Biology subjects

Su, Y.

Publications and source records attributed to Su, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2023

Probing Molecular Diversity and Ultrastructure of Brain Cells with Fluorescent Aptamers

Detergent-free immunolabeling has been proven feasible for correlated light and electron microscopy, but its application is restricted by the availability of suitable affinity reagents. Here we introduce CAptVE, a method using slow off-rate modified aptamers for cell fluorescence labeling on ultrastructurally reconstructable electron micrographs. CAptVE provides labeling for a wide range of biomarkers, offering a pathway to integrate molecular analysis into recent approaches to delineate neural circuits via connectomics.

neuroscience↗

bioRxiv2023

The SARS-CoV-2 nucleoprotein associates with anionic lipid membranes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N) and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we find the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, lipid binding is shown to occur in the N protein C-terminal domain, which is supported by extensive in silico analysis. Anionic lipid binding occurs for both the free and N oligomeric forms suggesting N can associate with membranes in the nucleocapsid form. Herein we present a lipid-dependent model based on in vitro, cellular and in silico data for the recruitment of N to M assembly sites in the lifecycle of SARS-CoV-2.

microbiology↗

bioRxiv2023

A role for LGI1 in regulating pain sensitivity.

Chronic pain represents a major unmet clinical need. Neuropathic pain, that is pain arising due to damage or disease of the somatosensory nervous system, represents a sizeable proportion of chronic pain cases, affecting around 8% of the general population. Neuronal hyperexcitability is a key driver of neuropathic pain. Leucine glioma inactivated 1 (LGI1), is a secreted protein known to regulate excitability within the nervous system and is the target of autoantibodies from neuropathic pain patients. Therapies that block or reduce antibody levels are highly effective at relieving pain in these patients, suggesting that LGI1 has an important role in clinical pain. Here we have studied the role of LGI1 in regulating pain using mouse models to specifically ablate LGI1 in neuron populations. LGI1 has been well studied at the level of the brain. Here we show that LGI1 is highly expressed in dorsal root ganglion (DRG) neurons and in dorsal horn neurons of the spinal cord. Using transgenic mice, we ablated LGI1, either specifically in nociceptors (LGI1fl/Nav1.8(+/-)), or in all DRG and spinal neurons (LGI1fl/Hoxb8(+/-)). On acute pain assays, mild phenotypes were observed when compared to littermate controls with limited changes in DRG neuron excitability. No differences were seen in the first phase of the formalin test, however LGI1fl/Hoxb8(+/-) mice displayed a significant increase in nocifensive behaviours in the second phase compared to littermate controls. Using the spared nerve injury model, we assessed the impact of LGI1 ablation on neuropathic pain-like behaviours. LGI1fl/Nav1.8(+/-) mice showed no differences in nerve injury induced mechanical hypersensitivity, brush-evoked allodynia or spontaneous pain behaviour compared to controls. However, LGI1fl/Hoxb8(+/-) mice showed a significant exacerbation of mechanical hypersensitivity and allodynia. These data show that LGI1 has a role in regulating pain sensitivity particularly in the context of nerve injury. We suggest this effect is likely mediated at the spinal level since no differences were observed following specific ablation of LGI1 in nociceptors. Neurons in dorsal horn of the spinal cord are important in gating nerve injury induced mechanical pain and our findings suggest that LGI1 plays an important role in this process.

neuroscience↗

bioRxiv2023

The longitudinal single-cell panorama of cynomolgus monkey ovary throughout lifespan revealed a conserved ovarian clock between primates

Ovarian function is critical for female fertility and impacts reproductive longevity. It is of great importance to accurately predict the aging process within the ovary for fertility assessment and disease diagnosis. However, cell metrics for evaluating the ovarian aging rate are still in urgent need, and molecular insights into ovarian development and dysfunction during the primate life course are also limited. Here, we reported large-scale ovarian cell atlas of consecutive development of cynomolgus monkeys across 22 years with 20 time points, covering the foetal, newborn, prepubertal, pubertal, adult, perimenopausal and menopausal stages. We characterized and validated distinct molecular signatures of each cluster of cells within primate ovaries, and uncovered a previously undocumented RHOXF1-positive oocyte type during primordial follicle assembly in primates. Furthermore, the constitution and developmental trajectories of primate germ cells, granulosa cells and stromal / theca cells were also elucidated, and their precursors were identified. More importantly, dynamics of cellular compositions were unravelled through the ovarian development, featured by granulosa, epithelial, stromal, and immune cells that showed strong temporal heterogeneity spanning lifetime, whilst referred to the key function during the corresponding stages. Based on the correlations of each cell type with age and stage-specific molecular dynamics, we further constructed a transcriptomic ovarian clock which could perceive an effective biological age prediction of the ovary and further applied to humans. The findings reveal granulosa, epithelial, and stromal cells as the highest performance predictors of ovarian biological age, while highlighting the crucial role of AGE-RAGE and Relaxin signaling pathways in regulating ovarian aging. Our work not only provide valuable resource for obtaining insights into the development, aging and dysfunction of key organs, but also establish a transcriptomic clock to predict biological ovarian aging thus to be potential clinical implementation in future.

developmental biology↗

bioRxiv2023

Local interneurons in the murine visual thalamus have diverse receptive fields and can provide feature selective inhibition to relay cells.

By influencing the type and quality of information that relay cells transmit, local interneurons in thalamus have a powerful impact on cortex. To define the sensory features that these inhibitory neurons encode, we mapped receptive fields of optogenetically identified cells in the murine dorsolateral geniculate nucleus. Although few in number, local interneurons had diverse types of receptive fields, like their counterpart relay cells. This result differs markedly from visual cortex, where inhibitory cells are typically less selective than excitatory cells. To explore how thalamic interneurons might converge on relay cells, we took a computational approach. Using an evolutionary algorithm to search through a library of interneuron models generated from our results, we show that aggregated output from different groups of local interneurons can simulate the inhibitory component of the relay cells receptive field. Thus, our work provides proof-of-concept that groups of diverse interneurons can supply feature-specific inhibition to relay cells.

neuroscience↗

bioRxiv2023

Cell division machinery drives cell-specific gene activation during bacterial differentiation

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor {sigma}F is exclusively activated in the smaller daughter cell. Compartment specific activation of {sigma}F requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates {sigma}F in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that a unique feature of the sporulation septum, defined by the cell division machinery, drives the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

microbiology↗

bioRxiv2023

Comprehensive Assessment of Isoform Detection Methods for Third-Generation Sequencing Data

The advancement of Third-Generation Sequencing (TGS) techniques has significantly increased the length of sequencing to several kilobases, thereby facilitating the identification of alternative splicing (AS) events and isoform expressions. Recently, numerous computational methods for isoform detection using long-read sequencing data have been developed. However, there is lack of prior comparative studies that systemically evaluates the performance of these software tools, implemented with different algorithms, under various simulations that encompass potential influencing factors. In this study, we conducted a benchmarking analysis of eleven methods implemented in eight computational tools capable of identifying isoform structures from TGS RNA sequencing data. We evaluated their performances using simulated data, which represented diverse sequencing platforms generated by an in-house simulator, as well as experimental data. Our comprehensive results demonstrate the guided mode of StringTie2 and Bambu achieved the best performance in sensitivity and precision, respectively. This study provides valuable guidance for future research on AS analysis and the ongoing improvement of tools for isoform detection using TGS data.

bioinformatics↗

bioRxiv2023

Design of SARS-CoV-2 protease inhibitors with improved affinity and reduced sensitivity to mutations

Inhibitors of the SARS-CoV-2 main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function on these mutants are thus urgently needed. We hypothesized that the covalent HCV protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more tightly than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to NTV. A crucial feature of ML2006a4 is a novel derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Finally, ML2006a4 is less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory drug design can preemptively address potential resistance mechanisms.

microbiology↗

bioRxiv2023

Augmented Super-Resolution Radial Fluctuations (aSRRF) Pushing the Limits of Structured Illumination Microscopy

Structured illumination microscopy (SIM) is a versatile super-resolution technique known for its compatibility with a wide range of probes and fast implementation. While 3D SIM is capable of achieving a spatial resolution of [~]120 nm laterally and [~]300 nm axially, attempting to further enhance the resolution through methods such as nonlinear SIM or 4-beam SIM introduces complexities in optical configurations, increased phototoxicity, and reduced temporal resolution. Here, we have developed a novel method that combines SIM with augmented super-resolution radial fluctuations (aSRRF) utilizing a single image through image augmentation. By applying aSRRF reconstruction to SIM images, we can enhance the SIM resolution to [~]50 nm isotopically, without requiring any modifications to the optical system or sample acquisition process. Additionaly, we have incorporated the aSRRF approach into an ImageJ plugin and demonstrated its versatility across various fluorescence microscopy images, showcasing a remarkable two-fold resolution increase.

cell biology↗

bioRxiv2023

Transcription factor ScWRKY4 in sugarcane negatively regulates the resistance to pathogen infection through the JA signaling pathway

WRKY transcription factor, the transcriptional regulators unique to plants, plays an important role in plant defense response to pathogen infection. However, the disease resistance mechanism of WRKY gene in sugarcane remains unclear. Previously, we identified a ScWRKY4 gene, a member of class IIc of the WRKY gene family, from the sugarcane cultivar ROC22. This gene could be induced by the stresses of salicylic acid (SA) and methyl jasmonate (MeJA). Interestingly, the expression of the ScWRKY4 gene was down-regulated in smut-resistant sugarcane cultivars but up-regulated in smut-susceptible sugarcane cultivars under Sporisorium scitamineum stress. Besides, stable overexpression of the ScWRKY4 gene in Nicotiana benthamiana enhanced susceptibility to Fusarium solani var. coeruleum and caused the down-regulated expression of immune marker-related genes. Furthermore, transcriptome analysis indicated that, the expression of most JAZ genes was suppressed in plant signal transduction pathway. In addition, ScWRKY4 could interact with ScJAZ13 and repress the expression of ScJAZ13. We thus hypothesized that the ScWRKY4 gene was involved in the regulatory network of plant disease resistance, most probably through the JA signaling pathway. The present study depicted the molecular mechanism of the ScWRKY4 gene involved in sugarcane disease resistance and laid the foundation for the subsequent investigation. HighlightTransgenic plants overexpressing the ScWRKY4 gene negatively regulated resistance to pathogen by inhibiting the expression of the JAZ genes.

plant biology↗

bioRxiv2023

A Bayesian observer model reveals a prior for natural daylights in hue perception

Incorporating statistical characteristics of stimuli in perceptual processing can be highly beneficial for reliable estimation from noisy sensory measurements but may generate perceptual bias. According to Bayesian inference, perceptual biases arise from integrating internal priors with noisy sensory inputs. We used a Bayesian observer model to derive biases and priors in hue perception based on discrimination data for hue ensembles with varying levels of chromatic noise. For isoluminant stimuli with hue defined by azimuth angle in cone-opponent color space, discrimination thresholds showed a bimodal pattern, with lowest thresholds near a non-cardinal blue-yellow axis that aligns closely with the variation of natural daylights. Perceptual biases showed zero crossings around this axis, indicating repulsion away from yellow and attraction towards blue. The biases could be explained by the Bayesian observer model through a non-uniform prior with a preference for blue. Our results suggest that visual processing exploits knowledge of the distribution of colors in natural environments for hue perception.

neuroscience↗

bioRxiv2023

Simultaneous enhancement of multiple functional properties using evolution-informed protein design

Designing optimized proteins is important for a range of practical applications. Protein design is a rapidly developing field that would benefit from approaches that enable many changes in the amino acid primary sequence, rather than a small number of mutations, while maintaining structure and enhancing function. Homologous protein sequences contain extensive information about various protein properties and activities that have emerged over billions of years of evolution. Evolutionary models of sequence co-variation, derived from a set of homologous sequences, have proven effective in a range of applications including structure determination and mutation effect prediction. In this work we apply one of these models (EVcouplings) to computationally design highly divergent variants of the model protein TEM-1 {beta}-lactamase, and characterize these designs experimentally using multiple biochemical and biophysical assays. Nearly all designed variants were functional, including one with 84 mutations from the nearest natural homolog. Surprisingly, all functional designs had large increases in thermostability and most had a broadening of available substrates. These property enhancements occurred while maintaining a nearly identical structure to the wild type enzyme. Collectively, this work demonstrates that evolutionary models of sequence co-variation (1) are able to capture complex epistatic interactions that successfully guide large sequence departures from natural contexts, and (2) can be applied to generate functional diversity useful for many applications in protein design.

synthetic biology↗

bioRxiv2023

Pannexin 1 modulates angiogenic activities of human endothelial colony-forming cells through IGF-1 mechanism and is a marker of senescence

BACKGROUNDWe examined the role of pannexins in human endothelial progenitor cell (EPC) senescence. METHODSYoung and replication-induced senescent endothelial colony-forming cells (ECFCs) derived from human circulating EPCs were used to examine cellular activities and senescence-associated indicators after transfection of siRNA specific to Panx1 or lentivirus-mediated Panx1 overexpression. Hindlimb ischemia mice were used as in vivo angiogenesis model. Protein and phospho-kinase arrays were used to determine underlying mechanisms. RESULTSPanx1 was the predominant pannexin isoform in human ECFCs and up-regulated in both replication-induced senescent ECFCs and circulating EPCs from aged mice and humans. Cellular activities of the young ECFCs were enhanced by Panx1 down-regulation, but attenuated by its up-regulation. In addition, reduction of Panx1 in the senescent ECFCs could rejuvenate cellular activities with reduced senescence-associated indicators, including senescence-associated {beta}-galactosidase activity, p16INK4a, p21, acetyl-p53, and phospho-Histone H2A.X. In mouse ischemic hindlimbs injected senescent ECFCs, blood perfusion ratio, salvaged limb outcome, and capillary density were all improved by Panx1 knockdown. Insulin-like growth factor 1 (IGF-1) was significantly increased in the supernatant from senescent ECFCs after Panx1 knockdown. The enhanced activities and paracrine effects of Panx1 knockdown senescent ECFCs were completely inhibited by anti-IGF-1 antibodies. FAK, ERK and STAT3 were activated in senescent ECFCs with Panx1 knockdown, in which the intracellular calcium level was reduced, and the activation was inhibited by supplemented calcium. The increased IGF-1 in Panx1-knockdown ECFCs was abrogated respectively by inhibitors of FAK (PF562271), ERK (U0126), and STAT3 (NSC74859), and supplemented calcium. CONCLUSIONSPanx1 expression is up-regulated in human ECFCs/EPCs with replication-induced senescence and during aging. Angiogenic potential of senescent ECFCs is improved by Panx1 reduction through increased IGF-1 production via activation of FAK-ERK axis following calcium influx reduction. Our findings provide new strategies to evaluate EPC activities and rejuvenate senescent EPCs for therapeutic angiogenesis.

cell biology↗

bioRxiv2023

Evolution of the Telencephalon Anterior-Posterior Patterning by Core Endogenous Network Bifurcation

How did the complex structure of telencephalon evolve? Existing explanations are based on phenomena and lack the first principle. The Darwinian dynamics and the endogenous network theory established a few years ago provide a mathematical and theoretical framework of a dynamical structure, and a general constitutive structure for theory-experiment coupling, respectively, for answering this question from the first principle perspective. By revisiting a gene network that explains the anterior-posterior patterning of the vertebrate telencephalon, we found that with the increase of the cooperative effect in this network, the fixed points gradually evolve, accompanied by the occurrence of two bifurcations. The dynamic behavior of this network consists with the knowledge obtained from experiments on telencephalon evolution. Furtherly, our work drew an answer quantitatively of how the telencephalon anterior-posterior patterning evolved from the pre-vertebrate chordate to the vertebrate and gave a series of verifiable predictions in a first principle manner. Figure Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

systems biology↗

bioRxiv2023

Single-Soma Deep RNA sequencing of Human DRG Neurons Reveals Novel Molecular and Cellular Mechanisms Underlying Somatosensation

The versatility of somatosensation arises from heterogeneous dorsal root ganglion (DRG) neurons. However, soma transcriptomes of individual human DRG (hDRG) neurons - critical information to decipher their functions - are lacking due to technical difficulties. Here, we developed a novel approach to isolate individual hDRG neuron somas for deep RNA sequencing (RNA-seq). On average, >9,000 unique genes per neuron were detected, and 16 neuronal types were identified. Cross-species analyses revealed remarkable divergence among pain-sensing neurons and the existence of human-specific nociceptor types. Our deep RNA-seq dataset was especially powerful for providing insight into the molecular mechanisms underlying human somatosensation and identifying high potential novel drug targets. Our dataset also guided the selection of molecular markers to visualize different types of human afferents and the discovery of novel functional properties using single-cell in vivo electrophysiological recordings. In summary, by employing a novel soma sequencing method, we generated an unprecedented hDRG neuron atlas, providing new insights into human somatosensation, establishing a critical foundation for translational work, and clarifying human species-specific properties.

neuroscience↗

bioRxiv2023

The Haemophilus influenzae in vivo gene expression reveals major clues about bacterial central metabolism, acquisition of trace elements, and other essential pathways during infection of the human lung

Haemophilus influenzae is a major cause of community and hospital acquired pneumonia. While extensively studied in various laboratory models, less is known about how this species persists and causes infection inside the human lung. We present the first study on the H. influenzae in vivo transcriptome during pneumonia, and contrast this with isolates cultured in vitro under standard laboratory conditions. Patients with pneumonia were recruited from emergency departments and intensive care units in a Swedish referral hospital during 2018-2020 (n=102). Duplicates of lower respiratory samples were collected for bacterial culture and RNA-extraction. Patient samples with H. influenzae (n=18) from which bacterial mRNA of adequate quantity and quality could be extracted (n=8) underwent RNA-sequencing, along with duplicates of lab-cultured counterparts (n=7). The transcripts were aligned to core and pan genomes created from 15 reference strains. While in vitro bacteria clustered tightly in principal component analyses of core genome (n=1067) expression, the in vivo samples displayed diverse transcriptomic signatures and did not group with their lab-grown counterparts. In total, 328 core genes were significantly differentially expressed between in vitro and in vivo conditions. The most upregulated genes in vivo included the transferrin-acquisition genes tbp1 and fbpA and the reductase gene msrAB involved in stress response pathways. Biosynthesis of nucleotides/purines, response-to-heat systems, and molybdopterin-scavenging processes were also significantly upregulated in vivo. Major metabolic pathways and iron-sequestering processes were downregulated in vivo. In conclusion, extensive transcriptomic differences were found between bacteria collected in the human lung during pneumonia and isogenic bacteria cultured in vitro.

microbiology↗

bioRxiv2023

Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EV-ISF) reveals sex-dependent changes in microglial EV proteome in response to Aβ pathology

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimers disease (AD), relaying important physiological information about their host tissues. Circulating EVs are protected from degradation, making them attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labeling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g. APPswe,PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with A{beta} deposition. Genotype, age, and A{beta} deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse-EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD. Graphical AbstractHippocampal EVISF response to amyloid beta (A{beta}) is sexually dimorphic and related to the microglial EVISF proteome. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/532133v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1a275d6org.highwire.dtl.DTLVardef@e63da5org.highwire.dtl.DTLVardef@1d93cecorg.highwire.dtl.DTLVardef@12ed0db_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Rotamer Density Estimator is an Unsupervised Learner of the Effect of Mutations on Protein-Protein Interaction

AO_SCPLOWBSTRACTC_SCPLOWProtein-protein interactions are crucial to many biological processes, and predicting the effect of amino acid mutations on binding is important for protein engineering. While data-driven approaches using deep learning have shown promise, the scarcity of annotated experimental data remains a major challenge. In this work, we propose a new approach that predicts mutational effects on binding using the change in conformational flexibility of the protein-protein interface. Our approach, named Rotamer Density Estimator (RDE), employs a flow-based generative model to estimate the probability distribution of protein side-chain conformations and uses entropy to measure flexibility. RDE is trained solely on protein structures and does not require the supervision of experimental values of changes in binding affinities. Furthermore, the unsupervised representations extracted by RDE can be used for downstream neural network predictions with even greater accuracy. Our method outperforms empirical energy functions and other machine learning-based approaches.

bioinformatics↗