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bioRxiv2023

The Design of Brainstem Interfaces: Characterisation of Physiological Artefacts and Implications for Closed-loop Algorithms

Surgical neuromodulation through implantable devices allows for stimulation delivery to subcortical regions, crucial for symptom control in many debilitating neurological conditions. Novel closed-loop algorithms deliver therapy tailor-made to endogenous physiological activity, however rely on precise sensing of signals such as subcortical oscillations. The frequency of such intrinsic activity can vary depending on subcortical target nucleus, while factors such as regional anatomy may also contribute to variability in sensing signals. While artefact parameters have been explored in more standard and commonly used targets (such as the basal ganglia, which are implanted in movement disorders), characterisation in novel candidate nuclei is still under investigation. One such important area is the brainstem, which contains nuclei crucial for arousal and autonomic regulation. The brainstem provides additional implantation targets for treatment indications in disorders of consciousness and sleep, yet poses distinct anatomical challenges compared to central subcortical targets. Here we investigate the region-specific artefacts encountered during activity and rest while streaming data from brainstem implants with a cranially-mounted device in two patients. Such artefacts result from this complex anatomical environment and its interactions with physiological parameters such as head movement and cardiac functions. The implications of the micromotion-induced artefacts, and potential mitigation, are then considered for future closed-loop stimulation methods.

bioengineering↗

bioRxiv2023

Structural stability of the oyster gut microbiome contributes to the host's physiological tolerance to temperature fluctuations on tropical rocky shores

Extreme high thermal conditions on tropical rocky shores are challenging to the survival of intertidal ectotherms. Yet, many species are highly successful in these environments in part due to their ability to regulate intrinsic mechanisms associated with physiological stress and their metabolic demand. More recently, there has been a growing awareness that other extrinsic mechanisms, such as animal-associated microbial communities, can also influence the tolerance and survival of ectotherms under stressful conditions. However, the extent to which the intrinsic and extrinsic mechanisms are functionally linked as part of the overall adaptive response of intertidal animals to temperature change and stress is poorly understood. Here, we examined the dynamics and potential interactions of intrinsic and extrinsic mechanisms in the tropical high-supratidal oyster, Isognomon nucleus. We found that oysters modulate their internal biochemistry (oxidized PUFA products, including 5-F2t-IsoP, 10-F4t-NeuroP, 13-F4t-NeuroP, and 16-F1t-PhytoP) as part of their adaptive regulation to cope with physiological stress during periods of extreme high temperatures when emersed. However, while we detected variation in alpha diversity (ASV richness and Shannon diversity index), dominant microbial taxa and microbial functions across time, no association was found with the host biochemical profiles. The findings here suggest that the thermal condition within oysters can independently influence their intrinsic biochemical responses and extrinsic microbiome profiles. Together, these mechanisms may contribute to the thermal tolerance and survival of the oysters in the challenging conditions of the tropical high-supratidal zone.

microbiology↗

bioRxiv2023

Optimization of chickpea irrigation in a semi-arid climate based on morpho-physiological parameters

While the world population is steadily growing, the demand for plant-based protein in general, and chickpea in particular, is rising. Heatwaves and terminal drought are the main environmental constraints on chickpea production worldwide. Thus, developing better irrigation management for the chickpea agro-system can promote higher and more sustainable yields. Supplemental irrigation at the right timing and dose can increase yield dramatically. Here, we studied the response of a modern Kabuli chickpea cultivar to supplemental irrigation during the critical pod-filling period over three growing seasons (2019-2021) in northern Negev, Israel, under semi-arid conditions. Six irrigation treatments were applied based on irrigation factors of 0, 0.5, 0.7, 1.0, 1.2, and 1.4 of crop evapotranspiration (ET0) as measured by an on-site meteorological station. Morpho-physiological parameters and above-ground biomass accumulation were monitored throughout the cropping seasons, and the final grain yield was determined at maturation. Irrigation onset was determined based on plants leaf water potential ({Psi}LWP > 15 bar) in the field. Our results indicate that optimal water status (as reflected by pressure chamber values) was 12-14 bar during the irrigation period. Irrigation according to evapotranspiration (ET0) with an irrigation factor of 1.2 resulted in the highest grain yields over the three years. To ensure optimal water supply during the reproductive phase compatible with the crop water requirements, maintaining a 25 mm node length above the last fully developed pod and a 90 mm distance between the last fully developed pod to the stem apex is recommended. In conclusion, irrigation onset when the crop is already at mild drought stress, followed by sufficient irrigation while following the indicated morphology and water potential values, may help farmers optimize irrigation and maximize chickpea crop production. HighlightsO_LIChickpea irrigation is optimized based on meteorological data (ETc) and morphological traits. C_LIO_LIMorphological- not only physiological- traits can capture the dynamics of crop water status. C_LIO_LIIrrigation onset should be at mild drought stress (>15 bar) during the reproductive stage. C_LIO_LIExtending the reproductive phase is essential for grain yield improvement under semi-arid conditions. C_LIO_LIIF=1.2 provides the most productive irrigation regime for chickpea in Mediterranean conditions. C_LI

plant biology↗

bioRxiv2023

Physiological studies on pH and salt tolerance and the utilization of diverse carbon sources by Listeria monocytogenes

The foodborne pathogen Listeria monocytogenes can grow in a wide range of environmental conditions. For the study of the physiology of this organism, several chemically defined media have been developed over the past decades. Here, we examined the ability of L. monocytogenes wildtype strains EGD-e and 10403S to grow under salt and pH stress in Listeria synthetic medium (LSM). Furthermore, we determined that a wide range of carbon sources could support growth of both wildtype strains in LSM. However, for hexose phosphate sugars such as glucose-1-phosphate, both L. monocytogenes strains need to be pre-grown under conditions, where the major virulence regulator PrfA is active. In addition, growth of both L. monocytogenes strains was observed when LSM was supplemented with the amino acid sugar N-acetylmannosamine (ManNAc). We were able to show that some of the proteins encoded in the operon lmo2795-nanE, such as the ManNAc-6-phosphate epimerase NanE, are required for growth in presence of ManNAc. The first gene of the operon, lmo2795, encodes a transcriptional regulator of the RpiR family. Using electrophoretic mobility shift assays and quantitative real time PCR analysis, we were able to show that Lmo2795 binds to the promoter region of the operon lmo2795-nanE and activates its expression. Originality-Significance StatementCurrent knowledge of growth and survival of the human pathogen Listeria monocytogenes under diverse stress conditions is mostly generated in complex medium, a condition that is rarely found in the environment or host of the pathogen. Our work contributes to the characterization of the physiology of L. monocytogenes grown under nutrient limiting conditions and its growth requirements with regards to metabolizable carbon sources.

microbiology↗

bioRxiv2023

Fever like temperature impacts on Staphylococcus aureus and Pseudomonas aeruginosa interaction, physiology, and virulence both in vitro and in vivo

BackgroundStaphylococcus aureus and Pseudomonas aeruginosa cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39{degrees}C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37{degrees}C, by using RNAseq and physiological assays including in-vivo experiments. ResultsIn general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes on both species. Altered metabolic features at 39{degrees}C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39{degrees}C they upregulated ethanol oxidation related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pms and pms{beta} genes at 39{degrees}C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphylxantin production and a decrease in biofilm formation at 39{degrees}C. On the side of PA monocultures, we observed increase in extracellular lipase and protease and biofilm formation at 39{degrees}C along with a decrease in motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in-vitro and in-vivo. S. aureus monocultured at 39{degrees}C showed a decrease in cellular invasion and an increase in IL-8 -but not in IL-6- production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39{degrees}C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37{degrees}C and 39{degrees}C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39{degrees}C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39{degrees}C comparing with 37{degrees}C. ConclusionOur results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.

microbiology↗

bioRxiv2023

Rapamycin-sensitive mechanisms confine the growth of fission yeast below the temperatures detrimental to cell physiology

Cells cease to proliferate above their growth-permissible temperatures, a ubiquitous phenomenon generally attributed to protein denaturing and heat damage to other cellular macromolecules. We here report that, in the presence of the macrolide compound rapamycin, the fission yeast Schizosaccharomyces pombe can proliferate at high temperatures that normally arrest its growth. Rapamycin is a potent inhibitor of the protein kinase complex TOR Complex 1 (TORC1), and consistently, mutations to the TORC1 subunit RAPTOR/Mip1 and the TORC1 substrate Sck1 significantly improve cellular heat resistance. These results suggest that TORC1, a well-established growth promoter, restricts the high-temperature growth of fission yeast and that compromised TORC1 signaling allows cell proliferation at higher temperatures. Aiming for a more comprehensive understanding of the negative regulation of high-temperature growth, we conducted genome-wide screens in S. pombe, which identified Sck1 and additional factors that appear to suppress cell proliferation at high temperatures. Our study has uncovered unexpected mechanisms of growth restraint even below the temperatures deleterious to cell physiology. Thus, growth arrest at high temperatures may not directly result from heat damage to cellular components essential for proliferation and viability. Significance StatementThe immunosuppressant rapamycin is a specific inhibitor of the protein kinase Target Of Rapamycin (TOR), and the drug is known to extend the lifespan of diverse eukaryotic organisms. In this study, we have found that rapamycin confers heat resistance on fission yeast, allowing its proliferation above the normal permissive temperatures. This unexpected observation suggests that TOR, which is known as a growth-promoting kinase, is inhibitory to cell proliferation at high temperatures. Our genome-wide screens have identified additional genes whose deletion leads to improved growth under heat stress. Thus, cells may have mechanisms that restrict proliferation even below the temperatures deleterious to their physiology.

cell biology↗

bioRxiv2023

Towards substitution of invasive telemetry: An integrated home cage concept for unobstrusive monitoring of objective physiological parameters in rodents

This study presents a novel concept for a smart cage designed to monitor the physiological parameters of mice and rats in animal-based experiments. The system focuses on monitoring key clinical parameters, including heart rate, respiratory rate, body temperature, activity, and circadian rhythm. To create the smart home cage system, an in-depth analysis of the requirements was performed, including camera positioning, imaging system types, resolution, frame rates, external illumination, video acquisition, data storage, and synchronization. Two different camera perspectives were considered, and specific camera models, including two near-infrared and two thermal cameras, were selected to meet the requirements. During the first testing phase, the system demonstrated the potential of extracting vital parameters such as respiratory and heart rate. This technology has the potential to reduce the need for implantable sensors while providing reliable and accurate physiological data, leading to refinement and improvement in laboratory animal care.

bioengineering↗

bioRxiv2023

Microhabitat acclimatization shifts physiological baselines and thermal tolerance of the symbiotic anemone, Anthopleura elegantissima

Contemporary organisms in extreme environments can give insight into how species will respond to environmental change. The intertidal forms an environmental gradient where stress increases with tidal height. Here, we explore the contribution of fixed genotypic and plastic environmental effects on thermal tolerance of the intertidal anemone Anthopleura elegantissima and its algal symbionts using a laboratory-based tank experiment. High intertidal anemones had lower baseline symbiont-to-host cell ratios under control conditions, but their symbionts had higher baseline maximum quantum yield compared to low intertidal anemone symbionts, despite identical symbiont communities. High intertidal anemones maintained greater maximum quantum yield and symbiont-to-host cell ratios under heat stress compared to low intertidal anemones, suggesting that high intertidal holobionts have greater thermal tolerance. However, thermal tolerance of clonal anemones acclimatized to different zones was not explained by tidal height alone, indicating emersion duration is not the sole environmental driver of physiological variation. Fixed genotypic effects also influenced physiological baselines, but did not modulate thermal tolerance, demonstrating thermal tolerance is largely driven by environmental history. These results indicate that this symbiosis is highly plastic and may be able to rapidly acclimatize to climate change, defying the convention that symbiotic organisms are more susceptible to environmental stress.

ecology↗

bioRxiv2023

Gene Expression Profiling and Physiological Adaptations of Pearl Spot (Etroplus suratensis) under Varying Salinity Conditions

Eutroplus suratensis (Pearl spot) is naturally found in estuarine environments and has been noted to have a high salinity tolerance, with the ability to thrive in freshwater as well as seawater. By examining the impact of various salinity levels on the growth and survival of Pearl spot, the present study aims to enhance aquaculture profitability by assessing their adaptability and physiological adjustments to changes in salinity, as well as determining their potential to acclimate to a broad range of salinity regimes. Pearl spot fingerlings were placed in tanks with varying salinities (15, 25, 35, 45, 60, and 75ppt) and monitored for mortality at 24-hour intervals up to 120 hours. Results revealed no mortality in the control group (0ppt), as well as in the 15, 25, and 35ppt treatment groups. However, the remaining groups (45, 60, and 75ppt) showed differing levels of mortality, with 44% mortality observed in the 45ppt group and 100% mortality in both the 60 and 75ppt groups. The impact of different salinity levels on the expression of pearl spot genes such as IGF-1, SOD, CAT, NaKATPase, OSTF-1, and HSP70 was investigated, along with a histological examination of the gills. The results showed significant physiological and cellular damage caused by the salinity levels. The expression analysis showed that liver IGF-1 mRNA expression increased by 2.6-fold at 15ppt, and HSP70 mRNA expression in the liver also showed a significant increase with rising salinity levels. In addition, OSTF1 expression exhibited an increase at 15ppt, whereas SOD and CAT expression reached their highest levels at 25ppt. At 15ppt, the expression of NKA mRNA increased significantly by 2.8-fold. The studys overall findings suggested that the fish demonstrated strong molecular-level performance between 15 to 25ppt salinity levels, with the best results observed at 15ppt. These findings suggest that utilizing a salinity level of 15ppt for Pearl spot production could be viable for profitable aquaculture.

animal behavior and cognition↗

bioRxiv2023

The mechanical force with the physiological movement works as a biological cue in tendon development during the postnatal phase in mice

The musculoskeletal system provides structural stability and coordination to enable movement. Tendons have the essential role of efficiently transmitting force generated from muscle contraction to bone to enable ambulation. In doing so, they resist high external forces. In fact, muscle contraction during embryonic development is required to maintain tendon growth and differentiation. Nonetheless, defining the types and magnitudes of loads that act on tendons during embryonic and early postnatal periods is quite difficult. In this study, we aimed to define the physiologic limb movement and forces experienced during these stages in the murine model. We found that late-stage embryos had limited amniotic space, which attenuated limb movement. In the neonatal phase, physical ability, as measured by rollover function and locomotion, increased. These changes, which likely corresponded to increased forces applied to the tendons, corresponded with the expression of tenogenic markers during the embryo to postnatal phase. In particular, we found that the upregulation of Scx and Tnmd correlated with increased movement during the two weeks after birth. Our results help define the spatiotemporal role of mechanical force, including internal and external factors, in tendon growth and development. HighlightsO_LIAssessed limb movement in amnion. C_LIO_LISpace limitation attenuated limb movement in the late-stage embryos. C_LIO_LIDefined the mechanical force from the limbs physiological environment. C_LIO_LIScx and Tnmd were upregulated synchronically with rollover function and locomotion. C_LIO_LIMechanical forces may work as the cue of tendon development C_LI

developmental biology↗

bioRxiv2023

Motile bacteria leverage bioconvection for eco-physiological benefits in a natural aquatic environment

Bioconvection, the active self-sustaining transport phenomenon triggered by the accumulation of motile microbes under competing physico-chemical cues, has been long studied, with recent reports suggesting its role in driving ecologically-relevant fluid flows. Yet, how this collective behaviour impacts the ecophysiology of swimming microbes remains unexplored. Here, through physicochemical profiles and physiological characterizations analysis of the permanently stratified meromictic Lake Cadagno, we characterize the community structure of a dense layer of anaerobic phototrophic sulfur bacteria, and report that the associated physico-chemical conditions engender bioconvection when bulk of the motile purple sulfur bacterium Chromatium okenii synchronize their movement against the gravity direction. The combination of flow cytometry and fluorescent in situ hybridization (FISH) techniques uncover the eco-physiological effects resulting from bioconvection, and simultaneous measurements using dialysis bags and 14C radioisotope, allowed us to quantify in situ the diurnal and nocturnal CO2 fixation activity of the three co-existing species in the bacterial layer. The results provide a direct measure of the cellular fitness, with comparative transcriptomics data - of C. okenii populations present in regions of bioconvection vis-a-vis populations in bioconvection-free regions - indicating the transcripts potentially involved in the bioconvection process. This work provides direct evidence of the impact of bioconvection on C. okenii metabolism, and highlights the functional role of bioconvection in enhancing the metabolic advantage of C. okenii relative to other microbial species inhabiting the microbial layer.

microbiology↗

bioRxiv2023

Human sodium current voltage-dependence at physiological temperature measured by coupling patch-clamp experiment to a mathematical model.

Voltage-gated sodium channels are crucial to action potential propagation in excitable tissues. Voltage-clamp measurements of sodium current are very challenging and are usually performed at room temperature due to the high amplitude and fast activation of the current. In this study, we measured sodium currents voltage dependence in stem-cell-derived cardiomyocytes at physiological temperature. Although apparent activation and inactivation curves measured as the sodium current amplitude dependence on voltage step is within the range reported in previous studies, we demonstrate a systematic error in our measurements that is due to deviation of membrane potential from the command potential of the amplifier. We show how this artifact can be accounted for by the computer simulation of the patch-clamp experiment. This patch-clamp model optimization technique yields a surprising result: -11.5 mV half-activation and -87 mV half-inactivation of the sodium current. Although the half-activation is strikingly different from what was previously believed to be typical for the cardiac sodium current, we show that this estimate explains conduction velocity dependence on extracellular potassium in hyperkalemic conditions. Key pointsO_LIVoltage gated sodium currents play a crucial role in excitable tissues including neurons, cardiac and skeletal muscles. C_LIO_LIMeasurement of sodium current is challenging because of its high amplitude and rapid kinetics, especially at physiological temperature. C_LIO_LIWe have used the patch-clamp technique to measure human sodium current voltage-dependence in human induced pluripotent stem cell-derived cardiomyocytes. C_LIO_LIThe patch-clamp data was processed by optimization of the model accounting for voltage-clamp experiment artifacts, revealing a large difference between apparent parameters of sodium current and the results of the optimization. C_LIO_LIWe conclude that actual sodium current activation is extremely depolarized in comparison to previous studies. C_LIO_LIThe new sodium current model provides a better understanding of action potential propagation, we demonstrate that it explains propagation in hyperkalemic conditions. C_LI

biophysics↗

bioRxiv2023

Molecular physiological characterization of the dynamics of persister formation in Staphylococcus aureus.

Bacteria possess the ability to enter a growth arrested state known as persistence in order to survive antibiotic exposure. Clinically, persisters are regarded as the main causative agents for chronic and recurrent infectious diseases. To combat this antibiotic-tolerant population, a better understanding of the molecular physiology of persisters is required. In this study, we collected samples at different stages of the biphasic kill curve to reveal the dynamics of the cellular molecular changes that occur in the process of persister formation. After exposure to antibiotics with different modes of action, namely vancomycin and enrofloxacin, similar persister levels were obtained. Both shared and distinct stress responses were enriched for the respective persister populations. However, the dynamics of the presence of proteins linked to the persister phenotype throughout the biphasic kill curve and the molecular profiles in a stable persistent population did show large differences depending on the antibiotic used. This suggests that persisters at the molecular level are highly stress specific, emphasizing the importance of characterizing persisters generated under different stress conditions. Additionally, although generated persisters exhibited cross-tolerance toward tested antibiotics, combined therapies were demonstrated to be a promising approach to reduce persister levels. In conclusion, this investigation sheds light on the stress-specific nature of persisters, highlighting the necessity of tailored treatment approaches and the potential of combined therapy. ImportanceBy monitoring proteome and metabolites during Staphylococcus aureus persister formation under vancomycin and enrofloxacin exposure, we revealed the dynamic information of the molecular physiology of persister formation upon exposure to two different antibiotics with different modes of action. The data shows that cells that phenotypically are similarly classified as persisters, do have several molecular characteristics in common but, remarkably so, differ substantially in a significant number of other aspects of their molecular makeup. These contrasts provided valuable insights into persister eradication, which holds considerable clinical relevance.

microbiology↗

bioRxiv2023

Physiological roles of endocytosis and presynaptic scaffold in vesicle replenishment at fast and slow central synapses

After exocytosis, release sites are cleared of vesicular residues to replenish with transmitter-filled vesicles. Endocytic and scaffold proteins are thought to underlie this site-clearance mechanism. However, the physiological significance of this mechanism at diverse mammalian central synapses remains unknown. Here, we tested this in a physiologically optimized condition using action potential evoked EPSCs at fast calyx synapse and relatively slow hippocampal CA1 synapse, in post-hearing mice brain slices at 37{degrees}C and in 1.3 mM [Ca2+]. Pharmacological block of endocytosis enhanced synaptic depression at the calyx synapse, whereas it attenuated synaptic facilitation at the hippocampal synapse. Block of scaffold protein activity likewise enhanced synaptic depression at the calyx but had no effect at the hippocampal synapse. At the fast calyx synapse, block of endocytosis or scaffold protein activity significantly enhanced synaptic depression as early as 10 ms after the stimulation onset. Unlike previous reports, neither endocytic blockers nor scaffold protein inhibitors prolonged the recovery from short-term depression. We conclude that the release-site clearance by endocytosis can be a universal phenomenon supporting vesicle replenishment at both fast and slow synapses, whereas the presynaptic scaffold mechanism likely plays a specialized role in vesicle replenishment predominantly at fast synapses.

neuroscience↗

bioRxiv2023

Declining old pole physiology gradually enhances gene expression asymmetry in bacteria

Gene expression is a heterogeneous process at the single-cell level. This heterogeneity is often coupled to individual growth rates, which are also highly stochastic, leading to the emergence of multiple physiological states within bacterial populations. Although recent advances have shown that cellular aging acts as a deterministic driver of growth asymmetry, the relationship between aging and gene expression heterogeneity remains elusive. Here we show that old poles undergo a progressive decline in gene expression as mother cells age, contributing to enhance phenotypic heterogeneity in bacterial populations. We quantified the activity of promoters with distinct activity profiles: a constitutive promoter, whose expression positively correlates with growth, and the promoter of RpoS, the general stress response sigma factor, for which growth and expression are mutually inhibitory. We demonstrate that mother cells have lower gene expression for both promoters. This asymmetry could not be explained by metabolic rate differences, but rather by the increasing intracellular heterogeneity of mother cells. As a mother ages, the declining activity of its old pole produces intracellular gradients in gene expression. This intracellular asymmetry manifests in the next generation as mother-daughter asymmetry, thus representing a source of phenotypic heterogeneity for the population. Our results show that bacterial asymmetry is built into the declining physiology of mother cells across generations, illustrating the deterministic nature of aging in bacterial systems. These findings provide further evidence for cellular aging as a mechanism to enhance the variance of metabolic states found in bacterial populations, with possible consequences for stress response and survival.

evolutionary biology↗

bioRxiv2023

Knockout studies using CD34+ hematopoietic cells suggest that CD44 is a physiological human neutrophil E-selectin ligand

The recruitment of peripheral blood neutrophils at sites of inflammation involves a multistep cascade, starting with E- and P-selectin expressed on the inflamed vascular endothelium binding sialofucosylated glycans on leukocytes. As the glycoconjugate biosynthesis pathways in different cells are distinct, the precise carbohydrate ligands of selectins varies both across species, and between different immune cell populations in a given species. To study this aspect in human neutrophils, we developed a protocol to perform CRISPR/Cas9 gene-editing on CD34+ hHSCs (human hematopoietic stem/progenitor cells) as they are differentiated towards neutrophil lineage. This protocol initially uses a cocktail of SCF (stem-cell factor), IL-3 (interleukin-3) and FLT-3L (FMS-like tyrosine kinase 3 ligand) to expand the stem/progenitor cells followed by directed differentiation to neutrophils using G-CSF (granulocyte colony-stimulating factor). Microfluidics based assays were performed on a confocal microscope platform to characterize the rolling phenotype of each edited cell type in mixed populations. These studies demonstrated that CD44, but not CD43, is a major E-selectin ligand on human neutrophils. The loss of function results were validated by developing sialofucosylated recombinant CD44. This glycosylated protein supported both robust E-selectin binding in a cell-free assay, and it competitively blocked neutrophil adhesion to E-selectin on inflamed endothelial cells. Together, the study establishes important methods to study human neutrophil biology and determines that sialoflucosylated-CD44 is a physiological human E-selectin ligand. KEY POINTSO_LIA CRISPR-Cas9 based protocol was optimized to knockout genes in primary human neutrophils derived from CD34+ hHSCs C_LIO_LIUnder physiological fluid shear conditions, a sialofucosylated form of CD44 expressed on human neutrophils is a major E-selectin ligand C_LI VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/553923v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1891653org.highwire.dtl.DTLVardef@118e074org.highwire.dtl.DTLVardef@38052aorg.highwire.dtl.DTLVardef@1db0882_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Actomyosin-mediated apical constriction promotes physiological germ cell death in C. elegans

Germ cell apoptosis in C. elegans hermaphrodites is a physiological process eliminating around 60% of all cells in meiotic prophase to maintain tissue homeostasis. In contrast to programmed cell death in the C. elegans soma, the selection of germ cells undergoing apoptosis is stochastic. By live-tracking individual germ cells at the pachytene stage, we found that germ cells smaller than their neighbors are selectively eliminated through apoptosis before differentiating into oocytes. Thus, cell size is a strong predictor of physiological germ cell death. The RAS/MAPK and ECT/RHO/ROCK pathways together regulate germ cell size by controlling actomyosin constriction at the apical rachis bridges, which are cellular openings connecting the syncytial germ cells to a shared cytoplasmic core. Enhancing apical constriction reduces germ cell size and increases the rate of cell death while inhibiting the actomyosin network in the germ cells prevents their death. We propose that actomyosin contractility at the rachis bridges of the syncytial germ cells amplifies intrinsic disparities in cell size. Through this mechanism, animals can adapt the rate of germ cell death and differentiation to changing environmental conditions.

developmental biology↗

bioRxiv2023

Epigenetic and physiological alterations in zebrafish subjected to hypergravity

Gravity is one of the most constant environmental factors across Earths evolution and all organisms are adapted to it. Consequently, spatial exploration has captured the interest in studying the biological changes that physiological alterations are caused by gravity. In the last two decades, epigenetics has explained how the environmental cues are able to altered gene functions in the organisms. Although many studies addressed gravity, the underlying biological and molecular mechanisms that are occurred in altered gravity for those epigenetics-related mechanisms, are mostly inexistent. The present study addressed the effects of hypergravity on development, behavior, gene expression, and most importantly, on the epigenetic changes in a world-wide animal model, the zebrafish (Danio rerio). To perform hypergravity experiments, a custom-centrifuge simulating the large diameter centrifuge (100 rpm [~] 3 g) were designed and zebrafish embryos were exposed during 5 days post fertilization (dpf). Results showed a significant decrease of survival at 2 dpf but not significance in the hatching rate. Physiological and morphological alterations including fish position, movement frequency and swimming behavior showed significant changes due to hypergravity. Epigenetic studies showed a significant hypermethylation of the genome of the zebrafish larvae subjected to 5 days of hypergravity. A downregulation of the gene expression of three epigenetic-related genes (dnmt1, dnmt3, and tet1), although not significant, were further observed. Taken altogether, gravity alterations affected biological responses including epigenetics in fish, providing a valuable roadmap of the putative hazards of living beyond Earth.

cell biology↗