Biology subjects

Xiang, K.

Publications and source records attributed to Xiang, K..

18 recordsLinked to original sources

bioRxiv2026

Structure-guided discovery and engineering of miniature CRISPR-Cas12m for epigenome editing

CRISPR-based epigenome editing represents a programmable strategy to precisely modulate gene expression, holding immense promise for therapeutic applications. However, the large size of the dCas proteins substantially impedes the delivery via adeno-associated virus (AAV) vectors. Here, through iterative bioinformatics analysis, structure-guided predictions, and functional assays, we identified and characterized PmCas12m, a novel miniature subtype V-M CRISPR-Cas12m. PmCas12m exhibited flexible 5-YTN-3 PAM-dependent recognition and robust double-stranded DNA binding properties, while lacking DNA cleavage activity, thus positioning it as an ideal tool for epigenome editing. Cryogenic electron microscopy (cryo-EM) structures of PmCas12m unveiled its unique molecular mechanism of DNA binding facilitating interference. Guided by these structural insights, we employed deep mutational scanning (DMS) and protein engineering to develop xCas12m, a hypercompact variant with highly potent and specific epigenome editing capabilities in human cells. We further constructed the xCas12m-CRISPRoff platform in a single AAV vector, which achieved durable epigenetic silencing and effective inhibition of hepatitis B virus (HBV) infection in a mouse model. Collectively, these findings establish xCas12m as a versatile epigenome editing platform with transformative potential for treating diseases, paving the way for clinical translation of epigenetic therapies.

bioengineering↗

bioRxiv2025

5' untranslated regions tune Toxoplasma translation

Some of the longest 5' untranslated regions (UTRs) documented in eukaryotes belong to parasites of the phylum Apicomplexa. Translational regulation plays prominent roles in the development of these parasites, including the agents of toxoplasmosis (Toxoplasma gondii) and malaria. To understand the function of 5' UTRs in apicomplexan translation, we performed high-resolution ribosome profiling of T. gondii in human fibroblasts. We show that parasite translation efficiency (TE) is largely controlled by 5' UTR features and tuned by the number of upstream AUGs (uAUGs). Examination of ribosome occupancy reveals that, despite widespread assembly of parasite monosomes on uAUGs, ribosomes seldom translate uORFs. These determinants of translation are reaffirmed in a massively parallel reporter assay examining the effect of more than 30,000 synthetic 5' UTRs in T. gondii. A model trained on these results accurately predicted the TE of newly designed 5' UTRs. Together, this work defines the regulatory language of T. gondii translation, providing a framework to understand the evolution of exceptionally long 5' UTRs in apicomplexans.

molecular biology↗

bioRxiv2025

Selenoprotein P Deficiency Drives Hepatocellular Carcinoma Progression via Induction of Neutrophil Senescence and Immunosuppressive Microenvironment

The tumor immune microenvironment (TIME) is critically shaped by immunosuppressive neutrophils, yet the molecular mechanisms underlying the functional reprogramming of these neutrophils remain poorly understood. Here, through integrative analysis of single-cell RNA sequencing (scRNA-seq) we showed the presence, in hepatocellular carcinoma (HCC) mice, of a novel subpopulation of senescent-like neutrophils that exhibit hepatic depletion of Sepp1 in concert with the accumulation of the cellular senescence marker Cdkn1a, the immunosuppressive proteins S100a8 and 9, and the pro-angiogenic factor Vegfa. Furthermore, we demonstrated that the hepatic depletion of Sepp1, in combination with this unique subset of senescent neutrophils, drives immunosuppressive activity and further accelerates tumor growth. Mechanistically, the loss of tumor cell-derived Sepp1 impaired selenium uptake in tumor-infiltrating neutrophils (TINs) by disrupting Lrp8 receptor-mediated transport, thereby suppressing intracellular selenium metabolism. This metabolic perturbation led to reduced hydrogen selenide (H2Se) production and accumulation of S-adenosylmethionine (SAM), resulting in increased levels of trimethylation of lysine 4 on histone H3 protein (H3K4me3) modification in neutrophils and collectively establishing a pro-senescence chromatin landscape in tumor-associated neutrophils. Furthermore, we demonstrated that selenium supplementation not only restores Sepp1 expression but also is essential to reshaping anti-tumor immunity in HCC mice, including reversal of the senescent-like phenotype and providing synergistic effects with anti-Programmed cell death 1 (anti-PD-1) therapy. Collectively, our findings identify SEPP1 as a master regulator acting against neutrophil senescence and the potent immunosuppressive effects of senescence in HCC. We discuss the therapeutic potential of strategies to modulate senescent-like neutrophils and underscore the potential utility of selenium supplementation as an adjuvant to enhance immunotherapy efficacy in liver cancer.

cancer biology↗

bioRxiv2025

Amyloid-β precursor protein promotes tumor growth by establishing an immune-exclusive tumor microenvironment

During initiation and progression, cancerous tissue hijacks a series of elaborate tissue homeostatic mechanisms to avoid immune surveillance, including neuro-immune interactions. Here, we show that amyloid-{beta} precursor protein (APP) and its {beta}-cleavage product amyloid-{beta}1-42 (A{beta}1-42), well-known in the pathogenesis of Alzheimers disease (AD), are expressed in multiple cancer tissues. However, the oncogenic activity of APP is due to its E1 domain, instead of A{beta}1-42. Mechanistically, APP restricts immune cells influx into tumor microenvironment (TME) and impairs CD8+ T cell and NK cell-based immunity, by dampening type I interferon (IFN) response in TME. We also provide proof-of-concept that vaccination targeting APP is effective for cancer prevention. Our current study reveals a previously unrecognized role of APP in cancer immune surveillance, and provides a new strategy for cancer prevention and treatment by targeting APP.

cancer biology↗

bioRxiv2025

Neoadjuvant anti-4-1BB confers protection against spontaneous metastasis through low-affinity intratumor CD8+ T cells in triple-negative breast cancer

Neoadjuvant immunotherapy seeks to harness the primary tumor as a source of relevant tumor antigens to enhance systemic anti-tumor immunity through improved immunological surveillance. Despite having revolutionized the treatment of patients with high-risk early-stage triple-negative breast cancer (TNBC), a significant portion of patients remain unresponsive and succumb to metastatic recurrence post-treatment. Here, we found that optimally scheduled neoadjuvant administration of anti-4-1BB monotherapy was able to counteract metastases and prolong survival following surgical resection. Phenotypic and transcriptional profiling revealed enhanced 4-1BB expression on tumor-infiltrating intermediate (Tint), relative to progenitor (Tprog) and terminally exhausted (Tterm) T cells. Furthermore, Tint was enriched in low-affinity T cells. Treatment with anti-4-1BB drove clonal expansion of Tint, with reduced expression of tissue-retention marker CD103 in Tprog. This was accompanied by increased TCR clonotype sharing between paired tumors and pre-metastatic lungs. Further interrogation of sorted intratumor T cells confirmed enhanced T cell egress into circulation following anti-4-1BB treatment. In addition, gene signature extracted from anti-4-1BB treated Tint was consistently associated with improved clinical outcomes in BRCA patients. Combinatorial neoadjuvant anti-4-1BB and ablation of tumor-derived CXCL16 resulted in enhanced therapeutic effect. These findings illustrate the intratumor changes underpinning the efficacy of neoadjuvant anti-4-1BB, highlighting the reciprocity between local tissue-retention and distant immunologic fortification, suggesting treatment can reverse the siphoning of intratumor T cells to primary tumor, enabling redistribution to distant tissues and subsequent protection against metastases.

immunology↗

bioRxiv2024

Increased versatility and convenience: advances and strategy optimization of ReMOT-mediated genetic modification in insects

Genetic modification via gene editing has become a widely adopted and demonstrably effective method in functional gene research within entomology. However, the optimal efficiency and simplicity of delivering exogenous guide RNA-clustered regularly interspaced short palindromic repeats-associated protein 9 (gRNA-Cas9) complexes into target tissues are crucial for successful gene editing. The Receptor-Mediated Ovary Transduction of Cargo (ReMOT) strategy, which simplifies the delivery process, target site selection, technical requirements, and delivery cost compared with embryonic microinjection, enabling efficient editing at the germline level, is gaining increasing attention. Although the feasibility and advantages of this technique have been demonstrated in various insect species, further optimization of operational details and the overcoming of further bottlenecks are still required. This review focuses on advances in developing ReMOT as a valuable technology, exploring its applicability, rationale for selecting the ovary as a delivery target site, factors influencing its efficiency, and recommendations for improvement. The versatility and effectiveness of ReMOT make it a promising method for researchers looking to make precise genetic modifications with greater ease and efficiency.

genetics↗

bioRxiv2024

Cis-Regulatory Evolution of CCNB1IP1 Driving Gradual Increase of Cortical Size and Folding in primates

Neocortex expansion has a concerted relationship with folding, underlying evolution of human cognitive functions. However, molecular mechanisms underlying this significant evolutionary process remains unknown. Here, using tree shrew as an outgroup of primates, we identify a new regulator CCNB1IP1, which acquired its expression before the emergence of primates. Following the evolution of cis-regulatory elements, the CCNB1IP1 expression has steadily increased over the course of primate brain evolution, mirroring the gradual increase of neocortex. Mechanistically, we elucidated that CCNB1IP1 expression can cause an increase in neural progenitors through shortening G1 phase. Consistently, the CCNB1IP1 knock-in mouse model exhibited traits associated with enhanced learning and memory abilities. Together, our study reveals how changes in CCNB1IP1 expression may have contributed to the gradual evolution in primate brain.

evolutionary biology↗

bioRxiv2024

DNA replication fork speed Acts as an Engine in Cortical Neurogenesis

DNA replication fork speed, which controls the rate of genome duplication, has emerged as a key regulator of cellular plasticity. However, its role in neurogenesis remains unexplored. Mini-chromosome maintenance complex (MCMs)-binding protein (MCMBP) functions as a chaperone for newly synthesized MCMs, increasing chromatin coverage to restrain fork speed. We demonstrate that selective deletion of MCMBP in neural progenitor radial glial cells (RGCs) accelerates fork speed, triggering widespread apoptosis, DNA damage, and micronuclei formation, ultimately activating p53 and causing microcephaly. Unexpectedly, concurrent deletion of Trp53 and Mcmbp further increased fork speed, leading to extensive RGC detachment from the ventricular zone and acquisition of outer-RGC characteristics. Mechanistically, we found that the MCM3 subunit coordinates DNA and centrosome duplication, thereby mediating RGC attachment. Behavioral analysis revealed that disruption of fork speed results in anxiety-like behavior in mice. These findings unveil a previously unrecognized role for replication fork speed in neurogenesis.

neuroscience↗

bioRxiv2024

PAL-AI reveals genetic determinants that control poly(A)-tail length during oocyte maturation with relevance to human fertility

In oocytes of mammals and other animals, gene regulation is mediated primarily through changes in poly(A)-tail length1-9. Here, we introduce PAL-AI, an integrated neural network machine-learning model that accurately predicts tail-length changes in maturing oocytes of frogs and mammals. We show that PAL-AI learned known and previously unknown sequence elements and their contextual features that control poly(A)-tail length, enabling it to predict tail-length changes resulting from 3'-untranslated region (3'-UTR) single-nucleotide substitutions. It also predicted tail-length-mediated translational changes, allowing us to nominate genes important for oocyte maturation. When comparing predicted tail-length changes in human oocytes with genomic datasets of the All of Us Research Program10 and gnomAD11, we found that genetic variants predicted to disrupt tail lengthening have been under negative selection in the human population, thereby linking mRNA tail lengthening to human female fertility.

bioinformatics↗

bioRxiv2024

Nuclear release of eIF1 globally increases stringency of start-codon selection to preserve mitotic arrest physiology

Regulated start-codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms. However, conditions under which start-codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling, we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This increased stringency of start-codon selection during mitosis results from increased interactions between the key regulator of start-codon selection, eIF1, and the 40S ribosome. We find that increased eIF1-40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in altered mitotic proteome composition. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage following treatment with anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.

molecular biology↗

bioRxiv2023

Control of poly(A)-tail length and translation in vertebrate oocytes and early embryos

During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3'-UTR sequence variants) in frog oocytes and embryos, and fish embryos. We found that the UUUUA element, together with the polyadenylation signal (AAUAAA or AUUAAA), specifies cytoplasmic polyadenylation, and identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans. HighlightsO_LIUUUUA, modulated by contextual features, specifies most cytoplasmic polyadenylation C_LIO_LIStage-specific sequence motifs drive waves of tail-length shortening in embryos C_LIO_LIUUUUA and C-rich motifs can direct tail-length-independent translational repression C_LIO_LITail-length control is conserved in oocytes of frogs, mice, and humans C_LI

molecular biology↗

bioRxiv2023

High-efficiency gene editing in Anopheles sinensis using ReMOT control

CRISPR/Cas9-mediated gene editing provides an effective method for deciphering the molecular mechanisms underlying mosquito development and mosquito-borne disease transmission, as well as for exploring genetic control strategies. However, delivering the Cas9 ribonucleoprotein complex by embryo injection to produce genetic modifications is challenging, is mostly confined to model mosquitoes and specialized laboratories, and has low editing efficiency. Here, we established an effective Receptor-Mediated Ovary Transduction of Cargo (ReMOT) control method, enabling the introduction of heritable mutations into Anopheles sinensis, the major malaria vector in China and Southeast Asia, via the injection of female adult mosquitoes. Injection of a mixture of P2C-DsRed and saponin resulted in red fluorescence in the ovaries, with a 100% success rate. Using this system, we knocked-out the pigment synthesis genes, Aswhite and Asyellow, using injected wild-type (WT) females mated with WT males, resulting in the highest efficiency of gene editing among mosquitoes under the same mating conditions. Furthermore, the gene-editing efficiency was increased by at least 2.1-fold using injected WT females mated with mutant males. This improved ReMOT control method exhibits high editing efficiency, with important benefits in terms of functional genomics research and genetic control strategies in An. sinensis. Moreover, this represents a convenient method for gene manipulation in laboratories that are unable to perform embryo injection or that lack embryo-injection equipment.

genetics↗

bioRxiv2022

Chromatin Remodeling in Patient-Derived Colorectal Cancer Models

Patient-Derived Organoids (PDO) and Xenografts (PDX) are the current gold standards for patient derived models of cancer (PDMC). Nevertheless, how patient tumor cells evolve in these models and the impact on drug response remains unclear. Herein, we compared the transcriptomic and chromatin accessibility landscapes of six matched sets of colorectal cancer (CRC) PDO, PDX, PDO-derived PDX (PDOX), and original patient tumors (PT) and discovered two major remodeling axes. The first axis delineates PDX and PDO from PT, and the second axis distinguishes PDX and PDO. PDOX were more similar to PDX than they were to PDO, indicating that the growth environment is a driving force for chromatin adaptation. Using bivariate genomic footprinting analysis, we identified transcription factors (TF) that differentially bind to open chromatins between matched PDO and PDOX. Among them, KLF14 and EGR2 footprints were enriched in all six PDOX relative to matched PDO, and silencing of KLF14 or EGR2 promoted tumor growth. Furthermore, EPHA4, a shared downstream target gene of KLF14 and EGR2, altered tumor sensitivity to MEK inhibitor treatment. Altogether, patient-derived CRC cells undergo both common and distinct chromatin remodeling in PDO and PDX/PDOX, driven largely by their respective microenvironments, which results in differences in growth and drug sensitivity and needs to be taken into consideration when interpreting their ability to predict clinical outcome.

cancer biology↗

bioRxiv2021

Inhibition of SAR S-CoV-2 infection and replication by lactoferrin, MUC1 and α-lactalbumin identified in human breastmilk

The global pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection confers great threat to the public health. Human breastmilk is an extremely complex with nutritional composition to nourish infants and protect them from different kinds of infection diseases and also SARS-CoV-2 infection. Previous studies have found that breastmilk exhibited potent antiviral activity against SARS-CoV-2 infection. However, it is still unknown which component(s) in the breastmilk is responsible for its antiviral activity. Here, we identified Lactoferrin (LF), MUC1 and -Lactalbumin (-LA) from human breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and in vitro confirmation that inhibited SARS-CoV-2 infection and analyzed their antiviral activity using the SARS-CoV-2 pseudovirus system and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP) in the Huh7.5, Vero E6 and Caco-2-N cell lines. Additionally, we found that LF and MUC1 could inhibit viral attachment, entry and post-entry replication, while -LA just inhibit viral attachment and entry. Importantly, LF, MUC1 and -LA possess potent antiviral activities towards not only wild-type but also variants such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.1 (kappa). Moreover, LF from other species (e.g., bovine and goat) is still capable of blocking viral attachment to cellular heparan sulfate. Taken together, our study provided the first line of evidence that human breastmilk components (LF, MUC1 and -LA) are promising therapeutic candidates warranting further development or treatingVID-19 given their exceedingly safety levels.

microbiology↗

bioRxiv2021

The molecular basis of coupling between poly(A)-tail length and translational efficiency

In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in frog oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic cells had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, in which this protein promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.

molecular biology↗

bioRxiv2020

SARS-CoV-2 and Malayan pangolin coronavirus infect human endoderm, ectoderm and induced lung progenitor cells

Since the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in several somatic cells, little is known about the infection of SASRS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that SARS-CoV-2 pseudovirus and GX_P2V could infect lung progenitor and even anterior foregut endoderm cells causing these cells death, which differentiated from human embryonic stem cells (hESCs). The infection and replication of SARS-CoV-2 and GX_P2V were inhibited when treated with whey protein of breastmilk and Remdesivir, confirming that these two viruses could infect lung progenitor and even anterior foregut endoderm. Moreover, we found that SARS-CoV-2 pseudovirus could infect endoderm and ectoderm. We found that whey protein blocked SARS-CoV-2 infecting these cells. In line with the SARS-CoV-2 results, GX_P2V could also infected endoderm and ectoderm, and also was inhibited by Remdesivir treatment. Although expressing coronavirus related receptor such as ACE2 and TMPRSS2, mesoderm cells are not permissive for SARS-CoV-2 and GX_P2V infection, which needed further to study the mechanisms. Interestingly, we also found that hESCs, which also express ACE2 and TMPRSS2 markers, are permissive for GX_P2V but not SARS-CoV-2 pseudovirus infection and replication, indicating the widespread cell types for GX_P2V infection. Heparin treatment blocked efficiently viral infection. These results provided insight that these stem cells maybe provided a stable repository of coronavirus function or genome. The potential consequence of SARS-CoV-2 and animal coronavirus such as GX_P2V infection in hESCs, germ layer and induced progenitors should be closely monitored.

microbiology↗

bioRxiv2020

The effect of whey protein on viral infection and replication of SARS-CoV-2 and pangolin coronavirus in vitro

Since the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human breastmilk, little is known about the antiviral property of human breastmilk to SARS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that whey protein from human breastmilk effectively inhibited both SARS-CoV-2 and GX_P2V by blocking viral attachment, entry and even post-entry viral replication. Moreover, human whey protein inhibited infectious virus production proved by the plaque assay. We found that whey protein from different species such as cow and goat also showed anti-coronavirus properties. And commercial bovine milk also showed similar activity. Interestingly, the main antimicrobial components of breastmilk, such as Lactoferrin and IgA antibody, showed limited anti-coronavirus activity, indicating that other factors of breastmilk may play the important anti-coronavirus role. Taken together, we reported that whey protein inhibits SARS-CoV-2 and its related virus of GX_P2V. These results rule out whey protein as a direct-acting inhibitor of SARS-CoV-2 and GX_P2V infection and replication and further investigation of its molecular mechanism of action in the context of COVID-19.

microbiology↗

bioRxiv2019

In-Depth Characterization and Validation in BRG1-Mutant Lung Cancers Define Novel Catalytic Inhibitors of SWI/SNF Chromatin Remodeling

Members of the ATP-dependent SWI/SNF chromatin remodeling complexes are among the most frequently mutated genes in cancer, suggesting their dysregulation plays a critical role. The synthetic lethality between SWI/SNF catalytic subunits BRM/SMARCA2 and BRG1/SMARCA4 has instigated great interest in targeting BRM. Here we have performed a critical and in-depth investigation of novel dual inhibitors (BRM011 and BRM014) of BRM and BRG1 in order to validate their utility as chemical probes of SWI/SNF catalytic function, while obtaining insights into the therapeutic potential of SWI/SNF inhibition. In corroboration of on-target activity, we discovered compound resistant mutations through pooled screening of BRM variants in BRG1-mut cancer cells. Strikingly, genome-wide transcriptional and chromatin profiling (ATAC-Seq) provided further evidence of pharmacological perturbation of SWI/SNF chromatin remodeling as BRM011 treatment induced specific changes in chromatin accessibility and gene expression similar to genetic depletion of BRM. Finally, these compounds have the capacity to inhibit the growth of tumor-xenografts, yielding important insights into the feasibility of developing BRM/BRG1 ATPase inhibitors for the treatment of BRG1-mut lung cancers. Overall, our studies not only establish the feasibility of inhibiting SWI/SNF catalytic function, providing a framework for SWI/SNF therapeutic targeting, but have also yielded successful elucidation of small-molecule inhibitors that will be of importance in probing SWI/SNF function in various disease contexts.

cancer biology↗