Biology subjects

Fu, X.

Publications and source records attributed to Fu, X..

At least 127 records · Page 7Linked to original sources

bioRxiv2022

Deletion of 12-lipoxygenase normalizes platelet function after storage and transfusion in thrombocytopenic mice

ObjectivePlatelets for transfusion are stored for 5-7 days. During storage, platelets undergo numerous detrimental functional changes. In the current study, we sought to understand how genetic deletion of 12 -lipoxygenase (12-LOX) affects platelets during storage, before, and after transfusion. Approach and ResultsWe obtained platelets from wild-type (WT) and 12-LOX-/-mice and performed storage studies for 24 and 48 hours. Using LC-MS/MS-MRM, we showed that {omega}-3 and {omega}-6 fatty acids increased significantly in stored platelets from 12-LOX-/-mice, while oxylipins were significantly lower than in WT platelets. The circulation time of fresh 12-LOX-/-platelets was significantly shorter than that of fresh WT platelets, but no differences were observed after storage. Baseline IIb{beta}3 integrin activation was significantly lower before and after 24 hours of storage in 12-LOX-/-platelets than in WT platelets. Surprisingly, after transfusion, we observed more baseline IIb{beta}3 integrin activation in 12-LOX-/-platelets than in WT platelets. In line with this, transfusion of stored 12-LOX-/-platelets led to more frequent and significantly faster vessel occlusions than transfusion of stored WT platelets in a FeCl3-induced carotid artery injury model in thrombocytopenic mice. ConclusionDeleting 12-LOX improves the post-transfusion function of stored murine platelets. Pharmacologic inhibition of 12-LOX or dietary alterations of {omega}-3 and {omega}-6 PUFAs could significantly enhance human platelet quality and function after storage. Future studies must determine the feasibility and safety of 12-LOX inhibition in stored and transfused human platelets.

physiology↗

bioRxiv2022

Unbalanced response to growth variations reshapes the cell fate decision landscape

The global regulation of cell growth rate on gene expression perturbs the performance of gene networks, which would impose complex variations on the cell-fate decision landscape. Here, we utilize a simple synthetic circuit of mutual repression that allows a bistable landscape, to examine how such global regulation would affect the stability of phenotypic landscape and the accompanying dynamics of cell-fate determination. We show that the landscape experiences a growth-rate-induced bifurcation between monostability and bistability. Theoretical and experimental analyses reveal that this bifurcating deformation of landscape arises from the unbalanced response of gene expression to growth variations. The path of growth transition across the bifurcation would reshape cell-fate decisions. These results demonstrate the importance of growth regulation on cell-fate determination processes, regardless of specific molecular signalling or regulation.

systems biology↗

bioRxiv2022

Characterizing the Resolution and Throughput of the Apollo Direct Electron Detector

Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 [A] resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 [A] resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 [A] resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples.

biochemistry↗

bioRxiv2022

Differential gene expression analysis and physiological response characteristics of passion fruit (Passiflora edulis) buds under high-temperature stress

High temperature in summer is an unfavorable factor for passion fruit (Passiflora edulis), which can lead to restricted growth, short flowering period, few flower buds, low fruit setting rate, severe fruit drop, and more deformed fruit. To explore the molecular physiology mechanism of passion fruit responding to high-temperature stress, we use Zhuangxiang Mibao, a hybrid passion fruit cultivar, as the test material. Several physiological indicators were measured and compared between high-temperature (average temperature 38{degrees}C) and nonmoral temperature (average temperature 25{degrees}C) conditions, including photosynthesis, chlorophyll fluorescence parameters, POD, SOD activity and malondialdehyde content. We performed RNA-seq analysis combined with biochemistry experiment to investigate the gene and molecular pathways that respond to high-temperature stress. The results showed that some physiological indicators in the high-temperature group, including the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, and the maximum chemical quantum yield of PS||, were significantly lower than those of the control group. Malondialdehyde content was substantially higher than the control group, while superoxide dismutase and superoxide dismutase activities decreased to different degrees. Transcriptome sequencing analysis showed that 140 genes were up-regulated and 75 genes were down-regulated under high-temperature stress. GO and KEGG annotation analysis of differentially expressed genes revealed many metabolic pathways related to high-temperature stress. Further investigation revealed that 30 genes might be related to high-temperature stress, such as CAO, GSH, WRKY, and HSP, which have also been reported in other species. The results of real-time fluorescence quantitative PCR and RNA-seq of randomly selected ten genes are consistent, which suggests that the transcriptome sequencing results were reliable. Our study provides a theoretical basis for the mechanism of passion fruit response to high-temperature stress. Also, it gives a theoretical basis for the subsequent breeding of new heat-resistant passion fruit varieties.

plant biology↗

bioRxiv2022

Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast

Aneuploidy compromises genomic stability, often leading to embryo inviability, and is frequently associated with tumorigenesis and aging. Different aneuploid chromosome stoichiometries lead to distinct transcriptomic and phenotypic changes, making it helpful to study aneuploidy in tightly controlled genetic backgrounds. By deploying the engineered SCRaMbLE system to the newly synthesized Sc2.0 megabase chromosome VII (synVII), we constructed a synthetic disomic yeast and screened hundreds of SCRaMbLEd derivatives with diverse chromosomal rearrangements. Phenotypic characterization and multi-omics analysis revealed that fitness defects associated with aneuploidy could be restored by i) removing most of the chromosome content, or ii) modifying specific regions in the duplicated chromosome. These findings indicate that both chromosome copy number and chromosomal regions contribute to the aneuploidy-related phenotypes, and the synthetic yeast resource opens new paradigms in studying aneuploidy. In briefUse of SCRaMbLE and newly synthesized Mb-scale Sc2.0 chromosome VII enables insights into genotype/phenotype relationships associated with aneuploidy HighlightsO_LIDe novo design and synthesis of a Mb-scale synthetic yeast chromosome VII, carrying 11.8% sequence modifications and representing nearly 10% of the yeast genome. C_LIO_LIA disomic yeast (n + synVII) is constructed for dissecting the aneuploidy phenotype C_LIO_LISCRaMbLE enables systematic exploration of regions causing aneuploidy phenotypes C_LIO_LIChromosomal copy number and content both contribute to aneuploidy phenotypes C_LIO_LIA 20 Kb deletion on the right arm of synVII leads to fitness improvement linked to up-regulation of protein synthesis C_LI

synthetic biology↗

bioRxiv2022

Stereoretentive Post-Translational Protein Editing

Chemical post-translational methods now allow convergent side-chain editing of proteins as a form of direct chemical mutagenesis without needing to resort to genetic intervention. Current approaches that allow the creation of constitutionally native side-chains via C-C formation using off-protein carbon-centred C* radicals added to unnatural amino acid radical acceptor SOMOphile tags such as dehydroalanine are benign and wide-ranging. However, they also typically create epimeric mixtures of D-/L-residues. Here we describe a light-mediated desulfurative method that, through the creation and reaction of stereoretained on-protein L-alanyl C{beta}* radicals, allows C{beta}-H{gamma}, C{beta}-O{gamma}, C{beta}-Se{gamma}, C{beta}-B{gamma} and C{beta}-C{gamma} bond formation to flexibly generate site-selectively edited proteins with full retention of native stereochemistry under mild conditions from a natural amino acid. This methodology shows great potential to explore protein side-chain diversity and construct useful bioconjugates. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=32 SRC="FIGDIR/small/504816v1_ufig1.gif" ALT="Figure 1"> View larger version (7K): org.highwire.dtl.DTLVardef@1ccd60corg.highwire.dtl.DTLVardef@f8ca72org.highwire.dtl.DTLVardef@1b33835org.highwire.dtl.DTLVardef@12f32e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Simultaneous transcriptome and proteome profiling in a single mouse oocyte with a deep single-cell multi-omics approach

Nowadays, although single-cell multi-omics technologies are undergoing rapid development, simultaneous transcriptome and proteome analysis of a single-cell individual still faces great challenges. Here, we developed a single-cell simultaneous transcriptome and proteome (scSTAP) analysis platform based on microfluidics, high-throughput sequencing and mass spectrometry technology, to achieve deep and joint quantitative analysis of transcriptome and proteome at the single-cell level for the first time. This platform was applied to analyze single mouse oocytes at different meiotic maturation stages, reaching an average quantification depth of 19948 genes and 2663 protein groups in single mouse oocytes. This reliable quantitative two-omics dataset of single cells provided an important resource for understanding the relationship between the transcriptome and the proteome in cells. Based on the correlation analysis of RNAs and proteins in the same single cell, we demonstrated the expressive heterogeneity of transcriptome and proteome during the cellular biological process. Specially, we analyzed the meiosis regulatory network during oocyte maturation with an unprecedented depth at the single-cell level, and identified 30 transcript-protein pairs as specific oocyte maturational signatures, providing crucial insights into the regulatory features of transcription and translation during oocyte meiotic maturation.

cell biology↗

bioRxiv2022

Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde trafficking

Mutations in the microtubule (MT)-binding protein doublecortin (DCX) or in the MT- based molecular motor dynein result in lissencephaly. However, a functional link between DCX and dynein has not been defined. Here, we demonstrate that DCX negatively regulates dynein-mediated retrograde transport by reducing dyneins association with MTs and by disrupting the composition of the dynein motor complex. Previous work showed an increased binding of the adaptor protein C-Jun-amino-terminal kinase-interacting protein 3 (JIP3) to dynein in the absence of DCX. Using purified components, we demonstrate that JIP3 forms an active motor complex with dynein and its cofactor dynactin with two dyneins per complex. DCX competes with the binding of the second dynein, resulting in a velocity reduction of the complex. We conclude that DCX negatively regulates dynein-mediated retrograde transport through two critical interactions by regulating dynein binding to MTs and by regulating the association of JIP3 to the dynein motor complex.

cell biology↗

bioRxiv2022

Bayesian models for event-related potentials time-series and electrode correlations estimation

Recent developments on Bayesian inference and prediction for effects on event-related potentials (ERPs) stem from a wide variety of methods, including machine learning, multilevel models, and others. However, few of these approaches make use of clear estimates of the ERP voltage across time and across space (scalp). In the present article, via an iterative process, we propose Gaussian random walk (GRW) models that can estimate voltage uncertainty across time and also provide correlation matrices across electrodes. We apply these models to real ERP data from a P3b paradigm as an example. We discuss results in terms of past and current literature of both ERP estimation and electroencephalography analysis in general.

neuroscience↗

bioRxiv2022

Coronaviruses using different strategies to antagonize antiviral responses and pyroptosis

Viral infection triggers inflammasome-mediated caspase-1 activation. Nevertheless, limited understanding exists regarding how viruses use the active caspase-1 to evade host immune response. Here, we use porcine epidemic diarrhea virus (PEDV) as a model of coronaviruses (CoVs) to illustrate the intricate regulation of CoVs to combat IFN-I signaling and pyroptosis. Our findings demonstrate that PEDV infection stabilizes caspase-1 expression via papain-like protease PLP2s deubiquitinase activity and the enhanced stabilization of caspase-1 disrupts IFN-I signaling by cleaving RIG-I at D189 residue. 6-Thioguanine (6TG), a PLP2 inhibitor, plays a critical role in reversing the inhibitory effect of IFN-I via PLP2 and also inhibiting PEDV replication. Meanwhile, PLP2 can degrade GSDMD-p30 by removing its K27-linked ubiquitin chain at K275 to restrain pyroptosis. Papain-like proteases from other genera of CoVs (PDCoV and SARS-CoV-2) have the similar activity to degrade GSDMD-p30. We further demonstrate that SARS-CoV-2 N protein induced NLRP3 inflammasome activation also uses the active caspase-1 to counter IFN-I signaling by cleaving RIG-I. Therefore, our work unravels a novel antagonistic mechanism employed by CoVs to evade host antiviral response.

immunology↗

bioRxiv2022

Deep Multimodal Graph-Based Network for Survival Prediction from Highly Multiplexed Images and Patient Variables

The spatial architecture of the tumour microenvironment and phenotypic heterogeneity of tumour cells have been shown to be associated with cancer prognosis and clinical outcomes, including survival. Recent advances in highly multiplexed imaging, including imaging mass cytometry (IMC), capture spatially resolved, high-dimensional maps that quantify dozens of disease-relevant biomarkers at single-cell resolution, that contain potential to inform patient-specific prognosis. However, existing automated methods for predicting survival typically do not leverage spatial phenotype information captured at the single-cell level, and current methods tend to focus on a single modality, such as patient variables (PVs). There is no end-to-end method designed to leverage the rich information in whole IMC images and all marker channels, and aggregate this information with PVs in a complementary manner to predict survival with enhanced accuracy. We introduce a deep multimodal graph-based network (DMGN) that integrates entire IMC images and multiple PVs for end-to-end survival prediction of breast cancer. We propose a multimodal graph-based module that considers relationships between spatial phenotype information in all image regions and all PVs, and scales each region-PV pair based on its relevance to survival. We propose another module to automatically generate embeddings specialised for each PV to enhance multimodal aggregation. We show that our modules are consistently effective at improving survival prediction performance using two public datasets, and that DMGN can be applied to an independent validation dataset across the same antigens but different antibody clones. Our DMGN outperformed state-of-the-art methods at survival prediction.

bioinformatics↗

bioRxiv2022

A novel RHH family transcription factor aCcr1 and its viral homologs dictate cell cycle progression in archaea

Cell cycle regulation is of paramount importance for all forms of life. Here we report that a conserved and essential cell cycle-specific transcription factor (designated as aCcr1) and its viral homologs control cell division in Sulfolobales. We show that the transcription level of accr1 reaches peak during active cell division (D-phase) subsequent to the expression of CdvA, an archaea-specific cell division protein. Cells over-expressing the 58-aa-long RHH (ribbon-helix-helix) family cellular transcription factor as well as the homologs encoded by large spindle-shaped viruses Acidianus two-tailed virus (ATV) and Sulfolobus monocaudavirus 3 (SMV3) display significant growth retardation and cell division failure, manifested as enlarged cells with multiple chromosomes. aCcr1 over-expression results in downregulation of 17 genes (>4-folds) including cdvA. A conserved motif, aCcr1-box, located between the TATA-binding box and the translation initiation site in the promoters of 13 out of the 17 highly repressed genes, is critical for aCcr1 binding. The aCcr1-box is present in the promoters of cdvA genes across Sulfolobales, suggesting that aCcr1-mediated cdvA repression is an evolutionarily conserved mechanism by which archaeal cells dictate cytokinesis progression, whereas their viruses take advantage of this mechanism to manipulate the host cell cycle.

microbiology↗

bioRxiv2022

A diverse network of pericoerulear neurons control arousal states

As the primary source of norepinephrine (NE) in the brain, the locus coeruleus (LC) regulates arousal, avoidance and stress responses1,2. However, how local neuromodulatory inputs control LC function remains unresolved. Here we identify a population of transcriptionally, spatially and functionally diverse GABAergic neurons in the LC dendritic field that receive distant inputs and modulate modes of LC firing to control global arousal levels and arousal-related processing and behaviors. We define peri-LC anatomy using viral tracing and combine single-cell RNA sequencing with spatial transcriptomics to molecularly define both LC-NE and peri-LC cell types. We identify several neuronal cell types which underlie peri-LC functional diversity using a series of complementary neural circuit approaches in behaving mice. Our findings indicate that LC and peri-LC neurons are transcriptionally, functionally, and anatomically heterogenous neuronal populations which modulate arousal and avoidance states. Defining the molecular, cellular, and functional diversity of the LC and peri-LC provides a road map for understanding the neurobiological basis of arousal, motivation and neuropsychiatric disorders.

neuroscience↗

bioRxiv2022

A causal role of the human left temporoparietal junction in computing social influence during goal-directed learning

The human temporoparietal junction (TPJ) is a brain area crucial for processing social information. Although brain stimulation studies have started to explore the causal function of TPJ under social contexts, few have explicitly considered bilateral TPJ as target regions. Here, leveraging non-invasive continuous theta-burst stimulation (cTBS) and hierarchical Bayesian computational modeling, we tested whether left or right TPJ (with vertex as control) is causally involved in how dissenting choices by others influence individuals choice adjustments in goal-directed learning. In our social learning paradigm, participants (N = 31) first made their private decision, and then were allowed to re-adjust their choices after observing choices of four other players. Behaviorally, we show that disruption of the left, but not the right TPJ, weakened participants choice adjustment and delayed their response speed when confronted with dissenting information from the other players. Computationally, disrupting activity in the left TPJ attenuated the degree of computing social influence during choice adjustment, whereas the extent to which how observational learning from others choices was integrated into direct learning remained intact. Together, our results provide evidence for the causal role of left TPJ in social influence during goal-directed learning and shed light on the relational function (with respect to oneself) of the TPJ in social cognition.

neuroscience↗

bioRxiv2022

Improving and evaluating deep learning models of cellular organization

MotivationCells contain dozens of major organelles and thousands of other structures, many of which vary extensively in their number, size, shape and spatial distribution. This complexity and variation dramatically complicates the use of both traditional and deep learning methods to build accurate models of cell organization. Most cellular organelles are distinct objects with defined boundaries that do not overlap, while the pixel resolution of most imaging methods is not sufficient to resolve these boundaries. Thus while cell organization is conceptually object-based, most current methods are pixel-based. Using extensive image collections in which particular organelles were fluorescently-labeled, deep learning methods can be used to build conditional autoencoder models for particular organelles. A major advance occurred with the use of a U-net approach to make multiple models all conditional upon a common reference, unlabeled image, allowing the relationships between different organelles to be at least partially inferred. ResultsWe have developed improved GAN-based approaches for learning these models and have also developed novel criteria for evaluating how well synthetic cell images reflect the properties of real images. The first set of criteria measure how well models preserve the expected property that organelles do not overlap. We also developed a modified loss function that allows retraining of the models to minimize that overlap. The second set of criteria uses object-based modeling to compare object shape and spatial distribution between synthetic and real images. Our work provides the first demonstration that, at least for some organelles, deep learning models can capture object-level properties of cell images. AvailabilityA Reproducible Research Archive containing all source code, generated images and analysis results will be made available at http://murphylab.cbd.cmu.edu/Software upon publication. Contactmurphy@cmu.edu Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2022

Control of flight responses by a non-canonical corticolimbic pathway.

Survival requires the selection of appropriate behaviour in response to threats, and dysregulated defensive reactions are associated with psychiatric illnesses such as posttraumatic stress and panic disorder.1 Threat-induced behaviours, including freezing and flight, are controlled by neuronal circuits in the central amygdala (CeA)2; however, the source of neuronal excitation to the CeA that contributes to high-intensity defensive responses is unknown. Here we used a combination of neuroanatomical mapping, in-vivo calcium imaging, functional manipulations, and electrophysiology to characterize a previously unknown projection from the dorsal peduncular (DP) prefrontal cortex to the CeA. DP-to-CeA neurons are glutamatergic and specifically target the medial CeA, the main amygdalar output nucleus. Using a behavioural paradigm that elicits both freezing and flight, we found that CeA-projecting DP neurons are activated by high-intensity threats in a context-dependent manner. Functional manipulations revealed that the DP-to-CeA pathway is necessary and sufficient for both avoidance behaviour and flight. Furthermore, we found that DP projections target distinct medial CeA neuronal populations projecting to midbrain flight centres. These results elucidate a non-canonical top-down pathway regulating defensive responses.

neuroscience↗

bioRxiv2022

Identification of Transient Intermediates During Spliceosome Activation by Single Molecule Fluorescence Microscopy

Spliceosome activation is the process of creating the catalytic site for RNA splicing and occurs de novo on each intron following spliceosome assembly. Dozens of factors bind to or are released from the activating spliceosome including the Lsm2-8 heteroheptameric ring that binds the U6 small nuclear RNA (snRNA) 3-end. Lsm2-8 must be released to permit active site stabilization by the Prp19-containing complex (NineTeen Complex, NTC); however, little is known about the temporal order of events and dynamic interactions that lead up to and follow Lsm2-8 release. We have used colocalization single molecule spectroscopy (CoSMoS) to visualize Lsm2-8 dynamics during activation of yeast spliceosomes. Lsm2-8 is recruited as a component of the tri-snRNP and is released after integration of the Prp19-containing complex (NineTeen Complex, NTC). Despite Lsm2-8 and the NTC being mutually exclusive in existing cryo-EM structures of yeast B complex spliceosomes, we identify a transient intermediate containing both [Formula] and provide a kinetic framework for its formation and transformation during activation. Prior to [Formula] assembly, the NTC rapidly and reversibly samples the spliceosome suggesting a mechanism for preventing NTC sequestration by defective spliceosomes that fail to properly activate. In complementary ensemble assays, we show that a base-pairing dependent ternary complex can form between Lsm2-8 and U2 and U6 helix II RNAs. Together our data suggest a Hfq-like function for Lsm2-8 in maintaining U2/U6 helix II integrity before it can be transferred to the NTC by transient formation of the [Formula] spliceosome. Significance StatementThe spliceosome active site is created de novo during activation and involves numerous conformational and compositional changes. Here, we define a kinetic pathway for yeast spliceosome activation using single molecule fluorescence that includes transient intermediates not previously identified. Real-time measurements allow us to uncover rapid, reversible sampling interactions of the NineTeen Complex (NTC) that may prevent its accumulation on defective spliceosomes. By analogy with bacterial Hfq, we propose that the homologous Lsm2-8 proteins stabilize U2/U6 helix II during activation before the helix is transferred to the NTC in a short-lived spliceosome containing both Lsm2-8 and the NTC. Our data demonstrate how single molecule studies of activation can reveal kinetically-competent intermediates and complement cryo-EM studies of stalled or inhibited complexes.

biochemistry↗

bioRxiv2022

Population genomics unravels the Holocene history of Triticum-Aegilops species

Deep knowledge of crop biodiversity is essential to improve global food security. Despite bread wheat serving as a keystone crop worldwide, the population history of bread wheat and its wild relatives (a.k.a. wheats) remains elusive. By analyzing whole-genome sequences of 795 wheats, we found that bread wheat originated southwest of the Caspian Sea [~]11,700 years ago and underwent a slow speciation process, lasting [~]3,300 years due to persistent gene flow from wild relatives. Soon after, bread wheat spread across Eurasia and reached Europe, South Asia, and East Asia [~]7,000 to [~]5,000 years ago, shaping a diversified but occasionally convergent adaptive landscape of bread wheat in novel environments. Opposite to cultivated wheat, wild wheat populations have declined by [~]82% in the past [~]2,000 years due to the food choice shift of humans, and likely continue to drop because of the changing climate. These findings will guide future efforts in protecting and utilizing wheat biodiversity to improve global food security.

genetics↗