Spaco: A comprehensive tool for coloring spatial data at single-cell resolution.

Understanding tissue architecture and niche-specific microenvironments in spatially resolved transcriptomics (SRT) requires in situ annotation and labeling of cells. Effective spatial visualization of these data demands appropriate colorization of numerous cell types. However, current colorization frameworks often inadequately account for the spatial relationships between cell types. This results in perceptual ambiguity in neighboring cells of biological distinct types, particularly in complex environments such as brain or tumor. To address this, we introduce Spaco, a potent tool for spatially aware colorization. Spaco utilizes the Degree of Interlacement metric to construct a weighted graph that evaluates the spatial relationships among different cell types, refining color assignments. Furthermore, Spaco incorporates an adaptive palette selection approach to amplify chromatic distinctions. When benchmarked on four diverse datasets, Spaco outperforms existing solutions, capturing complex spatial relationships and boosting visual clarity. Spaco ensures broad accessibility by accommodating color vision deficiency and offering open-accessible code in both Python and R.

Genome-wide transcriptomics and microRNAomics analyses uncover multi-faceted mechanisms to cope with copper stress in ancient macrobenthos amphioxus.

The mechanisms underlying the toxicity of environmental stress are unclear for marine macrobenthos. Copper/Cu has posed the most serious threats to amphioxus, an ancient and model benthic cephalochordate. Herein, a dynamic change in the physiological parameters (GR, SOD, ATP, and MDA) was detected with ROS accumulation in Branchiostoma belcheri exposed to 0.3 mg·L-1 Cu. Transcriptomes and microRNAomes of B. belcheri were generated to investigate the molecular mechanisms by which this amphioxus copes with Cu exposure. Time-specific genes identified at different time points after exposure were involved in the stimulus and immune response, detoxification and ionic homeostasis, aging and the nervous system, sequentially, with prolongation of exposure time, forming a dynamic process of molecular response to Cu stress. In total, 57 differentially expressed miRNAs were identified under Cu stress. Transcriptomics-miRNAomics analyses indicate that these miRNAs targeted genes associated with many key biological processes such as xenobiotics degradation, oxidative stress, and energy metabolism. The constructed miRNA-mRNA-pathway network uncovered a broad post-transcriptional regulatory mechanism in B. belcheri to cope with Cu stress. Overall, this integrated analyses show that enhanced defense response, accelerated ROS elimination, and repressed ATP production constitute a comprehensive strategy to cope with Cu toxicity in the ancient macrobenthos.

Transcriptomics based identification of S100A3 as the key anti-hepatitis B virus factor of 16F16.

More than 250 million people worldwide have chronic hepatitis B virus (HBV) infections, resulting in over 1 million annual fatalities because HBV cannot be adequately treated with current antivirals. Hepatocellular carcinoma (HCC) risk is elevated in the presence of the HBV. Novel and powerful medications that specifically target the persistent viral components are needed to remove infection. This study aimed to use HepG2.2.15 cells and the rAAV-HBV1.3 C57BL/6 mouse model established in our laboratory to examine the effects of 16F16 on HBV. The transcriptome analysis of the samples was performed to examine the impact of 16F16 therapy on host factors. We found that the HBsAg and HBeAg levels significantly decreased in a dose-dependent manner following the 16F16 treatment. 16F16 also showed significant anti-hepatitis B effects in vivo. The transcriptome analysis showed that 16F16 regulated the expression of several proteins in HBV-producing HepG2.2.15 cells. As one of the differentially expressed genes, the role of S100A3 in the anti-hepatitis B process of 16F16 was further investigated. The expression of the S100A3 protein significantly decreased following the 16F16 therapy. And upregulation of S100A3 caused an upregulation of HBV DNA, HBsAg, and HBeAg in HepG2.2.15 cells. Similarly, knockdown of S100A3 significantly reduced the levels of HBsAg, HBeAg, and HBV DNA. Our findings proved that S100A3 might be a new target for combating HBV pathogenesis. 16F16 can target several proteins involved in HBV pathogenesis, and may be a promising drug precursor molecule for the treatment of HBV.

Evaluation of exosomal miRNAs as potential diagnostic biomarkers for acute myocardial infarction using next-generation sequencing.

BACKGROUND: Acute myocardial infarction (AMI) is one of the most common global causes of death. Although considerable progress has been made in AMI diagnosis, there remains an urgent need for novel diagnostic biomarkers for its prevention and treatment. Functional exosomal microRNAs (miRNAs) are recognized as potential biomarkers in many diseases. This study's objective was to identify specific plasma exosomal miRNAs with biomarker potential for early AMI detection. METHODS: Exosomes from the plasma of 26 coronary artery disease (CAD) patients, 55 AMI patients, and 37 healthy controls were isolated and characterized by transmission electron microcopy (TEM), western blotting, and nanoparticle tracking analysis (NTA). The miRNAs were purified from exosomes, and unique molecular identifier (UMI) small RNA sequencing was performed. The random forest (RF) model was trained to predict potential biomarkers. RESULTS: NTA demonstrated that nanoparticle concentration did not change after AMI, while nanoparticle size distribution significantly decreased. The CAD and AMI groups' miRNA expression profiles significantly differed from the healthy group's profile. The RF classifier could be used to distinguish the healthy group from the AMI group, but could not be used to distinguish the CAD group from the other groups, which caused a high classification error rate. Eighteen miRNAs were selected as biomarkers based on their RF classifier significance. The diagnostic accuracy of 18 miRNAs was evaluated using AUC values of 0.93, 0.87, and 0.75 to detect healthy controls, AMI, and CAD, respectively. CONCLUSIONS: Nanoparticle diameter and the 18 miRNAs may serve as simple and accessible fingerprints for early AMI diagnosis.

Genome-Wide Identification and Transcriptomic Analysis of MicroRNAs Across Various Amphioxus Organs Using Deep Sequencing.

Amphioxus is the closest living invertebrate proxy of the vertebrate ancestor. Systematic gene identification and expression profile analysis of amphioxus organs are thus important for clarifying the molecular mechanisms of organ function formation and further understanding the evolutionary origin of organs and genes in vertebrates. The precise regulation of microRNAs (miRNAs) is crucial for the functional specification and differentiation of organs. In particular, those miRNAs that are expressed specifically in organs (OSMs) play key roles in organ identity, differentiation, and function. In this study, the genome-wide miRNA transcriptome was analyzed in eight organs of adult amphioxus Branchiostoma belcheri using deep sequencing. A total of 167 known miRNAs and 23 novel miRNAs (named novel_mir), including 139 conserved miRNAs, were discovered, and 79 of these were identified as OSMs. Additionally, analyses of the expression patterns of eight randomly selected known miRNAs demonstrated the accuracy of the miRNA deep sequencing that was used in this study. Furthermore, potentially OSM-regulated genes were predicted for each organ type. Functional enrichment of these predicted targets, as well as further functional analyses of known OSMs, was conducted. We found that the OSMs were potentially to be involved in organ-specific functions, such as epidermis development, gonad development, muscle cell development, proteolysis, lipid metabolism, and generation of neurons. Moreover, OSMs with non-organ-specific functions were detected and primarily include those related to innate immunity and response to stimuli. These findings provide insights into the regulatory roles of OSMs in various amphioxus organs.

Author Correction: Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation.

In the Supplementary Information file originally published with this Letter, the authors mistakenly omitted Supplementary Table 14; this has now been amended.

Adaptive evidence of mitochondrial genomes in Dolycoris baccarum (Hemiptera: Pentatomidae) to divergent altitude environments.

Given mitochondrion is the 'energy and oxygen usage factories', adaptive signatures of mitochondrial genes have been extensively investigated in vertebrates from different altitudes, but few studies focus on insects. Here, we sequenced the complete mitochondrial genome (mitogenome) of Dolycoris. baccarum living in the Tibetan Plateau (DBHC, ∼3200 m above sea level (asl)) and conducted a detailed comparative analysis with another D. baccarum mitogenome (DBQY) from relatively low altitude (∼1300 m asl). All the 37 mitochondrial genes were highly conserved and under purifying selection, except for two mitochondrial protein-coding genes (MPCGs) (atp6 and nad5) that showed positively selected signatures. We therefore further examined non-synonymous substitutions in atp6 and nad5, by sequencing more individuals from three populations with different altitudes. We found that these non-synonymous substitutions were polymorphic in these populations, likely due to relaxed selection constraints in different altitudes. Purifying selection in all mitochondrial genes may be due to their functional importance for the precision of ATP production usually. Length difference in mitochondrial control regions between DBHC and DBQY was also conversed at the population level, indicating that sequence size adjustments in control region may be associated with adaptation to divergent altitudes. Quantitatively real-time PCR analysis for 12 MPCGs showed that gene expression patterns had a significant change between the two populations, suggesting that expression levels of MPCGs could be modulated by divergent environmental pressures (e.g. oxygen content and ambient temperature). These results provided an important guide for further uncovering genetic mechanisms of ecological adaptation in insects.

Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation.

The genus Liriodendron belongs to the family Magnoliaceae, which resides within the magnoliids, an early diverging lineage of the Mesangiospermae. However, the phylogenetic relationship of magnoliids with eudicots and monocots has not been conclusively resolved and thus remains to be determined1-6. Liriodendron is a relict lineage from the Tertiary with two distinct species-one East Asian (L. chinense (Hemsley) Sargent) and one eastern North American (L. tulipifera Linn)-identified as a vicariad species pair. However, the genetic divergence and evolutionary trajectories of these species remain to be elucidated at the whole-genome level7. Here, we report the first de novo genome assembly of a plant in the Magnoliaceae, L. chinense. Phylogenetic analyses suggest that magnoliids are sister to the clade consisting of eudicots and monocots, with rapid diversification occurring in the common ancestor of these three lineages. Analyses of population genetic structure indicate that L. chinense has diverged into two lineages-the eastern and western groups-in China. While L. tulipifera in North America is genetically positioned between the two L. chinense groups, it is closer to the eastern group. This result is consistent with phenotypic observations that suggest that the eastern and western groups of China may have diverged long ago, possibly before the intercontinental differentiation between L. chinense and L. tulipifera. Genetic diversity analyses show that L. chinense has tenfold higher genetic diversity than L. tulipifera, suggesting that the complicated regions comprising east-west-orientated mountains and the Yangtze river basin (especially near 30° N latitude) in East Asia offered more successful refugia than the south-north-orientated mountain valleys in eastern North America during the Quaternary glacial period.

Population genomics of finless porpoises reveal an incipient cetacean species adapted to freshwater.

Cetaceans (whales, dolphins, and porpoises) are a group of mammals adapted to various aquatic habitats, from oceans to freshwater rivers. We report the sequencing, de novo assembly and analysis of a finless porpoise genome, and the re-sequencing of an additional 48 finless porpoise individuals. We use these data to reconstruct the demographic history of finless porpoises from their origin to the occupation into the Yangtze River. Analyses of selection between marine and freshwater porpoises identify genes associated with renal water homeostasis and urea cycle, such as urea transporter 2 and angiotensin I-converting enzyme 2, which are likely adaptations associated with the difference in osmotic stress between ocean and rivers. Our results strongly suggest that the critically endangered Yangtze finless porpoises are reproductively isolated from other porpoise populations and harbor unique genetic adaptations, supporting that they should be considered a unique incipient species.

Comparative transcriptomic analysis provides insights into antibacterial mechanisms of Branchiostoma belcheri under Vibrio parahaemolyticus infection.

Amphioxus, a basal chordate, is widely considered to be an existing proxy of the invertebrate ancestor of vertebrates, and it exhibits susceptibility to various pathogen infections and pathogenic mimic challenges. Here, in order to understand more clearly its antibacterial mechanisms, we analyzed the ribosomal RNA (rRNA)-depleted transcriptome of Chinese amphioxus (Branchiostoma belcheri) infected with Vibrio parahaemolyticus (V. p.) via next-generation deep sequencing technology (RNA-seq). We identified a total of 3214 differentially expressed genes (DEGs) by comparing V. p.-infected and control transcriptome libraries, including 2219 significantly up-regulated and 995 significantly down-regulated DEGs in V. p.-infected amphioxus. The DEGs with the top 10 most dramatic expression fold changes after V. p. infection, as well as 53 immune-related DEGs (IRDs) belonging to four primary categories of innate immunity were analyzed further. Through gene ontology (GO) and pathway enrichment analysis, DEGs were found to be primarily related to immune processes, apoptosis, catabolic and metabolic processes, binding and enzyme activity, while pathways involving bacterial infection, immune signaling, immune response, cancer, and apoptosis were overrepresented. We validated the RNA-seq results by detecting the expression levels of 10 IRDs using qRT-PCR, and we surveyed the dynamic variation in gene expression for these IRDs at 0, 6, 12, 24, and 48 h after V. p. TREATMENT: Subsequently, according to the RNA-seq results, the presence of a primitive Toll-like receptor (TLR)-mediated antibacterial immune signaling pathway was predicted in B. belcheri. This study provides valuable information regarding antibacterial immunity for further research into the evolution of immunity in vertebrates and broadens our understanding of the innate immune response against bacterial invasion in amphioxus.

Transcriptome-wide analysis of immune-responsive microRNAs against poly (I:C) challenge in Branchiostoma belcheri by deep sequencing and bioinformatics.

Amphioxus is a key experimental animal for studying the evolution of vertebrate immune system. However, we still do not know about the roles of microRNAs (miRNAs) under viral stress in amphioxus. In this study, we sequenced six small RNA libraries (three biological replicates were included in the treatments challenged by the viral mimic, poly (I:C) (pIC) and control groups, respectively) from Branchiostoma belcheri. A total of 151 known miRNAs, 197 new miRNAs (named novel_mir, including nine conserved miRNAs) were identified by deep sequencing from the six libraries. We primarily focused on differentially expressed miRNAs (DEMs) after pIC challenge. Next, we screened a total of 77 DEMs, including 27 down- and 50 up-regulated DEMs in response to pIC challenge. Furthermore, we used real-time quantitative PCR (qRT-PCR) to verify the expression levels of 10 randomly selected DEMs. Target genes likely regulated by DEMs were predicted, and functional enrichment analyses of these targets were performed using bioinformatics approach. MiRNA targets of DEMs are primarily involved in immune response, diseases, cancer and regulation process, and could be largely linked to 14 immune-related signaling pathways, including NF-kappa B, NOD-like receptor, RIG-I-like receptor and endocytosis. The present study for the first time explores key regulatory roles of miRNAs in the innate antiviral immune response in amphioxus, and will provide insight into the molecular basis of antiviral immunity and evolution of immune-related miRNAs.

Genome-wide gene expression analysis of amphioxus (Branchiostoma belcheri) following lipopolysaccharide challenge using strand-specific RNA-seq.

Amphioxus is the closest living proxy for exploring the evolutionary origin of the immune system in vertebrates. To understand the immune responses of amphioxus to lipopolysaccharide (LPS), 5 ribosomal RNA (rRNA)-depleted libraries of amphioxus were constructed, including one control (0 h) library and 4 treatment libraries at 6, 12, 24, and 48 h post-injection (hpi) with LPS. The transcriptome of Branchiostoma belcheri was analyzed using strand-specific RNA sequencing technology (RNA-seq). A total of 6161, 6665, 7969, and 6447 differentially expressed genes (DEGs) were detected at 6, 12, 24, and 48 hpi, respectively, compared with expression levels at 0 h. We identified amphioxus genes active during the acute-phase response to LPS at different time points after stimulation. Moreover, to better visualize the resolution phase of the immune process during immune response, we identified 6057 and 5235 DEGs at 48 hpi by comparing with 6 and 24 hpi, respectively. Through real-time quantitative PCR (qRT-PCR) analysis of 12 selected DEGs, we demonstrated the accuracy of the RNA-seq data in this study. Functional enrichment analysis of DEGs demonstrated that most terms were related to defense and immune responses, disease and infection, cell apoptosis, and metabolism and catalysis. Subsequently, we identified 1330, 485, 670, 911, and 1624 time-specific genes (TSGs) at 0, 6, 12, 24, and 48 hpi. Time-specific terms at each of 5 time points were primarily involved in development, immune signaling, signal transduction, DNA repair and stability, and metabolism and catalysis, respectively. As this is the first study to report the transcriptome of an organism with primitive immunity following LPS challenge at multiple time points, it provides gene expression information for further research into the evolution of immunity in vertebrates.

Gene sequence variations and expression patterns of mitochondrial genes are associated with the adaptive evolution of two Gynaephora species (Lepidoptera: Lymantriinae) living in different high-elevation environments.

The adaptive evolution of animals to high-elevation environments has been extensively studied in vertebrates, while few studies have focused on insects. Gynaephora species (Lepidoptera: Lymantriinae) are endemic to the Qinghai-Tibetan Plateau (QTP) and represent an important insect pest of alpine meadows. Here, we present a detailed comparative analysis of the mitochondrial genomes (mitogenomes) of two Gynaephora species inhabiting different high-elevation environments: G. alpherakii and G. menyuanensis. The results indicated that the general mitogenomic features (genome size, nucleotide composition, codon usage and secondary structures of tRNAs) were well conserved between the two species. All of mitochondrial protein-coding genes were evolving under purifying selection, suggesting that selection constraints may play a role in ensuring adequate energy production. However, a number of substitutions and indels were identified that altered the protein conformations of ATP8 and NAD1, which may be the result of adaptive evolution of the two Gynaephora species to different high-elevation environments. Levels of gene expression for nine mitochondrial genes in nine different developmental stages were significantly suppressed in G. alpherakii, which lives at the higher elevation (~4800m above sea level), suggesting that gene expression patterns could be modulated by atmospheric oxygen content and environmental temperature. These results enhance our understanding of the genetic bases for the adaptive evolution of insects endemic to the QTP.

Selection of reliable reference genes for normalization of quantitative RT-PCR from different developmental stages and tissues in amphioxus.

Amphioxus is a closest living proxy to the ancestor of cephalochordates with vertebrates, and key animal for novel understanding in the evolutionary origin of vertebrate body plan, genome, tissues and immune system. Reliable analyses using quantitative real-time PCR (qRT-PCR) for answering these scientific questions is heavily dependent on reliable reference genes (RGs). In this study, we evaluated stability of thirteen candidate RGs in qRT-PCR for different developmental stages and tissues of amphioxus by four independent (geNorm, NormFinder, BestKeeper and deltaCt) and one comparative algorithms (RefFinder). The results showed that the top two stable RGs were the following: (1) S20 and 18 S in thirteen developmental stages, (2) EF1A and ACT in seven normal tissues, (3) S20 and L13 in both intestine and hepatic caecum challenged with lipopolysaccharide (LPS), and (4) S20 and EF1A in gill challenged with LPS. The expression profiles of two target genes (EYA and HHEX) in thirteen developmental stages were used to confirm the reliability of chosen RGs. This study identified optimal RGs that can be used to accurately measure gene expression under these conditions, which will benefit evolutionary and functional genomics studies in amphioxus.

Spatiotemporal-specific lncRNAs in the brain, colon, liver and lung of macaque during development.

Genome-wide expression profiling during development provides useful information to uncover the potential molecular mechanisms of development in mammals. Recent studies have revealed that a subset of lncRNAs can regulate major biological processes during development. Here, we sequenced four tissues, including brain, colon, liver and lung, using RNA-seq across three developmental stages (early, middle and late stage), and then constructed genome-wide expression profiles during macaque development. In each tissue, we identified developmental time-specific lncRNA and mRNA clusters that displayed diverse expression alteration patterns, including a gradual increase, a gradual decrease, or a reversal of expression. These lncRNAs showed more specific functional associations with their corresponding tissues relative to the developmental time-specific mRNAs. Furthermore, we identified 20 spatiotemporal-specific co-modules including 101 lncRNAs and 609 mRNAs distributed at different developmental stages in different tissues. Our findings suggested that lncRNAs could play critical roles in the development of macaques through close cooperation with mRNAs. Finally, we predicted the functions of the spatiotemporal-specific lncRNAs by their spatiotemporal cooperation with mRNAs and further validated our findings using gene knockdown data of mouse. Our study reveals the spatiotemporal characteristics of lncRNAs and provides a functional map of the spatiotemporal-specific lncRNAs during the development of macaques.

Genomic Analysis of an Ascomycete Fungus from the Rice Planthopper Reveals How It Adapts to an Endosymbiotic Lifestyle.

A number of sap-sucking insects harbor endosymbionts, which are thought to play an important role in the development of their hosts. One of the most important rice pests, the brown planthopper (BPH), Nilaparvata lugens (Stål), harbors an obligatory yeast-like symbiont (YLS) that cannot be cultured in vitro. Genomic information on this YLS would be useful to better understand its evolution. In this study, we performed genome sequencing of the YLS using both 454 and Illumina approaches, generating a draft genome that shows a slightly smaller genome size and relatively higher GC content than most ascomycete fungi. A phylogenomic analysis of the YLS supported its close relationship with insect pathogens. We analyzed YLS-specific genes and the categories of genes that are likely to have changed in the YLS during its evolution. The loss of mating type locus demonstrated in the YLS sheds light on the evolution of eukaryotic symbionts. This information about the YLS genome provides a helpful guide for further understanding endosymbiotic associations in hemiptera and the symbiotic replacement of ancient bacteria with a multifunctional YLS seems to have been a successful change.

A host plant genome (Zizania latifolia) after a century-long endophyte infection.

Despite the importance of host-microbe interactions in natural ecosystems, agriculture and medicine, the impact of long-term (especially decades or longer) microbial colonization on the dynamics of host genomes is not well understood. The vegetable crop 'Jiaobai' with enlarged edible stems was domesticated from wild Zizania latifolia (Oryzeae) approximately 2000 years ago as a result of persistent infection by a fungal endophyte, Ustilago esculenta. Asexual propagation via infected rhizomes is the only means of Jiaobai production, and the Z. latifolia-endophyte complex has been maintained continuously for two centuries. Here, genomic analysis revealed that cultivated Z. latifolia has a significantly smaller repertoire of immune receptors compared with wild Z. latifolia. There are widespread gene losses/mutations and expression changes in the plant-pathogen interaction pathway in Jiaobai. These results show that continuous long-standing endophyte association can have a major effect on the evolution of the structural and transcriptomic components of the host genome.

Transcriptome profiling of brown adipose tissue during cold exposure reveals extensive regulation of glucose metabolism.

We applied digital gene expression profiling to determine the transcriptome of brown and white adipose tissues (BAT and WAT, respectively) during cold exposure. Male C57BL/6J mice were exposed to cold for 2 or 4 days. A notable induction of genes related to glucose uptake, glycolysis, glycogen metabolism, and the pentose phosphate pathway was observed in BAT from cold-exposed animals. In addition, glycerol-3-phosphate dehydrogenase 1 expression was induced in BAT from cold-challenged mice, suggesting increased synthesis of glycerol from glucose. Similarly, expression of lactate dehydrogenases was induced by cold in BAT. Pyruvate dehydrogenase kinase 2 (Pdk2) and Pdk4 were expressed at significantly higher levels in BAT than in WAT, and Pdk2 was induced in BAT by cold. Of notice, only a subset of the changes detected in BAT was observed in WAT. Based on changes in gene expression during cold exposure, we propose a model for the intermediary glucose metabolism in activated BAT: 1) fluxes through glycolysis and the pentose phosphate pathway are induced, the latter providing reducing equivalents for de novo fatty acid synthesis; 2) glycerol synthesis from glucose is increased, facilitating triacylglycerol synthesis/fatty acid re-esterification; 3) glycogen turnover and lactate production are increased; and 4) entry of glucose carbon into the tricarboxylic acid cycle is restricted by PDK2 and PDK4. In summary, our results demonstrate extensive and diverse gene expression changes related to glucose handling in activated BAT.

[Determination of impurity elements in the positive electrode material LiMn2O4 by inductively coupled plasma mass spectrometry].

Samples of LiMn2O4 were digested by microwave digestion, and impurity elements amounts of Na, Mg, Al, K, Ca, Ti, Cr, Fe, Cu, Zn, As, Ag, Cd and Pb in sample solutions were determined by inductively coupled plasma mass spectrometry (ICP-MS). Sample preparation was achieved by digestion with HNO3 + HCl in a closed-vessel microwave system. The effects of mass spectrum interference were studied. Sc, Rh and Tl as internal standard elements were used to compensate matrix effect and signal drift. Under the optimal conditions, the detection limits of the 14 elements are in the range of 0.007-0.209 microg x L(-1). The recovery was 92.66%-108.34% by adding standard recovery experiment, and the relative standard deviation (RSD) was less than 4.80% for all the elements. This method was simple, sensitive and precise, which could satisfy the sample examination request and provide scientific rationale for determining impurity elements of LiMn2O4.