Probiotic-derived nanoparticles inhibit ALD through intestinal miR194 suppression and subsequent FXR activation.

BACKGROUND AND AIMS: Intestinal farnesoid X receptor (FXR) plays a critical role in alcohol-associated liver disease (ALD). We aimed to investigate whether alcohol-induced dysbiosis increased intestinal microRNA194 (miR194) that suppressed Fxr transcription and whether Lactobacillus rhamnosus GG-derived exosome-like nanoparticles (LDNPs) protected against ALD through regulation of intestinal miR194-FXR signaling in mice. APPROACH AND RESULTS: Binge-on-chronic alcohol exposure mouse model was utilized. In addition to the decreased ligand-mediated FXR activation, alcohol feeding repressed intestinal Fxr transcription and increased miR194 expression. This transcriptional suppression of Fxr by miR194 was confirmed in intestinal epithelial Caco-2 cells and mouse enteriods. The alcohol feeding-reduced intestinal FXR activation was further demonstrated by the reduced FXR reporter activity in fecal samples and by the decreased fibroblast growth factor 15 (Fgf15) messenger RNA (mRNA) in intestine and protein levels in the serum, which caused an increased hepatic bile acid synthesis and lipogeneses. We further demonstrated that alcohol feeding increased-miR194 expression was mediated by taurine-upregulated gene 1 (Tug1) through gut microbiota regulation of taurine metabolism. Importantly, 3-day oral administration of LDNPs increased bile salt hydrolase (BSH)-harboring bacteria that decreased conjugated bile acids and increased gut taurine concentration, which upregulated Tug1, leading to a suppression of intestinal miR194 expression and recovery of FXR activation. Activated FXR upregulated FGF15 signaling and subsequently reduced hepatic bile acid synthesis and lipogenesis and attenuated ALD. These protective effects of LDNPs were eliminated in intestinal FxrΔIEC and Fgf15-/- mice. We further showed that miR194 was upregulated, whereas BSH activity and taurine levels were decreased in fecal samples of patients with ALD. CONCLUSIONS: Our results demonstrated that gut microbiota-mediated miR194 regulation contributes to ALD pathogenesis and to the protective effects of LDNPs through modulating intestinal FXR signaling.

A novel genome sequence of Jasminum sambac helps uncover the molecular mechanism underlying the accumulation of jasmonates.

Jasminum sambac is a well-known plant for its attractive and exceptional fragrance, the flowers of which are used to produce scented tea. Jasmonate (JA), an important plant hormone was first identified in Jasminum species. Jasmine plants contain abundant JA naturally, of which the molecular mechanisms of synthesis and accumulation are not clearly understood. Here, we report a telomere-to-telomere consensus assembly of a double-petal J. sambac genome along with two haplotype-resolved genomes. We found that gain-and-loss, positive selection, and allelic specific expression of aromatic volatile-related genes contributed to the stronger flower fragrance in double-petal J. sambac compared with single- and multi-petal jasmines. Through comprehensive comparative genomic, transcriptomic, and metabolomic analyses of double-petal J. sambac, we revealed the genetic basis of the production of aromatic volatiles and salicylic acid (SA), and the accumulation of JA under non-stress conditions. We identified several key genes associated with JA biosynthesis, and their non-stress related activities lead to extraordinarily high concentrations of JA in tissues. High JA synthesis coupled with low degradation in J. sambac results in accumulation of high JA under typical environmental conditions, similar to the accumulation mechanism of SA. This study offers important insights into the biology of J. sambac, and provides valuable genomic resources for further utilization of natural products.

A Semiautomated Luciferase Immunoprecipitation Assay for Rapid and Easy Detection of African Swine Fever Virus Antibody.

African swine fever (ASF) is a highly contagious viral disease of domestic pigs and wild boars. For disease surveillance and control, we developed a rapid and easy luciferase immunoprecipitation assay (MB-LIPS) to detect ASF virus (ASFV) antibody. The MB-LIPS is based on magnetic beads modified with protein A/G and the recombinant fusion protein of ASFV p30 and luciferase, where p30 functioned as the recognition element and luciferase as the signal component. Incubation and washing could be finished automatically on a machine with magnetic rods. Under optimal conditions, the MB-LIPS showed 96.3% agreement to a commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting ASFV antibody in swine sera. Analyzing serial dilutions of a swine serum sample showed that the MP-LIPS assay was 4 times more sensitive than the ELISA kit. The whole run of the MB-LIPS could be completed within 30 min. With its high sensitivity and simple operation, the MB-LIPS platform has great potential to be used for the detection of ASFV antibody and ASF control in small labs and farms.

Probiotic Lactobacillus rhamnosus GG Prevents Liver Fibrosis Through Inhibiting Hepatic Bile Acid Synthesis and Enhancing Bile Acid Excretion in Mice.

BACKGROUND AND AIMS: Cholestatic liver disease is characterized by gut dysbiosis and excessive toxic hepatic bile acids (BAs). Modification of gut microbiota and repression of BA synthesis are potential strategies for the treatment of cholestatic liver disease. The purpose of this study was to examine the effects and to understand the mechanisms of the probiotic Lactobacillus rhamnosus GG (LGG) on hepatic BA synthesis, liver injury, and fibrosis in bile duct ligation (BDL) and multidrug resistance protein 2 knockout (Mdr2-/- ) mice. APPROACH AND RESULTS: Global and intestine-specific farnesoid X receptor (FXR) inhibitors were used to dissect the role of FXR. LGG treatment significantly attenuated liver inflammation, injury, and fibrosis with a significant reduction of hepatic BAs in BDL mice. Hepatic concentration of taurine-ß-muricholic acid (T-ßMCA), an FXR antagonist, was markedly increased in BDL mice and reduced in LGG-treated mice, while chenodeoxycholic acid, an FXR agonist, was decreased in BDL mice and normalized in LGG-treated mice. LGG treatment significantly increased the expression of serum and ileum fibroblast growth factor 15 (FGF-15) and subsequently reduced hepatic cholesterol 7α-hydroxylase and BA synthesis in BDL and Mdr2-/- mice. At the molecular level, these changes were reversed by global and intestine-specific FXR inhibitors in BDL mice. In addition, LGG treatment altered gut microbiota, which was associated with increased BA deconjugation and increased fecal and urine BA excretion in both BDL and Mdr2-/- mice. In vitro studies showed that LGG suppressed the inhibitory effect of T-ßMCA on FXR and FGF-19 expression in Caco-2 cells. CONCLUSION: LGG supplementation decreases hepatic BA by increasing intestinal FXR-FGF-15 signaling pathway-mediated suppression of BA de novo synthesis and enhances BA excretion, which prevents excessive BA-induced liver injury and fibrosis in mice.

Cathelicidin-related antimicrobial peptide alleviates alcoholic liver disease through inhibiting inflammasome activation.

Alcoholic liver disease (ALD) is associated with gut dysbiosis and hepatic inflammasome activation. While it is known that antimicrobial peptides (AMPs) play a critical role in the regulation of bacterial homeostasis in ALD, the functional role of AMPs in the alcohol-induced inflammasome activation is unclear. The aim of this study was to determine the effects of cathelicidin-related antimicrobial peptide (CRAMP) on inflammasome activation in ALD. CRAMP knockout (Camp-/-) and wild-type (WT) mice were subjected to binge-on-chronic alcohol feeding and synthetic CRAMP peptide was administered. Serum/plasma and hepatic tissue samples from human subjects with alcohol use disorder and/or alcoholic hepatitis were analyzed. CRAMP deficiency exacerbated ALD with enhanced inflammasome activation as shown by elevated serum interleukin (IL)-1ß levels. Although Camp-/- mice had comparable serum endotoxin levels compared to WT mice after alcohol feeding, hepatic lipopolysaccharide (LPS) binding protein (LBP) and cluster of differentiation (CD) 14 were increased. Serum levels of uric acid (UA), a Signal 2 molecule in inflammasome activation, were positively correlated with serum levels of IL-1ß in alcohol use disorder patients with ALD and were increased in Camp-/- mice fed alcohol. In vitro studies showed that CRAMP peptide inhibited LPS binding to macrophages and inflammasome activation stimulated by a combination of LPS and UA. Synthetic CRAMP peptide administration decreased serum UA and IL-1ß concentrations and rescued the liver from alcohol-induced damage in both WT and Camp-/- mice. In summary, CRAMP exhibited a protective role against binge-on-chronic alcohol-induced liver damage via regulation of inflammasome activation by decreasing LPS binding and UA production. CRAMP administration may represent a novel strategy for treating ALD. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Origin and evolution of the triploid cultivated banana genome.

Most fresh bananas belong to the Cavendish and Gros Michel subgroups. Here, we report chromosome-scale genome assemblies of Cavendish (1.48 Gb) and Gros Michel (1.33 Gb), defining three subgenomes, Ban, Dh and Ze, with Musa acuminata ssp. banksii, malaccensis and zebrina as their major ancestral contributors, respectively. The insertion of repeat sequences in the Fusarium oxysporum f. sp. cubense (Foc) tropical race 4 RGA2 (resistance gene analog 2) promoter was identified in most diploid and triploid bananas. We found that the receptor-like protein (RLP) locus, including Foc race 1-resistant genes, is absent in the Gros Michel Ze subgenome. We identified two NAP (NAC-like, activated by apetala3/pistillata) transcription factor homologs specifically and highly expressed in fruit that directly bind to the promoters of many fruit ripening genes and may be key regulators of fruit ripening. Our genome data should facilitate the breeding and super-domestication of bananas.

Sequencing and Assembly of Polyploid Genomes.

Polyploidy has been observed throughout major eukaryotic clades and has played a vital role in the evolution of angiosperms. Recent polyploidizations often result in highly complex genome structures, posing challenges to genome assembly and phasing. Recent advances in sequencing technologies and genome assembly algorithms have enabled high-quality, near-complete chromosome-level assemblies of polyploid genomes. Advances in novel sequencing technologies include highly accurate single-molecule sequencing with HiFi reads, chromosome conformation capture with Hi-C technique, and linked reads sequencing. Additionally, new computational approaches have also significantly improved the precision and reliability of polyploid genome assembly and phasing, such as HiCanu, hifiasm, ALLHiC, and PolyGembler. Herein, we review recently published polyploid genomes and compare the various sequencing, assembly, and phasing approaches that are utilized in these genome studies. Finally, we anticipate that accurate and telomere-to-telomere chromosome-level assembly of polyploid genomes could ultimately become a routine procedure in the near future.

Assessment of the optical properties with physicochemical properties and cell wall polysaccharides of 'Korla' pear flesh during Alternaria alternata-induced disease development.

Cell wall polysaccharides and physicochemical properties are the major quality characteristics of fruit, but they are significantly affected by the postharvest disease. In this study, the influence of Alternaria alternata-induced disease on the contents of cell wall polysaccharides and physicochemical properties in 'Korla' pear flesh during storage, as well as their relationships of the optical absorption (µa) and reduced scattering (µs') were explored. The infected pear had lower individual sugars, covalent-soluble pectin, cellulose and hemicellulose contents than the healthy ones. The successive decreases of µa and increases of µs' in pears were observed while the process of pathogen infection. Path-coefficient analysis indicated the ionic-soluble pectin was the main reason responsible for the change of µs' in infected pear at 675 nm and 980 nm. This study indicated the optical properties have the possibility to present the physicochemical characteristics and cell wall polysaccharides of pears during postharvest pathogen infection.

Study on Black Spot Disease Detection and Pathogenic Process Visualization on Winter Jujubes Using Hyperspectral Imaging System.

In this work, the potential of a hyperspectral imaging (HSI) system for the detection of black spot disease on winter jujubes infected by Alternaria alternata during postharvest storage was investigated. The HSI images were acquired using two systems in the visible and near-infrared (Vis-NIR, 400-1000 nm) and short-wave infrared (SWIR, 1000-2000 nm) spectral regions. Meanwhile, the change of physical (peel color, weight loss) and chemical parameters (soluble solids content, chlorophyll) and the microstructure of winter jujubes during the pathogenic process were measured. The results showed the spectral reflectance of jujubes in both the Vis-NIR and SWIR wavelength ranges presented an overall downtrend during the infection. Partial least squares discriminant models (PLS-DA) based on the HSI spectra in Vis-NIR and SWIR regions of jujubes both gave satisfactory discrimination accuracy for the disease detection, with classification rates of over 92.31% and 91.03%, respectively. Principal component analysis (PCA) was carried out on the HSI images of jujubes to visualize their infected areas during the pathogenic process. The first principal component of the HSI spectra in the Vis-NIR region could highlight the diseased areas of the infected jujubes. Consequently, Vis-NIR HSI and NIR HSI techniques had the potential to detect the black spot disease on winter jujubes during the postharvest storage, and the Vis-NIR HSI spectral information could visualize the diseased areas of jujubes during the pathogenic process.

5mC DNA methylation modification-mediated regulation in tissue functional differentiation and important flavor substance synthesis of tea plant (Camellia sinensis L.).

In plants, 5mC DNA methylation is an important and conserved epistatic mark involving genomic stability, gene transcriptional regulation, developmental regulation, abiotic stress response, metabolite synthesis, etc. However, the roles of 5mC DNA methylation modification (5mC methylation) in tea plant growth and development (in pre-harvest processing) and flavor substance synthesis in pre- and post-harvest processing are unknown. We therefore conducted a comprehensive methylation analysis of four key pre-harvest tissues (root, leaf, flower, and fruit) and two processed leaves during oolong tea post-harvest processing. We found that differential 5mC methylation among four key tissues is closely related to tissue functional differentiation and that genes expressed tissue-specifically, responsible for tissue-specific functions, maintain relatively low 5mC methylation levels relative to non-tissue-specifically expressed genes. Importantly, hypomethylation modifications of CsAlaDC and TS/GS genes in roots provided the molecular basis for the dominant synthesis of theanine in roots. In addition, integration of 5mC DNA methylationomics, metabolomics, and transcriptomics of post-harvest leaves revealed that content changes in flavor metabolites during oolong tea processing were closely associated with transcription level changes in corresponding metabolite synthesis genes, and changes in transcript levels of these important synthesis genes were strictly regulated by 5mC methylation. We further report that some key genes during processing are regulated by 5mC methylation, which can effectively explain the content changes of important aroma metabolites, including α-farnesene, nerolidol, lipids, and taste substances such as catechins. Our results not only highlight the key roles of 5mC methylation in important flavor substance synthesis in pre- and post-harvest processing, but also provide epimutation-related gene targets for future improvement of tea quality or breeding of whole-tissue high-theanine varieties.

Detection of Alternaria alternata infection in winter jujubes based on optical properties and their correlation with internal quality parameters during storage.

In this work, a single integrating sphere system was applied to characterize the optical absorption (µa) and reduced scattering (µs') properties (550 - 1050 nm) in winter jujube flesh infected by Alternaria alternata during storage at 4 and 20 °C, respectively. Meanwhile, physical (L*, a*, weight loss) and biochemical characteristics (soluble solids content, titratable acids, chlorophyll, total phenolic, and ascorbic acid) of winter jujubes were measured. Among them, chlorophyll, weight loss and ascorbic acid were highly correlated with µa at 680 nm, 690 nm, while chlorophyll and a* had the best correlations with µs' at 700 - 920 nm. These optimal optical properties were proved efficiently contributed to the disease detection of winter jujubes after 12 days at 4 °C and 3 days at 20 °C during storage, with satisfactory discrimination accuracies (acc > 93.75 %). Consequently, optical properties in Vis-NIR region were available to detect the postharvest disease in winter jujubes.

The haplotype-resolved autotetraploid genome assembly provides insights into the genomic evolution and fruit divergence in wax apple (Syzygium samarangense (Blume) Merr. and Perry).

Wax apple (Syzygium samarangense) is an economically important fruit crop with great potential value to human health because of its richness in antioxidant substances. Here, we present a haplotype-resolved autotetraploid genome assembly of the wax apple with a size of 1.59 Gb. Comparative genomic analysis revealed three rounds of whole-genome duplication (WGD) events, including two independent WGDs after WGT-γ. Resequencing analysis of 35 accessions partitioned these individuals into two distinct groups, including 28 landraces and seven cultivated species, and several genes subject to selective sweeps possibly contributed to fruit growth, including the KRP1-like, IAA17-like, GME-like, and FLACCA-like genes. Transcriptome analysis of three different varieties during flower and fruit development identified key genes related to fruit size, sugar content, and male sterility. We found that AP2 also affected fruit size by regulating sepal development in wax apples. The expression of sugar transport-related genes (SWEETs and SUTs) was high in 'ZY', likely contributing to its high sugar content. Male sterility in 'Tub' was associated with tapetal abnormalities due to the decreased expression of DYT1, TDF1, and AMS, which affected early tapetum development. The chromosome-scale genome and large-scale transcriptome data presented in this study offer new valuable resources for biological research on S. samarangense and shed new light on fruit size control, sugar metabolism, and male sterility regulatory metabolism in wax apple.

A gap-free and haplotype-resolved lemon genome provides insights into flavor synthesis and huanglongbing (HLB) tolerance.

The lemon (Citrus limon; family Rutaceae) is one of the most important and popular fruits worldwide. Lemon also tolerates huanglongbing (HLB) disease, which is a devastating citrus disease. Here we produced a gap-free and haplotype-resolved chromosome-scale genome assembly of the lemon by combining Pacific Biosciences circular consensus sequencing, Oxford Nanopore 50-kb ultra-long, and high-throughput chromatin conformation capture technologies. The assembly contained nine-pair chromosomes with a contig N50 of 35.6 Mb and zero gaps, while a total of 633.0 Mb genomic sequences were generated. The origination analysis identified 338.5 Mb genomic sequences originating from citron (53.5%), 147.4 Mb from mandarin (23.3%), and 147.1 Mb from pummelo (23.2%). The genome included 30 528 protein-coding genes, and most of the assembled sequences were found to be repetitive sequences. Several significantly expanded gene families were associated with plant-pathogen interactions, plant hormone signal transduction, and the biosynthesis of major active components, such as terpenoids and flavor compounds. Most HLB-tolerant genes were expanded in the lemon genome, such as 2-oxoglutarate (2OG)/Fe(II)-dependent oxygenase and constitutive disease resistance 1, cell wall-related genes, and lignin synthesis genes. Comparative transcriptomic analysis showed that phloem regeneration and lower levels of phloem plugging are the elements that contribute to HLB tolerance in lemon. Our results provide insight into lemon genome evolution, active component biosynthesis, and genes associated with HLB tolerance.

Isoliquiritigenin alleviates the development of alcoholic liver fibrosis by inhibiting ANXA2.

The study aimed to investigate the effect of isoliquiritigenin (ISL) on model of alcoholic liver fibrosis (ALF). C57BL/6 mice were used to establish animal model of ALF, HSC-T6 cells were used to establish alcohol-activated cell model, and tandem mass tag (TMT) assays were used to analyze the proteome. The results showed that ISL obviously alleviated hepatic fibrosis in model mice. ISL visually improved the area of liver pathological stasis and deposition of fibrillar collagen (Sirius Red staining, Masson staining), inhibited the mRNA expression levels of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) in liver tissues. ISL down-regulated the mRNA expression levels of IL-6 and transforming growth factor-ß1(TGF-ß1) in activated hepatic stellate cells (HSCs). And ISL significantly reduced annexin A2 (ANXA2) in vitro detected by TMT proteomics technology. Interestingly, it was found for the first time that ISL could inhibit ANXA2 expression both in vivo and in vitro, block the sphingosine kinases (SPHKs)/sphingosine-1-phosphate (S1P)/interleukin 17 (IL-17) signaling pathway and regulate the expression of α-smooth muscle actin (α-SMA) by inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at the downstream signal to finally reverse HSCs activation and hepatic fibrosis. Thus, we demonstrated that ISL is a drug monomer with notable anti-hepatic fibrosis activity.

Chromosome-scale genome sequence of Suaeda glauca sheds light on salt stress tolerance in halophytes.

Soil salinity is a growing concern for global crop production and the sustainable development of humanity. Therefore, it is crucial to comprehend salt tolerance mechanisms and identify salt-tolerance genes to enhance crop tolerance to salt stress. Suaeda glauca, a halophyte species well adapted to the seawater environment, possesses a unique ability to absorb and retain high salt concentrations within its cells, particularly in its leaves, suggesting the presence of a distinct mechanism for salt tolerance. In this study, we performed de novo sequencing of the S. glauca genome. The genome has a size of 1.02 Gb (consisting of two sets of haplotypes) and contains 54 761 annotated genes, including alleles and repeats. Comparative genomic analysis revealed a strong synteny between the genomes of S. glauca and Beta vulgaris. Of the S. glauca genome, 70.56% comprises repeat sequences, with retroelements being the most abundant. Leveraging the allele-aware assembly of the S. glauca genome, we investigated genome-wide allele-specific expression in the analyzed samples. The results indicated that the diversity in promoter sequences might contribute to consistent allele-specific expression. Moreover, a systematic analysis of the ABCE gene families shed light on the formation of S. glauca's flower morphology, suggesting that dysfunction of A-class genes is responsible for the absence of petals in S. glauca. Gene family expansion analysis demonstrated significant enrichment of Gene Ontology (GO) terms associated with DNA repair, chromosome stability, DNA demethylation, cation binding, and red/far-red light signaling pathways in the co-expanded gene families of S. glauca and S. aralocaspica, in comparison with glycophytic species within the chenopodium family. Time-course transcriptome analysis under salt treatments revealed detailed responses of S. glauca to salt tolerance, and the enrichment of the transition-upregulated genes in the leaves associated with DNA repair and chromosome stability, lipid biosynthetic process, and isoprenoid metabolic process. Additionally, genome-wide analysis of transcription factors indicated a significant expansion of FAR1 gene family. However, further investigation is needed to determine the exact role of the FAR1 gene family in salt tolerance in S. glauca.

Gene mining and genomics-assisted breeding empowered by the pangenome of tea plant Camellia sinensis.

Tea is one of the world's oldest crops and is cultivated to produce beverages with various flavours. Despite advances in sequencing technologies, the genetic mechanisms underlying key agronomic traits of tea remain unclear. In this study, we present a high-quality pangenome of 22 elite cultivars, representing broad genetic diversity in the species. Our analysis reveals that a recent long terminal repeat burst contributed nearly 20% of gene copies, introducing functional genetic variants that affect phenotypes such as leaf colour. Our graphical pangenome improves the efficiency of genome-wide association studies and allows the identification of key genes controlling bud flush timing. We also identified strong correlations between allelic variants and flavour-related chemistries. These findings deepen our understanding of the genetic basis of tea quality and provide valuable genomic resources to facilitate its genomics-assisted breeding.

Pan-transcriptome assembly combined with multiple association analysis provides new insights into the regulatory network of specialized metabolites in the tea plant Camellia sinensis.

Specialized metabolites not only play important roles in biotic and abiotic stress adaptation of tea plants (Camellia sinensis (L.) O. Kuntze) but also contribute to the unique flavor of tea, the most important nonalcoholic beverage. However, the molecular networks and major genes that regulate specialized metabolites in tea plants are not well understood. Here, we constructed a population-level pan-transcriptome of the tea plant leaf using second-leaf transcriptome data from 134 accessions to investigate global expression differences in the population, expression presence or absence variations (ePAVs), and differentially expressed genes (DEGs) between pure Camellia sinensis var. assamica (CSA) and pure Camellia sinensis var. sinensis (CSS) accessions. Next, we used a genome-wide association study, a quantitative trait transcript study, and a transcriptome-wide association study to integrate genotypes, accumulation levels of specialized metabolites, and expression levels of pan-transcriptome genes to identify candidate regulatory genes for flavor-related metabolites and to construct a regulatory network for specialized metabolites in tea plants. The pan-transcriptome contains 30 482 expressed genes, 4940 and 5506 of which were newly annotated from a de novo transcriptome assembly without a reference and a genome reference-based assembly, respectively. DEGs and ePAVs indicated that CSA and CSS were clearly differentiated at the population transcriptome level, and they were closely related to abiotic tolerance and secondary metabolite synthesis phenotypes of CSA and CSS based on gene annotations. The regulatory network contained 212 specialized metabolites, 3843 candidate genes, and 3407 eQTLs, highlighting many pleiotropic candidate genes, candidate gene-rich eQTLs, and potential regulators of specialized metabolites. These included important transcription factors in the AP2/ERF-ERF, MYB, WD40, and bHLH families. CsTGY14G0001296, an ortholog of AtANS, appeared to be directly related to variation in proanthocyanins in the tea plant population, and the CsTGY11G0002074 gene encoding F3'5'H was found to contribute to the biased distribution of catechins between pure CSAs and pure CSSs. Together, these results provide a new understanding of the metabolite diversity in tea plants and offer new insights for more effective breeding of better-flavored tea varieties.

A triton X-100 assisted PMAxx-qPCR assay for rapid assessment of infectious African swine fever virus.

Introduction: African Swine Fever (ASF) is a highly infectious disease of pigs, caused by African swine fever virus (ASFV). The lack of vaccines and drugs makes strict disinfection practices to be one of the main measurements to curb the transmission of ASF. Therefore, it is important to assess if all viruses are inactivated after disinfection or after long time exposure in their natural conditions. Currently, the infectivity of ASFV is determined by virus isolation and culture in a biosafety level 3 (BSL-3) laboratory. However, BSL-3 laboratories are not readily available, need skilled expertise and may be time consuming. Methods: In this study, a Triton X-100 assisted PMAxx-qPCR method was developed for rapid assessment of infectious ASFV in samples. PMAxx, an improved version of propidium monoazide (PMA), can covalently cross-link with naked ASFV-DNA or DNA inside inactivated ASFV virions under assistance of 0.1% (v/v) TritonX-100, but not with ASFV-DNA inside live virions. Formation of PMAxx-DNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, the limit of detection of the PMAxx-qPCR assay was 2.32log10HAD50/mL of infectious ASFV. Testing different samples showed that the PMAxx-qPCR assay was effective to evaluate intact ASFV virions after treatment by heat or chemical disinfectants and in simulated samples such as swine tissue homogenate, swine saliva swabs, and environmental swabs. However, whole-blood and saliva need to be diluted before testing because they may inhibit the PCR reaction or the cross-linking of PMAxx with DNA. Conclusion: The Triton X-100 assisted PMAxx-qPCR assay took less than 3 h from sample to result, offering an easier and faster way for assessing infectious ASFV in samples from places like pig farms and pork markets.

Comprehensive Analysis of G-Quadruplexes in African Swine Fever Virus Genome Reveals Potential Antiviral Targets by G-Quadruplex Stabilizers.

African Swine Fever Virus (ASFV), a lethal hemorrhagic fever of the swine, poses a major threat to the world's swine population and has so far resulted in devastating socio-economic consequences. The situation is further compounded by the lack of an approved vaccine or antiviral drug. Herein, we investigated a novel anti-ASFV approach by targeting G-Quadruplexes (G4s) in the viral genome. Bioinformatics analysis of putative G-quadruplex-forming sequences (PQSs) in the genome of ASFV BA71V strain revealed 317 PQSs on the forward strand and 322 PQSs on the reverse strand of the viral genome, translating to a density of 3.82 PQSs/kb covering 9.52% of the entire genome, which means that 85% of genes in the ASFV genome have at least 1 PQS on either strand. Biochemical characterization showed that 8 out of 13 conserved PQSs could form stable G4s in the presence of K+, and 4 of them could be stabilized by G4 ligands, N-Methyl Mesoporphyrin (NMM), and pyridostatin (PDS) in vitro. An enhanced green fluorescent protein (EGFP)-based reporter system revealed that the expression of two G4-containing genes, i.e., P1192R and D117L, could be significantly suppressed by NMM and PDS in 293T cells. In addition, a virus infection model showed that NMM could inhibit the replication of ASFV in Porcine Alveolar Macrophages (PAM) cells with an EC50 value of 1.16 µM. Altogether, the present study showed that functional PQSs existent in the promoters, CDS, 3' and 5' UTRs of the ASFV genome could be stabilized by G4 ligands, such as NMM and PDS, and could serve as potential targets for antivirals.