Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

Gastrointestinal Characteristics of Constipation from the Perspectives of Microbiome and Metabolome.

BACKGROUND: Constipation is one of the most common gastrointestinal complaints. Yet, the underlying mechanisms of constipation remain to be explored deeply. Integration of microbiome and metabolome is powerful and promising to demonstrate characteristics of constipation. AIM OF STUDY: This study aimed to characterize intestinal microbiome and metabolome of constipation. In addition, this study revealed the correlations among behaviors, intestinal microbiota, and metabolites interrupted by constipation. METHODS: Firstly, the constipation model was successfully applied. At the macro level, the ability of learning, memory, locomotor activity, and the defecation index of rats with constipation-like phenotype were characterized. At the micro-level, 16S rRNA sequencing was applied to analyze the intestinal microbiota in rats with constipation-like phenotype. 1H nuclear magnetic resonance (NMR)-based metabolomics was employed to investigate the metabolic phenotype of constipation. In addition, we constructed a correlation network, intuitively showing the correlations among behaviors, intestinal microbiota, and metabolites. RESULTS: Constipation significantly attenuated the locomotor activity, memory recognition, and frequency of defecation of rats, while increased the time of defecation. Constipation significantly changed the diversity of intestinal microbial communities, which correspondingly involved in 5 functional pathways. Besides, 28 fecal metabolites were found to be associated with constipation, among which 14 metabolites were further screened that can be used to diagnose constipation. On top of this, associated networks intuitively showed the correlations among behaviors, intestinal microbiota, and metabolites. CONCLUSIONS: The current findings are significant in terms of not only laying a foundation for understanding characteristics of constipation, but also providing accurate diagnosis and treatments of constipation clinically.

Molecular barcoding of native RNAs using nanopore sequencing and deep learning.

Nanopore sequencing enables direct measurement of RNA molecules without conversion to cDNA, thus opening the gates to a new era for RNA biology. However, the lack of molecular barcoding of direct RNA nanopore sequencing data sets severely affects the applicability of this technology to biological samples, where RNA availability is often limited. Here, we provide the first experimental protocol and associated algorithm to barcode and demultiplex direct RNA nanopore sequencing data sets. Specifically, we present a novel and robust approach to accurately classify raw nanopore signal data by transforming current intensities into images or arrays of pixels, followed by classification using a deep learning algorithm. We demonstrate the power of this strategy by developing the first experimental protocol for barcoding and demultiplexing direct RNA sequencing libraries. Our method, DeePlexiCon, can classify 93% of reads with 95.1% accuracy or 60% of reads with 99.9% accuracy. The availability of an efficient and simple multiplexing strategy for native RNA sequencing will improve the cost-effectiveness of this technology, as well as facilitate the analysis of lower-input biological samples. Overall, our work exemplifies the power, simplicity, and robustness of signal-to-image conversion for nanopore data analysis using deep learning.

Fecal Metabolomics and Network Pharmacology Reveal the Correlations between Constipation and Depression.

Constipation and depression are tightly related and often co-occur and coexist in clinic. Yet, the relationships and the underlying mechanisms are still unclear. Fecal metabolomics and network pharmacology were, for the first time, applied to investigate the potential correlations from multiple levels including classic behaviors, metabolomics, and gene targets. The behavioral indicators were analyzed, providing behavioral correlations at a macrolevel. Besides, fecal samples were analyzed by nuclear magnetic resonance spectroscopy to screen the shared and the unique metabolites and pathways, revealing correlations from a metabolic perspective. Finally, the disease targets and the functional pathways were obtained via network pharmacology, demonstrating correlations at the molecular level. The correlations between constipation and depression were demonstrated and supported by four-level evidence: (1) general behaviors, (2) gastrointestinal functions, (3) fecal metabolites and pathways, and (4) common gene targets and functional pathways. Especially, the correlations of behaviors and common metabolites showed that metabolites, including choline, betaine, and glycine, were significantly associated with constipation and depression. Besides, inflammation and immune abnormalities and energy metabolism were significantly involved in the mechanisms. The current findings prove the correlations between constipation and depression, and provide a basis for deeply understanding the comorbidities of constipation and depression.

Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation.

RNA modifications are dynamic chemical entities that expand the RNA lexicon and regulate RNA fate. The most abundant modification present in mRNAs, N6-methyladenosine (m6A), has been implicated in neurogenesis and memory formation. However, whether additional RNA modifications may be playing a role in neuronal functions and in response to environmental queues is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m2,2G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNAAla(AGC) isodecoders at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes may play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.

[Mechanism of Xiaoyao San in treatment of depression,breast hyperplasia,and functional dyspepsia based on network pharmacology].

This study aimed to explore the mechanism of Xiaoyao San(XYS) in the treatment of three diseases of liver depression and spleen deficiency, ie, depression, breast hyperplasia, and functional dyspepsia, and to provide a theoretical basis for the interpretation of the scientific connotation of "treating different diseases with the same method" of traditional Chinese medicines. Traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP) was used to screen the active components of XYS which underwent principal component analysis(PCA) with the available drugs for these three diseases to determine the corresponding biological activities. The targets of XYS on depression, breast hyperplasia, and functional dyspepsia were obtained from GeneCards, TTD, CTD, and DrugBank databases. Cytoscape was used to plot the "individual herbal medicine-active components-potential targets" network. The resulting key targets were subjected to Kyoto encyclopedia of genes and genomes(KEGG) pathway analysis and gene ontology(GO) enrichment analysis. A total of 121 active components of XYS and 38 common targets in the treatment of depression, breast hyperplasia, and functional dyspepsia were collected. The key biological pathways were identified, including advanced glycation and products(AGEs)-receptor for advanced glycation and products(RAGE) signaling pathway in diabetic complications, HIF-1 signaling pathway, and cancer-related pathways. The key targets of XYS in the treatment of depression, breast hyperplasia, and functional dyspepsia included IL6, IL4, and TNF, and the key components were kaempferol, quercetin, aloe-emodin, etc. As revealed by the molecular docking, a strong affinity was observed between the key components and the key targets, which confirmed the results. The therapeutic efficacy of XYS in the treatment of diseases of liver depression and spleen deficiency was presumedly achieved by reducing the inflammatory reactions. The current findings are expected to provide novel research ideas and approaches to classify the scientific connotation of "treating different diseases with the same method" of Chinese medicines, as well as a theoretical basis for understanding the mechanism of XYS and exploring its clinical applications.

[Research progress in combination applications of antidepressant drugs].

Depression is a common affective disorder. The application of antidepressants can significantly alleviate the symptoms of depression, which is the most important way to treat depression in clinical practice. Due to the complex etiology, wide variety, as well as diversity and severity of serious concomitant symptoms, rational addition of other drugs into antidepressants can significantly improve the cure rates of depression, reduce adverse reactions, and improve patient compliances. Therefore, the combined applications of differential drugs have been commonly used in clinic. In this paper, more than 600 literatures about depression from 2010 to 2019 were collected based on the key words of antidepressant, depression, combined medication, synergism and increase efficiency. Based on this, by summarizing and classifying the existing combinations of antidepressant drugs, this paper systematically expounds the current combined applications of antidepressant drugs in three categories, i.e. western medicines combined with western medicines, western medicines combined with traditional Chinese medicines, and traditional Chinese medicines combined with traditional Chinese medicines, in the expectation of providing the direction and basis for the selection of rational combinations of antidepressant drugs in clinic.

Evolutionary origins of a bioactive peptide buried within Preproalbumin.

The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin; however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally diverse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1's additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure.

The Effect of a Direct Fed Microbial on Liveweight and Milk Production in Dairy Cattle.

This longitudinal study aimed to quantify the effects of dietary supplementation of a direct-fed microbial (DFM) consisting of three lactobacilli isolates on milk yield, milk fat and protein yields, somatic cell count (SCC), and liveweight in a single dairy herd in Australia. A total of 150 dairy cows were randomly selected based on parity and days in milk and divided into two groups: control (n = 75) and DFM treatment (n = 75). Throughout the study, the two groups of cows were housed separately in a dry lot yard, and each group had their own feeding area. For the DFM treatment group, selected cows in mid-lactation were supplemented with 10 mL/cow/day of the DFM via top dressing of the feed for the remainder of the lactation and through the dry period, extending into subsequent lactation. The control group had no supplementation. The milk yield and liveweight were recorded daily. Milk samples were collected every two months for milk component analysis (fat, protein, and somatic cell count [SCC]). The DFM-treated cows gained more liveweight across the study (19.40 kg, 95% CI 0.44 kg; 38.30 kg, p = 0.05) compared to the control cows. In the second production year, the DFM-treated cows mobilized more liveweight (-6.06 kg, 95% CI -10.49 kg; -1.61 kg, p = 0.01) and produced more milk (0.39 L/d 95% CI 0.10; 0.89, p = 0.05). Over a full lactation, DFM cows yielded at least 258 L (95% CI 252 L; 265 L) more milk than controls. No significant differences were found in fat and protein yield or SCC. This study suggests that consistent and ongoing supplementation with a Lacticaseibacillus- and Lentilactobacillus-based DFM could have a positive effect on milk production, but further research is needed to understand the underlying mechanism.

Tianhuang formula ameliorates liver fibrosis by inhibiting CCL2-CCR2 axis and MAPK/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In the progression of chronic liver diseases, liver fibrosis is a reversible pathophysiologic event for liver diseases prognosis and risk of cirrhosis. Liver injury factors of different etiologies mediate this process. There is still a lack of effective medications for treating liver fibrosis. Additionally, the ameliorative effects of traditional herbs on liver fibrosis have been commonly reported. Tianhuang formula (THF) is a drug combination consisting of 2 traditional Chinese herbs, which has been showing significant improvement in metabolic liver diseases. However, the hepatoprotective effect and mechanism of THF in ameliorating liver fibrosis are still unclear. AIM OF THE STUDY: This study aimed to investigate the effects of THF on carbon tetrachloride (CCl4)-induced and methionine-choline-deficient (MCD) diet-induced liver fibrosis model and to reveal the potential mechanisms. It can provide experimental evidence for THF as a therapeutic candidate for liver fibrosis. MATERIALS AND METHODS: In this study, CCl4-induced mice were treated with THF (80 mg/kg, 160 mg/kg) or Fuzheng Huayu (FZHY) capsules (4.8 g/kg) for 6 weeks. MCD-induced mice received the same doses of THF or FZHY for 4 weeks. FZHY is used as a comparative study in these two models. Following that, using kit reagents detected changes in relevant serum and liver biochemical indicators. Histological changes in mouse liver were measured by staining of H&E and Sirius Red. The markers expression of liver fibrosis and inflammation were detected using qRT-PCR, western blotting and immunohistochemical staining analysis. The potential regulatory mechanism of THF to ameliorate liver fibrosis was performed by RNA-sequencing analysis. Finally, the analysis results were verified by immunofluorescence co-staining, qRT-PCR and western blotting. RESULTS: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic triglyceride (TG) levels in CCl4 and MCD-induced liver fibrosis mice were significantly improved after THF treatment. Meanwhile, the expression of fibrosis and inflammation markers were significantly suppressed. Furthermore, THF downregulated the expression of the macrophage marker CD68. According to RNA-sequencing analysis, we found the CCL2-CCR2 axis and MAPK/NF-κB as the potential signaling pathway for THF against liver fibrosis. CONCLUSION: This study revealed that THF ameliorated liver injury, inflammation and fibrotic process by inhibiting CCL2-CCR2 axis and its downstream MAPK/NF-κB signaling pathway.

Targeting FTO induces colorectal cancer ferroptotic cell death by decreasing SLC7A11/GPX4 expression.

Ferroptosis is a newly identified iron-dependent form of death that is becoming increasingly recognized as a promising avenue for cancer therapy. N6-methyladenosine (m6A) is the most abundant reversible methylation modification in mRNA contributing to tumorigenesis. However, the crucial role of m6A modification in regulating ferroptosis during colorectal cancer (CRC) tumorigenesis remains elusive. Herein, we find that m6A modification is increased during ferroptotic cell death and correlates with the decreased m6A demethylase fat mass and obesity-associated protein (FTO) expression. Functionally, we demonstrate that suppressing FTO significantly induces CRC ferroptotic cell death, as well as enhancing CRC cell sensitivity to ferroptosis inducer (Erastin and RSL3) treatment. Mechanistically, high FTO expression increased solute carrier family 7 member 11 (SLC7A11) or glutathione peroxidase 4 (GPX4) expressions in an m6A-YTHDF2 dependent manner, thereby counteracting ferroptotic cell death stress. In addition, we identify Mupirocin as a novel inhibitor of FTO, and Mupirocin induces CRC ferroptosis and inhibits tumor growth. Clinically, the levels of FTO, SLC7A11, and GPX4, are highly correlated expression in CRC tissues. Our findings reveal that FTO protects CRC from ferroptotic cell death in promoting CRC tumorigenesis through triggering SLC7A11/GPX4 expression.

Genomic evidence for the parallel evolution of coastal forms in the Senecio lautus complex.

Instances of parallel ecotypic divergence where adaptation to similar conditions repeatedly cause similar phenotypic changes in closely related organisms are useful for studying the role of ecological selection in speciation. Here we used a combination of traditional and next generation genotyping techniques to test for the parallel divergence of plants from the Senecio lautus complex, a phenotypically variable groundsel that has adapted to disparate environments in the South Pacific. Phylogenetic analysis of a broad selection of Senecio species showed that members of the S. lautus complex form a distinct lineage that has diversified recently in Australasia. An inspection of thousands of polymorphisms in the genome of 27 natural populations from the S. lautus complex in Australia revealed a signal of strong genetic structure independent of habitat and phenotype. Additionally, genetic differentiation between populations was correlated with the geographical distance separating them, and the genetic diversity of populations strongly depended on geographical location. Importantly, coastal forms appeared in several independent phylogenetic clades, a pattern that is consistent with the parallel evolution of these forms. Analyses of the patterns of genomic differentiation between populations further revealed that adjacent populations displayed greater genomic heterogeneity than allopatric populations and are differentiated according to variation in soil composition. These results are consistent with a process of parallel ecotypic divergence in face of gene flow.

Microbiome-metabolomics deciphers the effects of Cistanche deserticola polysaccharides on aged constipated rats.

Chronic constipation is an extremely common gastrointestinal disorder that severely affects the life quality of the elderly. As an edible food and therapeutic medicine, Cistanche deserticola (CD) has been widely used not only as food in daily life, but also as a medicine to treat constipation. As the main component in CD, polysaccharide shows great potentials in improving constipation in the elderly. In this study, 16S rRNA analysis and fecal metabolomics were applied to investigate the impacts of constipation in an aged rat model, as well as the regulatory effects and the underlying mechanisms of CD polysaccharide (CDPS). Firstly, a classic constipation model of aged rats was constructed. The behavioral indicators of the rats were analyzed, providing behavioral correlations at the macro level. Meanwhile, the levels of SOD, GSH-Px, MDA, and CAT in serum samples of the rats were assessed. Additionally, the changes of gut microbiota, fecal metabolites and corresponding metabolic pathways in the aged constipated rats were demonstrated. On top of this, inter-and inner-layer networks of "behavioral indicators - intestinal bacteria - metabolites" were constructed to visually demonstrate the relationships among differential indicators. We found that CDPS significantly regulated the abnormalities of the behavioral indexes, the microbial richness and diversity, and the metabolite profiles that were induced by constipation in the aged rats. From the intestinal microbiological point of view, CDPS significantly increased the prevalence of beneficial bacteria while reducing the potentially pathogenic bacterial population. In terms of metabolomics, a total of 16 metabolites were finally identified as potential biomarkers of constipation in the aged rats. The mechanisms of CDPS were mainly involved in metabolic energy and the synthesis of amino acids. The current findings not only deepen our understanding about constipation in the elderly from the perspectives of microbiome and metabolomics, but also lay a solid foundation for the applications of polysaccharides in constipation in the elderly, the discovery of new medicines for constipation, and improving the life quality of the elderly.

Astragalus Polysaccharide Alleviates Constipation in the Elderly Via Modification of Gut Microbiota and Fecal Metabolism.

Constipation is one of the most common gastrointestinal disorders, whose incidence increasing with age. As one of the main components, Astragalus polysaccharide (APS) has been used to treat a variety of diseases. This study aimed to explore the effects of APS on the improvement of gastrointestinal functions and learning memory in elderly rats with constipation. In this study, both 16S rRNA sequencing-based microbiome and 1H NMR-based metabolomics were applied to demonstrate the effects of APS on host metabolism and gut microbiota of the elderly rats with constipation. On top of this, we constructed both inter- and inner-layer networks, intuitively showing the correlations among behavioral indicators, intestinal bacteria, and differential metabolites. Our results showed that APS significantly ameliorated the constipation and the cognitive dysfunctions of rats. Microbiome analysis revealed that APS raised the relative abundance of Blautia, whereas decreased the relative abundance of Lactobacillus in the elderly rats with constipation. In addition, APS decreased the levels of acetate, butyrate, and propionate in the fecal samples, correspondingly regulating glycolysis/gluconeogenesis metabolism and pyruvate metabolism. These findings lay solid foundations for understanding the pathogenesis of constipation in the elderly, and also offer a promising new treatment strategy for constipation in the elderly.

EpiNano: Detection of m6A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing.

RNA modifications play pivotal roles in the RNA life cycle and RNA fate, and are now appreciated as a major posttranscriptional regulatory layer in the cell. In the last few years, direct RNA nanopore sequencing (dRNA-seq) has emerged as a promising technology that can provide single-molecule resolution maps of RNA modifications in their native RNA context. While native RNA can be successfully sequenced using this technology, the detection of RNA modifications is still challenging. Here, we provide an upgraded version of EpiNano (version 1.2), an algorithm to predict m6A RNA modifications from dRNA-seq datasets. The latest version of EpiNano contains models for predicting m6A RNA modifications in dRNA-seq data that has been base-called with Guppy. Moreover, it can now train models with features extracted from both base-called dRNA-seq FASTQ data and raw FAST5 nanopore outputs. Finally, we describe how EpiNano can be used in stand-alone mode to extract base-calling "error" features and current intensity information from dRNA-seq datasets. In this chapter, we provide step-by-step instructions on how to produce in vitro transcribed constructs to train EpiNano, as well as detailed information on how to use EpiNano to train, test, and predict m6A RNA modifications in dRNA-seq data.

Integrating hippocampal metabolomics and network pharmacology deciphers the antidepressant mechanisms of Xiaoyaosan.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoyaosan (XYS), a classic description, has a history of thousands of years for treating depression through invigorating the liver and strengthening the spleen, which have been verified both clinically and experimentally. However, explanation of its underlying mechanisms remains a great challenge. AIM OF THE STUDY: The mechanisms of XYS in treating depression were investigated, with emphasis on the important biomarkers, targets and pathways. MATERIALS AND METHODS: In this study, taking the targeted organ of depression, hippocampus, as the object, a combination of GC-MS based metabolomics and network pharmacology was established to illustrate the abnormality of metabolic characteristics of hippocampus of depression rats and to demonstrate the antidepressant mechanisms of XYS. Hippocampal metabolomics demonstrated potential metabolites involving in the antidepressant effects of XYS, as well as the corresponding metabolic pathways. Network pharmacology screened the potential ingredients and the targets of XYS against depression. RESULTS: Metabolomics revealed that XYS significantly regulated the abnormal levels of lactic acid, glycerol, glutamine, glutamic acid, hypoxanthine, myo-inositol and cholesterol, which involved in the D-glutamine and D-glutamate metabolism, arginine biosynthesis and alanine, aspartate and glutamate metabolism. Network pharmacology showed that XYS exhibited anti-depression effects through paeoniflorin, quercetin, licochalcone a, naringenin, ß-sitosterol, formononetin and kaempferol acting on interleukin-6 (IL6), mitogen-activated protein kinase 1 (MAPK1), signal transducer and activator of transcription 3 (STAT3) and transcription factor AP-1 (JUN). CONCLUSION: Based on hippocampal metabolomics and network pharmacology, this study proved that the actions of XYS in treating depression depend on multi-components, multi-targets and multi-pathways, the unique characteristics of TCMs.

Phenotypic and genotypic parallel evolution in parapatric ecotypes of Senecio.

The independent and repeated adaptation of populations to similar environments often results in the evolution of similar forms. This phenomenon creates a strong correlation between phenotype and environment and is referred to as parallel evolution. However, we are still largely unaware of the dynamics of parallel evolution, as well as the interplay between phenotype and genotype within natural systems. Here, we examined phenotypic and genotypic parallel evolution in multiple parapatric Dune-Headland coastal ecotypes of an Australian wildflower, Senecio lautus. We observed a clear trait-environment association in the system, with all replicate populations having evolved along the same phenotypic evolutionary trajectory. Similar phenotypes have arisen via mutational changes occurring in different genes, although many share the same biological functions. Our results shed light on how replicated adaptation manifests at the phenotypic and genotypic levels within populations, and highlight S. lautus as one of the most striking cases of phenotypic parallel evolution in nature.

Hepatic metabolomics of the compatibility effect of Xiaoyaosan on CUMS-induced depression based on the TCM theory of "Treating Diseases via Regulating the Liver's Function".

This study aimed to demonstrate the scientific connotations and compatibility effects of Xiaoyaosan (XYS) based on the theory of "Treating Diseases via Regulating the Liver's Function" by hepatic metabolomics. XYS was divided into two efficacy groups, i.e. the Shugan (SG) and the Jianpi (JP) groups, according to the strategy of "Efficacy Compositions". The chronic unpredictable mild stress (CUMS) depression model was constructed. A 1H NMR-based hepatic metabolomics approach coupled with multivariate data (MVD) analysis was performed. Meanwhile, relative distance (RD) and Efficacy Index (EI) were calculated. XYS and its efficacy groups significantly reversed the abnormality of behavior and hepatic metabolomics of depression rats, but to different degrees. The results of ethology and metabolomics showed the same order, i.e. XYS > JP > SG. Two metabolites, i.e. tyrosine and malate, were regulated by all the treatment groups. Four metabolites were significantly regulated only by XYS group. Of note, the results showed the two efficacy groups of XYS exhibited synergistic anti-depression effects, and glutamate, malate and taurine could be the key hepatic metabolites for these synergistic effects. The current study not only complements and consummates the mechanisms of depression and the anti-depression effects of XYS from the perspective of hepatic metabolomics, but also lays a solid foundation for comprehensively and deeply understanding the compatibility effects of XYS against depression, especially from the points of view of compatibility in Traditional Chinese medicine (TCM) theory and synergism in modern medicine theory.

Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing.

Nanopore RNA sequencing shows promise as a method for discriminating and identifying different RNA modifications in native RNA. Expanding on the ability of nanopore sequencing to detect N6-methyladenosine, we show that other modifications, in particular pseudouridine (Ψ) and 2'-O-methylation (Nm), also result in characteristic base-calling 'error' signatures in the nanopore data. Focusing on Ψ modification sites, we detected known and uncovered previously unreported Ψ sites in mRNAs, non-coding RNAs and rRNAs, including a Pus4-dependent Ψ modification in yeast mitochondrial rRNA. To explore the dynamics of pseudouridylation, we treated yeast cells with oxidative, cold and heat stresses and detected heat-sensitive Ψ-modified sites in small nuclear RNAs, small nucleolar RNAs and mRNAs. Finally, we developed a software, nanoRMS, that estimates per-site modification stoichiometries by identifying single-molecule reads with altered current intensity and trace profiles. This work demonstrates that Nm and Ψ RNA modifications can be detected in cellular RNAs and that their modification stoichiometry can be quantified by nanopore sequencing of native RNA.

Integrative analyses of the RNA modification machinery reveal tissue- and cancer-specific signatures.

BACKGROUND: RNA modifications play central roles in cellular fate and differentiation. However, the machinery responsible for placing, removing, and recognizing more than 170 RNA modifications remains largely uncharacterized and poorly annotated, and we currently lack integrative studies that identify which RNA modification-related proteins (RMPs) may be dysregulated in each cancer type. RESULTS: Here, we perform a comprehensive annotation and evolutionary analysis of human RMPs, as well as an integrative analysis of their expression patterns across 32 tissues, 10 species, and 13,358 paired tumor-normal human samples. Our analysis reveals an unanticipated heterogeneity of RMP expression patterns across mammalian tissues, with a vast proportion of duplicated enzymes displaying testis-specific expression, suggesting a key role for RNA modifications in sperm formation and possibly intergenerational inheritance. We uncover many RMPs that are dysregulated in various types of cancer, and whose expression levels are predictive of cancer progression. Surprisingly, we find that several commonly studied RNA modification enzymes such as METTL3 or FTO are not significantly upregulated in most cancer types, whereas several less-characterized RMPs, such as LAGE3 and HENMT1, are dysregulated in many cancers. CONCLUSIONS: Our analyses reveal an unanticipated heterogeneity in the expression patterns of RMPs across mammalian tissues and uncover a large proportion of dysregulated RMPs in multiple cancer types. We provide novel targets for future cancer research studies targeting the human epitranscriptome, as well as foundations to understand cell type-specific behaviors that are orchestrated by RNA modifications.