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bioRxiv2024

Organ Chips with integrated multifunctional sensors enable continuous metabolic monitoring at controlled oxygen levels

Despite remarkable advances in Organ-on-a-chip (Organ Chip) microfluidic culture technology, recreating tissue-relevant physiological conditions, such as the region-specific oxygen concentrations, remains a formidable technical challenge, and analysis of tissue functions is commonly carried out using one analytical technique at a time. Here, we describe two-channel Organ Chip microfluidic devices fabricated from polydimethylsiloxane and gas impermeable polycarbonate materials that are integrated with multiple sensors, mounted on a printed circuit board and operated using a commercially available Organ Chip culture instrument. The novelty of this system is that it enables the recreation of physiologically relevant tissue-tissue interfaces and oxygen tension as well as non-invasive continuous measurement of transepithelial electrical resistance, oxygen concentration and pH, combined with simultaneous analysis of cellular metabolic activity (ATP/ADP ratio), cell morphology, and tissue phenotype. We demonstrate the reliable and reproducible functionality of this system in living human Gut and Liver Chip cultures. Changes in tissue barrier function and oxygen tension along with their functional and metabolic responses to chemical stimuli (e.g., calcium chelation, oligomycin) were continuously and noninvasively monitored on-chip for up to 23 days. A physiologically relevant microaerobic microenvironment that supports co-culture of human intestinal cells with living Lactococcus lactis bacteria also was demonstrated in the Gut Chip. The integration of multi-functional sensors into Organ Chips provides a robust and scalable platform for the simultaneous, continuous, and non-invasive monitoring of multiple physiological functions that can significantly enhance the comprehensive and reliable evaluation of engineered tissues in Organ Chip models in basic research, preclinical modeling, and drug development.

bioengineering↗

bioRxiv2024

Differences in energy storage in sympatric salmonid morphs with contrasting lifestyles

Although physiology ensures homeostasis and fitness in a particular environment, and ecological shifts cannot be realized without physiological changes, metabolic transformations during animal adaptive radiations still remain unexplored. We present a study of energy reserve storage in the salmonid assemblage inhabiting a cold-water Lake Kronotskoe. This assemblage diversified from Salvelinus malma and includes eight distinct ecomorphs with contrasting lifestyles and trophic specializations. We hypothesized that ecomorphs differ in energy storage and expenditure, and that their metabolic phenotypes should be among the primary targets of natural selection. To test this hypothesis, we compared the stored amount and ratio of carbohydrates, lipids, circulating peptides that supply the citric acid cycle, as well as proxy indicators of metabolic rate, the blood levels of plasma proteins (including albumin) and hemoglobin. Among ecomorphs, numerous significant differences in physiological parameters were found, closely related to the composition of food, the depth of habitat, and determined by internal factors, probably genetics. Each ecomorph has a specific metabolic phenotype corresponding to its tropho-ecological specialization and lifestyle. Metabolically advanced predators accumulate lipids; littoral insectivorous morphs grow slower and accumulate glycogen; amphipod feeders do not accumulate spare substances; the deepwater consumer of silt benthos differs in the most divergent physiological characteristics. We assume a specific selection on endocrine regulators of energy metabolism during the adaptive radiation of the assemblage, among which the most plausible candidates are thyroid hormones and leptin.

evolutionary biology↗

bioRxiv2024

Synaptic connectome of a neurosecretory network in the Drosophila brain

Hormones mediate inter-organ signaling which is crucial in orchestrating diverse behaviors and physiological processes including sleep and activity, feeding, growth, metabolism and reproduction. The pars intercerebralis and pars lateralis in insects represent major hubs which contain neurosecretory cells (NSC) that produce various peptide hormones. To obtain insight into how hormonal signaling is regulated, we have characterized the synaptic connectome of NSC in the adult Drosophila brain. Identification of neurons providing inputs to multiple NSC classes implicate diuretic hormone 44-expressing NSC as a major coordinator of physiology and behavior. Surprisingly, despite most NSC having dendrites in the subesophageal zone (primary taste processing center), inputs from peripheral gustatory neurons to NSC are largely indirect. We also deciphered pathways via which diverse olfactory inputs are relayed to NSC. Linear dynamical modeling of signal propagation through the connectome identifies enteric neurons as the strongest influencers of NSC activity compared to other sensory modalities. Further, our analyses revealed substantial inputs from brain descending neurons to NSC, suggesting that descending neurons regulate both endocrine and motor output to synchronize physiological changes with appropriate behaviors. In contrast to NSC inputs, synaptic output from NSC is sparse and mostly mediated by corazonin NSC. We show that both corazonin-expressing NSC and their downstream synaptic partner DNg27 influence egg-laying. We additionally explore putative paracrine interconnectivity between NSC classes and peptide hormone pathways from NSC to peripheral tissues by analyzing single-cell transcriptomic datasets. Our comprehensive characterization of the Drosophila neurosecretory network connectome provides a platform to understand complex hormonal networks and how they orchestrate animal behaviors and physiology.

neuroscience↗

bioRxiv2024

Copper impedes calcification of human aortic vascular smooth muscle cells through inhibition of osteogenic transdifferentiation and promotion of extracellular matrix stability

Vascular calcification (VC), a common pathological condition, is a strong predictor of cardiovascular events and associated mortality. Development and progression of VC heavily rely on vascular smooth muscle cells (VSMCs) and are closely related to oxidative stress, inflammation, and remodelling of extracellular matrix (ECM). Copper (Cu), an essential microelement, participates in these processes, however its involvement in pathophysiology of VC and VSMCs physiology remains poorly investigated. In the present study we analysed Cu impact on the calcification of human aortic primary VSMCs induced in vitro by treatment with high calcium and phosphate levels. Supplementation with physiological micromolar Cu significantly reduced the amount of calcium deposited on VSMCs as compared to moderate deficiency, Cu restriction with chelators or Cu excess. Moreover, optimal concentrations of Cu ions increased protein production by VSMCs, stimulated their metabolic activity, inhibited alkaline phosphatase activity associated with cell-conditioned medium and cellular lysates, and prevented osteogenic differentiation of VSMCs. RNA-seq results indicated that high calcium and phosphate treatments activated many pathways related to oxidative stress and inflammation in VSMCs at the initial stage of calcification. At the same time, expression of VSMC-specific markers and certain components of ECM were downregulated. Supplementation of calcifying cells with 10 M Cu prevented most of the transcriptomic alterations induced by high calcium and phosphate while chelation-mediated restriction of Cu greatly aggravated them. In summary, physiological concentration of Cu impedes in vitro calcification of VSMCs, prevents their osteogenic transition and minimises early phenotypic alterations induced by high calcium and phosphate, thereby underlining the importance of Cu homeostasis for the physiology of VSMCs, one of the cornerstones of cardiovascular health. Our data suggest that peculiarities of Cu metabolism and its status should be considered when developing preventive and therapeutic approaches for cardiovascular diseases.

biochemistry↗

bioRxiv2024

CGGBP1 from higher amniotes restricts cytosine methylation and drives a GC-bias in transcription factor binding sites at repressed promoters

CGGBP1, a 20 kDa protein, has several functions associated with its DNA-binding through a C2H2 zinc finger. A range of studies have shown that GC richness, inter-strand G/C-skew and low cytosine methylation are associated with CGGBP1 occupancy. The non-preference of any sequence motif as CGGBP1 binding site suggests widespread association of CGGBP1 with DNA including at potent transcription factor binding sites (TFBSs) in promoter regions. The evolutionary advantage of such a design remains unclear. The regulatory interference by human CGGBP1 at TFBSs is supported by purifying selection in the DNA-binding domain of CGGBP1 and its requirement for gene repression as well as restriction of cytosine methylation at GC-rich TFBSs. Here we describe an evolutionary trajectory of this property of CGGBP1 by combining global gene expression and cytosine methylation analyses on human cells expressing CGGBPs from four different vertebrates (representatives of coelacanth, reptiles, aves and mammals). We discover a potent cytosine methylation restriction by human CGGBP1 at some GC-rich TFBSs in repressed promoters. Further, we combine a high-throughput analysis of GC compositional bias of these CGGBP-regulated TFBSs from available orthologous sequences from a pool of over 100 species. We show that cytosine methylation restriction by CGGBP1 is tightly linked to GC retention in a set of TFBSs. Orthology analyses demonstrate that this property of CGGBPs has evolved in higher amniotes (aves and mammals) with lineage-specific heterogeneities in lower amniotes (reptiles). CGGBP1 ChIP-seq data suggest that occupancy of CGGBP1 at these target TFBSs plays a crucial role in their low methylation, GC-biased evolution and associated functions in gene repression. HighlightsO_LIResemblances in gene repression by overexpression of CGGBP1 from higher amniotes (Homo sapiens and Gallus gallus) is enhanced upon heat stress and differs from the non-repressive effects of lower amniotic CGGBPs (Anolis carolinensis and Latimeria chalumnae). C_LIO_LIGene repression by higher amniotic CGGBP1 is associated with restriction of cytosine methylation at specific GC-rich TFBSs in 1 kb promoters of target genes. Lower amniotic CGGBPs allow TFBS cytosine methylation and C-T transitions. C_LIO_LIOrthologs of CGGBP1-repressed genes from >100 vertebrates show signs of accelerated C-T losses explicitly in the TFBSs at which higher amniotic CGGBP1 restricts cytosine methylation. Such a TFBS GC-loss difference between lower and higher amniotes is restricted to genes repressed by higher amniotic CGGBP1 at physiological temperature, not heat stress. C_LIO_LIThis higher amniote-specific cytosine methylation restriction by CGGBP1 has likely influenced the differences between GC-rich TFBS composition and their abundance in target gene promoters throughout vertebrate evolution. C_LI SummaryEvolution of transcription factor binding sites (TFBSs) depends on a variety of factors including cytosine methylation-associated C-T transition rates. Most of our understanding of TFBS evolution is based on omic-scale sequence comparisons with only circumstantial evidence for the relationship between the TFBSs and physiological adaptation. We report a TFBS landscaping function for CGGBP1 by expressing its different taxon-derived forms in human cells through profiling of global gene expression and cytosine methylation alongside a meta-analysis of C-T transition rates from over 100 vertebrae genomes. We show that CGGBP1 from higher amniotes restricts cytosine methylation and maintains GC-rich TFBSs in target gene promoters for repression. This epigenetic affection of TFBS evolution by CGGBP1 is selectively seen at genes repressed at physiological temperature only and not under heat stress when gene repression by CGGBP1 becomes largely transcription factor binding site independent. Our findings connect epigenetic mechanisms to cellular physiology through TFBS evolution linked with changes in CGGBP1.

evolutionary biology↗

bioRxiv2024

An algorithm for predicting per-cell proteomic properties

Proteomic studies have traditionally focused on population-level analyses with an emphasis on the relative abundance of various proteins. Such studies have been useful in uncovering physiological differences across diverse species, the physiological response of individual species to distinct environmental conditions, and the function of individual proteins in the context of cellular networks. However, the absolute value of protein abundance in a cell is important for understanding single-cell physiology, detailed biophysical considerations, connecting diverse quantitative data, and for comparisons across species. Such detailed quantification will naturally occur as singlecell proteomics becomes more prevalent, but it is also of current interest to leverage population studies. There are several challenges here. First, most population studies do not measure the quantity of cells associated with a proteome. Second, recent work has shown that cell physiology radically shifts with cell size, and these effects need to be accounted for in going from population to single-cell estimates. Here we develop and implement a method to estimate the basic properties of proteomes, based on well-established scaling relationships among cell components, including genome size, cell size, and proteome volume. Our method estimates similar but higher total proteins per cell compared to previous theoretical and empirical estimations. Our algorithm has applications for interpreting proteomes, analyzing environmental samples, and designing artificial cells. While focusing on prokaryotes, we discuss how the method can be extended to unicellular and multicellular eukaryotes.

bioinformatics↗

bioRxiv2024

Knockout of all nematode-specific NSPC genes expressed exclusively in the excretory gland cell results in transcriptomic signatures indicating an affected insulin signaling

The nematode Caenorhabditis elegans is one of the best-studied model organisms in molecular biology; however, many aspects of its physiology and the functions of many genes remain poorly understood. In this study, we investigated the role of nematode-specific NSPC proteins, whose mRNAs were recently identified as primary targets of the poly(A) polymerase TENT-5. Surprisingly, we found that NSPCs are exclusively expressed in the excretory gland cell, a cell with still unclear functionality. Using an optogenetic approach, we precisely ablated the excretory gland cell and observed that nematodes exhibited no transcriptomic or physiological changes in its absence. Additionally, we generated and thoroughly studied a strain with a deletion of all 18 nspc genes, which revealed that, despite previous indications, NSPCs do not influence the worms defense response. Instead, the transcriptomic analysis showed that the absence of NSPCs strongly impacts DAF-2/DAF-16 insulin signaling, suggesting that NSPCs may function as neuropeptides influencing key C. elegans signaling pathways. Although further studies are required to elucidate the physiological effects of this regulation, our findings provide new insights into this unexplored part of nematode physiology.

molecular biology↗

bioRxiv2024

Missing what is right under your nose: failed appetitive and aversive audio-olfactory conditioning in humans

The comparison of physiological mechanisms underlying appetitive and aversive conditioning is often challenging due to the involvement of stimuli from different modalities with potentially disparate effective mechanisms (e.g., pain stimuli versus monetary rewards). The olfactory system offers a unique opportunity to examine both types of conditioning in humans, as isointense odors can serve as comparably pleasant and unpleasant stimuli. To study physiological and behavioral responses during appetitive and aversive learning, we employed odors as unconditioned stimuli (US) in a within-subjects design, measuring various conditioned physiological responses including skin conductance, heart rate, pulse wave amplitude, respiration, fear-potentiated startle, postauricular reflex, facial electromyography as well as event-related potentials, and auditory steady-state responses (ASSR) derived from electroencephalography. We conducted four experiments with a total of 95 participants, presenting three neutral sounds paired with either a pleasant odor, unpleasant odor, or odorless air. The first experiment involved uninstructed participants and frequency-modulated conditioned stimuli (CS) for ASSR analysis. In the second experiment, we omitted the frequency modulation and startle probe. The third experiment included pre-experiment instruction on CS-US contingencies, while the fourth employed a delayed conditioning paradigm in contrast to the other three experiments. Our results revealed differences between CS+ and CS-only in the fear-potentiated startle response in Experiment 3. No other effects were found. The minimal or absent learning effects observed across multiple peripheral and neural physiological measures may be attributed to the extra-thalamic nature of olfactory pathways and the subsequent difficulty in forming associations with auditory stimuli. Impact statementIn a series of 4 experiments, we explored the neurophysiological differences between appetitive and aversive conditioning. Yet, none of the experiments showed effective conditioning. We hypothesize that the lack of learning effects is attributed to the inherent difficulty in forming associations between auditory and olfactory inputs.

neuroscience↗

bioRxiv2024

G-quadruplexes catalyze protein folding by reshaping the energetic landscape

Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing G-quadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. As short nucleic acids can catalyze protein folding without the input of ATP, the ability of the cell to fold proteins is far higher than previously anticipated. Significance StatementHow folding of proteins occurs en masse in the cell is still a daunting unsolved problem. Many proteins have complicated and difficult folding trajectories, with in vitro folding times that are physiologically untenable. The acceleration of protein folding to physiologically relevant timescales is a biologically essential function thought to be accomplished by a small set of ATP-dependent chaperonins. In this work, we surprisingly show that small nucleic acid sequences containing G-quadruplexes can catalyze protein folding and reshape protein folding energy landscapes. As a result, the capacity for accelerating protein folding in the cells is far higher than previously suggested, potentially explaining the accommodation of large number of proteins with physiologically unreasonable folding times.

biophysics↗

bioRxiv2025

A formal model of anxiety disorders based on the neural circuit dynamics of the fear and extinction circuits

The pathophysiology of anxiety disorders is the outcome of an imbalance of the fear-anxiety circuit and the extinction circuit. We present a formal model using nonlinear dynamics and network theory, which captures the dynamic interactions of the key nodes of the anxiety and extinction networks. This rudimentary model can be modified by newer data. These core nodes consist of the cells of the paraventricular nucleus of the thalamus coding negative valence, the neurons of the basal-lateral amygdala coding negative valence (Rspo2+), the anterior cingulate cortex, the ventral hippocampus neurons coding fear memories, the somatostatin expressing cells of the lateral segment of the central amygdala, the medial segment of the central nucleus of the amygdala-bed nucleus of the stria terminalis and their target nodes. The extinction network consists primarily of the paraventricular thalamic cells coding positive valence, (Ppp1r1b+) cells of the basolateral amygdala coding positive valence, the ventromedial prefrontal cortex, the PKC{delta} cells of the lateral segment of the central amygdala, and the intercalated cells. Human and non-human animal genetic and epigenetic studies point to deficiencies in brain-derived neurotrophic factor and neurotrophic receptor kinase tyrosine 2 production in key nodes causing reduced plasticity extinction network plasticity and leading to a weakened extinction response. We rely primarily on the neurophysiological studies of non-human animal models since nodes generating fear/anxiety and extinction responses are highly conserved across species and equivalent nodes are present within analogous circuits of the human brain. The results are confirmed, where possible by human functional magnetic resonance imaging studies. We believe this simplified model is of heurist value and can lead to a more consistent focus on physiologically based pathophysiology. This would lead to treatments to reverse the pathologic physiology produced by genetic and epigenetic abnormalities, and greater efforts to directly correct pathologic circuit activity through direct interventions such as transcranial magnetic stimulation. SignificanceWe believe this simplified model is of heurist value and can lead to a more consistent focus on physiologically based pathophysiology. This would lead to treatments to reverse the pathologic physiology produced by genetic and epigenetic abnormalities, and greater efforts to directly correct pathologic circuit activity through direct interventions such as transcranial magnetic stimulation.

neuroscience↗

bioRxiv2025

A single dorsal vagal complex circuit mediates the aversive and anorectic responses to GLP1R agonists

GLP-1 receptor agonists (GLP1RAs) effectively reduce feeding to treat obesity, although nausea and other aversive side effects of these drugs can limit their use. Brainstem circuits that promote satiation and that mediate the physiologic control of body weight can be distinguished from those that cause aversion. It remains unclear whether brainstem Glp1r neurons contribute to the normal regulation of energy balance and whether GLP1RAs control appetite via circuits distinct from those that mediate aversive responses, however. Hence, we defined roles for AP and NTS Glp1r-expressing neurons (APGlp1r and NTSGlp1r neurons, respectively) in the physiologic control of body weight, the GLP1RA-dependent suppression of food intake, and the GLP1RA-mediated stimulation of aversive responses. While silencing non-aversive NTSGlp1r neurons interfered with the physiologic restraint of feeding and body weight, restoring NTSGlp1r neuron Glp1r expression on an otherwise Glp1r-null background failed to enable long-term body weight suppression by GLP1RAs. In contrast, selective Glp1r expression in APGlp1r neurons restored both aversive responses and long-term body weight suppression by GLP1RAs. Thus, while non-aversive NTSGlp1r neurons control physiologic feeding, aversive APGlp1r neurons mediate both the anorectic and weight loss effects of GLP1RAs, dictating the functional inseparability of these pharmacologic GLP1RA responses at a circuit level.

neuroscience↗

bioRxiv2025

Deciphering the metabolic details of L-lysine toxicity in cyanobacteria

L-lysine (Lys) has been explored as a potential cyanobactericide due to its inhibitory effects on cyanobacterial growth at micromolar concentrations, comparable to many antibiotics. Here, we investigated the early metabolic and physiological responses of the model cyanobacterium Synechocystis sp. PCC 6803 to Lys exposure. Physiological analyses revealed cell enlargement, oxidative stress, and photosynthesis inhibition, leading to growth arrest. Metabolomic profiling indicated disruptions in peptidoglycan biosynthesis, evidenced by the accumulation of L-/D-alanine, meso-diaminopimelate, and D-Ala-D-Ala, suggesting interference with cell wall integrity. Furthermore, levels of energy metabolites and other amino acids including tyrosine, tryptophan, valine, and iso-/leucine were significantly altered, implying broader metabolic impacts of Lys toxicity. To explore potential resistance mechanisms, we used a CRISPRi-based genetic screen to identify key genes involved in relieving Lys toxicity. The Bgt permease system, responsible for basic amino acid uptake, was essential for acquiring Lys-resistance, as a bgtA mutant exhibited a normal growth on elevated Lys concentrations, thereby validating our CRISPRi-screen. Additionally, UirR, a DNA-binding response regulator, and genes linked to c-di-AMP signaling, seemed implicated in Lys metabolism. Deletion of c-di-AMP synthase gene increased Lys sensitivity, supporting a role for c-di-AMP in cell wall homeostasis and osmotic stress regulation. Altogether, our findings explored the early metabolic responses and physiological consequences of Lys exposure in Synechocystis, demonstrating its effects on peptidoglycan biosynthesis, amino acid metabolism, and nucleotide biosynthesis. The identification of key genetic factors contributing to Lys resistance provides new insights into cyanobacterial physiology and the potential application of Lys in bloom control strategies.

microbiology↗

bioRxiv2025

The encoding of interoceptive-based predictions by the paraventricular nucleus of the thalamus D2+ neurons

Understanding how the brain integrates internal physiological states with external sensory cues to guide behavior is a fundamental question in neuroscience. This process relies on interoceptive predictions, which are internal models that anticipate changes in the bodys physiological state based on sensory inputs and prior experiences. Despite recent advances in identifying the neural substrates of interoceptive predictions, the precise neuronal circuits involved remain elusive. In our study, we demonstrate that Dopamine 2 Receptor (D2R+) expressing neurons in the paraventricular nucleus of the thalamus (PVT) play key roles in interoception and interoceptive predictions. Specifically, these neurons are engaged in behaviors leading to physiologically relevant outcomes, with their activity highly dependent on the interoceptive state of the mice and the expected outcome. Furthermore, we show that chronic inhibition of PVTD2R+ neurons impairs the long-term performance of interoceptive-guided motivated behavior. Collectively, our findings provide insights into the role of PVTD2R+ neurons in learning and updating state-dependent predictions by integrating past experiences with current physiological conditions to optimize goal-directed behavior.

neuroscience↗

bioRxiv2025

Airway smooth muscle--on-a-chip: a microfluidic approach to study alveolar smooth muscle remodelling

Respiratory illnesses, like chronic obstructive pulmonary disease (COPD) and asthma, pose significant global health challenges due to their chronic nature and limited treatment options. Airway smooth muscle (ASM) plays a vital role in respiratory diseases, particularly in airway remodelling and obstruction. ASM, which encircles the bronchial tree and extends to the trachea, plays a vital yet not fully understood role in lung physiology. However, its dysfunction is strongly associated with asthma and COPD progression, leading to excessive contraction, increased inflammatory mediator release, and ASM hypertrophy. However, identifying its precise function is challenging due to limitations in existing research models for assessing ASM contraction. In vivo models offer a comprehensive physiological perspective but possess ethical concerns and they do not allow for the direct measurement of ASM contraction. Meanwhile, ex vivo and in vitro models provide a more direct assessment; however, they lack crucial physiological factors. Understanding how ASM cells interact with their surroundings is essential for gaining deeper insights into respiratory disorders. To address this gap, we aimed to mimic the human airway smooth muscle-on-a-chip model, incorporating ASM cells in a 3D microenvironment. This microfluidic platform provides a physiologically relevant environment, allowing for studying complex mechanisms that drive airway remodelling and dysfunction in respiratory diseases. The ASM-on-a-chip is designed for long-term 3D cell culture of ASM cells that reorient itself to form a smooth muscle fibre. The design provides side channels for manipulating the constituent of the hydrogel to study the effect of compounds on AMS remodelling.

bioengineering↗

bioRxiv2025

Sub-lethal pesticide exposure facilitates the potential northward range shifts of ticks by increasing cold tolerance and overwintering survival

Pesticides are a primary tool for the control of harmful insects or other organisms throughout the world. Although most organisms are likely to encounter pesticides in the environment, little is known about the effects of these chemicals on the physiology of off-target species. We measured the impact of sublethal pesticide exposure on the cold tolerance of two common tick species. We predicted that sublethal pesticide exposure would make ticks more sensitive to extreme temperatures. In contrast, we found that exposure to sublethal pesticides had the opposite effect, increasing survival at low temperatures. Sublethal pesticide exposure decreased the LT50 (lethal temperature at which 50% mortality is observed) of adult and nymphal D. variabilis from -16{degrees}C to -19{degrees}C and improved overwintering survival. Evaluation of previously available RNA-seq data between rapid cold hardening and pesticide exposure indicated potential transcriptional shifts associated with cross-tolerance between cold and pesticide exposure. To investigate the population-level impacts of this physiological shift in cold tolerance, we developed a novel approach for incorporating physiological survival data into spatially explicit species distribution models (SDMs). Using this approach, we demonstrate that pesticide-induced increases in cold tolerance may permit faster northward range shifts of D. variabilis. We also demonstrate that incorporating physiological data into SDMs moderates the estimated impacts of environmental change and may provide more accurate predictions of species responses to changing environments. Although our study is limited to ticks, other studies have shown the effects of pesticides on thermal tolerance traits in diverse species. The extensive use of pesticides may drive complex interactions between species and their environments, leading to altered thermal tolerance traits and establishment in new habitats.

ecology↗

bioRxiv2025

Elevated GLUT4 Levels in Human Skeletal Muscle Microtissues is Accompanied by Functional Insulin Dependence

Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes mellitus (T2D). While two-dimensional myotube cultures offer a controlled environment for studying T2D-related metabolic dysfunction, insulin-dependent glucose transporter type 4 (GLUT4) levels are limited and insulin-independent glucose transporter type 1 (GLUT1) expression dominates; reducing physiological relevance. Three-dimensional skeletal muscle microtissue cultures offer a promising alternative, and unlike 2D myotubes, are amenable to repeated contractile stimulation. However, microtissue GLUT1 and GLUT4 glucose transporter profiles remain under-characterized, particularly under physiological glucose and insulin conditions, which is evaluated herein. We report that GLUT1 levels trended [~]3.0-fold lower in microtissues compared with myotubes in 2D culture, although not statistically significant (p = 0.072), while GLUT4 levels were [~]12-fold higher (p < 0.0001), leading to a [~]60-fold increase in the GLUT4:GLUT1 ratio (p = 0.023). Notably, the microtissue GLUT4:GLUT1 profile approached, but did not match that of native human muscle. Microtissues required supraphysiological insulin conditions for the development of maximal contractility, while physiological glucose levels were sufficient. Insulin withdrawal restored insulin responsiveness but impaired microtissue contractile strength (p < 0.0001) and fatigue resistance (p = 0.015). Our findings indicate that the glucose transporter profile of microtissues offers improved physiological relevance. However, their reliance on insulin to maintain contractile function limits their suitability for modeling T2D. The implementation of a robust, insulin-free differentiation protocol would facilitate the development of a microtissue-based T2D model which can be applied to study contraction-mediated increases in insulin sensitivity as a therapeutic approach.

cell biology↗

bioRxiv2025

Human Plasma-Like Medium Promotes Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

Maturing human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in vitro is critical for advancing drug discovery and cardiotoxicity screening applications of these cells. However, the metabolic compositions of basal media used for hPSC-CM culture typically offer limited relevance to human cardiac physiology. Here, we examined how culture in Human Plasma-Like Medium (HPLM) versus conventional basal media affects the behavior of hPSC-CMs. Starting with Day 16 hPSC-CMs, we cultured cells for two weeks in either HPLM or RPMI-based media and then assessed maturation outcomes at Day 30. Compared to RPMI/B27 media containing either RPMI-defined (11.1 mM) or physiologic glucose levels (5 mM), HPLM/B27 markedly enhanced hPSC-CM maturity as evinced by concerted transcriptomic, structural, functional, and metabolic phenotypes. These effects included a higher extent of myosin heavy chain isoform switching (-MHC to {beta}-MHC), accelerated ventricular-specific myosin light chain isoform switching (MLC2a to MLC2v), elongated sarcomeres, increased multinucleation, enhanced calcium transient kinetics, and coordinated activation of oxidative and glycolytic metabolism. Collectively, these findings demonstrate that medium composition has substantial effects on hPSC-CM biology and also establish HPLM as a tool for driving hPSC-CM maturation in vitro. Translational Impact StatementHPLM was designed to more closely recapitulate the metabolic composition of human plasma and thus provides a physiological platform to promote hPSC-CM maturation. By enhancing structural, functional, and metabolic maturity, HPLM-cultured hPSC-CMs better approximate cardiac physiology, positioning them as improved models for cardiovascular disease research, drug-induced cardiotoxicity screening, and personalized therapeutic testing. This medium can integrate with existing maturation strategies, accelerating the translation of basic cardiac research into clinically predictive tools for the drug development pipeline. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/650456v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a169eforg.highwire.dtl.DTLVardef@131e6f4org.highwire.dtl.DTLVardef@131d52dorg.highwire.dtl.DTLVardef@a2b248_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

The role of cytonuclear interactions to plant adaptation across a Populus hybrid zone

Co-adaptation of cytoplasmic and nuclear genomes are critical to physiological function for many species. Despite this understanding, hybridization can disrupt co-adaptation leading to a mismatch between maternally-inherited cytoplasmic genomes and biparentally inherited nuclear genomes. Few studies have examined the consequences of cytonuclear interactions to physiological function across environments. Here, we quantify the degree of co-introgression between chloroplast and nuclear-chloroplast (N-cp) genes across repeated hybrid zones and its consequences to physiological function across environments. We use whole-genome resequencing and common garden experiments with clonally replicated genotypes sampled across the natural hybrid zone between Populus trichocarpa and P. balsamifera. We use geographic clines to test for co-introgression of the chloroplast genome with N-cp and non-interacting nuclear genes. Co-introgression of chloroplast and N-cp genes was limited although contact zone-specific patterns suggest that local environments may influence co-introgression. Combining ancestry estimates with phenotypic data across common gardens revealed that mismatches between chloroplast and nuclear ancestry can influence physiological performance, but the strength and direction of these effects vary depending on the environment. Overall, this study highlights the importance of cytonuclear interactions to adaptation, and the role of environment in modifying the effect of those interactions.

evolutionary biology↗

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