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Yan, Y.

Publications and source records attributed to Yan, Y..

At least 19 recordsLinked to original sources

Compression Sequencing enables ultra-sensitive and scalable scRNA-seq

Current sequencing methods are inefficient and bottlenecked by repeated sampling of highly abundant molecules, which dominate sequencing reads, limit assay throughput and sensitivity for rare targets. For example, single-cell RNA sequencing (scRNA-seq) can profile up to millions of cells, but remains severely constrained by sequencing cost, resulting in shallow gene coverage and high dropout rate. Here we report an information science-inspired method, Compression Sequencing, that tackles this fundamental inefficiency and enables highly improved (>100x) sequencing power. Our method works by performing an accurate and unbiased logarithmic transform on molecular abundances over a wide (5 logs) dynamic range, thus suppressing high-abundance targets and enriching rare ones, while maintaining quantitative accuracy. Applied to scRNA-seq libraries, our method allows ultra-sensitive detection of low-abundance transcripts (2-5x more UMIs), ultra-low sequencing cost (200x reduction), preserves accurate cell types and differential expression analysis over a 500-2,000 gene panel. In AML clinical samples, Compression Sequencing reproduces clinical diagnosis and additionally allows transcriptomic profiling at affordable cost (est. $10 per sample). Our approach thus enables ultra-sensitive and scalable single-cell analysis for large-scale functional genomics studies, drug discovery screens, AI cell model training, as well as affordable single-cell disease diagnostics.

bioengineering

Drosophila scribble mutant tumors undergo a transition from a growth arrest state to a proliferative state over time

The Drosophila neoplastic tumor suppressor gene (nTSG) mutant tumors have successfully modeled many aspects of human tumor progression. However, the fly nTSG mutant tumors progress rapidly over days. This is in contrast with most human tumors which develop slowly, harbor heterogeneous cell populations for selection and undergo an evolution-like process. Whether the fast-growing fly nTSG mutant tumors have capacity for evolution remains unclear. Through quantitative analysis of the scrib mutant tumor growth, we found that the scrib mutant tumors evolve to display different growth rates and cell cycle profiles over time. Multiple growth-regulatory signaling pathways show quantitative differences in early versus late scrib mutant tumors. These data suggest that the scrib mutant tumors undergo a transition from a growth arrest state to a proliferative state. Through longitudinal single cell RNA (scRNA) data analysis we found that the scrib mutant tumors harbor heterogeneous cell populations likely of distinct proliferative states, which are available for potential selection. This study raises the possibility of studying tumor evolution in a genetically accessible and fast-growing invertebrate tumor model.

developmental biology

Quantitative Proteomic Analysis of Prostate Tissue Specimens Identifies Deregulated ProteinComplexes in Primary Prostate Cancer

Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and a leading cause of mortality among males in developed countries. However, our understanding of the global changes of protein complexes within PCa tissue specimens remains very limited, although it has been well recognized that protein complexes carry out essentially all major processes in living organisms and that their deregulation drives the pathogenesis and progression of various diseases. By coupling tandem mass tagging-synchronous precursor selection-mass spectrometry/mass spectrometry/mass spectrometry (TMT-SPSMS3) with differential expression and co-regulation analyses, the present study compared the differences between protein complexes in normal prostate, low-grade PCa, and high-grade PCa tissue specimens. Globally, a large downregulated putative protein-protein interaction (PPI) network was detected in both low-grade and high-grade PCa, yet a large upregulated putative PPI network was only detected in high-grade but not low-grade PCa, compared with normal controls. To identify specific protein complexes that are deregulated in PCa, quantified proteins were mapped to protein complexes in CORUM, a collection of experimentally verified mammalian protein complexes. Differential expression analysis suggested that mitochondrial ribosomes and the fibrillin-associated protein complex were significantly overexpressed, whereas the ITGA6-ITGB4-Laminin10/12 and the P2X7 receptor signaling complexes were significantly downregulated, in PCa compared with normal prostate. Moreover, differential co-regulation analysis indicated that the assembly levels of some nuclear protein complexes involved in RNA synthesis and processing were significantly increased in low-grade PCa, and those of mitochondrial complex I and its subcomplexes were significantly increased in high-grade PCa, compared with normal prostate. In summary, the study represents the first global and quantitative comparison of protein complexes in prostate tissue specimens. It is expected to enhance our understanding of the molecular mechanisms underlying PCa development and progression in human patients, as well as lead to the discovery of novel biomarkers and therapeutic targets for precision management of PCa.

cancer biology

Robust Algorithms for Capturing Population Dynamics and Transport in Oceanic Variables along Drifter Trajectories using Linear Dynamical Systems with Latent Variables

The blooms of Noctiluca in the Gulf of Oman and the Arabian Sea have been intensifying in recent years posing a threat to regional fisheries and the long-term health of an ecosystem supporting a coastal population of nearly 120 million people. We present the results of a microscopic data analysis to investigate the onset and patterns of the Noctiluca (mixotrophic dinoflagellate Noctiluca scintillans) blooms, which form annually during the winter monsoon in the Gulf of Oman and in the Arabian Sea. Our approach combines methods in physical and biological oceanography with machine learning techniques. In particular, we present a robust algorithm, the variable-length Linear Dynamic Systems (vLDS) model, that extracts the causal factors and latent dynamics at the microscopic population-level along each individual drifter trajectory, and demonstrate its effectiveness by using it to test and confirm previously benchmarked macroscopic scientific hypotheses. The test results provide microscopic statistical evidence to support and recheck the macroscopic physical and biological Oceanography hypotheses on the Noctiluca blooms; it also helps identify complementary microscopic dynamics that might not be visible or discoverable at the macroscopic scale. The vLDS model also exhibits a generalization capability (inherited from a machine learning methodology) to investigate important causal factors and hidden dynamics associated with ocean biogeochemical processes and phenomena at the population-level.

ecology

Functional importance of JMY expression by Sertoli cells in mediating mouse spermatogenesis

Sertoli cells are crucial for spermatogenesis in the seminiferous epithelium because their actin cytoskeleton supports vesicle transport, cell junction, protein anchoring and spermiation. Here, we show that junction-mediating and regulatory protein (JMY), an actin regulating protein, also affects endocytic vesicle trafficking and Sertoli cell junction remodeling since disruption of these functions induced male subfertility in Sertoli cell-specific Jmy knockout mice. Specifically, these mice have: a) impaired BTB integrity and spermatid adhesion in the seminiferous tubules; b) high incidence of sperm structural deformity; c) reduced sperm count and poor sperm motility. Moreover, the cytoskeletal integrity in Sertoli cell-specific Jmy knockout mice was compromised along with endocytic vesicular trafficking. These effects impaired junctional protein recycling and reduced Sertoli cell junctions. In addition, JMY interaction with -actinin1 and Sorbs2 was related to JMY activity and in turn actin cytoskeletal organization. In summary, JMY affects control of spermatogenesis through regulating actin filament organization and endocytic vesicle trafficking in Sertoli cells.

cell biology

Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae

The bacterial CRISPR/Cas genome editing system has provided a major breakthrough in molecular biology. One use of this technology is within a nuclease-based gene drive. This type of system can install a genetic element within a population at unnatural rates. Combatting of vector-borne diseases carried by metazoans could benefit from a delivery system that bypasses traditional Mendelian laws of segregation. Recently, laboratory studies in fungi, insects, and even mice, have demonstrated successful propagation of CRISPR gene drives and the potential utility of this type of mechanism. However, current gene drives still face challenges including evolved resistance, containment, and the consequences of application in wild populations. In this study, we use an artificial gene drive system in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences on Cas9 fusion proteins in vivo and demonstrate that appended signals can titrate gene drive activity and serve as a potential molecular safeguard.

synthetic biology

Cancer archetypes co-opt and adapt the transcriptional programs of existing cellular states

Transcriptional profiling has revealed a diverse range of cancer cell states, however an understanding of their function has remained elusive. Using a combination of zebrafish melanoma modeling and human validation, we have identified a conserved stress-like state that confers intrinsic drug resistance. The stress-like state expresses genes such as fos, hsp70 and ubb, all required for adaptation to diverse cellular stresses, and we confirmed its existence using immunofluorescence and spatial transcriptomics. We provide evidence that this state has a higher tumor seeding capabilities compared to non-stressed cells, and confers intrinsic resistance to MEK inhibitors, a commonly used melanoma therapeutic. Furthermore, the stress-like program can be induced by extrinsic processes such as heat shock, and confers resistance to both MEK and BRAF inhibitors in both zebrafish and human melanomas. Collectively, our study suggests that the transcriptional states associated with therapeutic failure are established during the earliest steps of tumorigenesis.

cancer biology

Development of a multi-locus CRISPR gene drive system in budding yeast

The discovery of CRISPR/Cas gene editing has allowed for major advances in many biomedical disciplines and basic research. One arrangement of this biotechnology, a nuclease-based gene drive, can rapidly deliver a genetic element through a given population and studies in fungi and metazoans have demonstrated the success of such a system. This methodology has the potential to control biological populations and contribute to eradication of insect-borne diseases, agricultural pests, and invasive species. However, there remain challenges in the design, optimization, and implementation of gene drives including concerns regarding biosafety, containment, and control/inhibition. Given the numerous gene drive arrangements possible, there is a growing need for more advanced designs. In this study, we use budding yeast to develop an artificial multi-locus gene drive system. Our minimal setup requires only a single copy of S. pyogenes Cas9 and three guide RNAs to propagate three separate gene drives. We demonstrate how this system could be used for targeted allele replacement of native genes and to suppress NHEJ repair systems by modifying DNA Ligase IV. A multi-locus gene drive configuration provides an expanded suite of options for complex attributes including pathway redundancy, combatting evolved resistance, and safeguards for control, inhibition, or reversal of drive action.

synthetic biology

Recent mixing of Vibrio parahaemolyticus populations

BackgroundHumans have profoundly affected the ocean environment but little is known about anthropogenic effects on the distribution of microbes. Vibrio parahaemolyticus is found in warm coastal waters and causes gastroenteritis in humans and economically significant disease in shrimps.\n\nResultsBased on data from 1,103 genomes, we show that V. parahaemolyticus is divided into four diverse populations, VppUS1, VppUS2, VppX and VppAsia. The first two are largely restricted to the US and Northern Europe, while the others are found worldwide, with VppAsia making up the great majority of isolates in the seas around Asia. Patterns of diversity within and between the populations are consistent with them having arisen by progressive divergence via genetic drift during geographical isolation. However, we find that there is substantial overlap in their current distribution. These observations can be reconciled without requiring genetic barriers to exchange between populations if dispersal between oceans has increased dramatically in the recent past. We found that VppAsia isolates from the US have an average of 1.01% more shared ancestry with VppUS1 and VppUS2 isolates than VppAsia isolates from Asia itself. Based on time calibrated trees of divergence within epidemic lineages, we estimate that recombination affects about 0.017% of the genome per year, implying that the genetic mixture has taken place within the last few decades.\n\nConclusionsThese results suggest that human activity, such as shipping and aquatic products trade, are responsible for the change of distribution pattern of this marine species.

microbiology

Base pair editing of goat embryos: nonsense codon introgression into FGF5 to improve cashmere yield

The ability to alter single bases without DNA double strand breaks provides a potential solution for multiplex editing of livestock genomes for quantitative traits. Here, we report using a single base editing system, Base Editor 3 (BE3), to induce nonsense codons (C-to-T transitions) at four target sites in caprine FGF5. All five progenies produced from microinjected single-cell embryos had alleles with a targeted nonsense mutation and yielded expected phenotypes. The effectiveness of BE3 to make single base changes varied considerably based on sgRNA design. Also, the rate of mosaicism differed between animals, target sites, and tissue type. PCR amplicon and whole genome resequencing analyses for off-target changes caused by BE3 were low at a genome-wide scale. This study provides first evidence of base editing in livestock, thus presenting a potentially better method to introgress complex human disease alleles into large animal models and provide genetic improvement of complex health and production traits in a single generation.

genetics

A novel histone H4 variant regulates rDNA transcription in breast cancer

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. H4G expression was found in a variety of cell lines and was particularly overexpressed in the tissues of breast cancer patients. H4G was found to localize primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha helix 3 of the histone fold motif with the histone chaperone, nucleophosphomin 1. In addition, we found that H4G nucleolar localization increased rRNA levels, protein synthesis rates, and cell cycle progression. Furthermore, micrococcal nuclease digestion of H4G-containing nucleosomes reconstituted in vitro indicated that H4G destabilizes the nucleosome, which may serve to alter nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

molecular biology

A Recurrent Neural Network Approach for Automated Neural Tracing in Multispectral 3D Images

Neuronal morphology reconstruction in fluorescence microscopy 3D images is essential for analyzing neuronal cell type and connectivity. Manual tracing of neurons in these images is time consuming and subjective. Automated tracing is highly desired yet is one of the foremost challenges in computational neuroscience. The multispectral labeling technique, Brainbow utilizes high dimensional spectral information to distinguish intermingled neuronal processes. It is particular interesting to develop new algorithms to include the spectral information into the tracing process. Recently, deep learning approaches achieved state-of-the-art in different computer vision and medical imaging applications. To benefit from the power of deep learning, in this paper, we propose an automated neural tracing approach in multispectral 3D Brainbow images based on recurrent neural net-work. We first adopt VBM4D approach to denoise multispectral 3D images. Then we generate cubes as training samples along the ground truth, manually traced paths. These cubes are the input to the recur-rent neural network. The proposed approach is simple and effective. The approach can be implemented with the deep learning toolbox Keras in 100 lines. Finally, to evaluate our approach, we computed the average and standard deviation of DIADEM metric from the ground truth results to our tracing results, and from our tracing results to the ground truth results. Extensive experimental results on the collected dataset demonstrate that the proposed approach performs well in Brainbow labeled mouse brain images.

neuroscience

B7-H1(PD-L1) confers chemoresistance through ERK and p38 MAPK pathway in tumor cells

Development of resistance to chemotherapy and immunotherapy is a major obstacle in extending the survival of patients with cancer. Although several molecular mechanisms have been identified that can contribute to chemoresistance, the role of immune checkpoint molecules in tumor chemoresistance remains underestimated. It has been recently observed that overexpression of B7-H1(PD-L1) confers chemoresistance in human cancers, however the underlying mechanisms are unclear. Here we show that the development of chemoresistance depends on the increased activation of ERK pathway in tumor cells overexpressing B7-H1. Conversely, B7-H1 deficiency renders tumor cells susceptible to chemotherapy in a cell-context dependent manner through activation of the p38 MAPK pathway. B7-H1 in tumor cells associates with the catalytic subunit of a DNA-dependent serine / threonine protein kinase (DNA-PKcs). DNA-PKcs is required for the activation of ERK or p38 MAPK in tumors expressing B7-H1, but not in B7-H1 negative or B7-H1 deficient tumors. Ligation of B7-H1 by anti-B7-H1 monoclonal antibody (H1A) increased the sensitivity of human triple negative breast tumor cells to cisplatin therapy in vivo. Our results suggest that B7-H1(PD-L1) expression in cancer cells modifies their chemosensitivity towards certain drugs and targeting B7-H1 intracellular signaling pathway is a new way to overcome cancer chemoresistance.

cancer biology

Acidic microenvironment shaped by lactate accumulation promotes pluripotency through multiple mechanisms

Enhanced glycolysis is a distinct feature associated with numerous stem cells and cancer cells. However, little is known about its regulatory roles in gene expression and cell fate determination. Here we show that acidic environment shaped by lactate accumulation promotes the self-renewal and pluripotency of both mouse and human embryonic stem cells (ESCs). Mechanistically, acidic pH reduces the tri-methylation of H3K27 globally at transcriptional start sites to partially prevent ESC differentiation. In addition, acidic pH stabilizes a large number of mRNAs including pluripotency genes. Furthermore, we found that AGO1 protein is downregulated at acidic conditions, leading to the de-repression of a subset of microRNA targets in low-pH treated ESCs. Altogether, our study provides insights into mechanisms whereby acidic microenvironment produced by enhanced glycolysis regulates gene expression to determine cell fate and has broad implications in the fields of regenerative medicine and cancer biology.

developmental biology

Widespread transcriptional scanning in testes modulates gene evolution rates

A long-standing question in molecular biology relates to why the testes express the largest number of genes relative to all other organs. Here, we report a detailed gene expression map of human spermatogenesis using single-cell RNA-Seq. Surprisingly, we found that spermatogenesis-expressed genes contain significantly fewer germline mutations than unexpressed genes, with the lowest mutation rates on the transcribed DNA strands. These results suggest a model of transcriptional scanning to reduce germline mutations by correcting DNA damage. This model also explains the rapid evolution in sensory- and immune-defense related genes, as well as in male reproduction genes. Collectively, our results indicate that widespread expression in the testes achieves a dual mechanism for maintaining the DNA integrity of most genes, while selectively promoting variation of other genes.

evolutionary biology

Nicotinic cholinergic receptors in VTA glutamate neurons modulate excitatory transmission

Ventral tegmental area (VTA) glutamate neurons are important components of brain reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used an array of molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. VTA neurons positive for the vesicular glutamate transporter 2 (VGLUT2+) are responsive to acetylcholine (ACh) released from mesopontine cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise typical heteromeric nAChRs. Electrophysiology, coupled with 2-photon microscopy and laser flash photolysis of a photoactivatable nicotine probe, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2+ neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits demonstrated that nicotine potently modulates excitatory transmission within the VTA. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.

neuroscience

bcGST - an interactive bias-correction method to identify over-represented gene-sets in boutique arrays

Motivation: Gene annotation and pathway databases such as Gene Ontology and Kyoto Encyclopedia of Genes and Genomes are important tools in Gene Set Test (GST) that describe gene biological functions and associated pathways. GST aims to establish an association relationship between a gene set of interest and an annotation. Importantly, GST tests for over-representation of genes in an annotation term. One implicit assumption of GST is that the gene expression platform captures the complete or a very large proportion of the genome. However, this assumption is neither satisfied for the increasingly popular boutique array nor the custom designed gene expression profiling platform. Specifically, conventional GST is no longer appropriate due to the gene set selection bias induced during the construction of these platforms.\n\nResults: We propose bcGST, a bias-corrected Gene Set Test by introducing bias correction terms in the contingency table needed for calculating the Fishers Exact Test (FET). The adjustment method works by estimating the proportion of genes captured on the array with respect to the genome in order to assist filtration of annotation terms that would otherwise be falsely included or excluded. We illustrate the practicality of bcGST and its stability through multiple differential gene expression analyses in melanoma and TCGA cancer studies.\n\nAvailability: The bcGST method is made available as a Shiny web application at http://shiny.maths.usyd.edu.au/bcGST/\n\nContact: kevin.wang@sydney.edu.au

bioinformatics

Neuronal tracing and analysis by multispectral tracing in densely labeled mouse brain

Accurate and complete neuronal wiring diagrams are necessary for understanding brain function at many scales from long-range interregional projections to microcircuits. Traditionally, light microscopy-based anatomical reconstructions use monochromatic labeling and therefore necessitate sparse labeling to eliminate tracing ambiguity between intermingled neurons. Consequently, our knowledge of neuronal morphology has largely been based on averaged estimations across many samples. Recently developed second-generation Brainbow tools promise to circumvent this limitation by revealing fine anatomical details of many unambiguously identifiable neurons in densely labeled samples. Yet, a means to quantify and analyze the information is currently lacking. Therefore, we developed nTracer, an ImageJ plugin capable of rapidly and accurately reconstructing whole-cell morphology of large neuronal populations in densely labeled brains.

neuroscience