Biology subjects

Han, J.

Publications and source records attributed to Han, J..

At least 163 records · Page 9Linked to original sources

bioRxiv2024

SKP2-mediated FBXO2 proteasomal degradation drives hepatocellular carcinoma progression via stabilizing Hsp47

Accumulating studies highlight that dysregulated E3 ubiquitin ligases are associated with the onset and advancement of cancers. Nevertheless, the impact and mechanism of most E3 ubiquitin ligases on tumorigenesis and tumor metastasis remain poorly understood. Here, we show that loss of FBXO2, an E3 ubiquitin ligase, accelerates hepatocellular carcinoma (HCC) tumor growth and metastasis to the lung through stabilizing heat shock protein 47 (Hsp47). Downregulation of FBXO2, caused by DNA-PKcs-medicated phosphorylation at serine 17 and E3 ligase SKP2-mediated ubiquitination at lysine 79 and subsequent proteasomal degradation, is observed in tumor tissues compared to their parallel non-tumor tissues resected from patients with HCC. Patients whose tumors are enriched for SKP2 or Hsp47 or express low levels of FBXO2 have poor median survival compared to those whose tumors have reversed levels of SKP2, FBXO2 and Hsp47. Together, FBXO2 acts as a tumor suppressor in HCC development. The components of the SKP2-FBXO2-Hsp47 axis provide newly prognostic and therapeutic factors for anti-HCC.

cancer biology↗

bioRxiv2024

A tale of two fusion proteins: understanding the metastability of human respiratory syncytial virus and metapneumovirus and implications for rational design of uncleaved prefusion-closed trimers

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause human respiratory diseases and are major targets for vaccine development. In this study, we designed uncleaved prefusion-closed (UFC) trimers for the fusion (F) proteins of both viruses by examining mutations critical to F metastability. For RSV, we assessed four previous prefusion F designs, including the first and second generations of DS-Cav1, SC-TM, and 847A. We then identified key mutations that can maintain prefusion F in a native-like, closed trimeric form (up to 76%) without introducing any interprotomer disulfide bond. For hMPV, we developed a stable UFC trimer with a truncated F2-F1 linkage and an interprotomer disulfide bond. Tens of UFC constructs were characterized by negative-stain electron microscopy (nsEM), x-ray crystallography (11 RSV-F and one hMPV-F structures), and antigenic profiling. Using an optimized RSV-F UFC trimer as bait, we identified three potent RSV neutralizing antibodies (NAbs) from a phage-displayed human antibody library, with a public NAb lineage targeting sites O and V and two cross-pneumovirus NAbs recognizing site III. In mouse immunization, rationally designed RSV-F and hMPV-F UFC trimers induced robust antibody responses with high neutralizing titers. Our study provides a foundation for future prefusion F-based RSV and hMPV vaccine development. ONE-SENTENCE SUMMARYThe metastability analysis of fusion proteins has informed rational design of uncleaved prefusion-closed trimers for RSV and hMPV vaccine development.

microbiology↗

bioRxiv2024

Salidroside and its in vivo metabolite tyrosol could act directly on dopamine D2 receptors: a study using RNAseq combined with Connectivity Map analysis

Salidroside is an active ingredient in traditional Chinese medicine such as Rhodiola. Its in vivo metabolite, tyrosol, exist in olive oil and red wine. For a long time, clinical practice and research have shown that both of them have many pharmacological activities, but their targets have not reached unanimous conclusion. The present study proposed that dopamine D2 receptor (DRD2) may be the target of salidroside and tyrosol, by using RNA sequencing combined with Connectivity Map (CMAP) analysis. On this basis, molecular docking, surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA) were used to verify that both salidroside and tyrosol directly bind to DRD2. The results of this study can serve as a guide for further pharmacological research on salidroside and tyrosol.

pharmacology and toxicology↗

bioRxiv2024

Spatiotemporal Changes in Netrin/Dscam1 Signaling Dictate Axonal Projection Direction in Drosophila Small Ventral Lateral Clock Neurons

Axon projection is a spatial and temporal-specific process in which the growth cone receives environmental signals guiding axons to their final destination. However, the mechanisms underlying changes in axonal projection direction without well-defined landmarks remain elusive. Here, we present evidence showcasing the dynamic nature of axonal projections in Drosophilas small ventral lateral clock neurons (s-LNvs). Our findings reveal that these axons undergo an initial vertical projection in the early larval stage, followed by a subsequent transition to a horizontal projection in the early-to-mid third instar larvae. The vertical projection of s-LNv axons correlates with mushroom body calyx expansion, while the s-LNv-expressed Down syndrome cell adhesion molecule (Dscam1) interacts with Netrins to regulate the horizontal projection. During a specific temporal window, locally newborn dorsal clock neurons (DNs) secrete Netrins, facilitating the transition of axonal projection direction in s-LNvs. Our study establishes a compelling in vivo model to probe the mechanisms of axonal projection direction switching in the absence of clear landmarks. These findings underscore the significance of dynamic local microenvironments in the complementary regulation of axonal projection direction transitions.

neuroscience↗

bioRxiv2024

Dynamic Lateralization in Contralateral-Projecting Corticospinal Neurons During Motor Learning

Understanding the adaptability of the motor cortex in response to bilateral motor tasks is crucial for advancing our knowledge of neural plasticity and motor learning. Here we aim to investigate the dynamic lateralization of contralateral-projecting corticospinal neurons (cpCSNs) during such tasks. Utilizing in vivo two-photon calcium imaging, we observe cpCSNs in mice performing a bilateral lever-press task. Our findings reveal a heterogeneous population dynamics in cpCSNs: a marked decrease in activity during ipsilateral motor learning, in contrast to maintained activity during contralateral motor learning. Notably, individual cpCSNs show dynamic shifts in their engagement with ipsilateral and contralateral movements, displaying an evolving pattern of activation over successive days. This suggests a flexible and learning-related reconfiguration in the cpCSN network, parallel to motor learning stages. Our findings suggest a complex reorganization in cpCSN activities, underscoring the dynamic nature of cortical lateralization in motor learning and offering insights for neuromotor rehabilitation.

neuroscience↗

bioRxiv2024

Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells

Type I CRISPR-Cas systems are widespread and have exhibited remarkable versatility and efficiency in genome editing and gene regulation in prokaryotes. However, due to the multi-subunit composition and large size, their application in eukaryotes has not been thoroughly investigated. Here, we demonstrate that the type I-F2 Cascade, the most compact among type I systems and significantly smaller than SpCas9, can be developed into programmable tools for use in human cells. For transcriptional activation, the efficiency of the tool based on the engineered I-F2 system can match or surpass that of dCas9. Besides, narrow editing windows limit the application of base editors. Although the R-loop formed by Cascade is much wider than that by Cas9 or Cas12, the potential of base editing with Cascade has not yet been explored. We successfully created a base editor with the I-F2 Cascade, which induces a considerably wide editing window ([~]30 nt) with a bimodal distribution. The wide editing window can expand the range of targetable sites and can be useful for disrupting functional sequences and genetic screening. The editing efficiency can achieve 50% in human cells. This research underscores the application potential of compact type I systems in eukaryotes and developed a new base editor with an extraordinary wide editing window.

molecular biology↗

bioRxiv2024

Bypassing PELO-mediated ATPase activation of NLR is a common pathogenic cause of NLR-associated autoinflammatory diseases

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) constitute the largest family of pattern recognition receptors. These receptors are master regulators of innate immunity. Recently, PELO-driven ATPase activation of NLRs was demonstrated as a critical step in NLR activation. Linkage studies in human with various inherited autoinflammatory conditions have revealed an extensive array of mutations and polymorphisms within NLRs. However, the precise mechanisms by which genetic variations in NLR genes contribute to disease onset remain largely elusive. Here we comprehensively analyze dozens of naturally occurring mutations across multiple human NLRs, and demonstrate that NLRs harboring pathogenic mutations in their NACHT domain--not those with non-pathogenic variants--exhibit spontaneous PELO-independent ATPase activation. This aberrant activation triggers corresponding NLR to mediate inflammatory responses. Thus, bypassing the PELO-checkpoint for ATPase activation is a major disease-causing mechanism underlying NLR mutations. Furthermore, quantifying this aberrant ATPase activation could serve as an assessment tool for classifying the pathogenesis of NLR-associated diseases.

immunology↗

bioRxiv2024

Diurnal rhythmicity in salivary effector expression and metabolism shapes aphid performance on wheat plants

Diurnal rhythms influence insect behavior, physiology, and metabolism, optimizing their performance by adapting to daily changes in the environment. While their impact on agricultural pests has been briefly explored, our understanding of how these rhythms drive adaptative responses in pest biology and influence host colonization remains elusive. Here, we show that a notorious global aphid pest, Rhopalosiphum padi, exhibits distinct diurnal patterns in feeding behavior, with elevated honeydew excretion at night and extended phloem salivation during early nighttime. Temporal aphid transcriptome profiling reveals four diurnally rhythmic clusters, two of which peak at night, exhibiting enrichment in carbohydrate and amino acid metabolism. Beyond the established role in manipulating host responses and allowing sustained feeding, our study reveals novel evidence of cyclical fluctuations in salivary effector expression in an insect species. Silencing key effector genes, peaking in expression during the increased nighttime salivation, results in a more pronounced reduction in aphid excretion activity on host plants during the night compared to the day, a phenomenon not observed on artificial diets. A better understanding of aphid diurnal rhythms and their roles in shaping aphid performance provides a promising avenue to refine and optimize pest management, granting a strategic advantage for minimizing crop damage.

systems biology↗

bioRxiv2024

Early evolution of the ecdysozoan body plan

Extant ecdysozoans (moulting animals) are represented by a great variety of vermiform or articulated organisms. However, controversies remain about the nature of their ancestral body plan although the vermiform hypothesis seems to prevail. We describe here Beretella spinosa gen et sp. nov. a tiny ecdysozoan from the early Cambrian, Yanjiahe Formation, South China, with an unusual sack-like appearance, single opening, and spiny ornament. Beretella has no equivalent among animals, except Saccorhytus from the basal Cambrian. Phylogenetic analyses resolve both forms as a sister group (Saccorhytida) to all known Ecdysozoa, thus suggesting that ancestral ecdysozoans may have been non-vermiform animals. Saccorhytids are likely to represent an early dead-end off-shot along the stem-line Ecdysozoa that possibly evolved through anatomical simplification (e.g. lack of anus). Although extinct during the Cambrian, this animal lineage provides precious insight into the early evolution of Ecdysozoa and the nature (possibly non-vermiform) of the earliest representatives of the group.

paleontology↗

bioRxiv2024

Comprehensive and accurate genome analysis at scale using DRAGEN accelerated algorithms

Research and medical genomics require comprehensive and scalable solutions to drive the discovery of novel disease targets, evolutionary drivers, and genetic markers with clinical significance. This necessitates a framework to identify all types of variants independent of their size (e.g., SNV/SV) or location (e.g., repeats). Here we present DRAGEN that utilizes novel methods based on multigenomes, hardware acceleration, and machine learning based variant detection to provide novel insights into individual genomes with [~]30min computation time (from raw reads to variant detection). DRAGEN outperforms all other state-of-the-art methods in speed and accuracy across all variant types (SNV, indel, STR, SV, CNV) and further incorporates specialized methods to obtain key insights in medically relevant genes (e.g., HLA, SMN, GBA). We showcase DRAGEN across 3,202 genomes and demonstrate its scalability, accuracy, and innovations to further advance the integration of comprehensive genomics for research and medical applications.

genomics↗

bioRxiv2024

Extinction Training Suppresses Activity of Fear Memory Ensembles Across the Hippocampus and Alters Transcriptomes of Fear-Encoding Cells

Contextual fear conditioning has been shown to activate a set of "fear ensemble" cells in the hippocampal dentate gyrus (DG) whose reactivation is necessary and sufficient for expression of contextual fear. We previously demonstrated that extinction learning suppresses reactivation of these fear ensemble cells and activates a competing set of DG cells - the "extinction ensemble." Here, we tested whether extinction was sufficient to suppress reactivation in other regions and used single nucleus RNA sequencing (snRNA-seq) of cells in the dorsal dentate gyrus to examine how extinction affects the transcriptomic activity of fear ensemble and fear recall-activated cells. Our results confirm the suppressive effects of extinction in the dorsal and ventral dentate gyrus and demonstrate that this same effect extends to fear ensemble cells located in the dorsal CA1. Interestingly, the extinction-induced suppression of fear ensemble activity was not detected in ventral CA1. Our snRNA-seq analysis demonstrates that extinction training markedly changes transcription patterns in fear ensemble cells and that cells activated during recall of fear and recall of extinction have distinct transcriptomic profiles. Together, our results indicate that extinction training suppresses a broad portion of the fear ensemble in the hippocampus, and this suppression is accompanied by changes in the transcriptomes of fear ensemble cells and the emergence of a transcriptionally unique extinction ensemble.

neuroscience↗

bioRxiv2023

Serine hydroxymethyl transferase is a binding target of caprylic acid: Uncovering a novel molecular target for a herbicide and for producing caprylic acid-tolerant crops

Identification of new binding targets is essential for the development of herbicides and phytotoxin-tolerant crops. Caprylic acid (CAP) is a safe and non-selective bio-herbicide in uncultivated areas. However, the herbicidal action of CAP remains unclear. Herein, metabolomic and proteomic profiling indicated that a serine hydroxymethyl transferase in Conyza canadensis (CcSHMT1) is a promising candidate binding targeted for CAP. The protein abundance and activity of CcSHMT1 were decreased in a time- and dosage-dependent manners after CAP treatment. CAP competes with phenyl-serine at the binding sites, decreasing the enzymatic activity of CcSHMT1. Overexpression of CcSHMT1, AtSHMT1 and OsSHMT1 in Arabidopsis or rice endowed plants with high tolerance to CAP treatment, whereas the knockout of osshmt1 led to death of plants under normal atmospheric conditions. Furthermore, T194A, T194A/ M195V and D209N Osshmt1 mutant plants derived from base editing exhibited tolerance to CAP. CAP bound to CcSHMT1 with high affinity, and Ala191 in the key domains of N-terminus was identified to be critical for the binding site of CAP. Collectively, our findings demonstrate that CcSHMT1 is a binding target for herbicidal activity of CAP. This study marks a key step in the druggability of SHMT inhibitors and represents an attractive target for phytotoxin-tolerant crops.

plant biology↗

bioRxiv2023

Not dasycladalean alga, but an Odyssey of the earliest Phanerozoic animal reef-builders

The compacted macrofossil Protomelission? sp. from the early Cambrian Xiaoshiba Lagerstatte was recently ascribed to early dasycladalean green algae and used to disprove the bryozoan affinity of coeval phosphatized microfossils, which made the puzzling question whether the bryozoans originated in early Cambrian pending again. Our new analyses of multiple specimens which are conspecific with Protomelission? from the Chengjiang Lagerstatte indicate that they are not dasycladaleans but one of the three groups of archaeocyath-like sponges that atypically inhabited siliciclastic substrates. All the archaeocyath-like fossils share the same preservation mode and exhibit archaeocyath-type external skeletal features. Particularly, the Protomellision?-like fossils preserve structures indicative of archaeocyath aquiferous system and ontogeny. They represent the first recognized one-walled archaeocyath sponges in South China and evidence the niche expansion of archaeocyaths on their way of global radiation from Siberia, 518 million years ago. The origin of the bryozoans remains a mystery.

paleontology↗

bioRxiv2023

CRISPR/Cas9-mediated genome editing of Frankliniella occidentalis, the western flower thrips, via embryonic microinjection

The western flower thrips, Frankliniella occidentalis, poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye-color genes, white (Fo-w) and cinnabar (Fo-cn), frequently used to assess Cas9 function in insects were identified in the F. occidentalis genome and targeted for knockout through embryonic microinjection of Cas9 complexed with Fo-w or Fo-cn specific guide RNAs. Homozygous Fo-w and Fo-cn knockout lines were established by crossing mutant females and males. The Fo-w knockout line revealed an age-dependent modification of eye-color phenotype. Specifically, while young larvae exhibit ivory-colored eyes, the color transitions to bright red as they age. Unexpectedly, loss of Fo-w function also altered body color, with Fo-w mutants having a lighter colored body than wild type, suggesting a dual role for Fo-w in thrips. In contrast, individuals from the Fo-cn knockout line consistently displayed bright red eyes throughout all life stages. Molecular analyses validated precise editing of both target genes. This study offers a powerful tool to investigate thrips gene functions and paves the way for the development of genetic technologies for population suppression and/or population replacement as a means of mitigating virus transmission by this vector.

genetics↗

bioRxiv2023

Alternative Polyadenylation Determines the Functional Landscape of Inverted Alu Repeats

Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' UTRs. Yet, the biological significance of 3' UTR IRAlus remains largely unknown. Here, we find that IRAlus induce the silencing of genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.

molecular biology↗

bioRxiv2023

Comprehensive profiling of L1 retrotransposons in mouse

L1 elements are retrotransposons currently active in mammals. Although L1s are typically silenced in most normal tissues, elevated L1 expression is associated with a variety of conditions, including cancer, aging, infertility, and neurological disease. These associations have raised interest in the mapping of human endogenous de novo L1 insertions, and a variety of methods have been developed for this purpose. Adapting these methods to mouse genomes would allow us to monitor endogenous in vivo L1 activity in controlled, experimental conditions using mouse disease models. Here we use a modified version of transposon insertion profiling, called nanoTIPseq, to selectively enrich young mouse L1s. By linking this amplification step with nanopore sequencing, we identified >95% annotated L1s from C57BL/6 genomic DNA using only 200,000 sequencing reads. In the process, we discovered 82 unannotated L1 insertions from a single C57BL/6 genome. Most of these unannotated L1s were near repetitive sequence and were not found with short-read TIPseq. We used nanoTIPseq on individual mouse breast cancer cells and were able to identify the annotated and unannotated L1s, as well as new insertions specific to individual cells, providing proof of principle for using nanoTIPseq to interrogate retrotransposition activity at the single cell level in vivo.

genomics↗

bioRxiv2023

Examination of the quantitative relationship between proteome- and transcriptome-level responses in larval guts of Frankliniella occidentalis infected with an orthotospovirus.

The western flower thrips, Frankliniella occidentalis, is the principal thrips vector of Orthotospovirus tomatomaculae (order Bunyavirales, family Tospoviridae), a devastating plant-pathogenic virus commonly referred to as tomato spotted wilt virus (TSWV). The larval gut is the gateway for virus transmission by F. occidentalis adults to plants. In a previous report, gut expression at the transcriptome-level was subtle but significant in response to TSWV in L1s. Since it has been well documented that the relationship between the expression of mRNA and associated protein products in eukaryotic cells is often discordant, we performed identical, replicated experiments to identify and quantify virus-responsive larval gut proteins to expand our understanding of insect host response to TSWV. While we documented statistically-significant, positive correlations between abundance of proteins (4,189 identified) and their cognate mRNAs expressed in first and second instar guts, there was virtually no alignment of individual genes identified to be differentially modulated by virus infection at the transcriptome and proteome levels. Predicted protein-protein interaction networks associated with clusters of co-expressed proteins revealed wide variation in correlation strength between protein and cognate transcript abundance, which appeared to be associated with the type of cellular processes, cellular compartments, and network connectivity represented by the proteins. In total, our findings indicate distinct and dynamic regulatory mechanisms of transcript and protein abundance (expression, modifications, and/or turnover) in virus-infected gut tissues. This study provides molecular candidates for future functional analysis of thrips vector competence and underscores the necessity of examining complex virus-vector interactions at a systems level.

systems biology↗

bioRxiv2023

Deep learning-guided selection of antibody therapies with enhanced resistance to current and prospective SARS-CoV-2 Omicron variants

Most COVID-19 antibody therapies rely on binding the SARS-CoV-2 receptor binding domain (RBD). However, heavily mutated variants such as Omicron and its sublineages, which are characterized by an ever increasing number of mutations in the RBD, have rendered prior antibody therapies ineffective, leaving no clinically approved antibody treatments for SARS-CoV-2. Therefore, the capacity of therapeutic antibody candidates to bind and neutralize current and prospective SARS-CoV-2 variants is a critical factor for drug development. Here, we present a deep learning-guided approach to identify antibodies with enhanced resistance to SARS-CoV-2 evolution. We apply deep mutational learning (DML), a machine learning-guided protein engineering method to interrogate a massive sequence space of combinatorial RBD mutations and predict their impact on angiotensin-converting enzyme 2 (ACE2) binding and antibody escape. A high mutational distance library was constructed based on the full-length RBD of Omicron BA.1, which was experimentally screened for binding to the ACE2 receptor or neutralizing antibodies, followed by deep sequencing. The resulting data was used to train ensemble deep learning models that could accurately predict binding or escape for a panel of therapeutic antibody candidates targeting diverse RBD epitopes. Furthermore, antibody breadth was assessed by predicting binding or escape to synthetic lineages that represent millions of sequences generated using in silico evolution, revealing combinations with complementary and enhanced resistance to viral evolution. This deep learning approach may enable the design of next-generation antibody therapies that remain effective against future SARS-CoV-2 variants.

immunology↗