Biology subjects

Nie, X.

Publications and source records attributed to Nie, X..

30 records · Page 2Linked to original sources

bioRxiv2021

Single-Cell Analysis of Human Testis Aging, and Impact of Elevated Body Mass Index

Aging human males display reduced reproductive health, however testis aging is poorly understood at the molecular and genomic level. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from both young and older men (>60 years old) - and examined age-related changes in germline development and in the somatic niche. Interestingly, age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and the somatic niche ranged from moderate to severe. Altered pathways included signaling and inflammation in multiple cell types, metabolic signaling in Sertoli cells, hedgehog signaling and testosterone production in Leydig cells, cell death and growth in testicular peritubular cells, and possible developmental regression in both Leydig and peritubular cells. Remarkably, the extent of dysregulation correlated with body mass index in older, but not younger men. Taken together, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging, and their exacerbation by concurrent chronic conditions such as obesity.

developmental biology↗

bioRxiv2021

Rapid visual CRISPR assay: a naked-eye colorimetric detection method for nucleic acids based on CRISPR/Cas12a and convolutional neural network

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with CRISPR/Cas12a based visual colorimetric assays. Among them, 9 ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for naked-eye read-out of assay results (no transducing component required for visualization). Subsequently, we developed a convolutional neural network algorithm standardize and to automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube towards an instrument-less colorimetric POCT format that requires only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the single-copy detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this novel RAVI-CRISPR system for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen-side testing.

molecular biology↗

bioRxiv2021

GPR39 Localization in Aging Human Brain and Correlation of Expression and Polymorphism with Vascular Cognitive Impairment

INTRODUCTIONThe pathogenesis of vascular cognitive impairment (VCI) is not fully understood. GPR39, an orphan G-protein coupled receptor, is implicated in neurological disorders but its role in VCI is unknown. METHODSWe performed GPR39 immunohistochemical analysis in postmortem brain samples from mild cognitive impairment (MCI) and control subjects. DNA was analyzed for GPR39 SNPs, and correlated with white matter hyperintensity (WMH) burden on premortem MRI. RESULTSGPR39 is expressed in aged human dorsolateral prefrontal cortex, localized to microglia and peri-capillary cells resembling pericytes. GPR39-capillary colocalization, and density of GPR39-expressing microglia was increased in aged brains compared to young. SNP distribution was equivalent between groups; however, homozygous SNP carriers were present only in the MCI group, and had higher WMH volume than WT or heterozygous SNP carriers. DISCUSSIONGPR39 may play a role in aging-related VCI, and may serve as a therapeutic target and biomarker for the risk of developing VCI.

neuroscience↗

bioRxiv2021

Genome-wide identification and characterization of DNA/RNA differences associated with Fusarium graminearum infection in wheat

RNA editing (DNA/RNA differences) as a post-transcriptional modification approach to enrich genetic information, plays the crucial role in regulating diverse biological processes in eukaryotes. Although it has been extensively studied in plant chloroplast and mitochondria genome, RNA editing in plant nuclear genome, especially those associated with Fusarium head blight (FHB), is not well studied at present. Here, we investigated the DNA/RNA differences associated with FHB through a novel method by comparing the RNA-seq data from Fusarium-infected and control samples from 4 wheat genotypes. A total of 187 DNA/RNA differences were identified in 36 wheat genes, representing the first landscape of the FHB-responsive RNA editome in wheat. Furthermore, all of these 36 edited genes were located in the FHB related co-expression gene modules, which may involve in regulating FHB response. Finally, the effects of DNA/RNA differences were systematically investigated to show that they could cause the change of RNA structure and protein structure in edited genes. In particular, the G to C editing (chr3A_487854715) in TraesCS3A02G263900, which is the orthology of OsRACK1, resulted that it was targeted by tae-miR9664-3p to control its expression in different genotype through different editing efficiency, suggesting RNA editing could mediate miRNA to participate in the regulation network of FHB tolerance. This study reported the first wheat DNA/RNA differences associated with FHB, which not only contribute to better understand the molecular basis underlying FHB tolerance, but also shed light on improving FHB tolerance through epigenetic method in wheat and beyond.

plant biology↗

bioRxiv2021

Establishment of Developmental Gene Silencing by Ordered Polycomb Complex Recruitment in Early Zebrafish Embryos

Vertebrate embryos achieve developmental competency during zygotic genome activation (ZGA) by establishing chromatin states that silence yet poise developmental genes for subsequent lineage-specific activation. Here, we reveal how developmental gene poising is established de novo in preZGA zebrafish embryos. Poising is established at promoters and enhancers that initially contain open/permissive chromatin with Placeholder nucleosomes (bearing H2A.Z, H3K4me1, and H3K27ac), and DNA hypomethylation. Silencing is initiated by the recruitment of Polycomb Repressive Complex 1 (PRC1), and H2Aub1 deposition by catalytic Rnf2 during preZGA and ZGA stages. During postZGA, H2Aub1 enables Aebp2-containing PRC2 recruitment and H3K27me3 deposition. Notably, preventing H2Aub1 (via Rnf2 inhibition) eliminates recruitment of Aebp2-PRC2 and H3K27me3, and elicits transcriptional upregulation of certain developmental genes during ZGA. However, upregulation is independent of H3K27me3 - establishing H2Aub1 as the critical silencing modification at ZGA. Taken together, we reveal the logic and mechanism for establishing poised/silent developmental genes in early vertebrate embryos. Impact StatementDe novo polycomb domains are formed in zebrafish early embryos by focal histone H2Aub1 deposition by Rnf2-PRC1 - which imposes transcription silencing - followed by subsequent recruitment of Aebp2-PRC2 and H3K27me3 deposition.

molecular biology↗

bioRxiv2020

A novel high-accuracy genome assembly method utilizing a high-throughput workflow

Across domains of biological research using genome sequence data, high-quality reference genome sequences are essential for characterizing genetic variation and understanding the genetic basis of phenotypes. However, the construction of genome assemblies for various species is often hampered by complexities of genome organization, especially repetitive and complex sequences, leading to mis-assembly and missing regions. Here, we describe a high-throughput gold standard genome assembly workflow using a large-scale bacterial artificial chromosome (BAC) library with a refined two-step pooling strategy and the Lamp assembler algorithm. This strategy minimizes the laborious processes of physical map construction and clone-by-clone sequencing, enabling inexpensive sequencing of several thousand BAC clones. By applying this strategy with a minimum tiling path BAC clone library for the short arm of chromosome 2D (2DS) of bread wheat, 98% of BAC sequences, covering 92.7% of the 2DS chromosome, were assembled correctly for this species with a highly complex and repetitive genome. We also identified 48 large mis-assemblies in the reference wheat genome assembly (IWGSC RefSeq v1.0) and corrected these large mis-assemblies in addition to filling 92.2% of the gaps in RefSeq v1.0. Our 2DS assembly represents a new benchmark for the assembly of complex genomes with both high accuracy and efficiency.

genomics↗

bioRxiv2020

Dissecting Mammalian Spermatogenesis Using Spatial Transcriptomics

Single-cell RNA sequencing has revealed extensive molecular diversity in gene programs governing mammalian spermatogenesis but fails to delineate their dynamics in the native context of seminiferous tubules -- the spatially-confined functional units of spermatogenesis. Here, we use Slide-seq, a novel spatial transcriptomics technology, to generate a comprehensive spatial atlas that captures the spatial gene expression patterns at near single-cell resolution in the mouse and human testis. By using Slide-seq data, we devise a computational framework that accurately localizes testicular cell types in individual seminiferous tubules. Unbiased spatial transcriptome analysis systematically identifies spatially patterned genes and gene programs, nominating genes with previously underappreciated but important functions in spermatogenesis. Using the human testicular spatial atlas, we identify two spatially segregated spermatogonial populations composed of stem cells at distinct transcriptional states. Finally, a comparison of the spatial atlas generated from the wild type and diabetic mouse testis reveals a disruption in the spatial cellular organization in diabetic seminiferous tubules.

genomics↗

bioRxiv2020

Effects of High-Grain Diet with Buffering Agent on the Milk Protein Synthesis in Lactating Goats

Feeding of straw as main roughage with numerous high-grain diets improves the performance of ruminants but it can easily lead to subacute ruminal acidosis. In recent years, buffering agent is applied to prevent the acid poisoning of ruminants and improve the production performance of ruminants in animal husbandry. it is necessary to understand feeding high-grain diet with buffering agent which transport carriers amino acids mainly take amino acids into the mammary gland and the signal mechanism of amino acids in the mammary gland synthesize milk proteins. To gain insight on the effects of a high-grain diet with buffering agent on the amino acids in the jugular blood, and the effects of amino acids on the synthesis of milk protein, commercial kit and high performance liquid chromatography (HPLC) were applied to determine the concentration of amino acids of jugular blood samples, quantitative real-time PCR, comparative proteomic approach and western blot were employed to investigate proteins differentially expressed in mammary tissues and the mechanism of amino acids on the synthesis of milk protein in mammary gland of lactating dairy goats fed high-grain diet with buffering agent or only high-grain diet. Results showed that feeding high-grain diet with buffering agent to lactating dairy goats could outstanding increase amino acid content of jugular blood (p<0.05), and mRNA transcriptional level of amino acid transporters in the mammary gland were also increased; the CSN2 and LF protein expression level were significant higher by 2-DE technique, MALDI-TOF/TOF proteomics analyzer and western blot analysis further validated in mammary of lactating dairy goats compared with high-grain group; the research on the mechanism of milk protein synthesis increasing suggested that it was related to the activation of mTOR pathway signaling. Feeding of high-grain diet with buffering agent promoted the jugular vein blood of amino acids concentration, and more amino acids flowed into the mammary. In addition, milk protein synthesis was increased and the increase of milk protein synthesis was related to the activation of mTOR pathway signalling.

biochemistry↗

bioRxiv2020

Establishing Prokaryotic Expression System of Angiotensin- Converting Enzyme 2 (ACE2) gene in pigs

In this paper, ACE2 gene of pigs was cloned and the purified protein was obtained via the prokaryotic expression system. Polyclonal antibody of high titer and sensitivity was obtained using Wastar rats immunization method and is then used to determine of the expression of ACE2 using immunohistochemistry. The sequence of ACE2 in pigs covered 2418 nucleotides and coded 805 amino acid (aa) residues. Sequence homology analysis showed that the ACE2 sequence in pigs is highly conserved among species at the nucleotide and amino acid levels. Genetic evolution analysis revealed that ACE2 gene in pigs has the shortest genetic distance with that in goats while residing in a totally different branch from that in zebra fishes. Analysis of protein structure predicted that ACE2 protein is a transmembrane secreted protein with high hydrophilicity, containing a signal peptide sequences locating between 1aa to 17aa. The ACE2 fusion protein expressed (under the induction with 1.0 mmol/L IPTG for 10 h) was of approximately 100 kDa and mainly existed in inclusion body. Wastar rats immunization showed that the titer of the anti-ACE2 antiserum in rats was 1: 3200. Western blot showed that the antibody binds specifically. Immunohistochemistry showed that the ACE2 protein was expressed in all major tissues of pigs. It is the first time that polyclonal antibody of ACE2 in pigs was obtained and the expression of ACE2 was confirmed. These results will provide a basis for investigating on ACE2s biological activity in pigs.

biochemistry↗

bioRxiv2020

Macromolecular and biochemical changes of G0 fission yeast nuclei

When starved of nitrogen, fission yeast Schizosaccharomyces pombe cells enter a quiescent "G0" state with smaller nuclei and transcriptional repression. The genomics of S. pombe G0 cells has been well studied, but much of its nuclear cell biology remains unknown. Here we use confocal microscopy, immunoblots, and electron cryotomography to investigate the cytological, biochemical, and ultrastructural differences between S. pombe proliferating, G1-arrested, and G0 cell nuclei, with an emphasis on the histone acetylation, RNA polymerase II fates, and macromolecular complex packing. Compared to proliferating cells, G0 cells have lower levels of histone acetylation, nuclear RNA polymerase II, and active transcription. The G0 nucleus has similar macromolecular crowding yet fewer chromatin-associated multi-megadalton globular complexes. Induced histone hyperacetylation in G0 results in cells that have larger nuclei and therefore less compact chromatin. However, these histone-hyperacetylated G0 cells remain transcriptionally repressed with similar nuclear crowding. Canonical nucleosomes - those that resemble the crystal structure - are rare in proliferating, G1-arrested, and G0 cells. Our study therefore shows that extreme changes in nucleus physiology are possible without extreme reorganisation at the macromolecular level. SummaryWe use multiple cell-biological techniques to compare proliferating and quiescent G0 fission yeast cell nucleus transcription, histone acetylation, macromolecular packing.

cell biology↗

bioRxiv2019

Characterization of Isoforms of the Ovine Granulocyte Colony Stimulating Factor

The granulocyte colony-stimulating factor (GCSF) regulates the maturation, proliferation, and differentiation of precursor cells of neutrophilic granulocytes, and has been widely studied in several species. To investigate the function of variants of sheep GCSF (sGCSF), this study compared difference in their mRNA expression levels. Both the activity and mRNA expression level of GCSFv2 were higher than those of GCSFv1. Their sequences were aligned, which showed that they had the highest homology with bovine GCSF. Then, predicted ovine GCSF isoforms and their constant C-terminals were cloned and expressed, which were stably expressed in mammalian cells. After purification, all GCSF functions were different both in vitro and in vivo, and the GCSF C-terminal was best. These results indicated that the ability to stimulate both the proliferation and differentiation of progenitor cells and to activate the maturation of neutrophils could be used for research of efficacious non-antibiotic protein drugs. Furthermore, GCSF can be used as candidate target of genetic breeding to specifically improve sheep immunity.

genetics↗

bioRxiv2019

CRISPR screening of porcine sgRNA library identified host factors essential for Japanese encephalitis virus replication

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, key host genes involved in the JEV life cycle and cell death are poorly understood. Here, we designed 85,674 single guide RNAs (sgRNAs) targeting 17,743 protein-coding genes, 11,053 long ncRNAs (lncRNAs), and 551 microRNAs (miRNAs) in the porcine genome, and subsequently developed a porcine sgRNA library and genome-scale CRISPR/Cas9 knockout (PigGeCKO) system. These sgRNAs were delivered into porcine kidney-15 (PK-15) cells that constitutively express Cas9, positive selection screening of the resulting PigGeCKO cell collection for resistance to JEV-induced cell death led to the identification of several previously unreported genes required for JEV infection. We conducted follow-up studies to verify the dependency of JEV on these genes, and identified functional contributions for six of the many candidate JEV-related host genes, including an endoplasmic reticulum membrane protein complex subunit 3 (EMC3) and calreticulin (CALR). Additionally, we identified that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPG sulfurylation, facilitated JEV entry into PK-15 cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this work deepens our basic understanding of flavivirus infection and identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to prevent and treat diseases caused by Japanese encephalitis virus.

genetics↗