ISSAAC-seq enables sensitive and flexible multimodal profiling of chromatin accessibility and gene expression in single cells.

Joint profiling of chromatin accessibility and gene expression from the same single cell provides critical information about cell types in a tissue and cell states during a dynamic process. Here, we develop in situ sequencing hetero RNA-DNA-hybrid after assay for transposase-accessible chromatin-sequencing (ISSAAC-seq), a highly sensitive and flexible single-cell multi-omics method to interrogate chromatin accessibility and gene expression from the same single nucleus. We demonstrated that ISSAAC-seq is sensitive and provides high quality data with orders of magnitude more features than existing methods. Using the joint profiles from over 10,000 nuclei from the mouse cerebral cortex, we uncovered major and rare cell types and cell-type specific regulatory elements and identified heterogeneity at the chromatin level within established cell types defined by gene expression. Finally, we revealed distinct dynamics and relationships of gene expression and chromatin accessibility during an oligodendrocyte maturation trajectory.

Serrate-Associated Protein 1, a splicing-related protein, promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) are essential regulators of gene expression in metazoans and plants. In plants, most miRNAs are generated from primary miRNA transcripts (pri-miRNAs), which are processed by the Dicer-like 1 (DCL1) complex along with accessory proteins. Serrate-Associated Protein 1 (SEAP1), a conserved splicing-related protein, has been studied in human and yeast. However, the functions of SEAP1 in plants remain elusive. Lack of SEAP1 results in embryo lethality and knockdown of SEAP1 by an artificial miRNA (amiRSEAP1 ) causes pleiotropic developmental defects and reduction in miRNA accumulation. SEAP1 associates with the DCL1 complex, and may promote the interaction of the DCL1 complexes with pri-miRNAs. SEAP1 also enhances pri-miRNA accumulation, but does not affect pri-miRNA transcription, suggesting it may indirectly or directly stabilize pri-miRNAs. In addition, SEAP1 affects the splicing of some pri-miRNAs and intron retention of messenger RNAs at global levels. Our findings uncover both conserved and novel functions of SEAP1 in plants. Besides the role as a splicing factor, SEPA1 may promote miRNA biogenesis by positively modulating pri-miRNA splicing, processing and/or stability.

Repeats in mitochondrial and chloroplast genomes characterize the ecotypes of the Oryza.

Mitochondria and chloroplast are very important organelles for organism, participating in basic life activity. Their genomes contain many repeats which can lead to a variation of genome structure. Oryza is an important genus for human beings' nutrition. Several mitochondrial and chloroplast genomes of Oryza have been sequenced, which help us to insight the distribution and evolution of the repeats in Oryza species. In this paper, we compared six mitochondrial and 13 chloroplast genomes of Oryza and found that the structures of mitochondrial genomes were more diverse than chloroplast genomes. Since repeats can change the structure of the genome, resulting in the structural diversity of the genome, we analyzed all repeats and found 31 repeats in mitochondrial and 13 repeats in chloroplast genomes. Further, we developed 21 pairs of MRS molecular markers and 12 pairs of CRS molecular markers based on mitochondrial repeats and chloroplast repeats, respectively. These molecular markers can be used to detect the repeat-mediated recombination in Oryza mitochondrial and chloroplast genomes by PCR or fluorescence quantification. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-020-01198-6.

Wide crossing diversify mitogenomes of rice.

BACKGROUND: In most angiosperms, the inheritance of the mitochondria takes place in a typical maternal manner. However, very less information is available about if the existence of structural variations or not in mitochondrial genomes (mitogenomes) between maternal parents and their progenies. RESULTS: In order to find the answer, a stable rice backcross inbred line (BIL) population was derived from the crosses of Oryza glaberrima/Oryza sativa//Oryza sativa. The current study presents a comparative analysis of the mitogenomes between maternal parents and five BILs. There were recorded universal structural variations such as reversal, translocation, fusion, and fission among the BILs. The repeat-mediated recombination and non-homologous end-joining contributed virtually equal to the rearrangement of mitogenomes. Similarly, the relative order, copy-number, expression level, and RNA-editing rate of mitochondrial genes were also extensively varied among BILs. CONCLUSIONS: These novel findings unraveled an unusual mystery of the maternal inheritance and possible cause for heterogeneity of mitogenomes in rice population. The current piece of work will greatly develop our understanding of the plant nucleo-cytoplasmic interaction and their potential role in plant growth and developmental processes.

Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize.

Genetic imprinting is a specific epigenetic phenomenon in which a subset of genes is expressed depending on their parent-of-origin. Two types of chromatin modifications, DNA methylation and histone modification, are generally believed to be involved in the regulation of imprinting. However, the genome-wide correlation between allele-specific chromatin modifications and imprinted gene expression in maize remains elusive. Here we report genome-wide high resolution allele-specific maps of DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3) in maize endosperm. For DNA methylation, thousands of parent-of-origin dependent differentially methylated regions (pDMRs) were identified. All pDMRs were uniformly paternally hypermethylated and maternally hypomethylated. We also identified 1131 allele-specific H3K27me3 peaks that are preferentially present in the maternal alleles. Maternally expressed imprinted genes (MEGs) and paternally expressed imprinted genes (PEGs) had different patterns of allele-specific DNA methylation and H3K27me3. Allele-specific expression of MEGs was not directly related to allele-specific H3K27me3, and only a subset of MEGs was associated with maternal-specific DNA demethylation, which was primarily located in the upstream and 5' portion of gene body regions. In contrast, allele-specific expression of a majority of PEGs was related to maternal-specific H3K27me3, with a subgroup of PEGs also associated with maternal-specific DNA demethylation. Both pDMRs and maternal H3K27me3 peaks associated with PEGs are enriched in gene body regions. Our results indicate highly complex patterns of regulation on genetic imprinting in maize endosperm.

Genome-wide Discovery of Circular RNAs in the Leaf and Seedling Tissues of Arabidopsis Thaliana.

BACKGROUND: Recently, identification and functional studies of circular RNAs, a type of non-coding RNAs arising from a ligation of 3' and 5' ends of a linear RNA molecule, were conducted in mammalian cells with the development of RNA-seq technology. METHOD: Since compared with animals, studies on circular RNAs in plants are less thorough, a genome-wide identification of circular RNA candidates in Arabidopsis was conducted with our own developed bioinformatics tool to several existing RNA-seq datasets specifically for non-coding RNAs. RESULTS: A total of 164 circular RNA candidates were identified from RNA-seq data, and 4 circular RNA transcripts, including both exonic and intronic circular RNAs, were experimentally validated. Interestingly, our results show that circular RNA transcripts are enriched in the photosynthesis system for the leaf tissue and correlated to the higher expression levels of their parent genes. Sixteen out of all 40 genes that have circular RNA candidates are related to the photosynthesis system, and out of the total 146 exonic circular RNA candidates, 63 are found in chloroplast.

Long-term outcomes of facial nerve schwannomas with favorable facial nerve function: tumor growth rate is correlated with initial tumor size.

OBJECTIVE: The study aimed to report long-term outcomes of facial nerve schwannomas (FNS) with favorable facial nerve function by observation, and to discuss about the relationship between initial tumor size and tumor growth. METHODS: 21 facial nerve schwannoma cases with favorable facial nerve function were managed by observation. They were divided into larger size group (size ≥10mm) and smaller size group (size <10mm) according to initial tumor size. RESULTS: They were followed up for 6.4±1.7years. 18 of 21 cases (85.7%) maintained House-Brackmann Grade III or better. Growth rate of the tumors in larger size group was 72.7%, much higher than 10% in smaller size group (p<0.05). CONCLUSIONS: Observation was feasible for most FNS with favorable facial nerve function, and growth rate of the tumors was associated with tumor size.

Short-term therapeutic effects of low-dose cytarabine plus surgical resection on elderly patients with trigeminal nerve tumor and safety observation.

OBJECTIVE: To evaluate the short-term therapeutic effects of low-dose cytarabine plus surgical resection on elderly patients with trigeminal nerve tumor and to observe the safety. METHODS: A total of 120 elderly patients with trigeminal nerve tumor were divided into a treatment group and a control group by random draw (n=60), and both groups were subjected to resection by stereotactic image-guided endoscopic nasal surgery. Afterwards, the control group was administered with high-dose cytarabine while the treatment group was given low-dose cytarabine for 14 days. RESULTS: Both groups completed treatment, but the effective rate of the treatment group (96.7%) was significantly higher than that of the control group (83.3%) (P < 0.05). The pain scores of the two groups were similar at T0, T1 and T2, but the score of the treatment group at T2 was significantly different from those at T0 and T1 (P < 0.05). During treatment, the treatment group was significantly less prone to complications such as headache, vomiting, vision impairment, nausea and local swelling than the control group (P < 0.05). During three months of follow-up, the appetite, sleep and daily living scores were significantly higher than those of the control group (P < 0.05). CONCLUSION: Stereotactic image-guided surgery was able to treat trigeminal nerve tumor well, and the effect was enhanced by low-dose cytarabine that improved postoperative outcomes and quality of life by dramatically decreasing complications.

Defining reference genes for quantitative real-time PCR analysis of anther development in rice.

Quantitative real-time polymerase chain reaction (qPCR) is one of the most accurate and widely used methods for gene expression analysis. However, the choice of reference genes for normalization is critical for accurate quantification of gene expression. As development of genomics, mining large-scale datasets such as microarray and RNA-sequencing data becomes a new approach for exploitation of new reference genes. In this study, we analyzed an RNA-sequencing dataset of rice anther and 167 microarray datasets involving different tissues and developing stages of rice anthers and pollens. We selected 12 candidate genes and other 5 reference genes, including ACT1, eEF-1α, GAPDH, Exp2, and CCDC72 used in previous studies, and evaluated their expression in eight tissues and different developmental stages of anthers in rice variety 9311 and Yuetai. UPF3, eIF4A-3, GAPDH, and PPP6 were identified as the most suitable reference genes for qPCR analysis of anther development in rice. The new candidate reference genes showed more stable expression than the traditionally used reference genes. These results provide a set of reliable reference genes for studies in rice anther developmental process.

Identification of RNA-based cell-type markers for stem-cell manufacturing systems with a statistical scoring function.

Cell-type biomarkers are useful in stem-cell manufacturing to monitor cell purity, quantity, and quality. However, the study on cell-type markers, specifically for stem cell manufacture, is limited. Emerging questions include which RNA transcripts can serve as biomarkers during stem cell culture and how to discover these biomarkers efficiently and precisely. We developed a scoring function system to identify RNA biomarkers with RNA-seq data for systems that have a limited number of cell types. We applied the method to two data sets, one for extracellular RNAs (ex-RNAs) and the other for intracellular microRNAs (miRNAs). The first data set has RNA-seq data of ex-RNAs from cell culture media for six different types of cells, including human embryonic stem cells. To get the RNA-seq data from intracellular miRNAs, we cultured three types of cells: human embryonic stem cells (H9), neural stem cells (NSC), hESC-derived endothelial cells (EC) and conducted small RNA-seq to their intracellular miRNAs. Using these data, we identified a set of ex-RNAs/smRNAs as candidates of biomarkers for different types of cells for cell manufacture. The validity of these findings was confirmed by the utilization of additional data sets and experimental procedures. We also used deep-learning-based prediction methods and simulated data to validate these discovered biomarkers.

Gentiopicroside alleviated epileptogenesis in immature rats through inactivation of NLRP3 inflammasome by inhibiting P2X7R expression.

OBJECTIVES: This study aimed to elucidate the effects of Gentiopicroside (Gent) on epileptogenesis and underlying mechanisms. METHODS: The status epilepticus (SE) model was established by intraperitoneal (i.p.) injection of lithium chloride (127 mg/kg) and pilocarpine (50 mg/kg) in immature rats. HAPI microglial cellular inflammation model was induced by lipopolysaccharide (LPS, 1 µg/ml) and adenosine triphosphate (ATP, 5 mM). The differential concentrations of Gent were used to pretreat animal (200, 400, and 800 mg/kg) and model cells (50, 100, and 200 µM). Epileptic discharges were assessed by electroencephalography (EEG) and Racine scale. Changes in spatial memory function were measured using the Morris water maze task test. Nissl and FJB staining were employed to assess the damage to hippocampus tissues. ELISA was used to detect the production of IL-1ß, IL-18, and TNF-α. The expressions of P2X7R and NLRP3 were detected by q-PCR, immunofluorescence staining, and Western blot, and cell viability was determined by cell counting kit-8 (CCK-8). RESULTS: Lithium chloride and pilocarpine (LICL-PILO) induced abnormal EEG activities, behavioral alterations, brain damage, and inflammatory responses in immature rats. However, Gent pretreatment significantly reduced the neuronal damage and spatial memory dysfunction induced by LICL-PILO. Additionally, Gent suppressed the production of inflammatory cytokines and inhibited the expression of P2X7R, NLRP3, ASC, and Caspase-1 in LPS/ATP-induced HAPI microglial cells. DISCUSSION: Gent intervention could improve epileptogenesis in immature rats partially due to suppressing P2X7R and NLRP3 inflammasome.

Variation in Rice Plastid Genomes in Wide Crossing Reveals Dynamic Nucleo-Cytoplasmic Interaction.

Plastid genomes (plastomes) of angiosperms are well known for their relative stability in size, structure, and gene content. However, little is known about their heredity and variations in wide crossing. To such an end, the plastomes of five representative rice backcross inbred lines (BILs) developed from crosses of O. glaberrima/O. sativa were analyzed. We found that the size of all plastomes was about 134,580 bp, with a quadripartite structure that included a pair of inverted repeat (IR) regions, a small single-copy (SSC) region and a large single-copy (LSC) region. They contained 76 protein genes, 4 rRNA genes, and 30 tRNA genes. Although their size, structure, and gene content were stable, repeat-mediated recombination, gene expression, and RNA editing were extensively changed between the maternal line and the BILs. These novel discoveries demonstrate that wide crossing causes not only nuclear genomic recombination, but also plastome variation in plants, and that the plastome plays a critical role in coordinating the nuclear-cytoplasmic interaction.

FDDM1 and FDDM2, Two SGS3-like Proteins, Function as a Complex to Affect DNA Methylation in Arabidopsis.

DNA methylation is an important epigenetic modification required for the specific regulation of gene expression and the maintenance of genome stability in plants and animals. However, the mechanism of DNA demethylation remains largely unknown. Here, we show that two SGS3-like proteins, FACTOR OF DNA DEMETHYLATION 1 (FDDM1) and FDDM2, negatively affect the DNA methylation levels at ROS1-dependend DNA loci in Arabidopsis. FDDM1 binds dsRNAs with 5' overhangs through its XS (rice gene X and SGS3) domain and forms a heterodimer with FDDM2 through its XH (rice gene X Homology) domain. A lack of FDDM1 or FDDM2 increased DNA methylation levels at several ROS1-dependent DNA loci. However, FDDM1 and FDDM2 may not have an additive effect on DNA methylation levels. Moreover, the XS and XH domains are required for the function of FDDM1. Taken together, these results suggest that FDDM1 and FDDM2 act as a heterodimer to positively modulate DNA demethylation. Our finding extends the function of plant-specific SGS3-like proteins.

Deciphering the Genetic Basis of Lodging Resistance in Wild Rice Oryza longistaminata.

The abuse of fertilizer results in tall rice plants that are susceptible to lodging and reduced plant yield. Hence, it is important to identify and utilize the quantitative trait loci (QTLs)/genes for lodging resistance breeding. Oryza longistaminata exhibits a strong stem and high biomass productivity, which could be a candidate gene pool for cultivars lodging resistance improvement. Here, a set of 152 BC2F20 lines derived from a cross between a cultivated line 93-11 and O. longistaminata was evaluated for lodging resistance. QTL mapping analysis combined with single-nucleotide polymorphism (SNP) marker derived from high-throughput sequencing identified 12 QTLs for stem diameter (SD), 11 QTLs for stem length (SL), and 3 QTLs for breaking strength (BS). Of which, 14 QTLs were first identified from O. longistaminata. A major QTL, qLR1, which was delimited to a region ∼80 kb on chromosome 1, increased stem diameter, stem length, and breaking strength. Another major QTL, qLR8, that was delimited in an interval ∼120 kb on chromosome 8, significantly enhanced the breaking strength. These results provide evidence that O. longistaminata can be exploited to develop lodging-resistant rice lines.

GABRG2 Deletion Linked to Genetic Epilepsy with Febrile Seizures Plus Affects the Expression of GABAA Receptor Subunits and Other Genes at Different Temperatures.

Mutations in γ-aminobutyric acid A receptor (GABAA) subunits and sodium channel genes, especially GABRG2 and SCN1A, have been reported to be associated with febrile seizures (FS) and genetic epilepsy with febrile seizures plus (GEFS+). GEFS+ is a well-known family of epileptic syndrome with autosomal dominant inheritance in children. Its most common phenotypes are febrile seizures often with accessory afebrile generalized tonic-clonic seizures, febrile seizures plus (FS+), severe epileptic encephalopathy, as well as other types of generalized or localization-related seizures. However, the pathogenesis of febrile seizures remains largely unknown. Here, we generated a GABRG2 gene knockout cell line (HT22GABRG2KO) by applying the CRISPR/Cas9-mediated genomic deletion in HT-22 mouse hippocampal neuronal cell line to explore the function of GABRG2 in vitro. With mRNA-seq, we found significant changes in the expression profiles of several epilepsy-related genes when GABRG2 was knockout, some of them showing temperature-induced changes as well. Kyoto Encyclopedia Gene and Genomic (KEGG) analysis revealed a significant alteration in the MAPK and PI3K-Akt signaling pathways. We also observed an up-regulation of the matrix metalloproteinases (MMPs) family after GABRG2 knockout. Furthermore, the significant decrease in expression of GABRA1 and CACNA1A (but not others) with an increase in temperature is a novel finding. In summary, mutations in the GABAA receptor can lead to a decrease in numbers of receptors, which may cause the impairment of GABAergic pathway signaling. This data has been the first time to reveal that GABRG2 mutations would affect the function of other genes, and based on this finding we hope this work would also provide a new direction for the research of GABRG2 in GEFS+. It also may provide a molecular basis for the severity of epilepsy, and guide the clinical medication for the treatment of the epilepsy focused on the function on GABAA receptors, which, might be a new strategy for genetic diagnosis and targeted treatment of epilepsy.

Transcriptome analysis-identified long noncoding RNA CRNDE in maintaining endothelial cell proliferation, migration, and tube formation.

Obesity is a leading risk factor for type-2 diabetes. Diabetes often leads to the dysregulation of angiogenesis, although the mechanism is not fully understood. Previously, long noncoding RNAs (lncRNAs) have been found to modulate angiogenesis. In this study, we asked how the expression levels of lncRNAs change in endothelial cells in response to excessive palmitic acid treatment, an obesity-like condition. Bioinformatics analysis revealed that 305 protein-coding transcripts were upregulated and 70 were downregulated, while 64 lncRNAs were upregulated and 46 were downregulated. Gene ontology and pathway analysis identified endoplasmic reticulum stress, HIF-1 signaling, and Toll-like receptor signaling as enriched after palmitic acid treatment. Moreover, we newly report enrichment of AGE-RAGE signaling pathway in diabetic complications, IL-17 signaling, and cysteine and methionine metabolism by palmitic acid. One lncRNA, Colorectal Neoplasia Differentially Expressed (CRNDE), was selected for further investigation. Palmitic acid induces CRNDE expression by 1.9-fold. We observed that CRNDE knockdown decreases endothelial cell proliferation, migration, and capillary tube formation. These decreases are synergistic under palmitic acid stress. These data demonstrated that lncRNA CRNDE is a regulator of endothelial cell proliferation, migration, and tube formation in response to palmitic acid, and a potential target for therapies treating the complications of obesity-induced diabetes.

Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T0 plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.

TRAF4 promotes tumorigenesis of breast cancer through activation of Akt.

Increasing evidence suggests that tumor necrosis factor receptor-associated factor 4 (TRAF4) is an oncogene which is frequently overexpressed in many human carcinomas. Although TRAF4 was originally identified in breast cancer, the underlying mechanism of TRAF4 in tumorigenesis remains largely unknown. In the present study, we found that TRAF4 was overexpressed in cancer cells, and RNA interference (RNAi)-mediated gene knockdown of TRAF4 decreased cell growth, cell migration and invasion. Next, we found that TRAF4 promoted cell survival kinase Akt membrane recruitment, which is essential for Akt activation. Furthermore, we demonstrated a direct interaction between Akt and TRAF4. Additionally, overexpression of constitutively activated Akt reversed cell growth arrest in TRAF4 gene-silenced cells. Taken together, our data indicate that TRAF4 plays an important role in tumorigenesis of breast cancer through direct interaction and activation of Akt, implying that TRAF4 may be a potential molecular target for breast cancer prevention and therapy.

Discriminative latent models for recognizing contextual group activities.

In this paper, we go beyond recognizing the actions of individuals and focus on group activities. This is motivated from the observation that human actions are rarely performed in isolation; the contextual information of what other people in the scene are doing provides a useful cue for understanding high-level activities. We propose a novel framework for recognizing group activities which jointly captures the group activity, the individual person actions, and the interactions among them. Two types of contextual information, group-person interaction and person-person interaction, are explored in a latent variable framework. In particular, we propose three different approaches to model the person-person interaction. One approach is to explore the structures of person-person interaction. Differently from most of the previous latent structured models, which assume a predefined structure for the hidden layer, e.g., a tree structure, we treat the structure of the hidden layer as a latent variable and implicitly infer it during learning and inference. The second approach explores person-person interaction in the feature level. We introduce a new feature representation called the action context (AC) descriptor. The AC descriptor encodes information about not only the action of an individual person in the video, but also the behavior of other people nearby. The third approach combines the above two. Our experimental results demonstrate the benefit of using contextual information for disambiguating group activities.