Biology subjects

Huang, Y.

Publications and source records attributed to Huang, Y..

At least 901 records · Page 50Linked to original sources

bioRxiv2020

A new role for histone demethylases in the maintenance of plant genome integrity

Histone modifications deposited by the Polycomb repressive complex 2 (PRC2) play a critical role in the control of growth, development and adaptation to environmental fluctuations in most multicellular eukaryotes. The catalytic activity of PRC2 is counteracted by Jumonji-type (JMJ) histone demethylases, which shapes the genomic distribution of H3K27me3. Here, we show that two JMJ histone demethylases in Arabidopsis, EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play distinct roles in H3K27me3 and H3K27me1 homeostasis. We show that failure to reset these chromatin marks during sexual reproduction results in the inheritance of epigenetic imprints, which cause a loss of DNA methylation at heterochromatic loci and transposon activation. Thus, Jumonji-type histone demethylases in plants contribute towards maintaining distinct transcriptional states during development and help safeguard genome integrity following sexual reproduction.

plant biology↗

bioRxiv2020

An in vitro vesicle formation assay to analyze protein sorting in the secretory transport pathway

The fidelity of protein transport in the secretory transport pathway relies on the accurate sorting of proteins to their correct destination. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on a specific cargo sorting machinery to be efficiently packaged into vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry analyses of the isolated vesicles revealed novel cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner or that interact with GTP-bound Sar1A on vesicle membranes. Functional analysis indicates that two of them, FAM84B and PRRC1, regulate anterograde trafficking. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Moreover, our results indicate that vesicles enriched with a specific cargo protein contain specific transmembrane cargo and SNARE proteins. A SNARE protein, Vti1B, is identified to be in vesicles enriched with a planar cell polarity protein, Frizzled6, and promotes vesicular release of Frizzled6. Our results indicate that the vesicle formation assay in combination with quantitative mass spectrometric analysis is a robust and powerful tool to reveal novel cytosolic and transmembrane proteins that regulate trafficking of a specific cargo protein.

cell biology↗

bioRxiv2020

Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles

Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

neuroscience↗

bioRxiv2020

Long-read Individual-molecule Sequencing Reveals CRISPR-induced Genetic Heterogeneity in Human ESCs

Accurately quantifying the genetic heterogeneity of a cell population is essential to understanding of biological systems. We develop a universal method to label individual DNA molecules for analyzing diverse types of rare genetic variants, with frequency as low as 4x10-5, using short- or long-read sequencing. It enables base-resolution haplotype-resolved quantitative characterization of rare variants. It provides the first quantitative evidence of persistent nonrandom large deletions and insertions following DNA repair of double-strand breaks induced by CRISPR-Cas9 in human pluripotent stem cells.

molecular biology↗

bioRxiv2020

Genome-wide molecular effects of the neuropsychiatric 16p11 CNVs in an iPSC-to-iN neuronal model

Copy number variants (CNVs), either deletions or duplications, at the 16p11.2 locus in the human genome are known to increase the risk for autism spectrum disorders (ASD), schizophrenia, and for several other developmental conditions. Here, we investigate the global effects on gene expression and DNA methylation using a 16p11.2 CNV patient-derived induced pluripotent stem cell (iPSC) to induced neuron (iN) cell model system. This approach revealed genome-wide and cell-type specific alterations to both gene expression and DNA methylation patterns and also yielded specific leads on genes potentially contributing to some of the known 16p11.2 patient phenotypes. PCSK9 is identified as a possible contributing factor to the symptoms seen in carriers of the 16p11.2 CNVs. The protocadherin (PCDH) gene family is found to have altered DNA methylation patterns in the CNV patient samples. The iPSC lines used for this study are available through a repository as a resource for research into the molecular etiology of the clinical phenotypes of 16p11.2 CNVs and into that of neuropsychiatric and neurodevelopmental disorders in general.

genetics↗

bioRxiv2020

Challenging a preconception: Optoacoustic spectrum differs from the absorption spectrum of proteins and dyes for molecular imaging

Optoacoustic (photoacoustic) imaging has seen marked technological advances in detection and data analysis, but there is less progress in understanding the photophysics of optoacoustic signal generation of commonly used contrast agents, such as dyes and chromoproteins. This gap blocks the precise development of novel agents and the accurate analysis and interpretation of Multispectral Optoacoustic Tomography (MSOT) images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. MLS provides reproducible, high-quality optoacoustic (non-radiative) spectra by using correction and referencing workflow. Herein, we employ MLS to analyze several common dyes (Methylene Blue, Rhodamine 800, Alexa Fluor 750, IRDye 800CW and Indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720 and smURFP) and shed light on their internal conversion properties. Our data shows that the optical absorption spectra do not correlate with the optoacoustic spectra for the majority of the analytes. We determine that for dyes, the transition underlying the high energy shoulder, which mostly correlates with an aggregation state of the dyes, has significantly more optoacoustic signal generation efficiency than the monomer transition. Our analyses for proteins point to a favored vibrational relaxation and optoacoustic signal generation that stems from the neutral or zwitterionic chromophores. We were able to crystalize HcRed in its optoacoustic state, confirming the change isomerization respect to its fluorescence state. Such data is highly relevant for the engineering of tailored contrast agents for optoacoustic imaging. Furthermore, discrepancies between absorption and optoacoustic spectra underline the importance of correct spectral information as a prerequisite for the spectral-unmixing schemes that are often required for in vivo imaging. Finally, optoacoustic spectra of some of the most commonly used proteins and dyes in optical imaging, recorded on our MLS, reveal previously unknown photophysical characteristics, such as unobserved photo-switching behavior.

biophysics↗

bioRxiv2020

Clinically applicable histopathological diagnosis system for gastric cancer detection using deep learning

Gastric cancer is among the most malignant tumours with the highest incidence and mortality rates. The early detection and accurate histopathological diagnosis of gastric cancer are essential factors that can help increase the chances of successful treatment. While the worldwide shortage of pathologists has imposed burdens on current histopathology services, it also offers a unique opportunity for the use of artificial intelligence assistance systems to alleviate the workload and increase diagnostic accuracy. To the best of our knowledge, there has not been a clinically applicable histopathological assistance system with high accuracy that can generalize to whole slide images created with diverse digital scanner models from different hospitals. Here, we report a clinically applicable artificial intelligence assistance system developed at the Chinese PLA General Hospital, China, using a deep convolutional neural network trained with 2,123 pixel-level annotated H&E-stained whole slide images. The model achieved a sensitivity near 100% and an average specificity of 80.6% on a real-world test dataset, which included 3,212 whole slide images digitalized with three scanner models. We showed that the system could aid pathologists in improving diagnostic accuracy and preventing misdiagnoses. Moreover, we demonstrated that our system could perform robustly with 1,582 whole slide images from two other medical centres. Our study suggests the feasibility and benefits of using histopathological artificial intelligence assistance systems in routine practice scenarios.Competing Interest StatementXiaoqing Zhang is the founder of Thorough Images. Shuhao Wang is the co-founder and chief technology officer (CTO) of Thorough Images. Calvin Ku, Cancheng Liu, Zhuo Sun, Gang Xu, Yuefeng Wang are algorithm researchers of Thorough Images. All remaining authors have declared no conflicts of interest.View Full Text

pathology↗

bioRxiv2020

Regulation of olfactory-based sex behaviors in the silkworm by genes in the sex-determination cascade

Insect courtship and mating depend on integration of olfactory, visual, and tactile cues. Compared to other insects, Bombyx mori, the domesticated silkworm, has relatively simple sexual behaviors as it cannot fly. Here by using CRISPR/Cas and electrophysiological techniques we found that courtship and mating behaviors are regulated in male silk moths by mutating genes in the sex determination cascade belonging to two conserved pathways. Loss of Bmdsx gene expression significantly reduced the peripheral perception of the major pheromone component bombykol by reducing expression of the product of the BmOR1 gene which completely blocked courtship in adult males. Interestingly, we found that mating behavior was regulated independently by another sexual differentiation gene, Bmfru. Loss of Bmfru completely blocked mating, but males displayed normal courtship behavior. Lack of Bmfru expression significantly reduced the perception of the minor pheromone component bombykal due to the down regulation of BmOR3 expression; further, functional analysis revealed that loss of the product of BmOR3 played a key role in terminating male mating behavior. Our results suggest that Bmdsx and Bmfru are at the base of the two primary pathways that regulate olfactory-based sexual behavior. Author SummaryThe fundamental insect sexual behaviors, courtship and mating, result from successful integration of olfactory, vision, tactile and other complex innate behaviors. In the widely used insect model, Drosophila melanogaster, the sex determination cascade genes fruitless and doublesex are involved in the regulation of courtship and mating behaviors; however, little is known about the function of these sexual differentiation genes in regulating sex behaviors of Lepidoptera. Here we combine genetics and electrophysiology to investigate regulation pathway of sexual behaviors in the model lepidopteran insect, the domesticated silk moth, Bombyx mori. Our results support the presence of two genetic pathways in B. mori, named Bmdsx-BmOR1-bombykol and Bmfru-BmOR3-bombykal, which control distinct aspects of male sexual behavior that are modulated by olfaction. This is the first comprehensive report about the role of sex differentiation genes in the male sexual behavior in the silk moth.

genetics↗

bioRxiv2020

Genome-wide discovery of lupus genetic risk variant allelic regulatory activity

Genome-wide association studies of Systemic Lupus Erythematosus (SLE) nominate 3,073 genetic variants at 91 risk loci. To systematically screen these variants for allelic transcriptional enhancer activity, we constructed a massively parallel reporter assay (MPRA) library comprising 12,396 DNA oligonucleotides containing the genomic context around every allele of each SLE variant. Transfection into the Epstein-Barr virus-transformed B cell line GM12878 revealed 482 variants with enhancer activity, with 51 variants showing genotype-dependent (allelic) enhancer activity at 27 risk loci. Comparison of MPRA results in GM12878 and Jurkat T cell lines highlights shared and unique allelic transcriptional regulatory mechanisms at SLE risk loci. In-depth analysis of allelic transcription factor (TF) binding at and around allelic variants identifies one class of TFs whose DNA-binding motif tends to be directly altered by the risk variant and a second, larger class of TFs that bind allelically without direct alteration of their motif by the variant. Collectively, our approach provides a blueprint for the discovery of allelic gene regulation at risk loci for any disease and offers insight into the transcriptional regulatory mechanisms underlying SLE.

genomics↗

bioRxiv2020

SMRT sequencing of the Oryza rufipogon genome reveals the genomic basis of rice adaptation

Asian cultivated rice is believed to have been domesticated from an immediate ancestral progenitor, Oryza rufipogon, which provides promising sources of novel alleles for world rice improvement. Here we first present a high-quality de novo assembly of the typical O. rufipogon genome through the integration of single-molecule sequencing (SMRT), 10x and Hi-C technologies. This chromosome-based reference genome allows a multi-species comparative analysis of the annual selfing O. sativa and its two wild progenitors, the annual selfing O. nivara and perennial outcrossing O. rufipogon, identifying massive numbers of dispensable genes that are functionally enriched in reproductive process. Comparative genomic analyses identified millions of genomic variants, of which large-effect mutations (e.g., SVs, CNV and PAVs) may affect the variation of agronomically significant traits. We demonstrate how lineage-specific expansion of rice gene families may have contributed to the formation of reproduction isolation (e.g., the recognition of pollen and male sterility), thus brightening the role in driving mating system evolution during the evolutionary process of recent speciation. We document thousands of positively selected genes that are mainly involved in flower development, ripening, pollination, reproduction and response to biotic- and abiotic stresses. We show that selection pressures may serve as crucial forces to govern substantial genomic alterations among the three rice species that form the genetic basis of rapid evolution of mating and reproductive systems under diverse habitats. This first chromosome-based wild rice genome in the genus Oryza will become powerful to accelerate the exploration of untapped genomic diversity from wild rice for the enhancement of elite rice cultivars.

genomics↗

bioRxiv2020

A global map of root biomass across the world's forests

Abstract (150 words limits)Root plays a key role in plant growth and functioning. Here we combine 10307 field measurements of forest root biomass worldwide with global observations of forest structure, climatic conditions, topography, land management and soil characteristics to derive a spatially-explicit global high-resolution (~ 1km) root biomass dataset, including fine and coarse roots. In total, 142 {+/-} 32 Pg of live dry matter biomass is stored below-ground, that is a global average root:shoot biomass ratio of 0.25 {+/-} 0.10. Our estimations of total root biomass in tropical, temperate and boreal forests are 44-226% smaller than earlier studies1-3. The smaller estimation is attributable to the updated forest area, spatially explicit above-ground biomass density used to predict the patterns of root biomass, new root measurements and upscaling methodology. We show specifically that the root shoot allometry is one underlying driver that leads to methodological overestimation of root biomass in previous estimations.

ecology↗

bioRxiv2020

Subcortical magnocellular visual system facilities object recognition by processing topological property

The Magnocellular (M) visual pathway is known as a fast route to convey coarse information and facilitates object recognition by initiating top-down processes. It is unclear what exact properties M pathway conveys to accelerate visual object processing. Previous studies suggest that visual systems are highly sensitive to the perception of topological property (TP), which remains unchanged under various shape changes, and the TP is probably processed through a fast subcortical pathway. Here we hypothesize that a subcortical M system contributes to the fast object recognition by processing TP first. We first demonstrate that the facilitation effect of TP processing on object perception occurs mainly in the M visual system, and then support the subcortical M hypothesis of TP processing by the evidence that the early processing of TP was not affected when cortical function was temporarily damaged by the transcranial magnetic stimulation and when stimuli were biased to M system.

neuroscience↗

bioRxiv2020

Accucopy: accurate and fast inference of allele-specific copy number alterations for heterogeneous tumor samples from high-throughput sequencing data

BackgroundCopy number alterations (CNAs), due to their large impact on the genome, have been an important contributing factor to oncogenesis and metastasis. Detecting genomic alterations from the shallow-sequencing data of a low-purity tumor sample remains a challenging task. ResultsWe introduce Accucopy, a method to infer total copy numbers (TCNs) and allele-specific copy numbers (ASCNs) from challenging low-purity and low-coverage tumor samples. Accucopy adopts many robust statistical techniques such as kernel smoothing of coverage differentiation information to discern signals from noise and combines ideas from time-series analysis and the signal-processing field to derive a range of estimates for the period in a histogram of coverage differentiation information. Statistical learning models such as the tiered Gaussian mixture model, the Expectation-Maximization (EM) algorithm, and Sparse Bayesian Learning (SBL) were customized and built into the model. Accucopy is implemented in C++/Rust, packaged in a docker image, and supports non-human samples, more at http://www.yfish.org/software/. ConclusionsWe describe Accucopy, a method that can predict both TCNs and ASCNs from low-coverage low-purity tumor sequencing data. Through comparative analyses in both simulated and real-sequencing samples, we demonstrate that Accucopy is more accurate than Sclust, ABSOLUTE, and Sequenza.

bioinformatics↗

bioRxiv2019

QTL-Seq Identifies QTL for Relative Electrical Conductivity Associated with Heat Tolerance in Bottle Gourd (Lagenaria Siceraria)

Heat is a major abiotic stressor seriously affects watermelon produce; however, its effects may be mitigated through grafting onto heat tolerant bottle gourd rootstock. To understand the genetic basis and select reliable markers are necessary in the variety breeding process. In this paper, the relative electric conductivity (REC) was used as a visual indicator for heat stress to study the genetics and SNP marker of heat tolerance in bottle gourd via QTL-seq approach. The results showed that recessive inheritance and a major QTL locus controlled REC related to heat tolerance. Seven heat-tolerant loci (qHT1.1, qHT2.1, qHT2.2, qHT5.1, qHT6.1, qHT7.1, and qHT8.1) exhibited high {Delta}(SNP-index) values (ranging from 0.19-0.38), and of these, the greatest value (0.32, P < 0.01) and greatest number of detected SNPs (9052) were found in chromosome 2 at qHT2.1 region (11.03 - 19.25 Mb); qHT2.1was taken as a promising major QTL for heat tolerance in bottle gourd. A total of 34 putative candidate genes related to heat stress were detected within the qHT2.1 region. Three polymorphic SNPs (BG_GLEAN_10022642, BG_GLEAN_10022727, and BG_GLEAN_10022589) that are involved in pollen sterility, intracellular transport, and signal recognition were validated and exhibited significant marker-trait association that could be used in heat tolerant molecular breeding for bottle gourd. The qHT2.1region is an important finding can be used for fine mapping and discovering new genes associated with heat tolerance in bottle gourd.

molecular biology↗

bioRxiv2019

NADPH diaphorase neuronal dystrophy in gracile nucleus, cuneatus nucleus and spinal trigeminal nucleus in aged rat

NADPH-diaphorase (N-d) activity is commonly used to identify NOS-ergic neurons. In our previous study, N-d positive neuritic dystrophy and spheroid termed aging-related N-d Body is discovered in the lumbosacral spinal cord in the normal aging rats. Histological studies also reveal that N-d positive neurodegenerative changes occur in the gracile nucleus. We re-examined N-d activity in gracile nucleus in aged rat. We found N-d positive neuritic dystrophy and spheroid also occurred in the cuneatus nucleus and spinal trigeminal nucleus. Besides regular coronal section, longitudinal oriented dystrophic neurites were detected in the sagittal and horizontal section in gracile nucleus and dorsal column. We fziurther examined the medullary oblongata with regular classical histology including Golgi staining, immunocytochemistry of NOS and phosphorylated tau protein, neuronal tracing method with wheat germ agglutinin conjugated alexa-fluor-488 through sciatic nerve, and spinal cord transection at thoracic level. Most of N-d positive neuritic dystrophy and spheroid did not showed colocalization with NOS or phosphorylated tau protein. Neuronal tracing and spinal cord transection revealed that N-d dystrophic neurites in gracile nucleus originated from terminal of sensory projection from spinal cord and peripheral somatic input. The results suggested that aging-related N-d dystrophy in the gracile nucleus was unique morphological feature. In conclusion, it was postulated that the N-d dystrophy as a morphological marker of aging degenerative damage in normal aged organisms.

neuroscience↗

bioRxiv2019

Nilotinib activates endothelial TLR4 to exacerbate atherosclerosis

The use of nilotinib (Tasigna(R)), a second-generation tyrosine kinase inhibitor for treating chronic myeloid leukemia, increases risks for atherosclerosis. Here, we demonstrate that in endothelial cells, nilotinib activated TLR4, triggerd expression of inflammatory molecules, and increased monocyte attachment, which were all inhibited by knockdown of TLR4 or TLR4 inhibitor, CLI-095. Orally administered nilotinib profoundly accelerated atherosclerotic lesion formation in ApoE-/- mice, while co-administration of CLI-095 effectively reduced lesion areas. Our findings reveal TLR4 activation as an underlying mechanism of the pro-atherosclerotic effect of nilotinib and suggest TLR4 inhibition as an effective therapeutic approach to address vascular safety issue of nilotinib.

cell biology↗

bioRxiv2019

dP75 safeguards oogenesis by preventing H3K9me2 spreading

Serving as a host factor for HIV integration, LEDGF/p75 has been under extensive study as a potential target for therapy. However, as a highly conserved protein, its physiological function remains to be thoroughly elucidated. Here we characterize the molecular function of dP75, the Drosophila homolog of p75, during oogenesis. dP75 binds to transcriptionally active chromatin with its PWWP domain. The C-terminus IBD domain-containing region of dP75 physically interacts with the histone kinase Jil-1 and stabilizes it in vivo. Together with Jil-1, dP75 prevents the spreading of the heterochromatin mark-H3K9me2-onto genes required for oogenesis and piRNA production. Without dP75, ectopically silencing of these genes disrupts oogenesis, activates transposons, and causes animal sterility. We propose that dP75, the homolog of an HIV host factor in Drosophila, partners with Jil-1 to ensure gene expression during oogenesis by preventing ectopic heterochromatin spreading.

developmental biology↗

bioRxiv2019

RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status but it relies on second strand cDNA synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol, and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods. Significance StatementRNA sequencing is widely used to measure gene expression in biomedical research; therefore, improvements in the simplicity and accuracy of the technology are desirable. All existing RNA sequencing methods rely on the conversion of RNA into double-stranded DNA through reverse transcription followed by second strand synthesis. The latter step requires additional enzymes and purification, and introduces sequence-dependent bias. Here, we show that Tn5 transposase, which randomly binds and cuts double-stranded DNA, can directly fragment and prime the RNA/DNA heteroduplexes generated by reverse transcription. The primed fragments are then subject to PCR amplification. This provides a new approach for simple and accurate RNA characterization and quantification.

molecular biology↗