Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 487 records · Page 27Linked to original sources

bioRxiv2023

An automated archival single-nucleus total RNA sequencing platform mapping integrative and retrospective cell atlas of gliomas

Single-cell RNA sequencing (scRNA-seq) has dramatically transformed biomedical research within laboratory settings. It has been extensively employed to investigate the heterogeneity and plasticity of glioma, the most prevalent brain tumor. However, the clinical diagnosis and treatment of glioma remain complex and challenging, highlighting the need for comprehensive cancer research. Currently available scRNA-seq platforms are insufficient to fulfill the demands posed by large-scale clinical applications. Here, we present an automated high-throughput single-nucleus total RNA sequencing platform, known as AAsnRandom-seq. This platform integrates automated single-nucleus isolation and droplet barcoding systems with the random primer-based scRNA-seq chemistry, designed to accommodate a diverse range of sample types. The performance and versatility of AAsnRandom-seq are validated using over one hundred clinical FFPE and frozen samples. AAsnRandom-seq was applied to archival FFPE samples of various glioma subtypes, including rare clinical samples, and matched primary-recurrent glioblastomas (GBMs), delving into the comprehensive molecular characteristic of glioma at single-cell level. Abundant non-coding RNAs (ncRNAs) with distinct expression profiles within different glioma clusters are detected. Promising recurrence-related targets and pathways are identified from the matched primary-recurrent GBMs. AAsnRandom-seq holds significant application value on large-scale integrative and retrospective clinical research using archived specimens.

bioengineering↗

bioRxiv2023

High-sensitivity whole-genome recovery of single viral species in environmental samples

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the methods efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.

bioengineering↗

bioRxiv2023

Heparan sulfate dependent phase separation of CCL5 and its chemotactic activity

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation (LLPS) and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin binding sequence) or by addition of heparin to wild type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

cell biology↗

bioRxiv2023

The in vitro functional profiles of fentanyl and nitazene analogs at the μ-opioid receptor - high efficacy is dangerous regardless of signaling bias

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as the -opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G protein biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at MOR using adenylate cyclase inhibition and {beta}-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and {beta}-arrestin2 recruitment pathways. Compared to the reference agonist DAMGO, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting {beta}-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and {beta}-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. Instead, our results show that, regardless of bias, opioids with sufficiently high intrinsic efficacy can be lethal, especially given the extremely high potency of many of these compounds that are now pervading the illicit drug market.

pharmacology and toxicology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 Omicron HK.3 variant harboring the "FLip" substitution

In November 2023, SARS-CoV-2 XBB descendants, including EG.5.1 (XBB.1.9.2.5.1), the currently predominant lineage, are circulating worldwide according to Nextstrain. EG.5.1 has a characteristic amino acid substitution in the spike protein (S), S:F456L, which contributes to its escape from humoral immunity. EG.5.1 has further evolved, and its descendant lineage harboring S:L455F (i.e., EG.5.1+S:L455F) emerged and was named HK.3 (XBB.1.9.2.5.1.1.3). HK.3 was initially discovered in East Asia and is rapidly spreading worldwide. Notably, the XBB subvariants bearing both S:L455F and S:F456L substitutions, including HK.3, are called the "FLip" variants. These FLip variants, such as JG.3 (XBB.1.9.2.5.1.3.3), JF.1 (XBB.1.16.6.1) and GK.3 (XBB.1.5.70.3), have emerged convergently, suggesting that the acquisition of these two substitutions confers a growth advantage to XBB in the human population. Here, we investigated the virological properties of HK.3 as a representative of the FLip variants.

microbiology↗

bioRxiv2023

Activation of glucocorticoid receptor signaling inhibits KSHV-induced inflammation and tumorigenesis

Hyperinflammation is the hallmark of Kaposis sarcoma (KS), the most common cancer in AIDS patients caused by Kaposis sarcoma-associated herpesvirus (KSHV) infection. However, the role and mechanism of induction of inflammation in KS remain unclear. In a screening for inhibitors of KSHV-induced oncogenesis, over half of the identified candidates were anti-inflammatory agents including dexamethasone functions by activating glucocorticoid receptor (GR) signaling. Here, we examined the mechanism mediating KSHV-induced inflammation. We found that numerous inflammatory pathways were activated in KSHV-transformed cells. Particularly, interleukin-1 alpha (IL-1) and IL-1 receptor antagonist (IL-1Ra) from the IL-1 family were the most induced and suppressed cytokines, respectively. We found that KSHV miRNAs mediated IL-1 induction while both miRNAs and vFLIP mediated IL-1Ra suppression. Furthermore, GR signaling was inhibited in KSHV-transformed cells, which was mediated by vFLIP and vCyclin. Dexamethasone treatment activated GR signaling, and inhibited cell proliferation and colony formation in soft agar of KSHV-transformed cells but had a minimal effect on matched primary cells. Consequently, dexamethasone suppressed the initiation and growth of KSHV-induced tumors in mice. Mechanistically, dexamethasone suppressed IL-1 but induced IL-1Ra expression. Treatment with recombinant IL-1 protein rescued the inhibitory effect of dexamethasone while overexpression of IL-1Ra caused a weak growth inhibition of KSHV-transformed cells. Furthermore, dexamethasone induced I{kappa}B expression resulting in inhibition of NF-{kappa}B pathway and IL-1 expression. These results reveal an important role of IL-1 pathway in KSHV-induced inflammation and oncogenesis, which can be inhibited by dexamethasone-activated GR signaling, and identify IL-1-mediated inflammation as a potential therapeutic target for KSHV-induced malignancies. ImportanceKaposis sarcoma (KS) is the most common cancer in HIV-infected patients caused by Kaposis sarcoma-associated herpesvirus (KSHV) infection. Hyperinflammation is the hallmark of KS. In this study, we have shown that KSHV mediates hyperinflammation by inducing IL-1 and suppressing IL-1Ra. Mechanistically, KSHV miRNAs and vFLIP induce hyperinflammation by activating the NF-{kappa}B pathway. A common anti-inflammatory agent dexamethasone blocks KSHV-induced hyperinflammation and tumorigenesis by activating glucocorticoid receptor signaling to suppress IL-1 and induce IL-1Ra. This work has identified IL-1-mediated inflammation as a potential therapeutic target and dexamethasone as a potential therapeutic agent for KSHV-induced malignancies.

cancer biology↗

bioRxiv2023

Dynamic RNA Polymerase II Recruitment Drives Differentiation of the Intestine under the direction of HNF4

Terminal differentiation requires a massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. We identified dynamic recruitment of RNA Polymerase II (Pol II) to gene promoters as the primary driver of transcriptomic shifts during intestinal differentiation in vivo. Changes in enhancer-promoter looping interactions accompany dynamic Pol II recruitment and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of-function, ChIP-seq and IP mass spectrometry, we demonstrate that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II recruitment at hundreds of genes pivotal to differentiation. We also explore alternate mechanisms which drive differentiation gene expression and find pause-release of Pol II and post-transcriptional mRNA stability regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in a renewing adult tissue. HIGHLIGHTSO_LIDynamic recruitment of Pol II largely drives the vast transcriptomic changes seen during differentiation of mouse intestinal epithelium. C_LIO_LISmaller groups of differentiated genes are subject to regulation through Pol II pause-release and post-transcriptional mechanisms such as differences in mRNA stability. C_LIO_LIIP-mass spectrometry analysis identifies the first interactome of HNF4 in the differentiated small intestine, finding interactions with chromatin looping and chromatin remodeling proteins. C_LIO_LIHNF4 transcription factors play a critical role in recruiting Pol II to the promoters of essential intestinal differentiation genes. C_LI

genetics↗

bioRxiv2023

The saccade-induced temporal attentional gradient: Duration compression of the post-saccadic second event

Rapid eye movements can distort our perception of time, as evidenced by the phenomenon of Chronostasis, where the first event following a saccade appears to last longer than it actually does. Despite extensive research on this phenomenon, the effect of saccades on sequential post-saccadic events, beyond the first one, has never been investigated. To address this, in the present study we compared the subjective estimates of the time of first and second post-saccadic events (with fixation conditions as controls). We found saccadic eye movements not only to affect the perceived duration of the first post-saccadic event (Chronostasis), but also that of the second event. Specifically, when the second event immediately following the first event, it was subjectively compressed. When the second event was used as the (constant) reference interval, Chronostasis was enhanced. Remarkably, the compression effect persisted even when potential "attentional-blink"-induced processes, that might affect timing at the transition from the first to the second event, were eliminated. To explain our findings, we propose that saccades induce a transient temporal attentional gradient that results in an overestimation of the first and an underestimation of the second interval when the two intervals are temporally (near-) contiguous.

neuroscience↗

bioRxiv2023

Pan-genome analysis of different morphotypes reveals genomic basis of Brassica oleracea domestication and differential organogenesis

The domestication of Brassica oleracea has resulted in diverse morphological types with distinct patterns of organ development. Here we report a graph-based pan-genome of B. oleracea constructed with high-quality genome assemblies of different morphotypes. The pan-genome harbors over 200 structural variant (SV) hotspot regions enriched with auxin and flowering-related genes. Population genomic analyses reveal that early domestication of B. oleracea focused on leaf or stem selection. Gene flows resulting from agricultural practices and variety improvement are detected among different morphotypes. Selective sweep analysis identifies an auxin-responsive SAUR gene and a CLE family gene as the crucial players in the leaf-stem differentiation during the early stage of B. oleracea domestication, and the BoKAN1 gene as instrumental in shaping the leafy heads of cabbage and Brussels sprouts. Our pan-genome and functional analyses further discover that variations in the BoFLC2 gene play key roles in the divergence of vernalization and flowering characteristics among different morphotypes, and variations in the first intron of BoFLC3 are involved in fine-tuning the flowering process in cauliflower. This study provides a comprehensive understanding of the pan-genome of B. oleracea and sheds light on the domestication and differential organ development of this globally important crop species.

plant biology↗

bioRxiv2023

Quantitative dynamics of intracellular NMN by genetically encoded biosensor

Nicotinamide mononucleotide (NMN) is a major precursor for NAD+ metabolism with promising effects in treating NAD+- and aging-related pathologies. However, measuring live cell NMN dynamics was not possible, leaving key questions in intracellular NMN uptake and regulation unanswered. Here we developed a genetically encoded bioluminescent sensor to quantify subcellular NMN in live cells by fusing engineered NMN-responsive binding domain to bioluminescent and fluorescent proteins from BRET pairs. The sensor dissected the multimechanistic uptake of extracellular NMN and precursors in live cells. We then captured the notably low mitochondrial NMN content and the thereafter vulnerable NMN/NAD+ ratio and SARM1 activation in mitochondria, establishing NMN/NAD+ ratio as an important parameter in evaluating NAD+ boosting strategies. Moreover, we characterized the signature of major NAD+ regulating enzymes on NMN and NMN/NAD+ ratios, in which Slc25a45 was identified to be a potential mitochondrial NMN transporter for its unique fingerprint on mitochondrial NMN/NAD+ ratio.

cell biology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant

In middle-late 2023, a sublineage of SARS-CoV-2 Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. Here, we performed multiscale investigations to reveal virological features of newly emerging EG.5.1 variant. Our phylogenetic-epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T, are critical to the increased viral fitness. Experimental investigations addressing the growth kinetics, sensitivity to clinically available antivirals, fusogenicity and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 is comparable to that of XBB.1.5. However, the cryo-electron microscopy reveals the structural difference between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible at least in our experimental setup. Our multiscale investigations provide the knowledge for understanding of the evolution trait of newly emerging pathogenic viruses in the human population.

microbiology↗

bioRxiv2023

The More Unclear the Hearing, the Faster the Feeling: Speech Comprehension Changes Subjective Speech Rate

Imagine being in a foreign country, surrounded by locals speaking a language entirely alien to you. Does their conversation seem to race like a machine gun? The perception of speech rate, as a facet of time perception, greatly impacts our daily lives. However, the factors influencing this perception remain poorly understood. Through a series of three experiments (with sample sizes of 31, 27, and 25 respectively), we discovered that speech comprehension significantly alters listeners perception of speech rate. As comprehensibility diminishes, listeners tend to overestimate the speech rate. Intriguingly, this effect is further influenced by the listeners language proficiency. Specifically, the better the listeners language proficiency, the more likely their perception of speech rate is influenced by the speech comprehension. Our study corroborates the notion that biases in time perception may be prevalent in our everyday communication. These subjective experiences could prompt listeners to modify their behavior or processing strategies.

molecular biology↗

bioRxiv2023

Probabilistic Graphical Modeling under Heterogeneity

Probabilistic graphical models are powerful and widely used tools to quantify, visualize and interpret dependencies in complex biological systems such as highthroughput genomics and proteomics. However, most existing graphical modeling methods assume homogeneity within and across samples which restricts their broad applicability to cases where sample-specific heterogeneity exists e.g. tumor heterogeneity. We propose a flexible Bayesian approach called Graphical Regression (GraphR) which (a) allows direct incorporation of intrinsic factors of sample heterogeneity at different scales through a regression-based formulation, (b) enables sparse network estimation at a sample-specific level, (c) allows identification and uncertainty quantification of potential effects of heterogeneity on network structures, and (d) is computationally efficient through the use of variational Bayes algorithms. We illustrate the comparative efficiency of GraphR against existing methods in terms of graph structure recovery and computational cost across multiple realistic simulation settings. We use GraphR to analyze four diverse multi-omics and spatial transcriptomics datasets to study inter- and intra-sample molecular networks and delineate biological discoveries that otherwise cannot be revealed by existing approaches. We have developed a GraphR R-package along with an accompanying Shiny App that provides comprehensive analysis and dynamic visualization functions.

genomics↗

bioRxiv2023

Dual Ethanolamine Head Groups in Ionizable Lipids Facilitate Phospholipid-free Stable Nanoparticle Formulation for Augmented and Safer mRNA Delivery

The accelerated advent of mRNA-based therapeutics and vaccines, highlighted by the battle against SARS-CoV-2, underscores the urgency to refine lipid nanoparticles (LNPs) for efficient mRNA delivery. In this work, we introduce a novel series of ionizable lipids characterized by double ethanolamine head groups, significantly amplifying mRNA binding affinity. A succinct three-component formulation is subsequently delineated, obviating the conventional dependency on phospholipids inherent in traditional four-component LNPs. Intriguingly, this formulation enables particle formation under neutral pH conditions, a notable departure from the acidic milieu traditionally required, attributable to the enhanced nonionic interactions predominating in mRNA encapsulation. The resultant particles exhibit exceptional stability, superior mRNA encapsulation efficiency, and maintain robust delivery efficacy. When deployed as a vaccine platform, the formulation elicited pronounced humoral and T-cell immune responses, concurrently exhibiting a favorable toxicity profile with a reduced induction of pro-inflammatory cytokines such as IL-6. Our exploration suggests that by fine-tuning the non-electrostatic interactions between the ionizable lipid and mRNA, the dynamics of particle formation can be considerably divergent from the prevailing paradigms of mRNA-LNP formation, hinting at a broader horizon for lipid optimization within the realm of mRNA delivery systems.

bioengineering↗

bioRxiv2023

Network level enrichment provides a framework for biological interpretation of machine learning results

Machine learning algorithms are increasingly used to identify brain connectivity biomarkers linked to behavior and clinical outcomes. However, non-standard methodological choices in neuroimaging datasets, especially those with families or twins, have prevented robust machine learning applications. Additionally, prioritizing prediction accuracy over biological interpretability has made it challenging to understand the biological processes behind psychopathology. In this study, we employed a linear support vector regression model to study the relationship between resting-state functional connectivity networks and chronological age using data from the Human Connectome Project. We examined the effect of shared variance from twins and siblings by using cross-validation, either randomly assigning or keeping family members together. We also compared models with and without a Pearson feature filter and utilized a network enrichment approach to identify predictive brain networks. Results indicated that not accounting for shared family variance inflated prediction performance, and the Pearson filter reduced accuracy and reliability. Enhancing biological interpretability was achieved by inverting the machine learning model and applying network-level enrichment on the connectome, while directly using regression coefficients as feature weights led to misleading interpretations. Our findings offer crucial insights for applying machine learning to neuroimaging data, emphasizing the value of network enrichment for comprehensible biological interpretation.

neuroscience↗

bioRxiv2023

Antibodies utilizing VL6-57 light chains target a convergent cryptic epitope on SARS-CoV-2 spike protein driving the genesis of Omicron variants

Continued evolution of SARS-CoV-2 generates variants to challenge antibody immunity established by infection and vaccination. A connection between population immunity and genesis of virus variants has long been suggested but its molecular basis remains poorly understood. Here, we identify a class of SARS-CoV-2 neutralising public antibodies defined by their shared usage of VL6-57 light chains. Although heavy chains of diverse genotypes are utilized, convergent HCDR3 rearrangements have been observed among these public antibodies to cooperate with germline VL6-57 LCDRs to target a convergent epitope defined by RBD residues S371-S373-S375. Antibody repertoire analysis identifies that this class of VL6-57 antibodies is present in SARS-CoV-2-naive individuals and is clonally expanded in most COVID-19 patients. We confirm that Omicron specific substitutions at S371, S373 and S375 mediate escape of antibodies of the VL6-57 class. These findings support that this class of public antibodies constitutes immune pressure promoting the introduction of S371L/F-S373P-S375F in Omicron variants. The results provide further molecular evidences to support that antigenic evolution of SARS-CoV-2 is driven by antibody mediated population immunity.

biochemistry↗

bioRxiv2023

Circadian photoreceptor CRYPTOCHROME promotes wakefulness under short winter-like days via a GABAergic circuitry

A cardinal symptom of seasonal affective disorder (SAD, also known as winter depression) is hypersomnolence, while the cause of this "winter sleepiness" is not known. Here we found that lack of the circadian photoreceptor cryptochrome (cry) leads to increased sleep under short winter-like days in fruit flies, reminiscent of the hypersomnolence in SAD. CRY functions in neurons that synthesize the major inhibitory neurotransmitter GABA, including the small ventral lateral neurons which are known to be circadian pacemakers, and down-regulates the GABAergic tone. This in turn leads to increased neural activity of the wake-promoting large ventral lateral neurons, a subset of circadian neurons that are inhibited by GABA-A receptor. CRY protein is known to be degraded by light, thus rendering CRY to be functional within this GABAergic circuitry to enhance wakefulness only under short day length. Taken together, we demonstrate a mechanism that specifically regulates wakefulness under short winter-like days, which may provide insights regarding the winter sleepiness in SAD.

neuroscience↗

bioRxiv2023

Disease-specific selective vulnerability and neuroimmune pathways in dementia revealed by single cellgenomics

Summary/AbstractThe development of successful therapeutics for dementias requires an understanding of their shared and distinct molecular features in the human brain. We performed single-nuclear RNAseq and ATACseq in Alzheimer disease (AD), Frontotemporal degeneration (FTD), and Progressive Supranuclear Palsy (PSP), analyzing 40 participants, yielding over 1.4M cells from three brain regions ranging in vulnerability and pathological burden. We identify 35 shared disease-associated cell types and 14 that are disease-specific, replicating those previously identified in AD. Disease-specific cell states represent molecular features of disease-specific glial-immune mechanisms and neuronal vulnerability in each disorder, layer 4/5 intra-telencephalic neurons in AD, layer 2/3 intra-telencephalic neurons in FTD, and layer 5/6 near-projection neurons in PSP. We infer intrinsic disease-associated gene regulatory networks, which we empirically validate by chromatin footprinting. We find that causal genetic risk acts in specific neuronal and glial cells that differ across disorders, primarily non-neuronal cells in AD and specific neuronal subtypes in FTD and PSP. These data illustrate the heterogeneous spectrum of glial and neuronal composition and gene expression alterations in different dementias and identify new therapeutic targets by revealing shared and disease-specific cell states.

neuroscience↗