Biology subjects

Xu, P.

Publications and source records attributed to Xu, P..

At least 127 records · Page 7Linked to original sources

bioRxiv2023

Inosine Induces Stemness Features in CAR T cells and Enhances Potency

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing. Statement of SignificanceAdenosine is well known to inhibit T cell function and substantial effort has focused on inhibiting adenosine generation and signaling. Here, we show that exhausted T cells are suppressed by adenosine, which is only modestly impacted by inhibiting adenosine generation or signaling. In contrast, metabolism of adenosine to inosine augmented T cell function and culture of T cells with inosine induced multi-level reprogramming leading to stemness and improved anti-tumor potency. We demonstrate the feasibility of introducing inosine during GMP cell manufacturing as a novel strategy to generate enhanced CAR-T cells.

immunology↗

bioRxiv2023

Reprogramming a high robust Geobacillus thermoglucosidasius for efficient synthesis of polymer-grade lactic acid under extremely high temperature (60℃)

Optically pure lactic acid is an important precursor for the synthesis of biodegradable plastic polylactic acid. Geobacillus thermoglucosidasius is a thermophilic bacterial chassis with industrial potential. In this study, we reprogrammed this chassis to efficiently produce polymer-grade lactic acid by combining rational and semi-rational design strategies. We obtained optically pure L-lactic acid- and D-lactic acid-producing strains, GTD17 and GTD7, respectively, via rational strategies: constructing a lactic acid synthesis module, deleting by-product synthesis, and enhancing lactic acid production. At an extremely high temperature (60{degrees}C), the engineered strain GTD17 produced 94.2 g L-1 of L-lactic acid with an overall yield and productivity of 91.5% and 2.0 g L-1 h-1, respectively, and the optical purity of L-lactic acid was 99.5%. We then performed semi-rational adaptive evolution on the engineered strain GTD7 to obtain an optically pure D-lactic acid-producing strain GTD7-144; at the extremely high temperature (60{degrees}C), it produced 153.1 g L-1 of D-lactic acid with an overall yield and productivity of 93.0% and 3.2 g L-1 h-1, respectively. The optical purity of the D-lactic acid was 99.6%. Genome resequencing and analysis of the strain GTD7-144 revealed potential genetic targets that may improve production performance. This study represents an advancement in the high-temperature production of polymer-grade lactic acid by G. thermoglucosidasius.

bioengineering↗

bioRxiv2023

Enriched zones of embedded ribonucleotides are associated with DNA replication and coding sequences in the human mitochondrial genome

Abundant ribonucleoside triphosphate (rNTP) incorporation in DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA structural change and genome instability. The rNMP distribution, characteristics, hotspots, and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in mtDNA of six different human cell types with wild-type or mutant ribonuclease (RNase) H2 genotype. The rNMPs are preferentially embedded in the DNA of the light strand in most cell types studied, but not in the liver-tissue cells, in which the rNMPs are dominant on the heavy strand of hmtDNA. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the replication-control region, which may result in the suppression of mtDNA replication. We also show that longer coding sequences have a significantly higher rNMP-embedment frequency per nucleotide. While the composition of the embedded rNMPs varies among the different cell types, by studying the genomic context of embedded rNMPs, we detected common rNMP-embedment patterns in hmtDNA. The genomic contexts of rNMPs found in hmtDNA are mainly distinct from those found in yeast mtDNA, highlighting a unique signature of rNTP incorporation by hmtDNA polymerase {gamma}.

genomics↗

bioRxiv2023

Loss-of-SIRT7 sensitizes hepatocellular carcinoma to sorafenib through the regulation of ERK Phosphorylation

The FDA-approved oral multi-kinase inhibitor, sorafenib (BAY 43-9006, Nexavar), is the first approved systemic therapy for patients with unresectable hepatocellular carcinoma (HCC). Although it has been shown to significantly improve the overall survival of patients with HCC, drug resistance limits the response rate to this therapeutic. Here, we report that acquired sorafenib resistance is associated with overexpression of the deacetylase, SIRT7, and a high level of ERK phosphorylation. Further, we identify that the hyperactivation of ERK is controlled by SIRT7-mediated deacetylation of DDX3X. The inhibition of SIRT7 combined with sorafenib resulted in a marked reduction of cell viability in vitro and of tumor growth in vivo. It seems plausible that SIRT7 is responsible for the acquired sorafenib resistance and its inhibition is most likely beneficial together in conjunction with sorafenib by suppressing ERK signaling. HighlightsO_LISorafenib resistance in HCC is associated with SIRT7 and ERK hyperactivation. C_LIO_LISuppression of SIRT7 combined with sorafenib restores sensitivity to sorafenib. C_LIO_LISIRT7 controls sorafenib resistance through ERK activation by mediating DDX3X deacetylation. C_LI

cancer biology↗

bioRxiv2023

An extra-circadian function for human CLOCK in the neocortex

Core circadian-related proteins such as the transcription factor CLOCK are ubiquitously expressed and important for regulating molecular pathways underlying circadian rhythms. Previous work has suggested that CLOCK has evolved human neocortex-specific gene regulation and therefore may have extra-circadian functions. To test this in vivo, we generated a mouse model that recapitulates human cortical expression of CLOCK. The CLOCK humanized (HU) mice show enhanced cognitive flexibility, which is associated with the alteration in spatiotemporal expression of CLOCK. Cell type specific genomic profiling of HU mice identified upregulated genes related to dendritic growth and spine formation in excitatory neurons. Consistent with this result, we found that excitatory neurons in HU mice have increased complexity of dendritic branching and spine density, as well as a greater frequency of excitatory postsynaptic currents, suggesting an increase in neural connectivity. In contrast, CLOCK knockout in human induced pluripotent stem cell-induced neurons showed reduced complexity of dendrites and lower density of presynaptic puncta. Together, our data demonstrate that CLOCK evolved extra-circadian gains of function via altered spatiotemporal gene expression and these functions may underlie human brain specializations.

neuroscience↗

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

Single-cell transcriptomic analyses reveal the cellular and genetic basis of aquatic locomotion in scyphozoan jellyfish

The authors have withdrawn their manuscript because the authors need to re-organize the data and writing. More experimental evidence from whole-mount ISH assay and RNAi assays will be added to support the viewpoints. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

ecology↗

bioRxiv2022

Cdkn2a regulates beige fat maintenance through BECN1-mediated autophagy

A potential therapeutic target to curb the obesity and diabetes epidemic is thermogenic beige adipocytes. However, beige adipocytes quickly transition into white adipocytes upon removing stimuli. Here, we define the critical role of Cdkn2a as a molecular pedal for the beige-to-white transition. Beige adipocytes lacking Cdkn2a exhibit prolonged lifespan, and mice are more resistant to diet-induced obesity, along with enhanced energy expenditure and improved glucose tolerance. Mechanistic studies demonstrate that Cdkn2a promotes the expression and activity of BECN1 by directly binding to its mRNA and its negative regulator BCL2L1, activating autophagy and accelerating the beige-to-white transition. Notably, reactivating autophagy by pharmacological or genetic methods abolishes beige adipocyte maintenance induced by Cdkn2a-ablation. Furthermore, hyperactive BECN1 alone significantly accelerates the beige-to-white transition. Collectively, these findings show that Cdkn2a-mediated autophagy serves as a brake system for beige adipocyte maintenance and is a highly promising target for anti-obesity and anti-diabetes therapy. HighlightsO_LICdkn2a ablation promotes beige fat maintenance and ameliorates diet-induced obesity C_LIO_LILoss of Cdkn2a retains beige adipocytes by inhibiting BECN1-mediated autophagy C_LIO_LICdkn2a modulates BECN1 by binding to its mRNA and its inhibitor BCL2L1, respectively C_LIO_LIHyperactive BECN1 is sufficient to accelerate the beige-to-white transition C_LI

cell biology↗

bioRxiv2022

Altered lipid homeostasis underlies selective neurodegeneration in SNX14 deficiency

Dysregulated lipid homeostasis is emerging as a potential cause of neurodegenerative disorders. However, evidence of errors in lipid homeostasis as a pathogenic mechanism of neurodegeneration remains limited. Here, we show that the cerebellar neurodegeneration caused by SNX14 deficiency is associated with lipid metabolism defects. Recent in vitro and in silico studies indicate that SNX14 is an inter-organelle lipid transfer protein that regulates lipid droplet biogenesis and fatty acid desaturation, suggesting that human SNX14 deficiency belongs to an expanding class of cerebellar neurodegenerative disorders caused by altered cellular lipid homeostasis. To test this hypothesis, we generated a mouse model that recapitulates the human SNX14 deficiency at genetic and phenotypic level. Through histological and transcriptomic analyses, we demonstrate that cerebellar Purkinje cells are selectively vulnerable to SNX14 deficiency, while forebrain regions preserve their neuronal content. Ultrastructure and lipidomic studies reveal widespread lipid storage and metabolism defects in SNX14 deficient mice. Furthermore, we identify a unique lipid metabolite profile that links the accumulation of acylcarnitines with the selective cerebellar neurodegeneration in SNX14 deficiency. These findings highlight the importance of lipid homeostasis for neuronal function and survival and suggest a mechanism for selective cerebellar vulnerability to altered lipid homeostasis.

neuroscience↗

bioRxiv2022

Comparative genomic analysis between Leonurus japonicus and Leonurus sibiricus

Leonurus japonicus Houtt. is an important medicinal plant in East Asia and is now widely recognized for its role in treating cerebral apoplexy and lowering blood lipids. Here, we report two sets of chromosome-level genome sequences for leonurine-producing Leonurus japonicus and for its closely related species leonurine-free Leonurus sibiricus, where 99.78% of 518.19 Mb of L. japonicus was assembled into ten pseudochromosomes with a contig N50 of 17.62 Mb and 99.33% of 472.29 Mb of L. sibiricus was assembled into nine pseudochromosomes with a contig N50 of 13.29 MB. The reference genomes of Leonurus will accelerate the decoding of novel bioactive molecules in medicinal plants, especially in the Lamiaceae family.

plant biology↗

bioRxiv2022

An image cryptography method in highly error-prone DNA storage channel

Rapid development in synthetic technologies has boosted DNA as a potential medium for large-scale data storage. Meanwhile, how to implement data security in DNA storage system is still an unsolved problem. In this paper, we propose an image encryption method based on the modulation-based storage architecture. The key idea is to take advantage of the unpredictable modulation signals to encrypt image in highly error-prone DNA storage channel. Numerical results demonstrate that our image encryption method is feasible and effective with excellent security against various attacks (statistical, differential, noise and data loss, etc.). Compared with other methods by DNA molecules hybridization reaction, the proposed method is more reliable and feasible for large-scale applications.

synthetic biology↗

bioRxiv2022

RNA-mediated double-strand break repair in human cells

Double-strand breaks (DSBs) in DNA are challenging lesions to repair. Human cells employ at least three DSB repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ)1,2. In contrast to HR, NHEJ and MMEJ do not utilize a DNA template molecule to recover damaged and/or lost nucleotides2. NHEJ directly ligates broken DNA ends, while MMEJ exploits the alignment of short microhomologies on the DSB sides and is associated with deletions of the sequence between the microhomologies3,4. It is unknown whether and to what extent a transcript RNA has a direct role in DSB-repair mechanisms in mammalian cells. Here, we show that both coding and non-coding transcript RNA facilitates DSB repair in a sequence-specific manner in human cells. Depending on its sequence complementarity with the broken DNA ends, the transcript RNA could promote the repair of a DSB or gap in its DNA gene via NHEJ or MMEJ, or mediate RNA-templated repair. The transcript RNA influences DSB repair by NHEJ and MMEJ even when the transcription level is low. The results demonstrate an unexpected role of transcript RNA in directing the way DSBs are repaired in human cells and maintaining genome stability.

molecular biology↗

bioRxiv2022

Coupled functional physiological phenotyping and simulation model to infer the transpiration sensitivity traits and its implications for transpiration ideotype design

As agricultural drought becomes more frequent worldwide, it is essential to improve crop productivity whilst reducing the water consumption to achieve a sustainable production. Plant transpiration rate and water use efficiency (WUE) collectively determine the yield performance, yet it is challenging to balance the two in breeding programs due to still insufficient mechanistic understanding of the traits. Here we demonstrate the feasibility and effectiveness of calculating dynamic and momentary WUE by coupling WUE model and the state-of-the-art functional physiological phenotyping (FPP). We also present the method of quantifying genotype-specific traits reflecting sensitivity of transpiration to radiation (STr-Rad) and vapor pressure deficit (STr-VPD), under evolving developmental stage and water availability. Using these methods, we revealed the genotypic difference of STr-Rad and STr-VPD in three watermelon accessions, the dramatic change in each of them across the drought treatment phases, and the quantitative impacts of them on dynamic WUE patterns. Based on our results and computational simulations, a general principle for transpiration ideotype design is proposed, which highlights the benefits of lowering STr-VPD to increase WUE and increasing STr-Rad to offset the decline of Tr. FPP-enabled phenomic selection will help screen for elite crops lines with desired transpiration sensitivities.

physiology↗

bioRxiv2022

Structural Genomics of the Human Dopamine Receptor System

The dopamine system, including five dopamine receptors (D1R to D5R), plays essential roles in the central nervous system (CNS) and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders, including Parkinsons Disease (PD) and schizophrenia. Here, we report five cryo-EM structures of all subtypes of human dopamine receptors in complex with G-protein and bound to the pan agonist, Rotigotine, which is used to treat PD and restless legs syndrome. The structures reveal the basis of Rotigotine binding modes to different dopamine receptors. Structural analysis together with functional assays illuminate determinants of ligand polypharmacology and selectivity. The structures also uncover the mechanisms of the dopamine receptor activation, unique structural features among the five receptor subtypes, and the basis of G-protein coupling specificity. Our works provide a comprehensive set of structural templates for the rational design of specific ligands to treat CNS diseases targeting the dopaminergic system.

biochemistry↗

bioRxiv2022

Understanding water conservation vs. profligation traits in vegetable legumes through a physio-transcriptomic-functional approach

Vegetable soybean and cowpea are related warm-season legumes showing contrasting leaf water use behaviors under similar root drought stresses, whose mechanisms are not well understood. Here we conducted an integrative phenomic-transcriptomic study on the two crops grown in a feedback irrigation system that enabled precise control of soil water contents. Continuous transpiration rate monitoring demonstrated that cowpea used water more conservatively under earlier soil drought stages, but tended to maintain higher transpiration under prolonged drought. Interestingly, we observed a soybean-specific transpiration rate increase accompanied by phase shift under moderate soil drought. Time-series transcriptomic analysis suggested a dehydration avoidance mechanism of cowpea at early soil drought stage, in which the VuHAI3 and VuTIP2;3 genes were suggested to be involved. Multifactorial gene clustering analysis revealed different responsiveness of genes to drought, time of day and their interactions between the two crops, which involved species-dependent regulation of the core clock genes. Gene network analysis identified two co-expression modules each associated with transpiration rate in cowpea and soybean, including a pair of negatively-correlated modules between species. Module hub genes, including the ABA-degrading gene GmCYP707A4 and the trehalose-phosphatase/synthase gene VuTPS9 were identified. Inter-modular network analysis revealed putative co-players of the hub genes. Transgenic analyses verified the role of VuTPS9 in regulating transpiration rate under osmotic stresses. These findings propose that species-specific transcriptomic reprograming in leaves of the two crops suffering similar soil drought was not only a result of the different drought resistance level, but a cause of it.

plant biology↗

bioRxiv2022

Estrogen signaling in the dorsal raphe regulates binge-like drinking in mice

The ovarian hormone estrogens promote binge alcohol drinking and contribute to sex differences in alcohol use disorder. However, the mechanisms for estrogen-induced binge drinking are largely unknown. This study aims to test if estrogens act on 5-hydroxytryptamine neurons in the dorsal raphe nucleus (5-HTDRN) to promote binge drinking. We used the drinking in the dark (DID) behavioral test in mice to mimic binge drinking in humans. We found that female mice drank more alcohol than male mice in chronic DID tests. This sex difference was associated with distinct alterations in mRNA expression of estrogen receptor (ER) and 5-HT-related genes in the DRN, suggesting a potential role of estrogen/ERs/5-HT signaling in binge alcohol drinking. In supporting this view, 5-HTDRN neurons from naive male mice had lower baseline neuronal firing activity but higher sensitivity to alcohol-induced excitation compared to 5-HTDRN neurons from naive female mice. Notably, this higher sensitivity was blunted by 17{beta}-estradiol treatment in males, indicating an estrogen-dependent mechanism. We further showed that both ER and ER{beta} are expressed in 5-HTDRN neurons, whereas ER agonist propyl pyrazole triol (PPT) depolarizes 5-HTDRN neurons and ER{beta} agonist diarylpropionitrile (DPN) hyperpolarizes 5-HTDRN neurons. Notably, both PPT and DPN treatments blocked the stimulatory effects of alcohol on 5-HTDRN neurons in males, despite the fact that they have antagonistic effects on the activity dynamics of 5-HTDRN neurons. These results suggest that ERs inhibitory effects on ethanol-induced burst firing of 5-HTDRN neurons may contribute to higher levels of binge drinking in females. Consistently, chemogenetic activation of ER- or ER{beta}-expressing neurons in the DRN reduced binge alcohol drinking. These results support a model in which estrogens act on ER/{beta} to prevent alcohol-induced activation of 5-HTDRN neurons, which in return leads to higher binge alcohol drinking.

neuroscience↗

bioRxiv2022

SOGO-SOFI, light-modulated super-resolution optical fluctuation imaging using only 20 raw frames for high-fidelity reconstruction

Taking advantage of the stochastic photoswitching of genetically encodable reversibly photoswitchable fluorescent proteins (RSFPs), super-resolution optical fluctuation imaging (SOFI) and its variant photochromic stochastic optical fluctuation imaging (pcSOFI) are valuable tools for wide field super- resolution (SR) imaging. Live-cell (pc)SOFI, which requires a small number of original frames to reconstruct an SR image, is prone to structural discontinuity artifacts and low spatial resolution. Herein, we developed a repeated synchronized on- and gradually off-switching SOFI (SOGO-SOFI) that maximized the photoswitching frequency of RSFPs by light modulation and required only 20 frames for high-quality reconstruction. Live-cell SOGO-SOFI imaging of the endoplasmic reticulum (ER) exhibited 10 times higher temporal resolution (100 fps) and fewer artifacts than pcSOFI. Moreover, a combination of SOGO-SOFI with Airyscan further increased the image contrast and the resolution of Airyscan by a factor of 1.5 from 140 nm to 91 nm. The capabilities of SOGO-SOFI were further demonstrated by dual- color imaging of nucleolar proteins in mammalian cells and deep imaging of ER structures in thick brain slices (20.6 {micro}m).

biophysics↗

bioRxiv2022

Enhanced Effector Activity of Mediator Kinase Module Deficient CAR-T Cells

Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes limiting T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were MED12 and CCNC, components of the cyclin-dependent kinase (CDK) module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. MED12 or CCNC deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. Chemical inhibition of CDK8/19 kinase activity recapitulated some features of genetic loss of MED12, including increased T cell expansion. MED12 deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation at enhancers used by transcription factors playing a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in MED12 deficient T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK module as a target for enhancing the potency of anti-tumor T cell responses. One Sentence SummaryThe Mediator kinase module is a primary regulator of T cell differentiation, and genetic or small molecule-based inhibition of this module enhances effector T cell potency.

immunology↗