Biology subjects

Yin, P.

Publications and source records attributed to Yin, P..

50 records · Page 3Linked to original sources

bioRxiv2020

A DNA nanoscope that identifies and precisely localizes over a hundred unique molecular features with nanometer accuracy

Techniques that can both spatially map out molecular features and discriminate many targets would be highly valued for their utility in studying fundamental nanoscale processes. In spite of decades of development, no current technique can achieve both nanoscale resolution and discriminate hundreds of targets. Here, we report the development of a novel bottom-up technology that: (a) labels a sample with DNA barcodes, (b) measures pairwise-distances between labeled sites and writes them into DNA molecules, (c) reads the pairwise-distances by sequencing and (d) robustly integrates this noisy information to reveal the geometry of the underlying sample. We demonstrate our technology on DNA origami, which are complex synthetic nanostructures. We both spatially localized and uniquely identified over a hundred densely packed unique elements, some spaced just 6 nm apart, with an average spatial localization accuracy (RMS deviation) of ~2 nm. The bottom-up, sequencing-enabled mechanism of the DNA nanoscope is fundamentally different from top-down imaging, and hence offers unique advantages in precision, throughput and accessibility.

biochemistry↗

bioRxiv2020

Molecular surveillance of HIV, HBV, and HCV amongst blood donors in five Chinese regions

Hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV) are transfusion transmissible infections (TTIs) agents that threaten the safety of the blood supply. Surveillance of the variance of those viruses is an important way to monitor their diversity and evolution to improve safety in blood transfusion. In this study, we characterized the specimens of blood donors from 13 blood centers located in 5 Chinese regions. Samples collected between 2014 and 2017 were screened with serological and molecular tests conducted on Abbott ARCHITECT and m2000 platforms. Sequencing was used to determine the classifications. The HBV immune escape mutations were also analyzed for assessing vaccine breakthrough risks and challenges for diagnostic tests. For HIV, 11 genotypes or recombinants were identified. The predominant genotype was C, which accounts for 42%. For HBV, the genotypes of B, C and D were identified, with B and C predominating. The major subgenotype was B2, comprising 84.1% of all infections. 79 out of 113 (69.9%) samples carried escape mutations in the "a" determinant region with 69 (87.3%) multiple mutants and 15 (19%) escape mutants which will affect HBsAg detection. For HCV, 7 genotypes or subtypes were identified. The major genotype was 1b (48%), followed by 6a (16.7%) and 2a & 3a (10%). This study provides the information of diversity of HBV, HCV and HIV strains circulating in blood centers from 5 regions in China. These data can also be scientific basis for development of detection assays that mitigate the impact of viral diversity on performance. ImportanceThe prevalence of TTIs in blood donations is important for evaluating blood safety and it can also reflect the burden of these disease among populations. Virus variance is threat to blood safety due to it may affect assays detection by nucleic acid, antigen and antibody-based methods in blood donors. HIV, HBV and HCV exhibit high degrees of genetic diversity, with different strains predominating in different geographic locations. The aim of this study is to assess the diversity of HBV, HCV and HIV among blood donors in China. In this study, 13 blood centers located in 5 Chinese regions were involved and the most informative phylogenetic regions of each virus had been sequenced. This will benefit for viral monitoring by subtype/genotype analyses to determine whether the distributions of variants are changing over time and geographically, and to speculate whether previously rare subtypes are becoming established in blood donors in China.

genetics↗

bioRxiv2019

Cryo-EM Structure of the Human Mitochondrial Translocase TIM22 Complex

Mitochondria play vital functions in cellular metabolism, homeostasis, and apoptosis1-3. Most of the mitochondrial proteins are synthesized as precursors in the cytosol and imported into mitochondria for folding or maturation4,5. The translocase TIM22 complex is responsible for the import of multiple hydrophobic carrier proteins that are then folded in the inner membrane of mitochondria6-8. In mammalian cells, the TIM22 complex consists of at least six components, Tim22, Tim29, AGK, and three Tim chaperones (Tim9, Tim10a and Tim10b)9-14. Here, we report the cryo-EM structure of the human translocase TIM22 complex at an overall resolution of 3.7 angstrom. The core subunit, Tim22, contains four transmembrane helices, forming a partial pore that is open to the lipid bilayer. Tim29 is a single transmembrane protein that provides an N-terminal helix to stabilize Tim22 and a C-terminal intermembrane space (IMS) domain to connect AGK and two TIM chaperone hexamers to maintain complex integrity. One TIM hexamer comprises Tim9 and Tim10a in a 3:3 molar ratio, and the other consists of two Tim9 units, three Tim10a units, and one Tim10b unit. The latter hexamer faces the intramembrane region of Tim22, likely providing the dock to load the precursors to the partial pore of Tim22. Our structure serves as a molecular basis for the mechanistic understanding of TIM22 complex function.

biophysics↗

bioRxiv2019

Integrating genome-wide association and transcriptome predicted model identify novel target genes with osteoporosis

Osteoporosis (OP) is a highly polygenetic disease which is usually characterized by low bone mineral density. Genome-wide association studies (GWAS) have identified hundreds of genetic loci associated with bone mineral density. However, the biological mechanisms of these loci remain elusive. To identify potential causal genes of the associated loci, we detected trait-gene expression associations by transcriptome-wide association study (TWAS) method. It directly imputes gene expression effects from GWAS data, using a statistical prediction model trained on GTEx reference transcriptome data, with blood and skeletal tissues data. Then we performed a colocalization analysis to evaluate the posterior probability of biological patterns: association characterized by a single shared causal variant or two distinct causal variants. The ultimate analysis identified 276 candidate genes, including 3 novel loci, 204 novel candidate genes and 69 replicated from GWAS. The 3 novel loci located at chr6: 72417543, chr15: 69601206, chr21: 30530692, mapping to gene RIMS1, SPESP1, MAP3K7CL. The results of colocalization analysis indicated that 142 of them showing strong evidence of a single shared causal variant and 134 of them showing evidence of joint causal variants. Their biological function was directly or indirectly associated with the occurrence of OP validated by VarElect tool. Several important OP-associated pathways were detected by protein-protein interaction and pathway enrichment analysis. Target genes were further enriched for differential expression genes in osteoblasts expression profiles, e.g. IBSP, affecting calcium and hydroxyapatite binding, and CD44, regulating alternative splicing of gene transcription. Transcriptome fine-mapping identifies more disease-related genes and provide additional insight into the development of novel targeted therapeutics to treat OP.

genetics↗

bioRxiv2019

Three dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval

Single-molecule localization microscopy is a powerful tool in visualizing organelle structures, interactions, and protein functions in biological research. However, whole-cell and tissue specimens challenge the achievable resolution and depth of nanoscopy methods. As imaging depth increases, photons emitted by fluorescent probes, the sole source of molecular positions, were scattered and aberrated, resulting in image artifacts and rapidly deteriorating resolution. We propose a method to allow constructing the in situ 3D response of single emitters directly from single-molecule dataset and therefore allow pin-pointing single-molecule locations with limit-achieving precision and uncompromised fidelity through whole cells and tissues. This advancement expands the routine applicability of super-resolution imaging from selected cellular targets near coverslips to intra- and extra-cellular targets deep inside tissues. We demonstrate this across a range of cellular-tissue architectures from mitochondrial networks, microtubules, and nuclear pores in 2D and 3D cultures, amyloid-{beta} plaques in mouse brains to developing cartilage in mouse forelimbs.

biophysics↗

bioRxiv2019

Rapid and scalable in vitro production of single-stranded DNA

We present a rapid, scalable, user-friendly method for in vitro production of high-purity single-stranded DNA (ssDNA) ranging from 89-3315 nucleotides in length. PCR with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology-directed repair in human cells, DNA-origami folding, and fluorescent in situ hybridization.

molecular biology↗

bioRxiv2018

Highly multiplexed in situ protein imaging with signal amplification by Immuno-SABER

Probing the molecular organization of tissues requires in situ analysis by microscopy. However current limitations in multiplexing, sensitivity, and throughput collectively constitute a major barrier for comprehensive single-cell profiling of proteins. Here, we report Immunostaining with Signal Amplification By Exchange Reaction (Immuno-SABER), a rapid, highly multiplexed signal amplification method that simultaneously tackles these key challenges. Immuno-SABER utilizes DNA-barcoded antibodies and provides a method for highly multiplexed signal amplification via modular orthogonal DNA concatemers generated by Primer Exchange Reaction. This approach offers the capability to preprogram and control the amplification level independently for multiple targets without in situ enzymatic reactions, and the intrinsic scalability to rapidly amplify and image a large number of protein targets. We validated our approach in diverse sample types including cultured cells, cryosections, FFPE sections, and whole mount tissues. We demonstrated independently tunable 5-180-fold amplification for multiple targets, covering the full signal range conventionally achieved by secondary antibodies to tyramide signal amplification, as well as simultaneous signal amplification for 10 different proteins using standard equipment and workflow. We further combined Immuno-SABER with Expansion Microscopy to enable rapid and highly multiplexed super-resolution tissue imaging. Overall, Immuno-SABER presents an effective and accessible platform for rapid, multiplexed imaging of proteins across scales with high sensitivity.

cell biology↗

bioRxiv2018

De-Novo-Designed Translational Repressors for Multi-Input Cellular Logic

Synthetic biology aims to apply engineering principles toward the development of novel biological systems for biotechnology and medicine. Despite efforts to expand the set of high-performing parts for genetic circuits, achieving more complex circuit functions has often been limited by the idiosyncratic nature and crosstalk of commonly utilized parts. Here, we present a molecular programming strategy that implements RNA-based repression of translation using de-novo-designed RNAs to realize high-performance orthogonal parts with mRNA detection and multi-input logic capabilities. These synthetic post-transcriptional regulators, termed toehold repressors and three-way junction (3WJ) repressors, efficiently suppress translation in response to cognate trigger RNAs with nearly arbitrary sequences using thermodynamically and kinetically favorable linear-linear RNA interactions. Automated in silico optimization of thermodynamic parameters yields improved toehold repressors with up to 300-fold repression, while in-cell SHAPE-Seq measurements of 3WJ repressors confirm their designed switching mechanism in living cells. Leveraging the absence of sequence constraints, we identify eight- and 15-component sets of toehold and 3WJ repressors, respectively, that provide high orthogonality. The modularity, wide dynamic range, and low crosstalk of the repressors enable their direct integration into ribocomputing devices that provide universal NAND and NOR logic capabilities and can perform multi-input RNA-based logic. We demonstrate these capabilities by implementing a four-input NAND gate and the expression NOT((A1 AND A2) OR (B1 AND B2)) in Escherichia coli. These features make toehold and 3WJ repressors important new classes of translational regulators for biotechnological applications.

synthetic biology↗

bioRxiv2018

SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

Fluorescent in situ hybridization (FISH) reveals the abun-dance and positioning of nucleic acid sequences in fixed sam-ples and can be combined with cell segmentation to produce a powerful single cell gene expression assay. However, it re-mains difficult to label more than a few targets and to visu-alize nucleic acids in environments such as thick tissue sam-ples using conventional FISH technologies. Recently, meth-ods have been developed for multiplexed amplification of FISH signals, yet it remains challenging to achieve high lev-els of simultaneous multiplexing combined with high sam-pling efficiency and simple workflows. Here, we introduce signal amplification by exchange reaction (SABER), which endows oligo-based FISH probes with long, single-stranded DNA concatemers that serve as targets for sensitive fluores-cent detection. We establish that SABER effectively ampli-fies the signal of probes targeting nucleic acids in fixed cells and tissues, can be deployed against at least 17 targets si-multaneously, and detects mRNAs with high efficiency. As a demonstration of the utility of SABER in assays involv-ing genetic manipulations, we apply multiplexed FISH of reporters and cell type markers to the identification of en-hancers with cell type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit to allow rapid and cost effective multiplexed imaging.

genetics↗

bioRxiv2018

Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

Chromosome structure is thought to be crucial for proper functioning of the nucleus. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. We begin by applying Oligopaint probes and the single-molecule localization microscopy methods of OligoSTORM and OligoDNA-PAINT to image 8 megabases of human chromosome 19, discovering that chromosomal regions contributing to compartments can form distinct structures. Intriguingly, our data also suggest that homologous maternal and paternal regions may be differentially organized. Finally, we integrate imaging data with Hi-C and restraint-based modeling using a method called integrative modeling of genomic regions (IMGR) to increase the genomic resolution of our traces to 10 kb.\n\nOne Sentence SummarySuper-resolution genome tracing, contact maps, and integrative modeling enable 10 kb resolution glimpses of chromosome folding.

genetics↗

bioRxiv2017

Task Engagement Enhances Population Encoding of Stimulus Meaning in Primary Auditory Cortex

The main functions of primary sensory cortical areas are classically considered to be the extraction and representation of stimulus features. In contrast, higher cortical sensory association areas are thought to be responsible for combining these sensory representations with internal motivations and learnt associations. These regions generate appropriate neural responses that are maintained until a motor command is executed. Within this framework, responses of the primary sensory areas during task performance are expected to carry less information about the behavioral meaning of the stimulus than higher sensory, association, motor and frontal cortices. Here we demonstrate instead that the neuronal population responses in the early primary auditory cortex (A1) display many aspects of responses generally associated with higher-level areas. A1 activity was recorded in awake ferrets while they were either passively listening or actively discriminating two periodic click trains of different rates in a Go/No-Go paradigm. By applying population-level dimensionality reduction techniques, we found that task-engagement induced a shift in the nature of the encoding from a sensory-driven representation of the two stimuli to a behaviorally relevant representation of the two categories that specifically enhances the target stimulus. We demonstrate that this shift in encoding relies partly on a novel mechanism of change in spontaneous activity patterns upon engagement in the task. We show that this population-level representation of stimuli in A1 population activity bears strong similarities to responses in the frontal cortex, but appears earlier following stimulus presentation. Analysis of neural activity recorded in various Go/No-Go tasks, with different sounds and reinforcement paradigms, reveals that this striking population-level enhancement of target representation is a general property of task engagement. These findings indicate that primary sensory cortices play a highly flexible role in the processing of incoming stimuli and implement a crucial change in the structure of population activity in order to extract task-relevant information during behavior.

neuroscience↗

bioRxiv2017

MeDEStrand: an improved method to infer genome-wide absolute methylation level from DNA enrichment experiment

BackgroundDNA methylation of dinucleotide CpG is an essential epigenetic modification that plays a key role in transcription. Bisulfite conversion method is a \"gold standard\" for DNA methylation profiling that provides single nucleotide resolution. However, whole-genome bisulfite conversion is very expensive. Alternatively, DNA enrichment-based methods offer high coverage of methylated CpG dinucleotides with the lowest cost per CpG covered genome-wide and have been used widely. They measure the DNA enrichment of methyl-CpG binding, therefore do not directly provide absolute methylation levels. Further, the enrichment is influenced by confounding factors besides the methylation status, e.g., CpG density. Computational models that can accurately derive the absolute methylation levels from the enrichment data are necessary.\n\nResultsWe present MeDEStrand, a method uses sigmoid function to estimate and correct the CpG bias from the numbers of reads that fell within bins that divide the genome. In addition, unlike the previous methods, which estimate CpG bias based on reads mapped at the same genomic loci, MeDEStrand processes the reads for the positive and negative DNA strands separately. We compare the performance of MeDEStrand with three other state-of-the-art methods MEDIPS, BayMeth and QSEA on four independent datasets generated using immortalized cell lines (GM12878 and K562) and human patient primary cells (foreskin fibroblast and mammary epithelial). Based on the comparison between the inferred absolute methylation levels from MeDIP-seq and the corresponding RRBS data, MeDEStrand shows the best performance at high resolution of 25, 50 and 100 base pairs.\n\nConclusions MeDEStrand benefits from the estimation of CpG bias with a sigmoid function and the procedure to process reads mapped to the positive and negative DNA strands separately. MeDEStrand is a tool to infer whole-genome absolute DNA methylation level at the cost of enrichment-based methods with adequate accuracy and resolution. R package MeDEStrand and its tutorial is freely available for download at https://github.com/jxu1234/MeDEStrand.git

bioinformatics↗

bioRxiv2017

OligoMiner: A rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes

Oligonucleotide (oligo)-based fluorescence in situ hybridization (FISH) has emerged as an important tool for the study of chromosome organization and gene expression and has been empowered by the commercial availability of highly complex pools of oligos. However, a dedicated bioinformatic design utility has yet to be created specifically for the purpose of identifying optimal oligo FISH probe sequences on the genome-wide scale. Here, we introduce OligoMiner, a rapid and robust computational pipeline for the genome-scale design of oligo FISH probes that affords the scientist exact control over the parameters of each probe. Our streamlined method uses standard bioinformatic file formats, allowing users to seamlessly integrate existing and new utilities into the pipeline as desired, and introduces a novel method for evaluating the specificity of each probe molecule that connects simulated hybridization energetics to rapidly generated sequence alignments using supervised learning. We demonstrate the scalability of our approach by performing genome-scale probe discovery in numerous model organism genomes and showcase the performance of the resulting probes with both diffraction-limited and single-molecule super-resolution imaging of chromosomal and RNA targets. We anticipate this pipeline will make the FISH probe design process much more accessible and will more broadly facilitate the design of pools of hybridization probes for a variety of applications.

bioinformatics↗

bioRxiv2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 {degrees}C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

bioengineering↗