sources of the reported changes in pupil size in the underlying biology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/595554v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@219856org.highwire.dtl.DTLVardef@1984694org.highwire.dtl.DTLVardef@41def7org.highwire.dtl.DTLVardef@953692_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance statementPhasic changes in pupil size are thought to reflect dynamic shifts between attentional states as instantiated by the locus-coeruleus noradrenaline system, and are crucial for adaptive behaviors. We found that the latent space of these changes is low-dimensional and remarkably similar across very different tasks, involving distinct cognitive processes. We therefore introduce the notion of pupillary manifolds as latent spaces that subtend the generative processes behind these changes. We suggest that manifolds arise due to hard constraints in the underlying physiological substrate - the relative balance between sympathetic and parasympathetic activity. In the framework outlined here, these mechanisms can be accessed and described directly, with only a handful of parameters, thus better informing computational modelling.

neuroscience↗

bioRxiv2024

Intercellular epigenomic signaling during B cell maturation.

B cell maturation is crucial for effective adaptive immunity. It requires a complex signaling network to mediate antibody diversification through mutagenesis. B cells also rely on queues from other cells within the germinal center. Recently, a novel class of intercellular signals mediated by extracellular vesicles (EVs) has emerged. Studies have shown B cell EV-mediated signaling is involved in immune response regulation and tumorigenesis. However, the mechanistic role of B cell EVs is not yet established. We herein study the biological properties and physiological function of B cell EVs during B cell maturation. We use emerging technologies to profile B cell EVs surface marker signatures at the single particle level, molecular cargo, and physiological roles in B cell maturation. EV ncRNA cargo, characterized by RNA-seq, identified an EV-mediated novel non-coding RNA regulatory network for B cell maturation. A previously uncharacterized micro-RNA (miR-5099) in combination with a set of long non-coding RNA carried within B cell EVs is shown to be important for antibody diversification. The physiological role of EVs in B cell maturation is investigated using EV blockade assays and complementation studies using diverse EV sources further confirmed the physiological role and mode of action of EVs in B cell maturation.

immunology↗

bioRxiv2024

Metabolomic Profiling and Characterization of a Novel 3D Culture System for Studying Chondrocyte Mechanotransduction

Background/ObjectiveArticular chondrocytes synthesize and maintain the avascular and aneural articular cartilage. In vivo these cells are surrounded by a 3D pericellular matrix (PCM) containing predominantly collagen VI. The PCM protects chondrocytes and facilitates mechanotransduction. PCM stiffness is critical in transmitting biomechanical signals to chondrocytes. Various culture systems with different hydrogels are used to encapsulate chondrocytes for 3D culture, but many lack either the PCM or the in vivo stiffness of the cartilage matrix. This study aimed at establishing a culture system to investigate a) if chondrocytes cultured in alginate will develop a PCM and b) study mechanotransduction via metabolic changes induced in 3D agarose-embedded chondrocytes upon mechanical stimulation. MethodsWe cultured primary human and bovine chondrocytes in monolayers or as alginate encapsulated cells in media containing sodium L-ascorbate. PCM expression was analyzed by immunofluorescence and western blots. We further characterized the response of chondrocytes embedded in physiologically stiff agarose to dynamic compression through metabolomic profiling. ResultsWe found that primary human and bovine chondrocytes, when cultured in alginate beads with addition of sodium L-ascorbate for 7 days, had a pronounced PCM, retained their phenotype, and synthesized both collagens VI and II. This novel culture system enables alginate-encapsulated chondrocytes to develop a robust PCM thereby creating a model system to study mechanotransduction in the presence of an endogenous PCM. We also observed distinct compression-induced changes in metabolomic profiles between the monolayer-agarose and alginate-released agarose-embedded chondrocytes indicating physiological changes in cell metabolism. Conclusion/SignificanceThese data show that 3D preculture of chondrocytes in alginate before encapsulation in physiologically stiff agarose leads to pronounced development of pericellular matrix that is sustained in the presence of ascorbate. This model can be useful in studying the mechanism by which chondrocytes respond to cyclical compression and other types of loading simulating in vivo physiological conditions.

bioengineering↗

bioRxiv2024

Interoceptive Signals Bias Decision Making in Rhesus Macaques

Several influential theories have proposed that interoceptive signals, sent from the body to the brain, contribute to neural processes that coordinate complex behaviors. We altered the physiological state of the body using compounds that have minimal effect on the brain and evaluated their effect on decision-making in rhesus monkeys. We used glycopyrrolate, a non-specific muscarinic (parasympathetic) antagonist, and isoproterenol, a beta-1/2 (sympathetic) agonist, to create a sympathetic-dominated state in the periphery, that was indexed by increased heart rate. Rhesus monkeys were trained on two variants of an approach-avoidance conflict task. The tasks offered a choice between enduring mildly aversive stimuli in exchange for a steady flow of rewards, or cancelling the aversive stimuli, forgoing the rewards. The delay to interrupt the aversive stimuli was used as a measure of monkeys tolerance for contact with a hot but not painful stimulus or airflow directed at their muzzle. Both drugs reduced tolerance for the aversive stimuli. To determine whether the drug-induced autonomic state reduced the subjective value of the reward, we tested the effects of glycopyrrolate on a food preference task. Food preference was unaltered, suggesting that the sympathetic dominated state in the periphery selectively reduces tolerance for aversive stimuli without altering reward-seeking behaviors. As the drugs used are expected to have little or no direct effect on the brain, the observed biases in decision making are likely induced by interoceptive afferents that signal to the brain the physiological state of the body. Significance statementThe brain adjusts body physiology to the behavioral agenda of the organism through autonomic efferents; concomitantly interoceptive afferents carry signals that inform the brain about the physiological state of the body, closing a homeostatic regulatory loop. Persuasive theories proposed that interoceptive afferents contribute to higher cognitive functions, including emotion. Empirical evidence that these signals are sufficient to bias complex behavior has been limited by the difficulty of isolating interoceptive afferents from the rest of the homeostatic loop. Here we selectively manipulated the autonomic state of the body using drugs with limited penetrance of the brain in macaques performing decision-making tasks. Sympathetic-dominated peripheral states significantly altered decision making, suggesting that changes in interoceptive afferent signals are sufficient to bias behavior.

neuroscience↗

bioRxiv2024

The cold-inducible RNA-binding protein RBM3 stabilises viral mRNA at the cooler temperatures of the upper respiratory tract.

Temperature is a critical determinant of host-pathogen interactions in the respiratory tract, where influenza A virus (IAV) has adapted to the cooler environment of the upper respiratory tract (URT) to enable efficient replication and transmission. The cold-inducible RNA-binding protein RBM3 is highly expressed in nasopharyngeal tissue and is known to stabilise mRNAs under hypothermic conditions; however, its role during viral infection has not been defined. Here, we identify RBM3 as a key host factor that facilitates IAV replication at sub-physiological temperatures. siRNA-mediated knockdown of RBM3 significantly impaired viral replication, while its constitutive overexpression at 37{degrees}C restored replication to levels typically observed at 33{degrees}C. Mechanistically, RBM3 binds directly to viral nucleoprotein (NP) mRNA, prolonging mRNA half-life and inevitably leading to increased viral production. This effect was abolished in an RNA-binding-deficient RBM3 mutant, confirming the requirement for direct RNA interaction, visualised by smiFISH-PLA. Crucially, this positive regulation of NP mRNA and interaction was validated in well-differentiated primary nasal epithelial cells, highlighting the physiological relevance of RBM3 in the human URT. These results reveal a temperature-sensitive host-virus interaction that promotes IAV replication in the cooler URT, a key site for viral shedding and transmission. By linking environmental temperature, host RNA-binding proteins, and viral mRNA stability, this study uncovers a novel mechanism of respiratory virus adaptation and identifies RBM3 as a potential therapeutic target for limiting early-stage viral replication and transmission. Author SummaryTo establish productive infection and ensure transmission, respiratory viruses such as influenza A virus (IAV) must adapt to the diverse environments of the human respiratory tract. One key challenge is the temperature gradient that exists between the upper and lower respiratory tract. The upper respiratory tract (URT), the main site of viral entry and transmission, maintains a lower physiological temperature ([~]33{degrees}C) than the lower respiratory tract ([~]37{degrees}C, LRT), creating a distinct cellular environment. In this study, we investigated how this cooler URT temperature alters the landscape of RNA-binding proteins (RBPs), which play a central role in regulating gene expression after transcription. Using mass spectrometry-based profiling and molecular virology approaches, we identified a cold-inducible RBP, RBM3, as significantly enriched at 33{degrees}C and acting as a key proviral host factor during IAV infection. We show that RBM3 binds directly to viral nucleoprotein (NP) mRNA, mainly in the cytoplasm, in both immortalised cell models and primary nasal epithelial cells. This interaction promotes the stability of NP mRNA thereby enhancing the overall production of infectious virions. Importantly, disrupting RBM3s RNA-binding ability abolished this proviral effect. These findings reveal that RBM3, elevated in the cooler URT environment, directly supports IAV replication. This work highlights how subtle physiological differences in host tissue can reshape post-transcriptional regulation and influence the outcome of respiratory virus infection.

microbiology↗

bioRxiv2024

Interactive effects of temperature acclimation and dietary fatty acids on metabolic rate and body composition of zebra finches (Taeniopygia guttata)

Climate change is contributing to geographic range shifts in many bird species, with possible exposure to novel diets. How individuals respond physiologically across chronic time frames to the interacting effects of diet and environmental temperature has been little explored. We acclimated zebra finches to either cool (20-24{degrees}C) or thermoneutral (35{degrees}C) temperatures over 6-months and provided them with diets enriched in either unsaturated or saturated fatty acids. We measured body mass throughout the study, and basal metabolic rate (BMR) and body composition at 3- and 6-months, respectively. Individuals held in cool conditions and fed a diet enriched with unsaturated fatty acids lost mass initially relative to the other groups, however, effects were reversible, and all individuals had a similar mass at 6- months. Chronic exposure to cool conditions increased BMR and the mass of the pectoral muscle and visceral organs. However, we could detect no long-term effect of diet on any physiological parameter. Our results contrast with those of birds studied over acute time frames, in which diet and temperature interact to determine energy expenditure. Over chronic time frames individuals appear to reach a new steady-state, with long-term physiological responses driven primarily by thermoregulatory responses to environmental temperature. Research HighlightsO_LIWith climate change, birds may encounter novel diets and temperatures C_LIO_LIIn zebra finches we show that chronic acclimation to cool temperatures increased energy expenditure and changed body composition. C_LIO_LIDietary fatty acid content had little long-term impact on the physiological parameters we measured. C_LI

zoology↗