Biology subjects

Xie, S.

Publications and source records attributed to Xie, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Expanding the CRISPR toolbox by engineering Cas12a orthologs of metagenomic discovery

Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility in genome editing and molecular diagnostic applications. However, the widespread adoption of CRISPR-Cas12a nucleases and their variants has been hindered by the requirement for a specific protospacer adjacent motif (PAM), relatively low CRISPR RNA (crRNA) activity and the inability to multiplex nucleic acid detection alone. To overcome these limitations, we employed a comprehensive framework combined with AlphaFold2 to de novo mine 1,261 previously unexploited Cas12a orthologs from the global microbiome. Following experimental analysis, we identified the most promising 21 Cas12a nuclease orthologs and designated them "Genie scissor 12" (Gs12). Our analysis uncovered two exceptional variants among these newly identified orthologs: Gs12-10, a first natural PAM-less Cas12a ortholog, which can recognize 52 distinct PAM types, representing a significant 1.8-fold expansion in recognition range compared to the relative LbCas12a PAM; and Gs12-7MAX, an engineered variant of Gs12-7 that exhibited 1.27-fold higher editing efficiency than enAsCas12a-HF. Furthermore, we harnessed Gs12-1, Gs12-4, Gs12-9, and Gs12-18, along with their corresponding engineered crRNAs, to develop a powerful four-channel multiplexed CRISPR-based nucleic acid detection system. The discovery of diverse functions in Cas12a offers a deeper understanding of the CRISPR/Cas12a family. Also, it holds great promise for expanding its applications and uncovering the untapped potential of other CRISPR/Cas systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/615316v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@bee75forg.highwire.dtl.DTLVardef@1480f61org.highwire.dtl.DTLVardef@1e9cbc9org.highwire.dtl.DTLVardef@4211b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

GPR30 in spinal cholecystokinin-positive neurons modulates neuropathic pain via mediating descending facilitation

Neuropathic pain, a major health problem affecting 7-10% of the global population, lacks effective treatment due to its elusive mechanisms. Cholecystokinin-positive (CCK+) neurons in the spinal dorsal horn (SDH) are critical for neuropathic pain, yet the underlying molecular mechanisms remain unclear. Here, we show that the membrane estrogen receptor G-protein coupled estrogen receptor (GPER/GPR30) in spinal neurons was significantly upregulated in chronic constriction injury (CCI) mice and that inhibition of GPR30 in CCK+ neurons reversed CCI-induced neuropathic pain. Furthermore, GPR30 in spinal CCK+ neurons was essential for the enhancement of AMPA-mediated excitatory synaptic transmission in CCI mice. Moreover, GPR30 was expressed in spinal CCK+ neurons that received direct projection from the primary sensory cortex (S1-SDH). Chemogenetic inhibition of S1-SDH post-synaptic neurons alleviated CCI-induced neuropathic pain. Conversely, chemogenetic activation of these neurons mimicked neuropathic pain symptoms, which were attenuated by spinal inhibition of GPR30. Finally, we confirmed that GPR30 in S1-SDH post-synaptic neurons was required for CCI-induced neuropathic pain. Taken together, our findings suggest that GPR30 in spinal CCK+ neurons and S1-SDH postsynaptic neurons is pivotal for neuropathic pain, thereby representing a promising therapeutic target for neuropathic pain.

neuroscience↗

bioRxiv2024

Unveiling allopolyploidization-driven genome duplications through progressive analysis of deep genome skimming data

Whole-genome duplication (WGD) events are widespread across the Web of Life (WoL). Given the prevalence of WGDs in the polyploid Prunus (Rosaceae), this economically- and agriculturally-important angiosperm lineage provides an excellent model for exploring this mode of reticulation. We used the polyploid Prunus to demonstrate a progressive strategy for analyzing Deep Genome Skimming (DGS) data in the presence of WGDs. Phylogenomic discordance analyses indicated that allopolyploidization, rather than Incomplete Lineage Sorting (ILS), played a dominant role in the origin and dynamics of polyploid Prunus. This study underscores how a progressive strategy to identify WGD events at different depths in a phylogenetic tree reveals the nuances of evolutionary mechanisms driving allopolyploidization. We inferred that the continued uplift of the Himalayas from the Middle to Late Miocene drove the rapid diversification of the Eastern Asia endemic Maddenia clade, by facilitating frequent hybridization and allopolyploidization, specifically introgression between the Himalayas-Hengduan and the Central-Eastern China clades.

systems biology↗

bioRxiv2024

Molecular recording of cellular protein kinase activity with chemical labeling

Protein kinases control most cellular processes and aberrant kinase activity is involved in numerous diseases. To investigate the link between specific kinase activities and cellular phenotypes in heterogeneous cell populations and in vivo, we introduce molecular recorders of kinase activities for later analysis. Based on split-HaloTag and a phosphorylation-dependent molecular switch, our recorders become rapidly labeled in the presence of a specific kinase activity and a fluorescent HaloTag substrate. The kinase activity in a given cell controls the degree of fluorescent labeling whereas the recording window is set by the presence of the fluorescent substrate. We have designed specific recorders for four protein kinases, including protein kinase A. We apply our protein kinase A recorder for the sorting of heterogeneous cell populations and subsequent transcriptome analysis, in genome-wide CRISPR screens to discover regulators of PKA activity and for the tracking of neuromodulation in freely moving mice.

biochemistry↗

bioRxiv2024

Exploring the effect of capsaicin on gene expression and chemotherapy sensitization in gastric cancer cells

PurposeCapsaicin has previously been demonstrated to exhibit anti-tumor effect in various cancer type. However, the effect of capsaicin on gene expression and its potential mechanism on chemotherapy sensitization were still uncertain. MethodHuman AGS gastric cancer cell line was treated with different concentrations of capsaicin, 5-fluorouracil and oxaliplatin. Cell viability was assessed using cell viability assay. High throughput RNA sequencing was used to screen differentially expressed genes triggered by capsaicin in AGS cells. qPCR and Western blotting were used to detect the expression of mRNAs and proteins induced by capsaicin. ResultCapsaicin could significantly inhibit cell viability at a dose-depend manner in AGS gastric cancer cell line. Through high-throughput RNA sequencing, genes regulating DNA repair, DNA replication and chromosome assemble pathways were analyzed to be down-regulated by capsaicin. qPCR and western blot assay demonstrated that capsaicin could inhibit expression of the key enzymes (FEN1, LIG1 and PARP1) which play critical roles in DNA damage response and chemotherapy resistance. In vitro assay demonstrated that capsaicin could significantly induce chemo-sensitivity of 5-FU and Oxaliplatin at low dose. ConclusionCapsaicin could inhibit DNA repair pathway, which might contribute to cell growth inhibition and improvement of chemotherapy sensitization. These results revealed a novel function of capsaicin in DNA damage repair and provided new potential targets in cancer therapy.

cancer biology↗

bioRxiv2024

BRAIN-METASTATIC MELANOMA MODELS UNCOVER IMMUNE AND MOLECULAR CORRELATES OF RESPONSE TO IMMUNOTHERAPY

Despite the promising results of immune checkpoint blockade (ICB) therapy, outcomes for patients with brain metastasis (BrM) remain poor. Identifying resistance mechanisms has been hindered by limited access to patient samples and relevant preclinical models. Here, we developed two mouse melanoma BrM models that recapitulate the disparate responses to ICB seen in patients. We demonstrate that these models capture the cellular and molecular complexity of human disease and reveal key factors shaping the tumor microenvironment and influencing ICB response. BR1-responsive tumor cells express inflammatory programs that polarize microglia into reactive states, eliciting robust T cell recruitment. In contrast, BR3-resistant melanoma cells are enriched in neurological programs and exploit tolerance mechanisms to maintain microglia homeostasis and limit T cell infiltration. In humans, BR1 and BR3 expression signatures correlate positively or negatively with T cell infiltration and BrM patient outcomes, respectively. Our study provides clinically relevant models and uncovers mechanistic insights into BrM ICB responses, offering potential biomarkers and therapeutic targets to improve therapy efficacy.

immunology↗

bioRxiv2024

MED26-enriched condensates drive erythropoiesis through modulating transcription pausing

The Mediator complex regulates various aspects of hematopoietic development, but whether composition of the Mediator complex undergoes dynamic changes for diversifying transcription and functional outputs is unknown. Here, we found that MED26, a subunit in the core Mediator complex, played a distinctive role in facilitating transcription pausing essential for erythroid development. While most Mediator subunits drastically decreased during this process, MED26 remained relatively abundant. Intriguingly, in the early stages, more than half of MED26 occupancy sites did not co-localize with MED1, a representative Mediator subunit, suggesting these subunits exert context-dependent gene regulation. We revealed that MED26-enriched loci were associated with RNA polymerase pausing. MED26 manifested a markedly preferential recruitment of pausing-related factors, leading to an increase in Pol pausing critical for genome-wide transcription repression during erythropoiesis. Moreover, MED26 exhibited pronounced condensate-forming capability, which was necessary for its function in promoting erythropoiesis and recruiting pausing-related factors. Collectively, this study provides mechanistic insights into the functional coordination of distinct Mediator subunits during development and highlights the switch of transcription condensates towards a MED26 enriched form, which modulates transcription pausing to facilitate transcription repression and erythroid development.

cell biology↗

bioRxiv2024

Tim-3 promotes viral infection by suppressing USP25-TRAF3-IRF7 signaling pathway

Tim-3, an immune checkpoint inhibitor, plays key roles in maintaining immune homeostasis and is involved in viral evasion. However, the precise role of Tim-3 in viral infection remains to be determined. USP25 is one of the deubiquitinating enzymes that serves to initiate antiviral immunity by deubiquitinating TRAF3 and triggering the anti-viral signaling pathway. Here we found that Tim-3 specific knockout in myeloid cells leads to enhanced anti-viral immunity in mice with VSV encephalitis by increasing type I interferon response. Mechanistically, Tim-3 inhibits the expression of USP25 via STAT1, interacts with USP25 but does not regulate its post-translational modification, and as a result, Tim-3 inhibits USP25 mediated deubiquitination of TRAF3, promotes k48-linked ubiquitination and degradation of TRAF3, and then inhibits the phosphorylation of IRF7, finally down-regulates the interferon response. These findings provide new insights into the function of Tim-3 in antiviral immunity and its related clinical significance.

immunology↗

bioRxiv2024

Optimized semi-specific PCR amplification using arbitrarily degenerate primer for genome-wide genotyping and its application in peanut genetic diversity study

Cultivated peanut (Arachis hypogaea L.) is an important legume crop with a large genome size but a limited genetic diversity. Evaluating the genetic variation among diverse peanut germplasms using genome-wide molecular markers is an effective strategy to explore its genetic diversity and thereby facilitate peanut improvement. In this study, we introduced a novel whole-genome genotyping (WGG) technique named dRAPD-seq (degenerate Random Amplification Polymorphic DNA and sequencing), which relies on semi-specific PCR amplification by arbitrarily degenerate (AD) primer and next-generation sequencing (NGS), and demonstrated its robust reproducibility and high accuracy. Subsequently, we applied dRAPD-seq to investigate the genetic relatedness within a population of 101 diverse peanut accessions and identified a low genetic diversity among these accessions. Our phylogenetic tree, population structure analyses, and principal component analysis (PCA) indicated that this population could be clustered into three subpopulations, largely corresponding to three botanical types. In conclusion, this research not only introduced a cost-effective and easy-to-conduct WGG method but also provided valuable insights for utilizing these peanut accessions in future breeding programs.

genomics↗

bioRxiv2024

Cyclin A/B RxL Macrocyclic Inhibitors to Treat Cancers with High E2F Activity

Cancer cell proliferation requires precise control of E2F1 activity; excess activity promotes apoptosis. Here, we developed cell-permeable and bioavailable macrocycles that selectively kill small cell lung cancer (SCLC) cells with inherent high E2F1 activity by blocking RxL-mediated interactions of cyclin A and cyclin B with select substrates. Genome-wide CRISPR/Cas9 knockout and random mutagenesis screens found that cyclin A/B RxL macrocyclic inhibitors (cyclin A/Bi) induced apoptosis paradoxically by cyclin B- and Cdk2-dependent spindle assembly checkpoint activation (SAC). Mechanistically, cyclin A/Bi hyperactivate E2F1 and cyclin B by blocking their RxL-interactions with cyclin A and Myt1, respectively, ultimately leading to SAC activation and mitotic cell death. Base editor screens identified cyclin B variants that confer cyclin A/Bi resistance including several variants that disrupted cyclin B:Cdk interactions. Unexpectedly but consistent with our base editor and knockout screens, cyclin A/Bi induced the formation of neo-morphic Cdk2-cyclin B complexes that promote SAC activation and apoptosis. Finally, orally-bioavailable cyclin A/Bi robustly inhibited tumor growth in chemotherapy-resistant patient-derived xenograft models of SCLC. This work uncovers gain-of-function mechanisms by which cyclin A/Bi induce apoptosis in cancers with high E2F activity, and suggests cyclin A/Bi as a therapeutic strategy for SCLC and other cancers driven by high E2F activity.

cancer biology↗

bioRxiv2024

Intraperitoneal programming of tailored CAR macrophages via mRNA-LNP to boost cancer immunotherapy

Therapeutic strategies for peritoneal metastasis in solid tumors are urgently needed in the clinic. Programming chimeric antigen receptor macrophages (CAR-Ms) in situ offers opportunities for an unmet demand. However, potential intracellular domains (ICDs) for CAR design and their antitumor mechanisms for macrophage empowerment remain to be explored systematically. By developing a targeted mRNA-LNP delivery system for macrophages, we have investigated 36 CAR combinations to determine the impact of CAR-Ms on immune regulation in vitro and in vivo. In two solid tumor mouse models, intraperitoneal programming of CAR-Ms was shown to elicit robust adaptive immune activation and significantly synergize with PD-1/L1 therapy. Single-cell RNA sequencing (scRNA-seq) analysis revealed that CAR-Ms could reshape the immunosuppressive tumor microenvironment (TME) and boost the TCF1+PD-1+ progenitor- exhausted CD8+ T cells (Tpex) population. Meanwhile, we found that tailored CAR-M with CD3{zeta}/TLR4 ICDs could favorably maintain proinflammatory phenotype and simultaneously upregulate MHC I and PD-L1 expression by perturbing NF-{kappa}B pathways. Moreover, the synergism between macrophage PD-L1 knockdown and CAR-M therapy highlighted the need to block the PD-1/L1 axis in antigen cross-presentation. In short, we developed an mRNA-LNP delivery system for intraperitoneal programming of tailored CAR-Ms in vivo and broadened understanding of both regulatory and feedback mechanisms for CAR-M therapies against solid tumors.

bioengineering↗

bioRxiv2024

The representational nature of spatio-temporal recurrent processing in visual object recognition

The human brain orchestrates object vision through an interplay of feedforward processing in concert with recurrent processing. However, where, when and how recurrent processing contributes to visual processing is incompletely understood due to the difficulties in teasing apart feedforward and recurrent processing. We combined a backward masking paradigm with multivariate analysis on EEG and fMRI data to isolate and characterize the nature of recurrent processing. We find that recurrent processing substantially shapes visual representations across the ventral visual stream, starting early on at around 100ms in early visual cortex (EVC) and in two later phases of around 175 and 300ms in lateral occipital cortex (LOC), adding persistent rather than transient neural dynamics to visual processing. Using deep neural network models for comparison with the brain, we show that recurrence changes the feature format in LOC from predominantly mid-level to more high-level features. Finally, we show that recurrence is mediated by four distinct spectro-temporal neural components in EVC and LOC, which span the theta to beta frequency range. Together, our results reveal the nature and mechanisms of the effects of recurrent processing on the visual representations in the human brain.

neuroscience↗

bioRxiv2024

VezA/vezatin facilitates proper assembly of the dynactin complex in vivo

Cytoplasmic dynein-mediated intracellular transport needs the multi-component dynactin complex for cargo binding and motor activation. However, cellular factors involved in dynactin assembly remain unexplored. Here we found in Aspergillus nidulans that the vezatin homolog VezA is important for dynactin assembly. VezA affects the microtubule plus-end accumulation of dynein before cargo binding and cargo adapter-mediated dynein activation, two processes that both need dynactin. The dynactin complex contains multiple components including an Arp1 (actin-related protein 1) mini-filament associated with a pointed-end sub-complex. VezA physically interacts with dynactin either directly or indirectly via the Arp1 mini-filament and its pointed-end sub-complex. Loss of VezA causes a defect in dynactin integrity, most likely by affecting the connection between the Arp1 mini-filament and its pointed-end sub-complex. Using various dynactin mutants, we further revealed that assembly of the dynactin complex must be highly coordinated. Together, these results shed important new light on dynactin assembly in vivo.

cell biology↗

bioRxiv2024

Systematic perturbation screens decode regulators of inflammatory macrophage states and identify a role for TNF mRNA m6A modification

Macrophages adopt dynamic cell states with distinct effector functions to maintain tissue homeostasis and respond to environmental challenges. During chronic inflammation, macrophage polarization is subverted towards sustained inflammatory states which contribute to disease, but there is limited understanding of the regulatory mechanisms underlying these disease-associated states. Here, we describe a systematic functional genomics approach that combines genome-wide phenotypic screening in primary murine macrophages with transcriptional and cytokine profiling of genetic perturbations in primary human monocyte-derived macrophages (hMDMs) to uncover regulatory circuits of inflammatory macrophage states. This process identifies regulators of five distinct inflammatory states associated with key features of macrophage function. Among these, the mRNA m6A writer components emerge as novel inhibitors of a TNF-driven cell state associated with multiple inflammatory pathologies. Loss of m6A writer components in hMDMs enhances TNF transcript stability, thereby elevating macrophage TNF production. A PheWAS on SNPs predicted to impact m6A installation on TNF revealed an association with cystic kidney disease, implicating an m6A-mediated regulatory mechanism in human disease. Thus, systematic phenotypic characterization of primary human macrophages describes the regulatory circuits underlying distinct inflammatory states, revealing post-transcriptional control of TNF mRNA stability as an immunosuppressive mechanism in innate immunity.

immunology↗

bioRxiv2024

The G1/S transition in mammalian stem cells in vivo is autonomously regulated by cell size

Cell growth and division must be coordinated to maintain a stable cell size, but how this coordination is implemented in multicellular tissues remains unclear. In unicellular eukaryotes, autonomous cell size control mechanisms couple cell growth and division with little extracellular input. However, in multicellular tissues we do not know if autonomous cell size control mechanisms operate the same way or whether cell growth and cell cycle progression are separately controlled by cell-extrinsic signals. Here, we address this question by tracking single epidermal stem cells growing in adult mice. We find that a cell-autonomous size control mechanism, dependent on the RB pathway, sets the timing of S phase entry based on the cells current size. Cell-extrinsic variations in the cellular microenvironment affect cell growth rates but not this autonomous coupling. Our work reassesses long-standing models of cell cycle regulation within complex metazoan tissues and identifies cell-autonomous size control as a critical mechanism regulating cell divisions in vivo and thereby a major contributor to stem cell heterogeneity.

cell biology↗

bioRxiv2024

GGCX promotes Eurasian avian-like H1N1 swine influenza virus adaption to interspecies receptor binding

The Eurasian avian-like (EA) H1N1 swine influenza virus (SIV) possesses the capacity to instigate the next influenza pandemic, owing to its heightened affinity for the human-type -2,6 sialic acid (SA) receptor. Nevertheless, the molecular mechanisms underlying the switch in receptor binding preferences of EA H1N1 SIV remain elusive. In this study, we conducted a comprehensive genome-wide CRISPR/Cas9 knockout screen utilizing EA H1N1 SIV in porcine kidney cells. Knocking out the enzyme gamma glutamyl carboxylase (GGCX) reduced virus replication in vitro and in vivo by inhibiting the carboxylation modification of viral haemagglutinin (HA) and the adhesion of progeny viruses, ultimately impeding the replication of EA H1N1 SIV. Furthermore, GGCX was revealed to be the determinant of the D225E substitution of EA H1N1 SIV, and GGCX-medicated carboxylation modification of HA 225E contributed to the receptor binding adaption of EA H1N1 SIV to the -2,6 SA receptor. Taken together, our CRISPR screen has elucidated a novel function of GGCX in the support of EA H1N1 SIV adaption for binding to -2,6 SA receptor. Consequently, GGCX emerges as a prospective antiviral target against the infection and transmission of EA H1H1 SIV.

microbiology↗

bioRxiv2024

A Functional Map of the Human Intrinsically Disordered Proteome

Intrinsically disordered regions (IDRs) represent at least one-third of the human proteome and defy the established structure-function paradigm. Because IDRs often have limited positional sequence conservation, the functional classification of IDRs using standard bioinformatics is generally not possible. Here, we show that evolutionarily conserved molecular features of the intrinsically disordered human proteome (IDR-ome), termed evolutionary signatures, enable classification and prediction of IDR functions. Hierarchical clustering of the human IDR-ome based on evolutionary signatures reveals strong enrichments for frequently studied functions of IDRs in transcription and RNA processing, as well as diverse, rarely studied functions, ranging from sub-cellular localization and biomolecular condensates to cellular signaling, transmembrane transport, and the constitution of the cytoskeleton. We exploit the information that is encoded within evolutionary conservation of molecular features to propose functional annotations for every IDR in the human proteome, inspect the conserved molecular features that correlate with different functions, and discover frequently co-occurring IDR functions on the proteome scale. Further, we identify patterns of evolutionary conserved molecular features of IDRs within proteins of unknown function and disease-risk genes for conditions such as cancer and developmental disorders. Our map of the human IDR-ome should be a valuable resource that aids in the discovery of new IDR biology.

bioinformatics↗

bioRxiv2024

Clearance of protein aggregates during cell division

Protein aggregates are spatially organized and regulated in cells to prevent deleterious effects of proteostatic stress. Misfolding of proteins in the endoplasmic reticulum (ER) results in aggregate formation, but how the aggregates are processed especially during cell division is not well understood. Here, we induced proteostatic stress and protein aggregation using a proteostasis reporter, which is prone to misfolding and aggregation in the ER. Unexpectedly, we detected solid- like protein aggregates deposited mainly in the nucleus and surrounded by the ER membrane. The membrane-bound aggregates were then cleared as cells progressed through mitosis and cytokinesis. Aggregate clearance depended on Hsp70 family chaperones in the ER, particularly BiP, and proteasomal activity. The clearance culminated at mitotic exit and required cyclin-dependent kinase 1 (Cdk1) inactivation but was independent of the anaphase-promoting complex (APC/C). The ER reorganization that is active during mitosis and cytokinesis was required for the aggregate clearance. Thus, dividing cells reorganize the ER networks to allow BiP to clear the protein aggregates to maintain proteostasis in the newly divided cells.

cell biology↗