Biology subjects

Chen, C.-Y.

Publications and source records attributed to Chen, C.-Y..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

VPS13A and VPS13C influence lipid droplet abundance

Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer proteins, which includes the VPS13 proteins (VPS13A-D), adopt a rod-like bridge conformation with an extended hydrophobic groove that enables the bulk transfer of membrane lipids for membrane growth. Loss of function mutations in VPS13A and VPS13C cause chorea acanthocytosis and Parkinsons disease, respectively. VPS13A and VPS13C localize to multiple organelle contact sites, including endoplasmic reticulum (ER) - lipid droplet (LD) contact sites, but the functional roles of these proteins in LD regulation remains mostly unexplored. Here, we employ CRISPR-Cas9 genome editing to generate VPS13A and VPS13C knockout cell lines in U-2 OS cells via deletion of exon 2 and introduction of an early frameshift. Analysis of LD content in these cell lines revealed that loss of either VPS13A or VPS13C results in reduced LD abundance under oleate-stimulated conditions. These data implicate VPS13A and VPS13C in LD regulation and raise the intriguing possibility that VPS13A and VPS13C-mediated lipid transfer facilitates LD biogenesis.

cell biology↗

bioRxiv2022

The high diversity of Scedosporium and Lomentospora species and their prevalence in human-disturbed areas in Taiwan

Scedosporium and Lomentospora are important opportunistic pathogens causing localized or disseminated infection in humans. Understanding their environmental distribution is critical for public hygiene and clinical management. We carried out the first environmental survey in urbanized and natural regions in Taiwan. Overall, Scedosporium and Lomentospora species were recovered in 130 out of 271 soil samples (47.9%) across Taiwan. We morphologically and molecularly identified five Scedosporium species and one Lomentospora species. All four major clinical relevant species were isolated with high frequency, i.e. S. apiospermum (42.5%), S. boydii (27.5%), L. prolificans (15.0%), and S. aurantiacum (8.8%); two clinically minor species, S. dehoogii (5.6%) and S. haikouense (0.6%), had moderate incidence. These fungal species have high incidence in urban (48%) and hospital (67.4%) soil samples, and have limited distribution in samples from natural regions (5%). Multivariate analysis of the fungal composition revealed strong evidence of the preferential distribution of these fungi in urban and hospital regions compared to natural sites. In addition, strong evidence suggested that the distribution and abundance of these fungal species are highly heterogeneous in the environment; samples in vicinity often yield varied fungal communities. Our results indicated that these fungal species are prevalent in soil in Taiwan and their occurrences are associated with human activities. Hygiene sensitive places such as hospitals should be particularly aware of the high frequency of the clinical relevant species.

microbiology↗

bioRxiv2022

The Network Zoo: a multilingual package for the inference and analysis of biological networks

Inference and analysis of cellular biological networks requires software tools that integrate multi-omic data from various sources. The Network Zoo (netZoo; netzoo.github.io) is an open-source software suite to model biological networks, including context-specific gene regulatory networks and multi-omics partial correlation networks, to conduct differential analyses, estimate community structure, and model the transitions between biological states. The netZoo builds on our ongoing development of network methods, harmonizing the implementations in various computing languages (R, Python, MATLAB, and C) and between methods to allow a better integration of these tools into analytical pipelines. To demonstrate the value of this integrated toolkit, we analyzed the multi-omic data from the Cancer Cell Line Encyclopedia (CCLE) by inferring gene regulatory networks for each cancer cell line and associating network features with other phenotypic attributes such as drug sensitivity. This allowed us to identify transcription factors that play a critical role in both drug resistance and cancer development in melanoma. We also used netZoo to build a pan-cancer, multi-tiered CCLE map and used it to identify known metabolic hallmarks of cancer and to estimate novel context-specific elements that mediate post-transcriptional regulation. Because the netZoo tools are open-source and there is a growing community of both users and developers, we built an ecosystem to support community contributions, share use cases, and visualize networks online. As additional data types become available and our suite of methods grows, we will expand "the zoo" to incorporate an increasingly sophisticated collection of tools for network inference and analysis.

genomics↗

bioRxiv2022

Detecting intragenic trans-splicing events with hybrid transcriptome sequencing in cancer cells

Trans-splicing can generate non-co-linear (NCL) transcripts that consist of exons in an order topologically inconsistent with the corresponding DNA template. Detecting trans-spliced RNAs (ts-RNAs) may be interfered by false positives from experimental artifacts, circular RNAs (circRNAs), and genetic rearrangements. Particularly, intragenic ts-RNAs, which are derived from separate precursor mRNA molecules of the same genes, are often mistaken for circRNAs through analyses of high-throughput transcriptome sequencing (RNA-seq) data. In addition, the biogenesis and function of ts-RNAs remain elusive. Here we developed a bioinformatics pipeline, NCLscan-hybrid, with the integration of long and short RNA-seq reads to minimize false positives and identify intragenic ts-RNAs. We utilized two features of long reads, out-of-circle and rolling circle, to distinguish intragenic ts-RNAs from circRNAs. We also designed multiple experimental validation steps to examine each type of false positives and successfully confirmed an intragenic ts-RNA (ts-ARFGEF1) in breast cancer cells. On the basis of ectopic expression and CRISPR-based endogenous genome modification experiments, we confirmed that ts-ARFGEF1 formation was significantly dependent on the reverse complementary sequences in the flanking introns of the NCL junction. Subsequent in vitro and in vivo experiments demonstrated that ts-ARFGEF1 silencing can significantly inhibit tumor cell growth. We further showed the regulatory role of ts-ARFGEF1 in p53-mediated apoptosis through affecting the PERK/eIF2a/ATF4/CHOP signaling pathway in breast cancer cells. This study thus described both bioinformatics procedures and experimental validation steps for rigorous characterization of transcriptionally non-co-linear RNAs, expanding the discovery of this important but understudied class of RNAs.

bioinformatics↗

bioRxiv2022

Evaluating the potential benefits and pitfalls of combining protein and expression quantitative trait loci in evidencing drug targets

Molecular quantitative trait loci (molQTL), which can provide functional evidence on the mechanisms underlying phenotype-genotype associations, are increasingly used in drug target validation and safety assessment. In particular, protein abundance QTLs (pQTLs) and gene expression QTLs (eQTLs) are the most commonly used for this purpose. However, questions remain on how to best consolidate results from pQTLs and eQTLs for target validation. In this study, we combined blood cell-derived eQTLs and plasma-derived pQTLs to form QTL pairs representing each gene and its product. We performed a series of enrichment analyses to identify features of QTL pairs that provide consistent evidence for drug targets based on the concordance of the direction of effect of the pQTL and eQTL. We repeated these analyses using eQTLs derived in 49 tissues. We found that 25-30% of blood-cell derived QTL pairs have discordant effects. The difference in tissues of origin for molecular markers contributes to, but is not likely a major source of, this observed discordance. Finally, druggable genes were as likely to have discordant QTL pairs as concordant. Our analyses suggest combining and consolidating evidence from pQTLs and eQTLs for drug target validation is crucial and should be done whenever possible, as many potential drug targets show discordance between the two molecular phenotypes that could be misleading if only one is considered. We also encourage investigating QTL tissue-specificity in target validation applications to help identify reasons for discordance and emphasise that concordance and discordance of QTL pairs across tissues are both informative in target validation.

genetics↗

bioRxiv2022

Insertion of YFP at the Arabidopsis AFL1 and P5CS1 loci shows the potential, and potential limitations, of gene targeting for functional analysis of stress-related genes

Crispr/CAS9-enabled homologous recombination to insert a tag in frame with an endogenous gene can circumvent difficulties such as context-dependent promoter activity that complicate analysis of gene expression and protein accumulation patterns. However, there have been few reports examining whether such Gene Targeting/Gene Tagging (GT) can alter expression of the target gene. The enzyme encoded by {Delta}1-pyrroline-5-carboxylate synthetase 1 (P5CS1) is key for stress-induced proline synthesis and drought resistance, yet its expression pattern and protein localization have been difficult to assay. We used GT to insert YFP in frame with the 5 or 3 ends of the endogenous P5CS1 and At14a-Like 1 (AFL1) coding regions. Insertion at the 3 end of either gene generated homozygous lines with expression of the gene-YFP fusion indistinguishable from the wild type allele. However, for P5CS1 this occurred only after selfing and advancement to the T5 generation allowed initial homozygous lethality of the insertion to be overcome. Once this was done, the GT-generated P5CS1-YFP plants revealed new information about P5CS1 localization and tissue-specific expression. In contrast, insertion of YFP at the 5 end of either gene blocked expression. The results demonstrate that GT can be useful for functional analyses of genes that are problematic to properly express by other means but also show that, in some cases, GT can disrupt expression of the target gene. Summary statementGene tagging of Arabidopsis thaliana P5CS1 and AFL1 shows the potential of GT for functional analysis of stress-related genes, but also provides examples of how GT can dramatically disrupt expression of the target gene.

plant biology↗

bioRxiv2022

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1/{beta}. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

molecular biology↗

bioRxiv2022

Genomic prediction with whole-genome sequence data in intensely selected pig lines

BackgroundEarly simulations indicated that whole-genome sequence data (WGS) could improve genomic prediction accuracy and its persistence across generations and breeds. However, empirical results have been ambiguous so far. Large data sets that capture most of the genome diversity in a population must be assembled so that allele substitution effects are estimated with high accuracy. The objectives of this study were to use a large pig dataset to assess the benefits of using WGS for genomic prediction compared to using commercial marker arrays, to identify scenarios in which WGS provides the largest advantage, and to identify potential pitfalls for its effective implementation. MethodsWe sequenced 6,931 individuals from seven commercial pig lines with different numerical size. Genotypes of 32.8 million variants were imputed for 396,100 individuals (17,224 to 104,661 per line). We used BayesR to perform genomic prediction for eight complex traits. Genomic predictions were performed using either data from a marker array or variants preselected from WGS based on association tests. ResultsThe prediction accuracy with each set of preselected WGS variants was not robust across traits and lines and the improvements in prediction accuracy that we achieved so far with WGS compared to marker arrays were generally small. The most favourable results for WGS were obtained when the largest training sets were available and used to preselect variants with statistically significant associations to the trait for augmenting the established marker array. With this method and training sets of around 80k individuals, average improvements of genomic prediction accuracy of 0.025 were observed in within-line scenarios. ConclusionsOur results showed that WGS has a small potential to improve genomic prediction accuracy compared to marker arrays in intensely selected pig lines in some settings. Thus, although we expect that more robust improvements could be attained with a combination of larger training sets and optimised pipelines, the use of WGS in the current implementations of genomic prediction should be carefully evaluated on a case-by-case basis against the cost of generating WGS at a large scale.

genomics↗

bioRxiv2022

Rare and population-specific functional variation across pig lines

BackgroundIt is expected that functional, mainly missense and loss-of-function (LOF), and regulatory variants are responsible for phenotypic differences among breeds, genetic lines, and varieties of livestock and crop species that have undergone diverse selection histories. However, there is still limited knowledge about the existing missense and LOF variation in livestock commercial populations, in particular regarding population-specific variation and how it can affect applications such as across-breed genomic prediction. MethodsWe re-sequenced the whole genome of 7,848 individuals from nine commercial pig breeding lines (average sequencing coverage: 4.1x) and imputed whole-genome genotypes for 440,610 pedigree-related individuals. The called variants were categorized according to predicted functional annotation (from LOF to intergenic) and prevalence level (number of lines in which the variant segregated; from private to widespread). Variants in each category were examined in terms of distribution along the genome, minor allele frequency, Wrights fixation index (FST), individual load, and association to production traits. ResultsOf the 46 million called variants, 28% were private (called in only one line) and 21% were widespread (called in all nine lines). Genomic regions with low recombination rate were enriched with private variants. Low-prevalence variants (called in one or a few lines only) were enriched for lower allele frequencies, lower FST, and putatively functional and regulatory roles (including loss-of-function and deleterious missense variants). Only a small subset of low-prevalence variants was found at intermediate allele frequencies and had large estimated effects on production traits. Individuals on average carried less private deleterious missense alleles than expected compared to other predicted consequence types. A small subset of low-prevalence variants with intermediate allele frequencies and higher FST were detected as significantly associated to the production traits and explained small fractions of phenotypic variance (up to 3.2%). These associations were tagged by other more widespread variants, including intergenic variants. ConclusionsMost low-prevalence variants are kept at very low allele frequency and only a small subset contributed detectable fractions of phenotypic variance. Not accounting for low-prevalence variants is therefore unlikely to hinder across-breed analyses, in particular for genomic prediction of breeding values using reference populations of a different genetic background.

genomics↗

bioRxiv2021

Fungal auxin is a quorum-based modulator of blast disease severity

Auxin is an important phytohormone regulating plant growth and development, and can also be produced by microbial pathogens including the rice-blast fungus Magnaporthe oryzae. However, the detailed biosynthesis pathway, biological function(s), and cellular distribution of such fungal auxin in M. oryzae remain largely unknown. Here, we report a sequential accumulation of intrinsic auxin in the three conidial cells, the infection structure (appressorium), and the invasive hyphae in M. oryzae. Such fungus-derived auxin was also secreted out and perceived by the host plants. A mitochondria-associated Indole-3-pyruvate decarboxylase, Ipd1, is essential for auxin/Indole-3-acetic acid biosynthesis in M. oryzae. The ipd1{Delta} was defective in pathogenicity whereas overexpression of IPD1 led to enhanced virulence in rice. Chemical inhibition of fungal IAA biosynthesis, or its increase via external supplementation decreased or increased the severity of blast disease, respectively, in a dose-dependent manner. Furthermore, the IAA produced and secreted by M. oryzae governed the incidence and severity of blast disease in a quorum-dependent manner. Appressorium formation, conidial cell death critical for appressorium function, and the transcription of infection-related genes, MPG1 and INV1, directly correlated with cell density and/or IAA levels within the conidial population at the early stages of pathogenic development. Overall, our study revealed that the severity of blast disease is regulated via quorum sensing with intrinsic IAA serving as an associated signal transducer in rice blast.

microbiology↗

bioRxiv2021

Spatial differences in stoichiometry of EGR phosphatase and Microtubule-Associated Stress Protein 1 control root meristem activity during drought stress.

During moderate severity drought and low water potential ({psi}w) stress, poorly understood signaling mechanisms restrict both meristem cell division and subsequent cell expansion. We found that the Clade E Growth-Regulating 2 (EGR2) protein phosphatase and Microtubule Associated Stress Protein 1 (MASP1) differed in their stoichiometry of expression across the root meristem and had opposing effects on root meristem activity at low {psi}w. Ectopic MASP1 or EGR expression increased or decreased, respectively, root meristem size and root elongation during low {psi}w stress. This, along with the ability of phosphomimic MASP1 to overcome EGR suppression of root meristem size and observation that ectopic EGR expression had no effect on unstressed plants, indicated that during low {psi}w EGR activation and attenuation of MASP1 phosphorylation in their overlapping zone of expression determines root meristem size and activity. Ectopic EGR expression also decreased root cell size at low {psi}w. Conversely, both the egr1-1egr2-1 and egr1-1egr2-1masp1-1 mutants had similarly increased root cell size; but, only egr1-1egr2-1 had increased cell division. These observations demonstrated that EGRs affect meristem activity via MASP1 but affect cell expansion via other mechanisms. Interestingly, EGR2 was highly expressed in the root cortex, a cell type important for growth regulation and environmental response. One Sentence SummarySpatial differences in EGR-MASP1 expression and control of MASP1 phosphorylation adjust root meristem activity to regulate growth during drought stress. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Paul E. Verslues (paulv@gate.sinica.edu.tw).

plant biology↗

bioRxiv2021

Response of Endozoicomonas montiporae to heat stress and coral host lysates

Endozoicomonas, a core bacterial group in corals, may also be a coral symbiont. Endozoicomonas communities often decrease rapidly in corals under heat stress. However, how the bacteria respond to changes in temperature and coral host during heat stress is unknown. Here, we employed the cultivable, dominant species E. montiporae as a working organism to explore how Endozoicomonas responds to heat stress. We designed two experiments to clarify the extent to which E. montiporae is influenced by temperature and coral host. We detected differentially expressed protein (DEP) profiles in this bacterium at 31{degrees}C and 33{degrees}C compared to 25{degrees}C by tandem mass tags-based quantitative proteome analysis. Fifty DEPs, including many heat shock proteins, were detected when the temperature changed. The expression of antioxidant defense proteins and key pyruvate synthase proteins decreased, suggesting that E. montiporae were in a physiology of stress at 33{degrees}C. Furthermore, some proteins were differentially expressed because of the heat-stress-treated coral lysate specifically, suggesting that not only heat but also heat-induced host factors can affect the protein expression of the bacterium. This study provides an in-depth analysis of how the molecular mechanisms of Endozoicomonas are affected by heat stress and coral host.

microbiology↗

bioRxiv2021

Quantifying the impact of gut microbiota on inflammation and hypertensive organ damage

AimsHypertension (HTN) can lead to heart and kidney damage. The gut microbiota has been linked to HTN, although it is difficult to estimate its significance due to the variety of other features known to influence HTN. In the present study, we used germ-free (GF) and colonized (COL) littermate mice to quantify the impact of microbial colonization on organ damage in HTN. Methods and resultsFour-week-old male GF C57BL/6J littermates were randomized to remain GF or receive microbial colonization. HTN was induced by subcutaneous infusion with angiotensin (Ang) II (1.44mg/kg/d) and 1% NaCl in the drinking water; sham-treated mice served as control. Renal damage was exacerbated in GF mice, whereas cardiac damage was more comparable between COL and GF, suggesting that the kidney is more sensitive to microbial influence. Multivariate analysis revealed a larger effect of HTN in GF mice. Serum metabolomics demonstrated that the colonization status influences circulating metabolites relevant to HTN. Importantly, GF mice were deficient in anti-inflammatory fecal short-chain fatty acids (SCFA). Flow cytometry showed that the microbiome has an impact on the induction of anti-hypertensive myeloid-derived suppressor cells and pro-inflammatory Th17 cells in HTN. In vitro inducibility of Th17 cells was significantly higher for cells isolated from GF than conventionally raised mice. ConclusionsMicrobial colonization status of mice had potent effects on their phenotypic response to a hypertensive stimulus, and the kidney is a highly microbiota-susceptible target organ in HTN. The magnitude of the pathogenic response in GF mice underscores the role of the microbiome in mediating inflammation in HTN. Translation PerspectiveTo assess the potential of microbiota-targeted interventions to prevent organ damage in hypertension, an accurate quantification of microbial influence is necessary. We provide evidence that the development of hypertensive organ damage is dependent on colonization status and suggest that a healthy microbiota provides anti-hypertensive immune and metabolic signals to the host. In the absence of normal symbiotic host-microbiome interactions, hypertensive damage to the kidney in particular is exacerbated. We suggest that hypertensive patients experiencing perturbations to the microbiota, which are common in CVD, may be at a greater risk for target-organ damage than those with a healthy microbiome.

microbiology↗

bioRxiv2021

Comparative genomics of three Colletotrichum scovillei strains and genetic analysis revealed genes involved in fungal growth and virulence on chili pepper

Colletotrichum scovillei is a virulent pathogen and the dominant species causing anthracnose of chili pepper in many Asian countries. Three strains of this pathogen, Coll-524, Coll-153 and Coll-365, show varied virulence on chili pepper fruit. Among the three strains, Coll-365 showed significant defects in growth and virulence. To decipher the genetic variations among these strains and identify genes contributing to growth and virulence, in this study, comparative genomic analysis and gene transformation to verify gene function were applied. The genomes of the three strains were sequenced and Coll-524 had 1.3% and 1.5% more genes than Coll-153 and Coll-365, respectively. Compared to Coll-524 and Coll-153, Coll-365 had numerous gene losses including 33 effector genes that are distributed in different scaffolds and a cluster of 14 genes in a 34-kb genomic fragment. Through gene transformation, three genes in the 34-kb fragment were identified to have functions in growth and/or virulence of C. scovillei. Gene 15019 encoding a protein related to phospholipase A2-activating protein enhanced the growth of Coll-365. A combination of 15019 with one transcription factor gene 15022 and one C6 zinc finger domain-containing protein gene 15029 was found to enhance the pathogenicity of Coll-365. Introduction of gene 15215, which encodes a LysM domain-containing protein, into Coll-365 caused a reduction in the germination rate of Coll-365. In conclusion, the higher virulent strain Coll-524 had more genes and encoded more pathogenicity related proteins and transposable elements than the other two strains, which may contribute to the high virulence of Coll-524. In addition, the absence of the 34-kb fragment plays a critical role in the defects of growth and virulence of strain Coll-365. Author SummaryColletotrichum scovillei is a highly virulent and dominant pathogen causing anthracnose of chili that leads to significant economic loss in chili production in many Asia countries. In this study we focus on finding the gene differences of three C. scovillei strains with different pathogenicity in chili pepper infection and verifying the function of some genes in the lowest virulence strain. We sequenced them and did gene annotation and genome comparison. We setup a simple mathematical method to identify gene variations between strong and weak virulence strains. Our results show that the lowest virulence strain has less pathogenicity-related genes. We also found that the absence of 14 genes in a compact genomic fragment was part of the reason of growth and virulence defect of the lowest virulence strain. We identified four genes that play roles on fungal growth and/or virulence on chili pepper. We also found a group of effector genes that specifically appear in species collected form infected chili in C. acutatum species complex. Our research provides detailed information for why the three strains have different virulence on chili pepper.

genomics↗

bioRxiv2021

Dissecting errors of the exogenously-driven and endogenously-driven saccadic tasks in the common marmoset

Various visual paradigms in oculomotor research have been used for studying the neural processes of eye movement, cognitive control, attention and neurological disorders. However, we usually analyse data collected from humans or over-trained non-human primates (NHPs), focusing only on successful trials, whereas error trials are usually excluded. These errors may repetitively show up in diseases, but their interpretation would be difficult due to the absence of records taken from healthy controls. In the current study, we aimed to analyse both correctly and incorrectly performed trials in both marmosets and humans. We trained marmosets to perform the gap saccade task and the oculomotor delayed response task. We also collected data from human subjects who performed identical tasks. We categorised error trials into three different groups, based on the time when an incorrect response occurred. We also interpreted possible causes by analysing saccade reaction time, saccade landing position and task history. Despite the rareness of human error, we found that marmosets and humans showed remarkably similar behaviour in error and success. We also found that successful saccades in the gap saccade task had always the highest peak velocity in both species, reflecting faster sensorimotor processing for correct responses. Our results suggest that marmosets and humans might share similar neural processing for successful and unsuccessful oculomotor behaviour, making them a suitable model for studying human behaviour. More importantly, analysing error trials in sync with successful ones will provide further insights into the cognitive and sensorimotor processes. NEW AND NOTEWORTHYThis is the first detailed report focusing on analysing both error and successful trials in oculomotor tasks. We proposed nomenclatures and a generalizable way of grouping and analysing error trials. Our results also indicate that marmosets can be a promising experimental candidate for oculomotor research because they replicate the saccade properties of error and success seen in humans. This will help set the baseline measurements to study brain disorders using NHP and understand the neural mechanisms from a different perspective.

neuroscience↗

bioRxiv2021

Recurrent de novo single point mutation on the gene encoding Na+ /K+ pump results in epilepsy

The etiology of epilepsy remains undefined in two-thirds of patients. Here, we identified a de novo mutation of ATP1A2 (c.2426 T>G, p.Leu809Arg), which encodes the 2 subunit of Na+/K+-ATPase, from a family with idiopathic epilepsy. This mutation caused seizures in the study patients. We generated the point mutation mouse model Atp1a2L809R, which recapitulated the epilepsy observed in the study patients. In Atp1a2L809R/WT mice, convulsions were observed and cognitive and memory function was impaired. This mutation affected the potassium binding function of the protein, disabling its ion transport ability, thereby increasing the frequency of nerve impulses. Our work revealed that ATP1A2L809R mutations cause a predisposition to epilepsy. Moreover, we first provide a point mutation mouse model for epilepsy research and drug screening.

neuroscience↗

bioRxiv2021

TAS-Seq: a robust and sensitive amplification method for beads-based scRNA-seq

Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data. We developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq) for scRNA-seq based on a terminator, terminal transferase, and nanowell/bead-based scRNA-seq platform; TAS-Seq showed high tolerance to variations in the terminal transferase reaction, which complicate the handling of existing terminal transferase-based scRNA-seq methods. In murine and human lung samples, TAS-Seq yielded scRNA-seq data that were highly correlated with flow-cytometric data, showing higher gene-detection sensitivity and more robust detection of important cell-cell interactions and expression of growth factors/interleukins in cell subsets than 10X Chromium v2 and Smart-seq2. Expanding TAS-Seq application will improve understanding and atlas construction of lung biology at the single-cell level.

genomics↗

bioRxiv2021

Selective whole genome amplification as a tool to enrich specimens with low Treponema pallidum genomic DNA copies for whole genome sequencing

Downstream next generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA using the NEBNext(R) Microbiome DNA Enrichment Kit followed by MDA with the REPLI-g Single Cell Kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/l of sample for SWGA and >129 genomic copies/l for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now. ImportanceSyphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions, and if left untreated, can progress through multiple stages with varied clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past two decades, WGS data is needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of a SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

genomics↗