Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

DNA methylation atlas of the mouse brain at single-cell resolution.

Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing1,2 to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data3 enabled prediction of high-confidence enhancer-gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments4. By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum.

Epigenomic diversity of cortical projection neurons in the mouse brain.

Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain1, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

Genome-wide identification and expression patterns of the laccase gene family in response to kiwifruit bacterial canker infection.

BACKGROUND: Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a destructive disease worldwide. Resistance genes that respond to Psa infection urgently need to be identified for controlling this disease. Laccase is mainly involved in the synthesis of lignin in the plant cell wall and plays a prominent role in plant growth and resistance to pathogen infection. However, the role of laccase in kiwifruit has not been reported, and whether laccase is pivotal in the response to Psa infection remains unclear. RESULTS: We conducted a bioinformatics analysis to identify 55 laccase genes (AcLAC1-AcLAC55) in the kiwifruit genome. These genes were classified into five cluster groups (I-V) based on phylogenetic analysis, with cluster groups I and II having the highest number of members. Analysis of the exon-intron structure revealed that the number of exons varied from 1 to 8, with an average of 5 introns. Our evolutionary analysis indicated that fragment duplication played a key role in the expansion of kiwifruit laccase genes. Furthermore, evolutionary pressure analysis suggested that AcLAC genes were under purifying selection. We also performed a cis-acting element analysis and found that AcLAC genes contained multiple hormone (337) and stress signal (36) elements in their promoter regions. Additionally, we investigated the expression pattern of laccase genes in kiwifruit stems and leaves infected with Psa. Our findings revealed that laccase gene expression levels in the stems were higher than those in the leaves 5 days after inoculation with Psa. Notably, AcLAC2, AcLAC4, AcLAC17, AcLAC18, AcLAC26, and AcLAC42 showed significantly higher expression levels (p < 0.001) compared to the non-inoculated control (0 d), suggesting their potential role in resisting Psa infection. Moreover, our prediction indicated that 21 kiwifruit laccase genes are regulated by miRNA397, they could potentially act as negative regulators of lignin biosynthesis. CONCLUSIONS: These results are valuable for further analysis of the resistance function and molecular mechanism of laccases in kiwifruit.

Antifungal activity and mechanism of tetramycin against Alternaria alternata, the soft rot causing fungi in kiwifruit.

Kiwifruit rot caused by the fungus Alternaria alternata occurs in many countries, leading to considerable losses during kiwifruit production. In this study, we evaluated the antifungal activity and mechanism of tetramycin against kiwifruit soft rot caused by Alternaria alternata. Tetramycin exerted antifungal effects through the suppression of mycelial growth, conidial germination, and the pathogenicity of A. alternata. Scanning electron microscopic observations revealed that tetramycin destroyed the mycelial structure, causing the mycelia to twist, shrink, and even break. Furthermore, transmission electron microscopy revealed that tetramycin caused severe plasmolysis and a decrease in cell inclusions, and the cell wall appeared thinner with blurred boundaries. In addition, tetramycin destroyed cell membrane integrity, resulting in the leakage of cellular components such as nucleic acids and proteins in mycelial suspensions. Moreover, tetramycin also caused cell wall lysis by enhancing the activities of chitinase and ß-1,3-glucanase and inducing the overexpression of related chitinase gene (Chit) and ß-1,3-glucanase gene (ß-1,3-glu) in A. alternata. In field trials, tetramycin not only decreased the incidence of kiwifruit rot but also create a beneficial living space for kiwifruit growth. Overall, this study indicated that the application of tetramycin could serve as an alternative measure for the management of kiwifruit rot.

Histone modifications predispose genome regions to breakage and translocation.

Chromosome translocations are well-established hallmarks of cancer cells and often occur at nonrandom sites in the genome. The molecular features that define recurrent chromosome breakpoints are largely unknown. Using a combination of bioinformatics, biochemical analysis, and cell-based assays, we identify here specific histone modifications as facilitators of chromosome breakage and translocations. We show enrichment of several histone modifications over clinically relevant translocation-prone genome regions. Experimental modulation of histone marks sensitizes genome regions to breakage by endonuclease challenge or irradiation and promotes formation of chromosome translocations of endogenous gene loci. Our results demonstrate that histone modifications predispose genome regions to chromosome breakage and translocations.

Synthesis of Isoselenazoles and Isothiazoles from Demethoxylative Cycloaddition of Alkynyl Oxime Ethers.

A general method for the synthesis of isoselenazoles and isothiazoles has been developed by the base-promoted demethoxylative cycloaddition of alkynyl oxime ethers using the cheap and inactive Se powder and Na2S as selenium and sulfur sources. This transformation features the direct construction of N-, Se-, and S-containing heterocycles through the formation of N-Se/S and C-Se/S bonds in one-pot reactions with excellent functional group tolerance.

Base-promoted thioannulation of o-alkynyl oxime ethers with sodium sulfide for the general synthesis of isothiocoumarins.

A general and efficient strategy for the one-pot synthesis of isothiocoumarin-1-ones has been developed via the base-promoted 6-endo-dig thioannulation of o-alkynyl oxime ethers using the cheap and readily available Na2S as the sulfur source. Mechanistic studies disclosed that the reaction proceeded through two C-S bond formations, N-O bond cleavage and the final hydrolysis of imines.

An alternative pluripotent state confers interspecies chimaeric competency.

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.

Sulfur-Induced Resistance against Pseudomonas syringae pv. actinidiae via Triggering Salicylic Acid Signaling Pathway in Kiwifruit.

Sulfur has been previously reported to modulate plant growth and exhibit significant anti-microbial activities. However, the mechanism underlying its diverse effects on plant pathogens has not been elucidated completely. The present study conducted the two-year field experiment of sulfur application to control kiwifruit canker from 2017 to 2018. For the first time, our study uncovered activation of plant disease resistance by salicylic acid after sulfur application in kiwifruit. The results indicated that when the sulfur concentration was 1.5-2.0 kg m-3, the induced effect of kiwifruit canker reached more than 70%. Meanwhile, a salicylic acid high lever was accompanied by the decline of jasmonic acid. Further analysis revealed the high expression of the defense gene, especially AcPR-1, which is a marker of the salicylic acid signaling pathway. Additionally, AcICS1, another critical gene of salicylic acid synthesis, was also highly expressed. All contributed to the synthesis of increasing salicylic acid content in kiwifruit leaves. Moreover, the first key lignin biosynthetic AcPAL gene was marked up-regulated. Thereafter, accumulation of lignin content in the kiwifruit stem and the higher deposition of lignin were visible in histochemical analysis. Moreover, the activity of the endochitinase activity of kiwifruit leaves increased significantly. We suggest that the sulfur-induced resistance against Pseudomonas syringae pv. actinidiae via salicylic activates systemic acquired resistance to enhance plant immune response in kiwifruit.

CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

The accessible chromatin landscape of the human genome.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.

Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity.

The human body contains thousands of unique cell types, each with specialized functions. Cell identity is governed in large part by gene transcription programs, which are determined by regulatory elements encoded in DNA. To identify regulatory elements active in seven cell lines representative of diverse human cell types, we used DNase-seq and FAIRE-seq (Formaldehyde Assisted Isolation of Regulatory Elements) to map "open chromatin." Over 870,000 DNaseI or FAIRE sites, which correspond tightly to nucleosome-depleted regions, were identified across the seven cell lines, covering nearly 9% of the genome. The combination of DNaseI and FAIRE is more effective than either assay alone in identifying likely regulatory elements, as judged by coincidence with transcription factor binding locations determined in the same cells. Open chromatin common to all seven cell types tended to be at or near transcription start sites and to be coincident with CTCF binding sites, while open chromatin sites found in only one cell type were typically located away from transcription start sites and contained DNA motifs recognized by regulators of cell-type identity. We show that open chromatin regions bound by CTCF are potent insulators. We identified clusters of open regulatory elements (COREs) that were physically near each other and whose appearance was coordinated among one or more cell types. Gene expression and RNA Pol II binding data support the hypothesis that COREs control gene activity required for the maintenance of cell-type identity. This publicly available atlas of regulatory elements may prove valuable in identifying noncoding DNA sequence variants that are causally linked to human disease.

Dual RNA sequencing during Trichoderma harzianum-Phytophthora capsici interaction reveals multiple biological processes involved in the inhibition and highlights the cell wall as a potential target.

BACKGROUND: Phytophthora capsici is a destructive oomycete pathogen, causing huge economic losses for agricultural production. The genus Trichoderma represents one of the most extensively researched categories of biocontrol agents, encompassing a diverse array of effective strains. The commercial biocontrol agent Trichoderma harzianum strain T-22 exhibits pronounced biocontrol effects against many plant pathogens, but its activity against P. capsici is not known. RESULTS: T. harzianum T-22 significantly inhibited the growth of P. capsici mycelia and the culture filtrate of T-22 induced lysis of P. capsici zoospores. Electron microscopic analyses indicated that T-22 significantly modulated the ultrastructural composition of P. capsici, with a severe impact on the cell wall integrity. Dual RNA sequencing revealed multiple biological processes involved in the inhibition during the interaction between these two microorganisms. In particular, a marked upregulation of genes was identified in T. harzianum that are implicated in cell wall degradation or disruption. Concurrently, the presence of T. harzianum appeared to potentiate the susceptibility of P. capsici to cell wall biosynthesis inhibitors such as mandipropamid and dimethomorph. Further investigations showed that mandipropamid and dimethomorph could strongly inhibit the growth and development of P. capsici but had no impact on T. harzianum even at high concentrations, demonstrating the feasibility of combining T. harzianum and these cell wall synthesis inhibitors to combat P. capsici. CONCLUSION: These findings provided enhanced insights into the biocontrol mechanisms against P. capsici with T. harzianum and evidenced compatibility between specific biological and chemical control strategies. © 2024 Society of Chemical Industry.

Honokiol inhibits Botryosphaeria dothidea, the causal pathogen of kiwifruit soft rot, by targeting membrane lipid biosynthesis.

BACKGROUND: Kiwifruit soft rot is mainly caused by Botryosphaeria dothidea, representing a considerable threat to kiwifruit industry. This investigation assessed the inhibitory consequences and mechanisms of honokiol against B. dothidea, evaluating the inhibitory effects and underlying mechanism. RESULTS: A strain of B.dothidea (XFCT-2) was isolated from infected soft rot kiwifruit. The findings indicate that honokiol hindered the mycelial growth, conidial germination, and pathogenicity of B. dothidea in a dose-dependent manner, both in vitro and in vivo. Furthermore, ultrastructural examinations showed that honokiol impaired the integrity of B. dothidea, leading to an elevation in cell membrane permeability, engendering a multitude of intracellular substance extravasations and hampering energy metabolism. Transcriptome analysis exhibited that honokiol-regulated genes were related to membrane lipid biosynthesis, comprising ACC1, FAS2, Arp2, gk, Cesle, and Etnk1. These findings indicate that honokiol impedes B. dothidea by obstructing lipid biosynthesis within the cell membrane and compromising its integrity, halting the growth of the mycelia, which could potentially cause cellular demise. CONCLUSION: This investigation illustrates how honokiol functions as an eco-friendly approach to prevent the occurrence of soft rot in kiwifruits. © 2023 Society of Chemical Industry.

Regioselective Synthesis of Multisubstituted 2,3-Dihydrooxazoles by 1,3-Dithiane Umpolung.

Herein, we report a novel strategy for the regioselective synthesis of aromatic and heteroaromatic 2,3-dihydrooxazoles from 2-alkynyl-1,3-dithianes and nitrones. This method exploits the 1,3-dithiane umpolung, nucleophilic cycloaddition, and rearrangement processes to achieve the rapid assembly of dihydrooxazole molecules. The regioselective method is extremely mild, achieving complete conversion within one min under air at 25 °C with high efficiency.

Divergent 1,2-carboallylation of terminal alkynes enabled by metallaphotoredox catalysis with switchable triplet energy transfer.

We report a metallaphotoredox strategy for stereodivergent three-component carboallylation of terminal alkynes with allylic carbonates and alkyl trifluoroborates. This redox-neutral dual catalytic protocol utilizes commercially available organic photocatalyst 4CzIPN and nickel catalysts to trigger a radical addition/alkenyl-allyl coupling sequence, enabling straightforward access to functionalized 1,4-dienes in a highly chemo-, regio-selective, and stereodivergent fashion. This reaction features a broad substrate generality and a tunable triplet energy transfer control with pyrene as a simple triplet energy modulator, offering a facile synthesis of complex trans- and cis-selective skipped dienes with the same set of readily available substrates.

Activity of the botanical compound thymol against kiwifruit rot caused by Fusarium tricinctum and the underlying mechanisms.

BACKGROUND: Kiwifruit rot is an important disease caused by different fungal pathogens, which can lead to huge economic loss in the kiwifruit industry. The aims of this study were to discover an effective botanical compound that significantly inhibits the pathogens causing kiwifruit rot, evaluate its control efficacy against this disease, and reveal the underlying mechanisms. RESULTS: A strain of Fusarium tricinctum (GF-1), isolated from diseased kiwifruit, could cause fruit rot in both Actinidia chinensis var. chinensis and Actinidia chinensis var. deliciosa. Different botanical chemicals were used for antifungal activity test against GF-1 and thymol was the most effective one with a 50% effective concentration (EC50 ) of 30.98 mg L-1 . The minimal inhibitory concentration (MIC) of thymol against GF-1 was 90 mg L-1 . Control efficacy of thymol against kiwifruit rot was evaluated and the results indicated that thymol could effectively decrease the occurrence and spread of kiwifruit rot. The mechanisms underlying the antifungal activity of thymol against F. tricinctum were investigated, and it showed that thymol could significantly damage the ultrastructure, destroy the plasma membrane integrity, and instantaneously increase energy metabolisms of F. tricinctum. Further investigations indicated that thymol could extend shelf life of kiwifruit by increasing their storability. CONCLUSION: Thymol can effectively inhibit F. tricinctum that is one of the causal agents of kiwifruit rot. Multiple modes of action are involved in the antifungal activity. The results of this study indicate that thymol can be a promising botanical fungicide to control kiwifruit rot and provide useful references for thymol application in agriculture system. © 2023 Society of Chemical Industry.

Bearing surface defect detection based on improved convolutional neural network.

This paper addresses the issue of artificial visual inspection being overly reliant on subjective experience and the difficulty for the human eye to accurately identify dense and non-significant defects. To solve this problem, we have implemented an automatic object detection algorithm based on an improved version of YOLOv5.First, we use the K-means++ clustering algorithm to automatically calculate the Anchor of the model to reduce the effect of the close location of the initial clustering centers on the clustering of the sample data.Second, we add the Coordinate Attention (CA) attention mechanism to the model to allow the model to better capture and understand important features in the images. Then, we add a new detection layer with a downsampling multiplier of 4 to the Neck network to improve the precision of the model. Finally, we use the lightweight network MobileNetV3 instead of YOLOv5's backbone network to reduce the model detection time overhead.Our model achieves 85.87% mAP, which is 6.44% better than the YOLOv5 network, and the detection time for a single image is only 54ms, which is 50% faster than the YOLOv5 network. After testing, we have proven that our proposed algorithm can quickly and accurately detect the condition of bearing appearance defects, improving detection efficiency and reducing costs.