Biology subjects

Huang, Y.

Publications and source records attributed to Huang, Y..

At least 541 records · Page 30Linked to original sources

bioRxiv2023

A Fuzzy sequencer for rapid DNA fragment counting and genotyping

High-throughput sequencing technologies generate a vast number of DNA sequence reads simultaneously, which are subsequently analyzed using the information contained within these fragmented reads. The assessment of sequencing technology relies on information efficiency, which measures the amount of information entropy produced per sequencing reaction cycle. In this study, we propose a fuzzy sequencing strategy that exhibits information efficiency more than twice of currently prevailing cyclic reversible terminator sequencing methods. To validate our approach, we developed a fully functional and high-throughput fuzzy sequencer. This sequencer implements a highly efficient fluorogenic sequencing-by-synthesis chemistry and underwent testing across various application scenarios, including copy-number variation detection, noninvasive prenatal testing, transcriptome profiling, mutation genotyping, and metagenomic profling. Our findings unequivocally demonstrate that the fuzzy sequencing strategy outperforms existing methods in terms of information efficiency and delivers accurate resequencing results with faster turnaround times. One Sentence SummaryA fuzzy sequencer can exceed current limit of information efficiency of DNA sequencers for resequencing applications.

genomics↗

bioRxiv2023

Waveform-based classification of dentate spikes

Synchronous excitatory discharges from the entorhinal cortex (EC) to the dentate gyrus (DG) generate fast and prominent patterns in the hilar local field potential (LFP), called dentate spikes (DSs). As sharp-wave ripples in CA1, DSs are more likely to occur in quiet behavioral states, when memory consolidation is thought to take place. However, their functions in mnemonic processes are yet to be elucidated. The classification of DSs into types 1 or 2 is determined by their origin in the lateral or medial EC, as revealed by current source density (CSD) analysis, which requires recordings from linear probes with multiple electrodes spanning the DG layers. To allow the investigation of the functional role of each DS type in recordings obtained from single electrodes and tetrodes, which are abundant in the field, we developed an unsupervised method using Gaussian mixture models to classify such events based on their waveforms. Our classification approach achieved high accuracies (> 80%) when validated in 8 mice with DG laminar profiles. The average CSDs, waveforms, rates, and widths of the DS types obtained through our method closely resembled those derived from the CSD-based classification. As an example of application, we used the technique to analyze single-electrode LFPs from apolipoprotein (apo) E3 and apoE4 knock-in mice. We observed that the latter group, which is a model for Alzheimers disease, exhibited wider DSs of both types from a young age, with a larger effect size for DS type 2, likely reflecting early pathophysiological alterations in the EC-DG network, such as hyperactivity. In addition to the applicability of the method in expanding the study of DS types, our results show that their waveforms carry information about their origins, suggesting different underlying network dynamics and roles in memory processing. Author summaryThe entorhinal cortex and the dentate gyrus are regions of the brains hippocampal formation that play a crucial role in learning and memory. Their synchronous activation generates fast and high-amplitude electrophysiological patterns in the dentate gyrus, called dentate spikes (DSs). However, the functional role of DSs is still poorly understood. A technical limitation for the study of DSs is that their classification into types 1 and 2 is only possible through laminar profiles obtained by multicontact linear probes. In the present work, we propose a method to classify the different types of DSs through their waveforms, thus expanding the investigation of their significance in mnemonic processes by making possible their study in single-site recordings.

neuroscience↗

bioRxiv2023

CellContrast: Reconstructing Spatial Relationships in Single-Cell RNA Sequencing Data via Deep Contrastive Learning

A vast amount of single-cell RNA-seq (SC) data has been accumulated via various studies and consortiums, but the lack of spatial information limits its analysis of complex biological activities. To bridge this gap, we introduce cellContrast, a computational method for reconstructing spatial relationships among SC cells from spatial transcriptomics (ST) reference. By adopting a contrastive learning framework and training with ST data, cellContrast projects gene expressions into a hidden space where proximate cells share similar representation values. We performed extensive benchmarking on diverse platforms, including SeqFISH, Stereo-Seq, 10X Visium, and MERSCOPE, on mouse embryo and human breast cells. The results reveal that cellContrast substantially outperforms other related methods, facilitating accurate spatial reconstruction of SC. We further demonstrate cellContrasts utility by applying it to cell-type co-localization and cell-cell communication analysis with real-world SC samples, proving the recovered cell locations empower novel discoveries and mitigate potential false positives.

bioinformatics↗

bioRxiv2023

MetaQuad: Shared Informative Variants Discovery in Metagenomic Samples

MotivationStrain-level analysis of metagenomic data has garnered significant interest in recent years. Microbial single nucleotide polymorphisms (SNPs) are genomic variants that can reflect strain-level differences within a microbial species. The diversity and emergence of SNPs in microbial genomes may reveal evolutionary history and environmental adaptation in microbial populations. However, efficient discovery of shared polymorphic variants in a large collection metagenomic samples remains a computational challenge. ResultsMetaQuad employs a density-based clustering technique to efficiently differentiate shared variants from non-polymorphic sites using shotgun metagenomic data. Empirical comparisons with other state-of-the-art methods show that MetaQuad significantly reduces the number of false-positive SNPs without greatly affecting the true-positive rate. We used MetaQuad to identify antibiotic-associated variants in patients who underwent Helicobacter pylori eradication therapy. MetaQuad detected 7,591 variants across 529 antibiotic resistance genes. The nucleotide diversity of some genes is increased six weeks after antibiotic treatment, potentially indicating the role of these genes in specific antibiotic treatments. AvailabilityMetaQuad is an open-source Python package available via https://github.com/holab-hku/MetaQuad. Contactjwkho@hku.hk Supplementary informationSupplementary data are available at XXXX online.

genomics↗

bioRxiv2023

Lysosomal proteomics reveals mechanisms of neuronal apoE4-associated lysosomal dysfunction

ApoE4 is the primary risk factor for Alzheimer Disease (AD). Early AD pathological events first affect the neuronal endolysosomal system, which in turn causes neuronal protein aggregation and cell death. Despite the crucial influence of lysosomes upon AD pathophysiology, and that apoE4 localizes to lysosomes, the influence of apoE4 on lysosomal function remains unexplored. We find that expression of apoE4 in neuronal cell lines results in lysosomal alkalinization and impaired lysosomal function. To identify driving factors for these defects, we performed quantitative lysosomal proteome profiling. This revealed that apoE4 expression results in differential regulation of numerous lysosomal proteins, correlating with apoE allele status and disease severity in AD brains. In particular, apoE4 expression results in the depletion of lysosomal Lgals3bp and the accumulation of lysosomal Tmed5. We additionally validated that these lysosomal protein changes can be targeted to modulate lysosomal function. Taken together, this work thereby reveals that apoE4 causes widespread lysosomal defects through remodeling the lysosomal proteome, with the lysosomal Tmed5 accumulation and Lgals3bp depletion manifesting as lysosomal alkalinization in apoE4 neurons.

neuroscience↗

bioRxiv2023

Antigenicity and receptor affinity of SARS-CoV-2 BA.2.86 spike

Although the COVID-19 pandemic has officially ended1, SARS-CoV-2 continues to spread and evolve. Recent infections have been dominated by XBB.1.5 and EG.5.1 subvariants2. A new subvariant designated BA.2.86 has just emerged, spreading to 21 countries in 5 continents3. This virus contains 34 spike mutations compared to its BA.2 predecessor, thereby raising concerns about its propensity to evade existing antibodies. We examined its antigenicity using human sera and monoclonal antibodies (mAbs). Reassuringly, BA.2.86 was not more resistant to human sera than XBB.1.5 and EG.5.1, indicating that the new subvariant would not have a growth advantage in this regard. Importantly, sera from patients who had XBB breakthrough infection exhibited robust neutralizing activity against all viruses tested, suggesting that upcoming XBB.1.5 monovalent vaccines could confer added protection. The finding that the longer genetic distance of BA.2.86 did not yield a larger antigenic distance was partially explained by the mAb data. While BA.2.86 showed greater resistance to mAbs to subdomain 1 (SD1) and receptor-binding domain (RBD) class 2 and 3 epitopes, it was more sensitive to mAbs to class 1 and 4/1 epitopes in the "inner face" of RBD that is exposed only when this domain is in the "up" position. We also identified six new spike mutations that mediate antibody resistance, including E554K that threatens SD1 mAbs in clinical development. The BA.2.86 spike also had a remarkably high receptor affinity. The ultimate trajectory of this new SARS-CoV-2 variant will soon be revealed by continuing surveillance, but its worldwide spread is worrisome.

microbiology↗

bioRxiv2023

A potential acoustic role for CFTR ion channel in conductive hearing loss

Loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). The middle ear and eustachian tube could be adversely affected in CF. In this study, we provide evidence of the role of CFTR function in conductive hearing. We developed an in-situ model to determine CFTR dependent fluid secretion in the middle ear using native mouse auditory capsule. A unique middle ear-on-a-chip was developed to address the functional and molecular basis of conductive hearing impairment. Using single-cell transcriptomics, middle ear cell composition and the associated transcriptomic signature were compared between CF and WT groups. A specialized subset of epithelial cells expressed CFTR with an overlapping signature with secretory epithelial cells. Genes related to ciliogenesis, hearing and ossification were significantly altered in CF mice middle ear. Our data suggest that CF middle ear may be at higher risk for conductive hearing loss.

cell biology↗

bioRxiv2023

Perceptual priors add sensory detail to contextual feedback processing in V1

How do we develop models of the world? Contextualising ambiguous information with previous experience allows us to form an enriched perception. Contextual information and prior knowledge facilitate perceptual processing, improving our recognition of even distorted or obstructed visual inputs. As a result, neuronal processing elicited by identical sensory inputs varies depending on the context in which we encounter those inputs. This modulation is in line with predictive processing accounts of vision which suggest that the brain uses internal models of the world to predict sensory inputs, with cortical feedback processing in sensory areas encoding beliefs about those inputs. As such, acquiring knowledge should enhance our internal models that we use to resolve sensory ambiguities, and feedback signals should encode more accurate estimates of sensory inputs. We used partially occluded Mooney images, ambiguous two-tone images which are difficult to recognise without prior knowledge of the image content, in behavioural and 3T fMRI experiments to measure if contextual feedback signals in early visual areas are modulated by learning. We show that perceptual priors add sensory detail to contextual feedback processing in early visual areas in response to subsequent presentations of previously ambiguous images.

neuroscience↗

bioRxiv2023

Structural and Functional Disparities within the Human Gut Virome in terms of Genome Topology and Representative Genome Selection

Circularity confers protection to viral genomes where linearity falls short, thereby fulfilling the form follows function aphorism. Yet, a shift away from morphology-based classification towards molecular and ecological classification of viruses is currently underway within the field of virology. Recent years have seen drastic changes in the ICTV operational definitions of viruses, particularly for those tailed phages that inhabit the human gut. After the abolition of the order Caudovirales, these tailed phages are best defined as members of the class Caudoviricetes, with taxonomic uncertainty prevailing at more granular taxa. In order to determine the epistemological value of genome topology in the context of the human gut virome, we designed a set of seven experiments to assay the impact of genome topology and representative viral selection on biological interpretation. Using Oxford Nanopore long reads for viral genome assembly coupled with Illumina short read polishing, we show that circular and linear virus genomes differ remarkably in terms of genome quality, GC-skew, tRNA gene frequency, structural variant frequency, cross-reference functional annotation (COG, KEGG, Pfam, and TIGRfam), state-of-the-art marker-based classification, and phage-host interaction. The disparity profile furthermore changes during dereplication. In particular, our phage-host interaction results demonstrate that proportional abundances are incomparable without due regard for genome topology and dereplication threshold, which necessitates the need for standardized reporting. As a best practice guideline, we recommend that comparative studies of the human gut virome should report the ratio of circular to linear viral genomes ({Delta}CL) along with the dereplication threshold so that structural and functional metrics can be placed into context when assessing biologically relevant metagenomic properties such as proportional abundance.

genomics↗

bioRxiv2023

Single-cell mitochondrial variant enrichment resolved clonal tracking and spatial architecture in human embryonic hematopoiesis

The ability to perform lineage tracing at the single-cell level is critical to reconstructing dynamic transitions during cell differentiation. However, prospective tracing approaches inevitably encounter outstanding challenges including barcoding precision, barcode diversity, and detection efficiency, which can skew inferred lineage relationships. Human pluripotent stem cells (hPSCs) even face risks of DNA-damage-induced toxicity-related cell death. We explored the use of naturally occurring somatic mutations in mitochondrial transcripts detected in single-cell RNA-seq as genetic lineage barcodes in hPSCs. In this study, we used an enrichment of scRNA-seq mitochondrial reads and a robust computational method to identify clonally relevant mitochondrial variants as endogenous genetic barcodes for clonal tracking of early embryonic hematopoiesis from hPSC. We modeled the development of embryonic tissues from hPSCs and delineated cell fate specification by integrating synthetic barcoding with mitochondrial lineage tracing. Using a biophysical model, we reconstructed the sequential transcriptional logic of fate specification and its underlying regulatory network. We further applied mitochondrial lineage tracing to spatial transcriptomics, which enabled us to identify the spatial clonal architecture of human embryonic organoids. Our analysis revealed that this spatial zonation was orchestrated by NOTCH-mediated crosstalk between stromal cells and hematopoietic progenitors. Our multi-modal framework links clonal dynamics with niche-specific fate decisions, providing a generalizable approach to dissecting tissue organization in development and disease. This study underscores the utility of mitochondrial variants as endogenous markers for high-resolution spatial clonal tracking in stem cell-derived organoid models of human development.

developmental biology↗

bioRxiv2023

B Cells Promote T Cell Immunosenescence and Mammalian Aging Parameters

A dysregulated adaptive immune system is a key feature of aging, and is associated with age-related chronic diseases and mortality. Most notably, aging is linked to a loss in the diversity of the T cell repertoire and expansion of activated inflammatory age-related T cell subsets, though the main drivers of these processes are largely unknown. Here, we find that T cell aging is directly influenced by B cells. Using multiple models of B cell manipulation and single-cell omics, we find B cells to be a major cell type that is largely responsible for the age-related reduction of naive T cells, their associated differentiation towards pathogenic immunosenescent T cell subsets, and for the clonal restriction of their T cell receptor (TCR). Accordingly, we find that these pathogenic shifts can be therapeutically targeted via CD20 monoclonal antibody treatment. Mechanistically, we uncover a new role for insulin receptor signaling in influencing age-related B cell pathogenicity that in turn induces T cell dysfunction and a decline in healthspan parameters. These results establish B cells as a pivotal force contributing to age-associated adaptive immune dysfunction and healthspan outcomes, and suggest new modalities to manage aging and related multi-morbidity. One Sentence SummaryInsulin receptor signaling facilitates the induction of age associated B cell inflammatory changes, which drive phenotypic aging of the T cell compartment and adverse outcomes to mammalian healthspan parameters.

immunology↗

bioRxiv2023

Tree diversity increases forest temperature buffering

Global warming is increasing the frequency and intensity of climate extremes. Forests may buffer such extreme events by creating their own microclimate below their canopy via cooling hot and insulating against cold macroclimate air temperatures. This buffering capacity of forests may be increased by tree diversity and may itself maintain forest functioning and biodiversity. However, despite its relevance for many ecosystem processes, the effect of tree diversity on temperature buffering is largely unexplored. Here, we show that tree species richness consistently increases forest temperature buffering across daily, monthly, and annual scales over six years. This finding is based on data from a large-scale tree diversity experiment covering a species richness gradient of 1 to 24 tree species. We found that species richness strengthened both components of forest temperature buffering: the attenuation of hot and of cold macroclimate air temperatures, with the cooling effect being more pronounced. The buffering effect of tree species richness was mediated by canopy density and structural diversity, assessed as leaf area index and stand structural complexity index, respectively. Safeguarding and planting diverse forests may thus mitigate negative effects of global warming and climate extremes on ecosystem functions and communities below the tree canopy.

ecology↗

bioRxiv2023

Simplicity: web-based visualization and analysis of high-throughput cancer cell line screens

High-throughput drug screens are a powerful tool for cancer drug development. However, the results of such screens are often made available only as raw data, which is intractable for researchers without informatic skills, or as highly processed summary statistics, which can lack essential information for translating screening results into clinically meaningful discoveries. To improve the usability of these datasets, we developed Simplicity, a robust and user-friendly web interface for visualizing, exploring, and summarizing raw and processed data from high-throughput drug screens. Importantly, Simplicity allows for easy recalculation of summary statistics at user-defined drug concentrations. This allows Simplicitys outputs to be used with methods that rely on statistics being calculated at clinically relevant doses. Simplicity can be freely accessed at https://oncotherapyinformatics.org/simplicity/.

pharmacology and toxicology↗

bioRxiv2023

Resolution of ring chromosomes, Robertsonian translocations, and complex structural variants from long-read sequencing and telomere-to-telomere assembly

The capacity to resolve structural variants (SVs) at sequence resolution in highly repetitive genomic regions has long been intractable. Consequently, the properties, origins, and functional effects of multiple classes of complex rearrangement are unknown. To resolve these challenges, we leveraged recent technical milestones: 1) Oxford-Nanopore (ONT) sequencing; 2) the gapless Telomere-to-Telomere (T2T) genome assembly; and 3) a novel tool to discover large-scale rearrangements from long-reads. We applied these technologies across 13 patients with ring chromosomes, Robertsonian translocations, and complex balanced SVs that were unresolved by short-read sequencing. We resolved 10 of 13 events, including ring chromosomes, the complex SVs, and a Robertsonian translocation. Multiple breakpoints were localized to highly repetitive regions inaccessible to short-read alignment, such as acrocentric p-arms, ribosomal DNA arrays, and telomeric repeats, and involved complex structures such as a deletion-inversion and interchromosomal dispersed duplications. We also leveraged ONT native methylation detection to discover phased differential methylation in a gene promoter proximal to a ring fusion site, suggesting a long-range positional effect with heterochromatin spreading. Breakpoint sequences were consistent with common mechanisms of SV formation, including microhomology-mediated mechanisms, non-homologous end-joining, and non-allelic homologous recombination. These methods provide some of the first glimpses into the sequence resolution of ring chromosomes and Robertsonian translocations and illuminate the structural diversity of chromosomal rearrangements with implications for molecular diagnosis and genome biology. HighlightsO_LICryptic rearrangements revealed in repetitive and previously inaccessible regions C_LIO_LIAligning long-reads to telomere-to-telomere assembly enabled breakpoint discovery C_LIO_LIFirst sequence resolution of a Robertsonian translocation breakpoint C_LIO_LIHaplotype-specific methylation changes associated with ring formation C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/555775v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@188cef8org.highwire.dtl.DTLVardef@cc2ed5org.highwire.dtl.DTLVardef@116b7e6org.highwire.dtl.DTLVardef@837f73_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

High-throughput, microscopy-based screening, and quantification of genetic elements

Synthetic biology relies on the screening and quantification of genetic components to assemble sophisticated gene circuits with specific functions. Microscopy is powerful tool for characterizing complex cellular phenotypes with increasing spatial and temporal resolution to library screening of genetic elements. Microscopy-based assays are powerful tools for characterizing cellular phenotypes with spatial and temporal resolution, and can be applied to large-scale samples for library screening of genetic elements. However, strategies for high-throughput microscopy experiments remain limited. Here, we present a high-throughput, microscopy-based platform that can simultaneously complete the preparation of an 8x12-well agarose pads plate, allowing for the screening of 96 independent strains or experimental conditions in a single experiment. Using this platform, we screened a library of natural intrinsic promoters from Pseudomonas aeruginosa and identified a small subset of robust promoters that drives stable levels of gene expression under varying growth conditions. Additionally, the platform allowed for single-cell measurement of genetic elements over time, enabling the identification of complex and dynamic phenotypes to map genotype in high-throughput. We expected that the platform could be employed to accelerate the identification and characterization of genetic elements in various biological systems, as well as to understand the relationship between cellular phenotypes and internal states, including genotypes and gene expression programs. Impact statementThe high-throughput microscopy-based platform, presented in this study, enables efficient screening of 96 independent strains or experimental conditions in a single experiment, facilitating the rapid identification of genetic elements with desirable features, thereby advancing synthetic biology. The robust promoters identified through this platform, which provide predictable and consistent control over gene expression under varying growth conditions, can be utilized as reliable tools to regulate gene expression in various biological applications, including synthetic biology, metabolic engineering, and gene therapy, where consistent system performance is required.

molecular biology↗

bioRxiv2023

Cell wall fluidization by mechano-endopeptidases sets up a turgor-mediated volumetric pacemaker

The cell wall is an essential cellular component of bacteria and the target of many antibiotics. However, how bacteria regulate the rate of cell wall biosynthesis as growth rates change remains unresolved. In E. coli, cell wall growth was thought to proceed independently from turgor pressure1, the osmotic pressure that the cytoplasm exerts on the cell wall. Here, we uncover a striking increase of turgor pressure with growth rate. Modulating turgor pressure and measuring cell wall biosynthesis, we find that turgor pressure is directly controls the rate of cell wall biosynthesis. The picture that emerges is that turgor pressure is largely generated by counterions of negatively charged cellular biomass. The increase in turgor pressure with growth rates results from more ribosomes and therefore higher concentrations of negatively charged ribosomal RNA. Elegantly, the coupling between biomass composition, turgor pressure and cell wall biosynthesis simultaneously explains how bacteria achieve homeostasis of cytoplasmic crowding and how they regulate the rate of cell wall biosynthesis across growth rates.

microbiology↗

bioRxiv2023

CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers

Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimized a bacterial CRISPR-hybrid system coupled with FACS, and identified novel high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allowed effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.

synthetic biology↗

bioRxiv2023

Reply to: Contribution of carbon inputs to soil carbon accumulation cannot be neglected

In the accompanying Comment1, He et al. argue that the determinant role of microbial carbon use efficiency in global soil organic carbon (SOC) storage shown in Tao et al. (2023)2 was overestimated because carbon inputs were neglected in our data analysis while they suggest that our model-based analysis could be biased and model-dependent. Their argument is based on a different choice of independent variables in the data analysis and a sensitivity analysis of two process-based models other than that used in our study. We agree that both carbon inputs and outputs (as mediated by microbial processes) matter when predicting SOC storage - the question is their relative contributions. While we encourage further studies to examine how the evaluation of the relative importance of CUE to global SOC storage may vary with different model structures, He et al.s claims about Tao et al. (2023) need to be taken as an alternative, unproven hypothesis until empirical data support their specific parameterization. Here we show that an additional literature assessment of global data does not support He et al.s argument, in contrast to our study, and that further study on this topic is essential.

ecology↗