Biology subjects

Chen, X.

Publications and source records attributed to Chen, X..

At least 883 records · Page 49Linked to original sources

bioRxiv2023

Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor

SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an -ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (Mpro), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a Ki of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 Mpro:RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the -ketoamide warhead; more extensive interactions are identified between bound RAY1216 and Mpro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials (https://clinicaltrials.gov/ct2/show/NCT05620160) to test real-world therapeutic efficacy against COVID-19.

biochemistry↗

bioRxiv2023

Loss of kinase Atg1 increases yeast maintenance energy requirement

Maintenance of cellular homeostasis underlies healthy aging. The processes involved in homeostasis rely on the so-called maintenance energy requirement and changes in this maintenance energy requirement impact aging and survival. Among maintenance processes, autophagy plays a crucial role as it is involved in the turn-over and recycling of damaged cellular material, such as organelles or proteins. The contribution of autophagy to the maintenance energy requirement is however unknown. Taking advantage of the high degree of conservation of autophagy between humans and Saccharomyces cerevisiae, we have used this yeast as a model organism to study the impact of macroautophagy on the maintenance energy requirement. The combination of the GFP-Atg8 cleavage assay with yeast retentostat cultures showed that autophagy is highly active in chronologically aging yeast cells, in non-dividing, but non-starving conditions. Deletion of the autophagy-activating kinase ATG1, homolog of human ULK1, resulted in a 60% increase in the maintenance energy requirement and doubled the specific death rate. Both these increases cannot be solely attributed to an observed increase in loss of respiratory capacity. Intriguingly, loss of Atg1 only reduced GFP-Atg8 cleavage by 20% under these conditions, indicating that Atg1-indendent modes of autophagy might be active. Overall, we illustrate the importance of autophagy on the energetics of aging cells and propose an alternative system for the widely applied yeast stationary phase cultures in chronological aging studies.

cell biology↗

bioRxiv2023

Zero-shot learning enables instant denoising and super-resolution in optical fluorescence microscopy

Computational super-resolution (SR) methods, including conventional analytical algorithms and deep learning models, have substantially improved optical microscopy. Among them, supervised deep neural networks have demonstrated outstanding SR performance, however, demanding abundant high-quality training data, which are laborious and even impractical to acquire due to the high dynamics of living cells. Here, we develop zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary SR imaging conditions in an unsupervised manner without the need for either ground truths or additional data acquisition. We demonstrate the versatile applicability of ZS-DeconvNet on multiple imaging modalities, including total internal reflection fluorescence microscopy, three-dimensional (3D) wide-field microscopy, confocal microscopy, lattice light-sheet microscopy, and multimodal structured illumination microscopy (SIM), which enables multi-color, long-term, super-resolution 2D/3D imaging of subcellular bioprocesses from mitotic single cells to multicellular embryos of mouse and C. elegans.

bioengineering↗

bioRxiv2023

Keystone taxa responsible for the microbial community stability and performance of activated sludges

BackgroundThe functions and stability of a community depend on its species, which form complex interaction networks. The keystone taxa identified by network analysis are generally considered to play a vital role in the structure and function of microbial communities, but there is no uniformly accepted operational definition of such taxa. Further, what species and how they affect the communitys stability and function are still poorly understood. MethodsTo solve this problem, we performed a large-scale network analysis of the microbial communities residing in 1186 activated sludge (AS) samples. ResultsWe found that the AS co-occurrence network is a typical scale-free network. While most taxa in the AS co-occurrence network have little association, there are still a small number of taxa that are strongly interconnected. We defined a group of keystone taxa that have an important impact on network stability. Further analysis results indicate that the communities harboring the keystone taxa maintain higher stability, but these communities possess lower pollutant removal rates. In addition, we found that keystone taxa were more likely to appear in samples with lower sludge load. ConclusionsOur work identified the keystone taxa that maintain the stability of microbial communities in the AS systems but at the cost of reducing their function. This finding shed light on the relationship between composition, stability, and function within microbial communities. It also provides novel insights into manipulating the function of microbial communities by modifying their composition.

ecology↗

bioRxiv2023

High immigration rates critical for establishing emigration-driven diversity in microbial communities

Unraveling the mechanisms governing the diversity of ecological communities is a central goal in ecology. While microbial dispersal (including the emigration and immigration process) constitutes an important ecological process, the coupling effects of dispersal and microbial competition in microbial diversity are poorly understood. Here, we investigated how microbial dispersal affects the diversity of microbial communities in the presence of inter-species competition, using a generalized Lotka-Volterra model in combination with experimental investigations. Our model shows that emigration reduces the diversity induced by immigration at low immigration rates. We surprisingly find that it increases the diversity of the community when the immigration rates cross a defined threshold, which we identified as Ineutral. We also found that at high immigration rates, emigration weakens the relative abundance of fast-growing species, and thus enhances the mass effect and increases the diversity. We experimentally confirmed this finding using cocultures of 20 bacterial strains isolated from the soil. Our model further showed that Ineutral exists over a wide range of species pool sizes, growth rates, and interspecies interactions, and decreases with the increasing of species pool size, growth rate, and interspecies interaction. Our work deepens the understanding of the effects of dispersal on diversity of natural communities.

ecology↗

bioRxiv2023

Lower shear stress exacerbates atherosclerosis by inducing the generation of neutrophil extracellular traps via Piezo1-mediated mechanosensation

BACKGROUNDAtherosclerosis is a chronic lipid-driven inflammatory disease, largely influenced by hemodynamics. Neutrophil extracellular traps (NETs)-mediated inflammation plays an important role in atherosclerosis. However, little is known about the mechanism of the generation of NETs under different shear stress and subsequent damage to endothelial cells. We sought to identify a novel mechanical signal provokes NETs generation and to investigate its potential role in atherosclerosis. METHODSApoE-/- mice were fed with high-fat diet (HFD) to induce atherosclerosis. The model of lower shear stress (LSS) with a partial ligation of the left carotid artery was established to assess the role of LSS in NETs generation and atherosclerotic lesions development. Furthermore, the underlying mechanism of LSS promoting NETs generation and injuring endothelial cells was deciphered in neutrophil-like human promyelocytic leukemia (HL-60) cells in parallel-plate flow chamber. RESULTSWe found that LSS correlated spatially with both NETs and atherosclerosis, while inhibition of NETosis could significantly reduce plaque formation in ApoE-/- mice. In vitro, LSS could promote NETs generation directly through down-regulation of Piezo1, a mechanosensitive ion channel. downexpression of Piezol could activate neutrophils and promote NETosis in static. Conversely, Yoda1-evoked activation of Piezo1 attenuated LSS-induced NETosis. Mechanistically, the downexpression of Piezo1 resulted in decreased Ca2+ influx and increased histone deacetylase 2 (HDAC2), which increase reactive oxygen species levels, then led to NETosis. LSS-induced NETs generation promoted the apoptosis and adherence of endothelial cells. CONCLUSIONSLSS directly promotes NETosis through piezo1-HDAC2 axis in atherosclerosis progression. This study uncovers the essential role of Piezo1-mediated mechanical signaling in NETs generation and plaque formation, which provides a promising therapeutic strategy for atherosclerosis. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/529165v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@583911org.highwire.dtl.DTLVardef@aa3e2org.highwire.dtl.DTLVardef@8d83e5org.highwire.dtl.DTLVardef@ce55a0_HPS_FORMAT_FIGEXP M_FIG C_FIG Proposed mechanism for lower shear stress LSS exacerbating atherosclerosis. LSS stimuli decrease Piezo1 expression in the neutrophils, resulting in decreased intracellular Ca2+ concentration, as well as the higher expression level of HDAC2, which could activate oxidative stress and promote intracellular reactive oxygen species formation, and ultimately lead to NETs generation. NETs could aggravate endothelial cells injury and exasperate atherosclerosis. Highlights{blacksquare} Lower shear stress (LSS) promotes Neutrophil extracellular traps (NETs) formation, which is critical for lipid deposits and plaque formation in Atherosclerosis. {blacksquare}Atherosclerotic plaque formation was significantly reduced in the aorta of high fat diet fed ApoE-/- mice intraperitoneal injected with NETs inhibitor, GSK484, especially in the lower shear stress regions. {blacksquare}Piezo1 is a key molecule in the process of neutrophils sense lower shear stress. {blacksquare}lower shear stress inhibits the activation of Piezo1 and promotes NETosis through piezo1-HDAC2 axis. {blacksquare}LSS-induced NETs promote the apoptosis and adhesion of endothelial cells.

cell biology↗

bioRxiv2023

Protective effect of plasma neutralization from prior SARS-CoV-2 Omicron infection against BA.5 subvariant symptomatic reinfection

From December 2022 to January 2023, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections caused by BA.5 and BF.7 subvariants of B.1.1.529 (Omicron) swept across mainland China. It is crucial to estimate the protective effect of the neutralizing antibodies generated by such mass infections against the next potential SARS-CoV-2 reinfection wave, especially if driven by CH.1.1 or XBB.1.5. Previously, we recruited and continuously followed a cohort of individuals that experienced Omicron BA.1, BA.2, and BA.5 breakthrough infections, as well as a control cohort with no history of SARS-CoV-2 infection. In the previously uninfected cohort, the total symptomatic infection rate surveyed during the outbreak was 91.6%, while the symptomatic reinfection rate was 32.9%, 10.5%, and 2.8% among individuals with prior Omicron BA.1, BA.2 and BA.5 infection, respectively, with median intervals between infections of 335, 225 and 94 days. Pseudovirus neutralization assays were performed in plasma samples collected from previously Omicron BA.1-infected individuals approximately 3 months before the outbreak. Results indicate a robust correlation between the plasma neutralizing antibody titers and the protective effect against symptomatic reinfection. The geometric mean of the 50% neutralizing titers (NT50) against D614G, BA.5, and BF.7 were 2.0, 2.5, and 2.3-fold higher in individuals without symptomatic reinfection than in those with symptomatic reinfection (p < 0.01). Low plasma neutralizing antibody titer (below the geometric mean of NT50) was associated with an enhanced cumulative risk of symptomatic reinfection, with a hazard ratio (HR) of 23.55 (95% CI: 9.23-60.06) against BF.7 subvariant. Importantly, neutralizing antibodies titers post one month after BF.7/BA.5 breakthrough infections against CH.1.1 and XBB.1.5 are similar to that against BF.7 from individuals with prior BA.1 infection while not experiencing a symptomatic BF.7/BA.5 reinfection (plasma collected 3 months before the outbreak), suggesting that the humoral immunity generated by the current BF.7/BA.5 breakthrough infection may provide protection against CH.1.1 and XBB.1.5 symptomatic reinfection wave for 4 months. Of note, the higher hACE2 binding of XBB.1.5 may reduce the protection period since the potential increase of infectivity.

immunology↗

bioRxiv2023

Massive Multiplexing of Spatially Resolved Single Neuron Projections with Axonal BARseq

Neurons in the cortex are heterogeneous, sending diverse axonal projections to multiple brain regions. Unraveling the logic of these projections requires single-neuron resolution. Although a growing number of techniques have enabled high-throughput reconstruction, these techniques are typically limited to dozens or at most hundreds of neurons per brain, requiring that statistical analyses combine data from different specimens. Here we present axonal BARseq, a high-throughput approach based on reading out nucleic acid barcodes using in situ RNA sequencing, which enables analysis of even densely labeled neurons. As a proof of principle, we have mapped the long-range projections of >8,000 mouse primary auditory cortex neurons from a single brain. We identified major cell types based on projection targets and axonal trajectory. The large sample size enabled us to systematically quantify the projections of intratelencephalic (IT) neurons, and revealed that individual IT neurons project to different layers in an area-dependent fashion. Axonal BARseq is a powerful technique for studying the heterogeneity of single neuronal projections at high throughput within individual brains.

neuroscience↗

bioRxiv2023

SynEcoSys: a multifunctional platform of large-scale single-cell omics data analysis

Next generation sequencing technologies enable the analysis of the transcriptomes of individual cells, providing a higher resolution of gene expression and function at the single cell level. Various single-cell data are continuously generated every year, covering fields from scientific research to clinical development. The fast-growing public datasets are collected by distinctive platforms, which are designed to facilitate biological discoveries, disease diagnosis, and new treatments. However, these platforms are hard to meet the urgency of having a unified data integration pipeline to improve comparability between datasets. Here we present SynEcoSys, an online multifunctional platform for single-cell transcriptomic data analysis, visualization, and exploration. SynEcoSys by Singleron Biotechnologies currently provides a massive collection of publicly available single-cell sequencing dataset, involving 46,326,175 cells from 731 datasets across multiple platforms and species. All datasets are generated with a strict and uniform data analysis pipeline and cell marker-based manual annotation, thus facilitating more comprehensive and reliable data mining. SynEcoSys is available at: https://www.synecosys.com.

bioinformatics↗

bioRxiv2023

Measuring chondrocyte viability of articular cartilage based on label-free two-photon microscopy and deep learning image analysis

ObjectiveChondrocyte viability (CV) is an important indicator of articular cartilage health. Two-photon excitation autofluorescence (TPAF) and second harmonic generation (SHG) microscopy provide a label-free method for imaging chondrocytes. In this study, we propose an automated assessment of CV using deep learning cell segmentation and counting based on acquired TPAF/SHG images. DesignLabel-free TPAF/SHG images of cartilage samples from rats and porcine were acquired using both commercial and home-built two-photon microscopes, respectively. TPAF/SHG images were merged to form RGB color images with red, green, and blue channels assigned to TPAF (two channels) and SHG signals, respectively. To make the training datasets for the deep learning networks, individual chondrocyte areas on the RGB color images were manually circled and live or dead chondrocytes were validated by using Calcein-AM and Ethidium homodimer-1 dye labeling. We first built a chondrocyte viability network (MCV-Net) using the Mask R-CNN architecture, which could provide individual segmented cellular areas with live or dead status. Wiener deconvolution preprocessing was added before the input of MCV-Net to improve the accuracy of the CV analysis, forming the Wiener deconvolution CV network (wMCV-Net). ResultsTraining (300 images) and test (120 images) datasets were built for rats and porcine cartilage respectively. Wiener deconvolution could improve the Peak Signal-to-Noise Ratio (PSNR) for 30-40%. We demonstrated that both MCV-Net and wMCV-Net significantly improved the accuracy of the CV measurement. ConclusionA custom desktop TPAF/SHG microscope was used in collaboration with deep learning algorithm wMCV-Net based label-free method to assess the CV and get 95% accuracy with both rats and porcine samples.

bioengineering↗

bioRxiv2023

simCAS: an embedding-based method for simulating single-cell chromatin accessibility sequencing data

Single-cell chromatin accessibility sequencing (scCAS) technology provides an epigenomic perspective to characterize gene regulatory mechanisms at single-cell resolution. With an increasing number of computational methods proposed for analyzing scCAS data, a powerful simulation framework is desirable for evaluation and validation of these methods. However, existing simulators generate synthetic data by sampling reads from real data or mimicking existing cell states, which is inadequate to provide credible ground-truth labels for method evaluation. We present simCAS, an embedding-based simulator, for generating high-fidelity scCAS data from both cell-wise and peak-wise embeddings. We demonstrate simCAS outperforms existing simulators in resembling real data and show that simCAS can generate cells of different states with user-defined cell populations and differentiation trajectories. Additionally, simCAS can simulate data from different batches and encode user-specified interactions of chromatin regions in the synthetic data, which provides ground-truth labels more than cell states. We systematically demonstrate that simCAS facilitates the benchmarking of four core tasks in downstream analysis: cell clustering, trajectory inference, data integration, and cis-regulatory interaction inference. We anticipate simCAS will be a reliable and flexible simulator for evaluating the ongoing computational methods applied on scCAS data. AvailabilitysimCAS is freely available at https://github.com/Chen-Li-17/simCAS.

bioinformatics↗

bioRxiv2023

Astroglial exosome HepaCAM signaling and ApoE antagonization coordinates early postnatal cortical pyramidal neuronal axon growth and dendritic spine formation

Developing astroglia play important roles in regulating synaptogenesis through secreted and contact signals. Whether they regulate postnatal axon growth is unknown. By selectively isolating exosomes using size-exclusion chromatography (SEC) and employing cell-type specific exosome reporter mice, our current results define a secreted astroglial exosome pathway that can spread long-range in vivo and stimulate axon growth of cortical pyramidal neurons. Subsequent biochemical and genetic studies found that surface expression of glial HepaCAM protein essentially and sufficiently mediates the axon-stimulating effect of astroglial exosomes. Interestingly, apolipoprotein E (ApoE), a major astroglia-secreted cholesterol carrier to promote synaptogenesis, strongly inhibits the stimulatory effect of astroglial exosomes on axon growth. Developmental ApoE deficiency also significantly reduces spine density of cortical pyramidal neurons. Together, our study suggests a surface contact mechanism of astroglial exosomes in regulating axon growth and its antagonization by ApoE, which collectively coordinates early postnatal pyramidal neuronal axon growth and dendritic spine formation.

neuroscience↗

bioRxiv2023

cGAS-activating lupus autoantibody for cancer immunotherapy

Cytoplasmic DNA triggers a cGAS-mediated signaling cascade that promotes an innate immune response and is potentially actionable in cancer immunotherapy. Here we show that a cytoplasmic-localizing lupus anti-DNA autoantibody activates cGAS and facilitates an immune-mediated prolongation of survival in orthotopic models of glioblastoma (GBM). Mechanistically, cellular penetration and blood-brain barrier crossing by the anti-DNA autoantibody is linked to nucleoside transport. Pulldown, knockdown, signaling, and cytotoxicity assays demonstrate autoantibody association with and activation of cGAS. In orthotopic GBM models, the autoantibody localizes to brain tumor, increases tumor CD8+ T cell content, and prolongs survival in immunocompetent but not immunodeficient mice. This work introduces the new concept of a cGAS-activating anti-DNA autoantibody, which impacts theories on mechanisms of autoimmunity and has translational applications in cancer immunotherapy.

immunology↗

bioRxiv2023

TRAF4 is crucial for the propagation of ST2+ memory Th2 cells in IL-33-mediated type 2 airway inflammation

Tumor necrosis factor receptor (TNF)-associated factor 4 (TRAF4) is an important regulator of type 2 responses in the airway; however, the underlying cellular and molecular mechanisms remain elusive. Herein, we generated T cell-specific TRAF4-deficient (CD4cre-Traf4fl/fl) mice and investigated the role of TRAF4 in interleukin (IL)-33 receptor (ST2, suppression of tumorigenicity 2)-expressing memory Th2 cells (ST2+ mTh2) in IL-33-mediated type 2 airway inflammation. We found that in vitro polarized TRAF4-deficient (CD4cre-Traf4fl/fl) ST2+ mTh2 cells exhibited decreased IL-33-induced proliferation as compared with TRAF4-sufficient (Traf4fl/fl) cells. Moreover, CD4cre-Traf4fl/fl mice showed less ST2+ mTh2 cell proliferation and eosinophilic infiltration in the lungs than Traf4fl/fl mice in the preclinical models of IL-33-mediated type 2 airway inflammation. Mechanistically, we discovered that TRAF4 was required for the activation of AKT/mTOR and ERK1/2 signaling pathways as well as the expression of transcription factor Myc and nutrient transporters (Slc2a1, Slc7a1, and Slc7a5), signature genes involved in T cell growth and proliferation, in ST2+ mTh2 cells stimulated by IL-33. Taken together, the current study reveals a previously unappreciated role of TRAF4 in ST2+ mTh2 cells in IL-33-mediated type 2 pulmonary inflammation, opening up avenues for the development of new therapeutic strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/528093v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@18a8f48org.highwire.dtl.DTLVardef@1c4e29corg.highwire.dtl.DTLVardef@f98f72org.highwire.dtl.DTLVardef@38aacb_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

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

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

genetics↗

bioRxiv2023

A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3/{beta} affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3/{beta} signaling and solved a co-crystal structure of analog 2 bound to CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3/{beta}. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3/{beta} promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

cell biology↗

bioRxiv2023

Structure-dependent spontaneous calcium dynamics in cultured neuronal networks on microcontact-printed substrates

Microcontact printing (CP) is widely used in neuroscience research. However, CP yields reduced cell-substrate adhesion compared with directly coating cell adhesion molecules. Here, we demonstrate that the reduced cell-substrate adhesion caused by CP, high seeding density, and the local restriction would separately contribute to more aggregated (neurons closer to each other in separate clusters) neuronal networks. Calcium recordings revealed that more aggregated networks presented fewer spontaneous calcium activity patterns, and were more likely dominated by synchronized network-wide calcium oscillation (network bursts). First, on a uniform microcontact-printed substrate, densely seeded neurons were reaggregated into a Petri dish-wide network consisting of small clusters, of which the calcium dynamics were dominated by network bursts. Next, further analysis revealed this dominance was maintained since its appearance, and the initiation and propagation of bursts in the small-cluster network shared a similar mechanism with that of homogeneous networks. Then, sparsely seeded neurons formed several networks with different aggregation degrees, in which the less clustered ones presented abundant time-varying subnetwork burst patterns. Finally, by printing locally restricted patterns, highly clustered networks formed, where dominant network bursts reappeared. These findings demonstrate the existence of structure-dependent spontaneous calcium dynamics in cultured networks on microcontact-printed substrates, which provide important insights into designing cultured networks by using CP, and into deciphering the onset and evolution of network bursts in developmental nerve systems.

neuroscience↗

bioRxiv2023

Coupling Flux Balance Analysis with Reactive Transport Modeling through Machine Learning for Rapid and Stable Simulation of Microbial Metabolic Switching

Integration of genome-scale metabolic networks with reactive transport models (RTMs) is an advanced simulation technique that enables predicting the changes of microbial growth and metabolism in space and time. Despite promising demonstrations in the past, computational inefficiency has been pointed out as a critical issue to overcome because it requires repeated implementation of linear programming (LP) to get flux balance analysis (FBA) solutions in every time step and every spatial grid. To address this challenge, we propose a new simulation method where we train/validate artificial neural networks (ANNs) using FBA solutions randomly sampled and incorporate the resulting reduced-order FBA model (represented as algebraic equations) into RTMs as source/sink terms. We demonstrate the efficiency of our method via a case study of Shewanella oneidensis MR-1 strain. During the aerobic growth on lactate, S. oneidensis produces metabolic byproducts (such as pyruvate and acetate), which are subsequently consumed as alternative carbon sources when the preferred ones are depleted. Simulating such intricate dynamics posed a considerable challenge, which we overcame by adopting the cybernetic approach that describes metabolic switches as the outcome of dynamic competition among multiple growth options. In both zero-dimensional batch and one-dimensional column configurations, the ANN-based reduced-order models achieved substantial reduction of computational time by several orders of magnitude compared to the original LP-based FBA models. Importantly, the ANN models produced robust solutions without any special measures to prevent numerical instability. These developments significantly promote our ability to utilize genome-scale networks in complex, multi-physics, and multi-dimensional ecosystem modeling.

systems biology↗