Biology subjects

Gao, G.

Publications and source records attributed to Gao, G..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Potential limitations of micro-dystrophin gene therapy for Duchenne muscular dystrophy

Adeno-associated viruses (AAVs) expressing versions of truncated dystrophin (micro-dystrophins) are being delivered at high doses to patients with Duchenne muscular dystrophy (DMD) in clinical trials. We examined this strategy with two different micro-dystrophins, similar to those currently in clinical trials, in a severe mouse model of DMD, the D2.mdx mouse, using doses of AAV comparable to those used in the clinical trials. We achieved high levels of micro-dystrophin expression in striated muscle with cardiac expression [~]10 fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of the skeletal muscle disease is observed. Surprisingly, a lethal acceleration of cardiac disease progression occurs with one of the micro-dystrophins, while the second appears to benefit the heart. The detrimental impact on the heart in the first case appears to be caused by the high levels of micro-dystrophin in the heart resulting in competition between micro-dystrophin and utrophin at the cardiomyocyte membrane. While the significance of these observations for patients currently being treated with AAV-micro-dystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown, it suggests that micro-dystrophin treatments may need to be carefully titrated to avoid high levels of expression in the heart.

pharmacology and toxicology↗

bioRxiv2022

The generation of the first chromosome-level de-novo genome assembly and the development and validation of a 50K SNP array for North American Atlantic salmon

Given the genetic and genomic differences between Atlantic salmon of European origin and North American (N.A.) origin, it is crucial to develop unique genomic resources for each lineage. Here we describe the resources that we recently developed for genomic and genetic research in N.A. Atlantic salmon. Firstly, a new single nucleotide polymorphism (SNP) database for N.A. Atlantic salmon consisting of 3.1 million putative SNPs was generated using data from whole genome resequencing of 80 N.A. Atlantic salmon individuals; Secondly, a high density 50K SNP array enriched for the genic regions of the genome and containing three sex determination and 61 continent of origin markers was developed and validated; Thirdly, a genetic map composed of 27 linkage groups with 36K SNP markers, was generated from 2,512 individuals in 141 full-sib families; Finally, a chromosome level de-novo assembly of a male N.A. Atlantic salmon genome was generated using PacBio long-reads. Information from Hi-C proximity ligation sequences and Bionano optical mapping was used to concatenate the contigs into scaffolds. The assembly contains 1,755 scaffolds and only 1,253 gaps, with a total length of 2.83 Gb and N50 of 17.2 Mb. A BUSCO analysis detected 96.2% of conserved Actinopterygii genes in the assembly and the genetic linkage information was used to guide the formation of 27 chromosome sequences. In contrast, the karyotype of the European Atlantic salmon lineage is composed of 29 chromosomes. Comparative analysis with the reference genome assembly of the European Atlantic salmon confirmed that the karyotype differences between the two linages are caused by a fission in chromosome Ssa01 and three chromosome fusions including the p arm of chromosome Ssa01 with Ssa23, Ssa08 with Ssa29 and Ssa26 with Ssa28. The genomic resources we have generated for Atlantic salmon provide a crucial boost for genetic research and for management of farmed and wild populations in this highly valued species.

genomics↗

bioRxiv2022

Kiaa1024L/Minar2 is essential for hearing by regulating cholesterol distribution in hair bundles

Unbiased genetic screens implicated a number of uncharacterized genes in hearing loss, suggesting some biological processes required for auditory function remain unexplored. Loss of Kiaa1024L/Minar2, a previously understudied gene, caused deafness in mice, but how it functioned in hearing was unclear. Here we show that disruption of kiaa1024L/minar2 causes hearing loss in the zebrafish. Defects in mechanotransduction, longer and thinner hair bundles, and enlarged apical lysosomes in hair cells are observed in kiaa1024L/minar2 mutant. In cultured cells, Kiaa1024L/Minar2 is mainly localized to lysosomes and its overexpression recruits cholesterol and increases cholesterol labeling. Strikingly, an accessible pool of cholesterol is highly enriched in the hair bundle membrane, and loss of kiaa1024L/minar2 reduces cholesterol localization to the hair bundles. Decreasing cholesterol levels aggravates, while increasing cholesterol levels rescues hair cell defects in kiaa1024L/minar2 mutant. Therefore cholesterol plays an essential role in the hair bundles, and Kiaa1024L/Minar2 regulates cholesterol distribution and homeostasis to ensure normal hearing.

neuroscience↗

bioRxiv2022

Markonv: a novel convolutional layer with inter-positional correlations modeled

Deep neural networks equipped with convolutional neural layers have been widely used in omics data analysis. Though highly efficient in data-oriented feature detection, the classical convolutional layer is designed with inter-positional independent filters, hardly modeling inter-positional correlations in various biological data. Here, we proposed Markonv layer (Markov convolutional neural layer), a novel convolutional neural layer with Markov transition matrices as its filters, to model the intrinsic dependence in inputs as Markov processes. Extensive evaluations based on both synthetic and real-world data showed that Markonv-based networks could not only identify functional motifs with inter-positional correlations in large-scale omics sequence data effectively, but also decode complex electrical signals generated by Oxford Nanopore sequencing efficiently. Designed as a drop-in replacement of the classical convolutional layer, Markonv layers enable an effective and efficient identification for inter-positional correlations from various biological data of different modalities. All source codes of a PyTorch-based implementation are publicly available on GitHub for academic usage.

bioinformatics↗

bioRxiv2022

Preclinical evaluation of RQ3013, a broad-spectrum mRNA vaccine against SARS-CoV-2 variants

The global emergence of SARS-CoV-2 variants has led to increasing breakthrough infections in vaccinated populations, calling for an urgent need to develop more effective and broad-spectrum vaccines to combat COVID-19. Here we report the preclinical development of RQ3013, an mRNA vaccine candidate intended to bring broad protection against SARS-CoV-2 variants of concern (VOCs). RQ3013, which contains pseudouridine-modified mRNAs formulated in lipid nanoparticles, encodes the spike(S) protein harboring a combination of mutations responsible for immune evasion of VOCs. Here we characterized the expressed S immunogen and evaluated the immunogenicity, efficacy, and safety of RQ3013 in various animal models. RQ3013 elicited robust immune responses in mice, hamsters, and nonhuman primates (NHP). It can induce high titers of antibodies with broad cross-neutralizing ability against the Wild-type, B.1.1.7, B.1.351, B.1.617.2, and the omicron B.1.1.529 variants. In mice and NHP, two doses of RQ3013 protected the upper and lower respiratory tract against infection by SARS-CoV-2 and its variants. We also proved the safety of RQ3013 in NHP models. Our results provided key support for the evaluation of RQ3013 in clinical trials.

immunology↗

bioRxiv2022

Plant-on-Chip: core morphogenesis processes in the tiny plant Wolffia australiana

A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the tiniest known angiosperm to overcome this challenge and provide an ideal model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the Plant-on-Chip culture system and demonstrate the application of advanced technologies such as snRNA-seq, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can open a new horizon for deciphering the core regulatory mechanisms of plant morphogenesis. SignificanceWhat is the core morphogenetic process in angiosperms, a plant like a tree indeterminately growing, or a bud sequentially generating limited types of organs? Wolffia australiana, one of the smallest angiosperms in the world may help to make a distinction. Wolffia plantlet constitutes of only three organs that are indispensable to complete life cycle: one leaf, one stamen and one gynoecium. Before the growth tip is induced to flower, it keeps branching from the leaf axil and the branches separate from the main plantlet. Here we present a high-quality genome of W. australiana, detailed morphological description, a Plant-on-Chip cultural system, and some principle-proof experiments, demonstrating that W. australiana is a promising model system for deciphering core developmental program in angiosperms.

plant biology↗

bioRxiv2022

Development of a high-density 665 K SNP array for rainbow trout genome-wide genotyping

Single nucleotide polymorphism (SNP) arrays, also named << SNP chips >>, enable very large numbers of individuals to be genotyped at a targeted set of thousands of genome-wide identified markers. We used preexisting variant datasets from USDA, a French commercial line and 30X-coverage whole genome sequencing of INRAE isogenic lines to develop an Affymetrix 665 K SNP array (HD chip) for rainbow trout. In total, we identified 32,372,492 SNPs that were polymorphic in the USDA or INRAE databases. A subset of identified SNPs were selected for inclusion on the chip, prioritizing SNPs whose flanking sequence uniquely aligned to the Swanson reference genome, with homogenous repartition over the genome and the highest Minimum Allele Frequency in both USDA and French databases. Of the 664,531 SNPs which passed the Affymetrix quality filters and were manufactured on the HD chip, 65.3% and 60.9% passed filtering metrics and were polymorphic in two other distinct French commercial populations in which, respectively, 288 and 175 sampled fish were genotyped. Only 576,118 SNPs mapped uniquely on both Swanson and Arlee reference genomes, and 12,071 SNPs did not map at all on the Arlee reference genome. Among those 576,118 SNPs, 38,948 SNPs were kept from the commercially available medium-density 57K SNP chip. We demonstrate the utility of the HD chip by describing the high rates of linkage disequilibrium at 2 kb to 10 kb in the rainbow trout genome in comparison to the linkage disequilibrium observed at 50 kb to 100 kb which are usual distances between markers of the medium-density chip.

genomics↗

bioRxiv2022

Paradoxical activation of SREBP1c and de novo lipogenesis by hepatocyte-selective ACLY depletion in obese mice.

Hepatic steatosis associated with high fat diets, obesity and type 2 diabetes is thought to be the major driver of severe liver inflammation, fibrosis, and cirrhosis. Cytosolic acetyl-coenzyme A (AcCoA), a central metabolite and substrate for de novo lipogenesis (DNL), is produced from citrate by ATP-citrate lyase (ACLY) and from acetate through AcCoA synthase short chain family member 2 (ACSS2). However, the relative contributions of these two enzymes to hepatic AcCoA pools and DNL rates in response to high fat feeding is unknown. We report here that hepatocyte-selective depletion of either ACSS2 or ACLY caused similar 50% decreases in liver AcCoA levels in obese mice, showing that both pathways contribute to generation of this DNL substrate. Unexpectedly however, the hepatocyte ACLY depletion in obese mice paradoxically increased total DNL flux measured by D2O incorporation into palmitate, while in contrast ACSS2 depletion had no effect. The increase in liver DNL upon ACLY depletion was associated with increased expression of nuclear sterol regulatory element-binding protein 1c (SREBP1c) and of its target DNL enzymes. This upregulated DNL enzyme expression explains the increased rate of palmitate synthesis in ACLY depleted livers. Furthermore, this increased flux through DNL may also contribute to the observed depletion of AcCoA levels due to its increased conversion to Malonyl CoA (MalCoA) and palmitate. Together, these data indicate that in HFD fed obese mice, hepatic DNL is not limited by its immediate substrates AcCoA or MalCoA, but rather by activities of DNL enzymes.

molecular biology↗

bioRxiv2022

Fish oil Nano-emulsion Kills Macrophage: Ferroptosis and Autophagy Triggered by Catalase-catalysed Superoxide Eruption

Fish oil is increasingly utilized in the form of nano-emulsion as nutrient and function fortifier. The nano-emulsions high polyunsaturated fatty acids content and electron donors at oil/water interface could be a potential site of redox reaction, if a previously unrecognised trigger was provided. Here we report that a vigorous superoxide production occurred in fish oil nano-emulsion in the presence of mammalian catalase in both acellular and cellular systems. Furthermore, the resulting superoxide increased cytosolic ROS and membrane lipid peroxidation of murine macrophage, and eventually caused fatal oxidative damage, which involves autophagy and ferroptosis but not apoptosis. The cell death was significantly inhibited by a catalase-specific inhibitor. The ferroptosis was independent of protease Caspase-3 activation or glutathione peroxidase suppression. Our findings discover a hidden risk factor of the widely acclaimed fish oil emulsion and suggest a novel mechanism of cellular damage by dietary lipids on mucosal layer of the alimentary tract.

cell biology↗

bioRxiv2021

Multiplex, translaminar imaging in the spinal cord of behaving mice

While the spinal cord is known to play critical roles in sensorimotor processing, including pain-related signaling, corresponding activity patterns in genetically defined cell types across spinal laminae have remained elusive. Calcium imaging has enabled cellular activity measurements in behaving rodents but is currently limited to superficial regions. Using chronically implanted microprisms, we imaged sensory and motor evoked activity in regions and at speeds inaccessible by other high-resolution imaging techniques. To enable translaminar imaging in freely behaving animals through implanted microprisms, we additionally developed wearable microscopes with custom-compound microlenses. This new integrated system addresses multiple challenges of previous wearable microscopes, including their limited working distance, resolution, contrast, and achromatic range. The combination of these innovations allowed us to uncover that dorsal horn astrocytes in behaving mice show somatosensory program-dependent and lamina-specific calcium excitation. Additionally, we show that tachykinin precursor 1 (Tac1)-expressing neurons exhibit upper laminae-restricted activity to acute mechanical pain but not locomotion.

neuroscience↗

bioRxiv2021

Trans-segmental imaging in the spinal cord of behaving mice

Spinal cord circuits play crucial roles in transmitting and gating cutaneous somatosensory modalities, such as pain, but the underlying activity patterns within and across spinal segments in behaving mice have remained elusive. To enable such measurements, we developed a wearable widefield macroscope with a 7.9 mm2 field of view, subcellular lateral resolution, 2.7 mm working distance, and <10 g overall weight. We show that highly localized painful mechanical stimuli evoke widespread, coordinated astrocyte excitation across multiple spinal segments.

neuroscience↗

bioRxiv2021

Quantitative model suggests both intrinsic and contextual features contribute to the transcript coding ability determination in cells

Gene transcription and protein translation are two key steps of the "central dogma". It is still a major challenge to quantitatively deconvolute factors contributing to the coding ability of transcripts in mammals. Here, we propose Ribosome Calculator (RiboCalc) for quantitatively modeling the coding ability of RNAs in human genome. In addition to effectively predicting the experimentally confirmed coding abundance via sequence and transcription features with high accuracy, RiboCalc provides interpretable parameters with biological information. Large-scale analysis further revealed a number of transcripts with a variety of coding ability for distinct types of cells (i.e., context-dependent coding transcripts, CDCTs), suggesting that, contrary to conventional wisdom, a transcripts coding ability should be modeled as a continuous spectrum with a context-dependent nature.

bioinformatics↗

bioRxiv2021

OsMADS58 stabilizes gene regulatory circuits during rice stamen development

Rice (Oryza sativa) OsMADS58 is a C-class MADS box protein, and characterization of a transposon insertion mutant osmads58 suggested that OsMADS58 plays a role in stamen development. However, as no null mutation has been obtained, its role has remained unclear. Here, we report that the CRISPR knockout mutant osmads58 exhibits complex altered phenotypes, including anomalous diploid germ cell differentiation, aberrant meiosis, and delayed tapetum degeneration. This CRISPR mutant line exhibited stronger changes in expression of OsMADS58 target genes compared with the osmads58 dSpm (transposon insertion) line, along with changes in multiple pathways related to early stamen development. Notably, transcriptional regulatory circuits in young panicles covering the stamen at stages 4-6 were substantially altered in the CRISPR line compared to the dSpm line. These findings strongly suggest that the pleiotropic effects of OsMADS58 on stamen development derive from a potential role in stabilizing gene regulatory circuits during early stamen development. Thus, this work opens new avenues for viewing and deciphering the regulatory mechanisms of early stamen development from a network perspective.

plant biology↗

bioRxiv2021

Fetal neural progenitors process TLR signals from bacterial components to enhance proliferation and rework brain development

Bacterial cell wall, a universal pathogen-associated molecular pattern (PAMP), crosses the placenta into the fetal brain. We determined that PAMPs interact with TLR2/6 on murine fetal neural progenitor cells (NPCs) to induce overexpansion of all neocortical layers leading to a larger, folded cortex and abnormal postnatal behavior. The NPC overexpansion originated at E10 and targeted ventricular radial glia (vRG), the primary NPC, by shortening cell cycle and increasing self-renewal. The mechanism involved two novel signaling pathways in NPCs mediated by recognition of bacterial PAMPs by TLR2/6 including: a) loss of primary cilia, activation of hedgehog signaling, and increased FOXG1 and b) increased PI3K/AKT activity. These findings reveal PAMP/TLR2/6 acts as a morphogen in fetal neurodevelopment. In addition, the loss of Tlr2 or Tlr6 without pathogenic challenge, increased the number of neurons, establishing the requirement for an endogenous TLR2 signal for normal neurodevelopment in the embryo.

neuroscience↗

bioRxiv2021

Formation of multinucleated globoid cells in Krabbe iPSC-derived microglia cultures: a preliminary investigation

Globoid cell leukodystrophy (Krabbe disease) is a severe demyelinating, neurodegenerative lysosomal storage disorder caused by deficiency in glycosphingolipid catabolic enzyme galactosylceramidase (GALC). Histologically, Krabbe disease is characterized by the appearance of large multinucleated globoid cells that express classical macrophage markers (both of brain-resident microglia and peripheral monocyte-derived). Globoid cells reside near areas of degeneration; however, their functional significance in disease progression remains unclear. In the current study, we differentiated microglia-like cells from iPSCs from a donor with infantile Krabbe disease and compared them to microglia generated from two healthy controls and two donors with the lysosomal storage disorder metachromatic leukodystrophy (MLD), which is genetically distinct from Krabbe disease but presents similarly in terms of severity of demyelination and neurodegeneration. We report the novel finding of prominent formation of giant multinucleated globoid cells from the microglia derived from the Krabbe donor, but not from healthy control or MLD donors. The Krabbe microglia displayed reduced IL-6 protein expression upon stimulation with lipopolysaccharide, and the multinucleated globoid cells themselves appeared deficient in phagocytosis of both disease-relevant myelin debris and E. coli, together hinting at an impairment of normal function. The formation of the globoid cells could be attenuated by fully replacing the medium following passaging, suggesting that yet-to-be determined secreted factors are influencing cell fusion in our culture system. While preliminary, our results imply that globoid cells may be detrimental in Krabbe disease by hindering the normal function of brain-residing macrophages.

neuroscience↗

bioRxiv2021

Serine/arginine-rich splicing factor 7 plays oncogenic roles through specific regulation of m6A RNA modification

Serine/Arginine-Rich Splicing Factor 7 (SRSF7), which is previously recognized as a splicing factor, has been revealed to play oncogenic roles in multiple cancers. However, the mechanisms underlying its oncogenic roles have not been well addressed. Here, based on N6-methyladenosine (m6A) co-methylation network analysis across diverse cell lines, we found SRSF7 positively correlated with glioblastoma cell-specific m6A methylation. We then proved SRSF7 is a novel m6A regulator that specifically facilitates the m6A methylation near its binding sites on the mRNAs involved in cell proliferation and migration through recruiting methyltransferase complex. Moreover, SRSF7 promotes the proliferation and migration of glioblastoma cells largely dependent on the m6A methyltransferase. The two single-nucleotide m6A sites on PBK are regulated by SRSF7 and partially mediate the effects of SRSF7 on glioblastoma cells through recognition by IGF2BP2. Together, our discovery revealed a novel role of SRSF7 in regulating m6A and timely confirmed the existence and functional importance of RNA binding protein (RBP) mediated specific regulation of m6A.

molecular biology↗

bioRxiv2021

Gp78-mediated basal mitophagy promotes mitochondrial health and limits mitochondrial ROS production

Mitochondria are major sources of cytotoxic reactive oxygen species (ROS) that contribute to cancer progression. Mitophagy, the selective elimination of mitochondria by autophagy, monitors and maintains mitochondrial health and integrity, eliminating ROS-producing mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis under basal conditions remain poorly understood. Gp78 E3 ubiquitin ligase is an endoplasmic reticulum membrane protein that induces mitochondrial fission and mitophagy of depolarized mitochondria. Here, we report that CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells increased mitochondrial volume and rendered cells resistant to carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitophagy. These effects were phenocopied by knockdown of the essential autophagy protein ATG5 in wild-type HT-1080 cells. Use of the mito-Keima mitophagy probe confirmed that Gp78 promoted both basal and damage-induced mitophagy. Application of a spot detection algorithm (SPECHT) to GFP-mRFP tandem fluorescent-tagged LC3 (tfLC3)-positive autophagosomes reported elevated autophagosomal maturation in wild-type HT-1080 cells relative to Gp78 knockout cells, predominantly in proximity to mitochondria. Mitophagy inhibition by either Gp78 knockout or ATG5 knockdown reduced mitochondrial potential and increased mitochondrial ROS. Live cell analysis of tfLC3 in HT-1080 cells showed the preferential association of autophagosomes with mitochondria of reduced potential. Basal Gp78-dependent mitophagic flux is therefore selectively associated with reduced potential mitochondria promoting maintenance of a healthy mitochondrial population and limiting ROS production.

cell biology↗

bioRxiv2021

Multi-omics integration and regulatory inference for unpaired single-cell data with a graph-linked unified embedding framework

With the ever-increasing amount of single-cell multi-omics data accumulated during the past years, effective and efficient computational integration is becoming a serious challenge. One major obstacle of unpaired multi-omics integration is the feature discrepancies among omics layers. Here, we propose a computational framework called GLUE (graph-linked unified embedding), which utilizes accessible prior knowledge about regulatory interactions to bridge the gaps between feature spaces. Systematic benchmarks demonstrated that GLUE is accurate, robust and scalable. We further employed GLUE for various challenging tasks, including triple-omics integration, model-based regulatory inference and multi-omics human cell atlas construction (over millions of cells) and found that GLUE achieved superior performance for each task. As a generalizable framework, GLUE features a modular design that can be flexibly extended and enhanced for new analysis tasks. The full package is available online at https://github.com/gao-lab/GLUE for the community.

bioinformatics↗