Aspergillus niger is the main industrial workhorse for citric acid production. Since the release of the genome sequence, extensive multi-omic data are being rapidly obtained, which greatly boost our understanding of the citric acid accumulation mechanism in A. niger to a molecular and system level. Most recently, the rapid development of CRISPR/Cas9 system facilitates highly efficient genome-scale genetic perturbation in A. niger. In this review, we summarize the impact of systems biology on the citric acid molecular regulatory mechanisms, the advances in metabolic engineering strategies for enhancing citric acid production and discuss the development and application of CRISPR/Cas9 systems for genome editing in A. niger. We believe that future systems metabolic engineering efforts will redesign and engineer A. niger as a highly optimized cell factory for industrial citric acid production.Citric acid is the world’s largest consumed organic acid and is widely used in beverage, food and pharmaceutical industries. The online version of this article (10.1186/s12934-019-1064-6) contains supplementary material, which is available to authorized users. Citric acid  is known as an intermediate of the tricarboxylic acid cycle that is used to release energy from carbohydrates, fats, and proteins via the oxidation of acetyl-CoA [Absidia sp., Acremonium, Botrytis, Eupenicillium, Penicillium, and some Aspergillus sp., such as Aspergillus niger, Aspergillus awamori, Aspergillus nidulans, Aspergillus luchensis, and Aspergillus flavus [Bacillus sp., Brevibacterium sp., Corynebacterium sp., Klebsiella sp., and Pseudomonas sp. amongst others [Candida sp. and Yarrowia sp. [A. niger [The high commercial interest has attracted numerous scientists to devote themselves to citric acid over-producing strain development since the last century. Many microorganisms have been discovered to accumulate citric acid including s flavus . Besidest others . Yeast sowia sp. . Howeverowia sp. , approxiA. niger .A. niger strains excreted large amounts of citric acid at the initial pH of 2.5 [A. niger has been the main industrial workhorse owing to its unique inherent physiological characters and better fitness for industrial fermentation [A. niger has powerful polymer degrading enzyme systems to hydrolyze many polymeric substrates, enabling to rapidly grow and ferment on a variety of low-cost raw materials such as corn meal and molasses [A. niger demonstrates great robustness to extreme acid environments, leading to outcompeting other rival microorganisms and reducing contamination risk. The high citric acid yield of 0.95 g/g supplied sugar can be achieved with the assistance of fermentation optimization [Thermotoga maritima by increasing the H2 yield even beyond the previously predicted biological limit [Although many microorganisms could be utilized for citric acid production, since 1917, Currie discovered that some H of 2.5 . A. nigeentation , 6. A. nmization , therebyal limit . Neverthal limit , 11.A. niger strains brought the study of A. niger into the post-genomic era [A. niger citric acid fermentation can gradually be unveiled, and systems metabolic engineering is currently being used to redesign and optimize A. niger as a cell factory. Until now, no review has focused on how advances in systems biology and metabolic engineering of A. niger are enhancing citric acid production. In this review, we summarize the impact of systems biology on understanding the citric acid molecular regulatory mechanisms, the existing metabolic engineering strategies implemented to improve citric acid production and review the development of CRISPR/Cas9 systems for genome editing in A. niger. We also proposed future prospects in the systems metabolic engineering cycle, combining the genome information, modern bioinformatics approaches and efficient molecular genetic manipulation tools, to design and engineer A. niger as a highly optimized cell factory for improving the yield, titer and productivity with reduced costs and improved environmental sustainability.Since 2007, the public release of genome data for omic era . With th2 released in the mitochondria, while oxaloacetate is formed by pyruvate carboxylation from pyruvate with 1 mol CO2 fixation in the cytoplasm. Oxaloacetate is subsequently converted into malic acid and enters the mitochondria through a malate–citrate shuttle. Malic acid is reconverted into oxaloacetate and oxaloacetate takes part in citric acid synthesis. One mole glucose is converted into 1 mol citric acid with 1 mol of ATP and 3 mol of nicotinamide adenine dinucleotide (NADH), resulting in the maximum theoretical yield of 1.067 g/g glucose [Citric acid is the first intermediate of the TCA cycle, and is synthesized by the condensation of acetyl-coenzyme A (acetyl-CoA) and oxaloacetate moiety . Acetyl- glucose .A. niger is capable of citric acid accumulation in large amounts by an active glycolytic pathway. The unique citric acid metabolism regulation in A. niger has attracted much interest, and several excellent reviews have discussed the biochemical mechanisms before the A. niger genome release [A. niger, including genomics, transcriptomics, proteomics and metabolomics, which are being rapidly obtained and boost our understanding of A. niger further to a system and molecular level. The impact of these datasets is discussed in detail in the subsequent section.As an intermediate of the TCA cycle, citric acid is commonly catabolized by cis-aconitase, and citrate and ATP usually have feedback inhibition against the glycolysis pathway. Nevertheless,  release , 2, 4. MA. niger stains with different phenotypes have been submitted in the Genome database of National Center for Biotechnology Information  [A. niger strains with different citrate production efficiencies and mycelial pellets morphology was analyzed [A. niger H915-1 exhibited the highest citrate titer of 157 g/L and the yield of 0.98 g/g total sugar in 85 h with compact pellets and short, swollen hyphal branches, whereas the degenerated isolates A1 and L2 produced 117 g/L in 92 h with less hyphal branch in compact pellets, and 76 g/L in 160 h with mycelial clumps, respectively [These comparative genomic studies between different industrial isolates have been further supplemented by genomic profiling of mutant isolates and progenitor strains, which has also been useful for predicting key molecular aspects of citric acid production. For example, comparative genomics of three analyzed . A. nigeectively . Compareectively . The mycectively . These dA. niger. Before the availability of genome data, the first DNA microarray study in A. niger only investigated the transcription change of 15 genes [Aspergillus microarray for comparative transcriptomics of A. niger, A. nidulans and A. oryzae. With the availability of microarrays, Salazar et al. [Aspergilli species. Comparative transcriptome has also been applied for unveiling the notable diversity between CBS513.88 and ATCC1015 [Transcriptomics is an important technique in functional genomics of 15 genes  developer et al.  elucidatAspergillus genus using RNA-seq was performed in A. oryzae [A. niger via RNA-seq. Since then, RNA-seq has been widely used in global gene expression profiling to investigate the transcription response and regulation of A. niger, including carbon source utilization and regulation [A. niger.Compared to DNA microarray technologies, RNA sequencing (RNA-seq) is increasingly used for transcriptome analysis, due to its higher sensitivity, accuracy and resolution . After t. oryzae , Delmas . oryzae  also assgulation –24, conigulation , 26, celgulation , 28, secgulation , 30 and gulation , 31–33. gulation  obtainedA. niger proteome under different carbon substrate using 2-D gel electrophoresis/MALDI-TOF and nano-HPLC MS/MS and found that the secretome was dramatically influenced by the extracellular carbon substrate. Elsewhere, Adav et al. [A. niger protein secretion profile using iTRAQ quantitative proteomics and demonstrated that 102 secreted enzymes ensured the powerful capability and potential of polymer degradation. Moreover, membrane-associated proteomic analysis has been developed to identify the new transporters. Sloothaak analyzed the plasmalemma proteomics under different glucose concentration with a hidden Markov model (HMM) and identified two high-affinity glucose transporter MstG and MstH [l-rhamnose transporter RhtA [Similar to transcriptomics, proteomics is an essential component of systems biology, which enables the qualitative and quantitative assessment of the entire proteins of an organism under different conditions. Lu et al.  collectev et al.  analyzedand MstH . They futer RhtA . These sA. niger metabolomics [Metabolomics is an important potential tool for industrial biotechnology: uncovering global metabolite profiles, identifying the biosynthetic intermediates and metabolic bottlenecks, elucidating phenotype differentiation, and also previously unknown pathways . A signibolomics –42.A. niger metabolomics adopted − 45 °C 60% methanol quenching which had previously been applied in yeast [A. niger, given its minimal cell damage, high intracellular metabolite recovery and relatively efficient quenching efficiency [Early investigations into in yeast . Recentlin yeast . Howeverficiency .A. niger, such as chloroform/methanol/buffer (CM) [A. niger isolate. The metabolomics analyses suggest that high Embden-Meyerhof pathway (EMP) flux and high level of citric acid precursors ensure citrate accumulation [Few intracellular metabolite extraction methods have been used in fer (CM) , or boilfer (CM) . A limitfer (CM) . Given tmulation . For examulation .A. niger. 13C metabolic flux analysis, for example, has been utilized to investigate the metabolic difference in mutant enzyme over-producing strains [oahA gene encoding oxaloacetate acetylhydrolase in a glucoamylase-producing strain did not influence the central carbon metabolism and metabolic flux distribution, while Driouch et al. [Similar to the metabolomics, fluxomics analysis is also a powerful strategy to unveil the metabolic properties and in vivo flux distribution in filamentous fungi such as  strains –47. Pede strains  found thh et al.  discoverA. niger have been developed to uncover the key aspects of citric acid over-production [A. niger based on the genome information of CBS513.88 and ATCC9029, including enzymes with 988 unique EC numbers, 2443 reactions and 2349 metabolites. Additional copies of alternative mitochondrial oxidoreductase (AOX) and citrate synthase (CS) encoding genes were found in A. niger, which might contribute to the citric acid accumulation. Consequently, these open reading frames constitute outstanding candidates for rational strain engineering using the extensive A. niger toolkit [With the availability of massive multi-omics data , genome-oduction –55. Sun oduction  develope toolkit .iMA871 of A. niger was reconstructed based on the genome of ATCC1015, including 1190 reactions [Another genome-scale metabolic model eactions . Compareeactions . Upton eeactions  demonstrA. niger can be summarized as follows: efficient carbon utilization and transport resulted from powerful hydrolytic enzyme and glucose transport system, high glycolysis flux resulted from the relief of feedback inhibition of ATP and citrate, high C4 anaplerotic activity catalyzed by pyruvate carboxylase to secure precursor supplement, low cis-aconitase and isocitrate dehydrogenase activity to prevent citrate degradation, efficient alternative respiratory chain mediated by AOX to accelerate NADH oxidation and NAD+ regeneration with less energy production, ATP futile cycle and consumption catalyzed by ACL, Mn2+ deficiency to keep high glycolysis flux but low citrate degradation flux via TCA cycle, and compact mycelial pellets to secure the oxygen transfer by lower the viscosity of the fermentation broth, and high acid resistance mediate by GABA shunt. With the assistance of systems biology, especially the genome-scale metabolic modeling, it is now possible to identify bottlenecks as targets for A. niger metabolic engineering, which efforts to design and optimize new stains capable of enhanced citric acid production on low-cost feedstocks, including agro-industrial wastes and lignocellulose biomass, with reduced energy consumption and environmental contamination.In summary, the combination of systems biology datasets from the highlighted studies, key attributes of citric acid accumulation mechanism in Y. lipolytica  into mono-saccharides. However, when liquefied corn starch was utilized for citric acid production, about 2%–3% of the residual sugar remains at the end of the fermentation process. Owing to citric acid production scale of about 1.7 million tons, the residual sugar would account for the annual loss of 150 thousand tons of corn and lead to great environmental pressure world-wide [agdA efficiently reduced the isomaltose concentration [glaA, the residual sugar reduced about 88.2% and the citric acid production increased 16.9%, reaching up to 185.7 g/L [glaA gene under the native PagdA promoter improved the extracellular glucoamylase activity by 34.5% [rld-wide . Thereforld-wide . Deletiontration . Combine85.7 g/L . The mulby 34.5% . The gluby 34.5% . Thus, tacl1 in A. niger AB4.1 increased the organic acids including succinic acid and citric acid. Chen et al. [A. niger ATCC1015 resulted in decreasing citric acid production, concomitant with diminished asexual conidiogenesis, conidial germination and cell growth. In contrast, the overexpression exhibited reverse effects, suggesting that ACL is beneficial for citric acid accumulation. It was consistent with the time-series transcriptome analyses of citric acid fermentation, which speculated that the cytosolic ACL may involve in the ATP futile cycle [Acetyl-CoA and oxaloacetate are the two direct substrates for citric acid synthesis. Acetyl-CoA is generated by pyruvate dehydrogenase (PDH), cytosolic acetyl-CoA synthetase (ACS) and ATP-citrate lyase (ACL), and fatty acids beta-oxidization . The pron et al.  found thle cycle .A. niger, and highlight how novel industrial capabilities can be developed using systems metabolic engineering and synthetic biology. Indeed, the citric acid metabolic pathway may be entirely re-routed in the future and even synthesized in the cytoplasm instead of mitochondria.Oxaloacetate is formed by pyruvate carboxylation in the cytoplasm and subsequently converted into malic acid. After entering the mitochondria through a malate–citrate shuttle, malic acid is reconverted into oxaloacetate and oxaloacetate takes part in citric acid synthesis. Therefore, de Jongh and Nielsen engineered the cytosol reductive TCA (rTCA) cycle by inserting heterogeneous malate dehydrogenase, fumarase and fumarate reductase . It was ggsA) only slightly led to the earlier initiation of citric acid accumulation and the final citric acid production was even reduced compared with the parent strain or multicopy transformant [Hexokinase is strongly inhibited by trehalose 6-phosphate . Howeversformant . Legisa sformant .A. niger PFK1 [mt-pfkA10 with T89D single site mutation to elude the phosphorylation requirement. A. niger TE23, constructed by overexpressing the active shorter PFK1 fragment in A. niger A158, exhibited citric acid production of 120 g/L at 300 h, about 70% higher than the control strain [PFK is another crucial controlling step for glycolysis metabolic flux via the allosteric inhibition or activation. ATP and citric acid are the inhibitors of PFK. Spontaneous post-translational modification plays a vital role in keeping the high activity of ger PFK1 . In the ger PFK1  designedl strain .2+ deficiency, whereas the AOX activity increases. Recently, some oxidative phosphorylation inhibitors, such as succinate-cytochrome c inhibitor antimycin A or the oxidative phosphorylation uncoupler 2,4-dinitrophenol (DNP) [aox1 gene improve citric acid production up to 169.1 g/L in the fermentation medium with antimycin A. Obviously, these studies pave the way for combined engineering of the cytochrome-dependent respiratory chain and alternative respiratory chain by promoter engineering.In the citric acid synthesis pathway, the equivalent quantitative conversion of glucose to citric acid generated 1 mol of ATP and 3 mol of NADH. The NADH oxidization cycle by cytochrome-dependent respiration usually generated excess ATP, which strongly feedback inhibited PFK and impaired the glycolysis flux. Thus, when citric acid begins to accumulate, cytochrome-dependent respiration is replaced by the alternative route, which enables the NADH oxidization without concomitant ATP production . Wallratol (DNP) . Hou et ol (DNP)  uncovere2+ deficiency plays a crucial role in citric acid accumulation. Mn2+ interferes with A. niger metabolism in several ways, for example by preventing citrate reutilization, suppressing macromolecular  synthesis, enhancing protein degradation and intracellular NH4+ concentration, altering the ratio of saturated: unsaturated fatty acid in the plasma membrane, modifying the polysaccharide concentration of the cell wall, and influencing morphology [Brsa-25 gene, which encodes a putative amino acid transporter, is involved in the regulation of morphology formation in response to Mn2+. Down-regulation of the Brsa-25 expression by antisense RNA reshaped the mycelial pellets and enhanced the citric acid production by 10% [chsC) also caused lower proportion of dispersed mycelia in the mycelial pellets and improved citric acid production about 42.6% [2+ response and morphology regulation are highly complex, and involve a large number of genes with different functions. Therefore, an efficient multiplex gene editing technology is in an urgent necessity for testing the synergistic effect and interaction of individual genes in a network.Mnn by 10% . Similarut 42.6% . Mn2+ reA. niger [Genetic and genomic manipulations exert a crucial influence in metabolic engineering of A. niger . The rapA. niger , 81, theA. niger  and tested the gene disruption efficiency of CRIPSR/Cas9 system based on this PanU6 and other two heterologous U6 promoters (PhU6 and PyU6)  homologous donor DNA  systems have become a very powerful genome editing technique , 83. Recger Fig. , Table 2n et al. , 86 adopn et al. . Moreoven et al. . To fillg et al.  identifiA. niger for citric acid production that is more environmentally friendly, with better food safety, and improved cost-effectiveness. A Learn-Design-Build-Test (LDBT) cycle has been gradually established for customized metabolic engineering on a large-scale of A. niger, combining multi-omics analysis, computational biology approaches, molecular genetic manipulation toolbox and high through-put platform (Fig. A. niger strains then would be constructed by genome editing and regulation toolboxes (Build, Fig. As mentioned above, the development of systems biology and genome editing technology paves the way to systemically engineer orm Fig. . Namely,orm Fig. . The metorm Fig. . The desA. niger at a systems-level. On one hand, to construct the stoichiometric and/or dynamic biological network, more absolutely quantitative omics data are required. On the other hand, the more efficient integrated approaches for multi-omics data are required to uncover the interactions among multi-omics data and the molecular regulation mechanism at different molecular levels, and ultimately to achieve the holistic design of new citric acid producing isolates meeting various requirements.First, the massive multi-omics data provide the feasibility for understanding the A. niger have been developed for steady state, usually as stoichiometric models. In the future, a dynamic/kinetic model is required to illustrate the multi-omics and process data and predict the behavior of A. niger responding to interior and exterior changes during citric acid fermentation. Therefore, genome-scale metabolic modeling would become the main systems approach to optimize the metabolic engineering design.Secondly, until now, three metabolic engineering strategies have been developed for target prediction, including current knowledge-driven design, comparative omics-driven design, and in silico modeling design. Common knowledge-driven design strategies mostly focus on the improvement of precursor supplements, reduction of by-product formation and feedback inhibition, which are usually limited by the complexity of metabolic regulation. The comparative omics-driven design approach is suitable to distinguish the key genes contributed to specific phenotypes, in which the selection of intercomparable strains and condition design is of vital importance for target discovery. Otherwise, it is hard to find the key genes from the numerous potential differences. In contrast, genome-scale models facilitate to integrate multi-omics data and construct the organism-specific metabolic maps, interpret the changes of transcriptional and metabolic profiles, ultimately to establish a full understanding of the cell regulation complexity at different levels . Most geA. niger to accelerate the systems metabolic engineering cycle for final industrialization.Thirdly, to construct on-demand well-designed strains, three aspects should be paid more attention, including construction of synthetic biological module, construction of robust chassis and development of multiplex genetic manipulation toolboxes. CRISPR/Cas9 genome editing technology facilitates the rapid verification of new hypotheses and the realization of target predictions. Multiplex genome engineering and marker-free base editing are required to be established in Finally, high throughput platforms, including the spore collection, strain cultivation, metabolite detection and fermentation optimization, should be developed to test and screen the well-designed strains in a large-scale. All the exhaustive measurement data would be applied for the next strategy design.A. niger biotechnology is the generation of designer strains and super cell factory with higher titre, yield and productivity. Toward this end, some directions for systems metabolic engineering can be summarized as follows: improving the substrate utilization, removing by-product synthesis, removing negative feedback effect, enhancing the precursor supplement, enhancing the transport efficiency of substrates and citric acid, optimizing the NADH regeneration by regulating the respiratory chain, enhancing the robustness and resistance against environmental stress, regulating the morphology to fit the process operation. Many genome editing strategies could be applied to achieve metabolic engineering, including promoter engineering of target genes with inducible promoters, transcription factor engineering, transporter engineering, and transcription regulation via CRSIPRi/CRSIPRa system or RNAi. In conclusion, holistic design from multi-omics analyses and dynamic modeling, genome editing combined with synthetic biology provide great promise for achieving rational design of A. niger at a system level.With the rapid development of systems biology and synthetic biology, a major goal for the future of Additional file 1: Table S1. Genome information of several A. niger strains. Table S2. Metabolic engineering strategies for enhancing citric acid production in Yarrowia lipolytica.


PPRs in Salvia miltiorrhiza, which is a well-known material of traditional Chinese medicine and an emerging model system for medicinal plant studies. Among 562 identified SmPPRs, 299 belong to the P subfamily while the others belong to the PLS subfamily. The majority of SmPPRs have only one exon and are localized in the mitochondrion or chloroplast. As many as 546 SmPPRs were expressed in at least one tissue and exhibited differential expression patterns, which indicates they likely play a variety of functions in S. miltiorrhiza. Up to 349 SmPPRs were salicylic acid-responsive and 183 SmPPRs were yeast extract and Ag+-responsive, which indicates these genes might be involved in S. miltiorrhiza defense stresses and secondary metabolism. Furthermore, 23 salicylic acid-responsive SmPPRs were co-expressed with phenolic acid biosynthetic enzyme genes only while 16 yeast extract and Ag+-responsive SmPPRs were co-expressed with tanshinone biosynthetic enzyme genes only. Two SmPPRs were co-expressed with both phenolic acid and tanshinone biosynthetic enzyme genes. The results provide a useful platform for further investigating the roles of PPRs in S. miltiorrhiza.The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants and plays important roles in posttranscriptional regulation. In this study, we combined whole genome sequencing and transcriptomes to systematically investigate  Salvia miltiorrhiza Bunge, which is a well-known traditional Chinese medicine (TCM), is widely used to treat the cardiovascular and cerebrovascular diseases [S. miltiorrhiza has been considered an ideal model species for genomic and genetic studies of medicinal plants. Hydrophilic phenolic acids and lipophilic diterpenoids are the main active pharmaceutical compounds of S. miltiorrhiza. Hydrophilic phenolic acids such as salvianolic acid A, salvianolic acid B, and rosmarinic acid are derived from the phenylpropanoid pathway and the tyrosine-derived pathway [d-erythritol 4-phosphate (MEP) pathway and/or the mevalonate (MVA) [S. miltiorrhiza have attracted widespread interest [S. miltiorrhiza quality improvement.diseases . With th pathway ,3. Lipopte (MVA) . Due to interest ,10,11,12interest ,13  gene family exists widely in most eukaryotes including plants, mammals, and protists . The num100 PPRs . Howeverectively ,17,18,19ectively . Poplar ectively . For insca napus ,24,25,26nd maize ,28. EMP4nd maize ,30,31. Wnd maize . OsPPR67nd maize . PPR40, bidopsis ,35,36,37ze cells ,39,40,41bidopsis ,43. AlthSmPPRs were genome-wide identified and characterized in S. miltiorrhiza. Different expression patterns were investigated. The majority of elicitor-responsive SmPPRs and subsets of SmPPRs co-expressed with phenolic acid and tanshinone biosynthetic genes were shown. Our results provide some references to further functional studies of this family gene in S. miltiorrhiza.In this study, 562 Arabidopsis thaliana PPRs against the current assembly of the S. miltiorrhiza genome and subsequent gene prediction of the retrieved genomic DNA sequences, a total of 562 SmPPR genes were identified. They were named SmPPR1–SmPPR562, respectively  containing no intron, about 10% (57/562) containing one intron, and only 2% (10/562) more than one  and the long (L) motifs). Furthermore, based on the C-terminal motifs, the PLS subfamily could be further divided into the P-L-S, E, Eubgroups . Motif aroup see . We failorrectly .The subcellular localization prediction showed that SmPPRs were widely located in chloroplast, mitochondrion, secretory pathway, and other locations see  with theSmPPRs in S. miltiorrhiza growth and development, the expression of SmPPRs in roots, stems, leaves, and flowers of S. miltiorrhiza was analyzed transcriptome-wide of which 546 were expressed  values less than one in all tissues analyzed. They are pseudogenes or expressed only at specific developmental stages or under special conditions [It has been shown that many PPR proteins are involved in plant developmental processes ,31,32,33ssed see . Among tions see a. These roup see . These group see b of whicroup see . It indinditions .SmPPR195 and SmPPR554 mainly expressed in roots, SmPPR158 and SmPPR401 mainly expressed in stems, SmPPR312 and SmPPR420 mainly expressed in leaves, and SmPPR271 and SmPPR278 mainly expressed in flowers  and yeast extract could improve the antioxidant defense of S. miltiorrhiza hairy roots [+ was more effective than single yeast extract or Ag+ in the hairy root cultures of S. miltiorrhiza [S. miltiorrhiza and stimulate the activities of HMGR and DXS enzymes [+ are effective elicitors for bioactive compound production and related gene expression in S. miltiorrhiza. In this study, 349 SmPPRs were responsive to salicylic acid treatment and 183 SmPPRs were responsive to yeast extract and Ag+ treatment, which indicates the importance of SmPPRs in S. miltiorrhiza stress responses and secondary metabolites. Co-expression analysis has previously been described to be a suitable tool for gene function identification [Arabidopsis starch metabolism-related genes [SmPPRs were co-expressed with phenolic acid biosynthetic genes and 18 yeast extract and Ag+-responsive SmPPRs were co-expressed with tanshinone biosynthetic genes  with whole genome sequence available was grown in a field nursery at the Institute of Medicinal Plant Development . Roots, stems, leaves, and flowers were collected from two-year-old plants and stored in liquid nitrogen until use.Arabidopsis PPR (AtPPR) protein sequences were downloaded from TAIR (http://www.arabidopsis.org/). BLAST analysis of AtPPR proteins against the genome database of S. miltiorrhiza (line 993) [−10 was applied. Gene models of SmPPRs were predicted based on the alignments between the retrieved DNA sequences and PPR proteins from other plant species via BLASTx. The predicted gene models were examined and comparatively analyzed with the genome database of the other S. miltiorrhiza line . The domain information of SmPPR proteins was analyzed using the software package HMMER 3.0 (http://hmmer.janelia.org/software/archive). The HMMbuild program in the HMMER 3.0 package was used for producing the matrices specific to each type of SmPPRs. Motifs were searched using the HMMsearch program in the HMMER 3.0 package. The gene structures were analyzed using the Gene Structure Display Server . Subcellular localization of SmPPR proteins was predicted with TargetP version 1.1 (http://www.cbs.dtu.dk/services/TargetP/). The molecular weight (MW) and theoretical isoelectric point (pI) were predicted via the compute pI/MW tool on the ExPASy server (http://web.expasy.org/compute_pi/).ine 993)  was carrSmPPR genes in different organs were analyzed using the transcriptome datasets of roots, stems, leaves, and flowers of S. miltiorrhiza downloaded from GenBank . The changes of SmPPR expression levels in response to stress treatments were analyzed using the transcriptome datasets of suspension cells treated with salicylic acid  (SRX1423774) for 0 h, 2 h, and 8 h and hairy roots treated with yeast extract (100 µg/mL) and Ag+ (30 µM) (SRR924662) for 0 h, 12 h, 24 h, and 36 h. RNA-sequence reads were mapped to SmPPRs using SOAP 2.0 [SmPPR genes with phenolic acid and tanshinone biosynthetic genes in S. miltiorrhiza was analyzed using the software package of Co-expression Pattern Clustering Analysis in the BMKCloud cloud server (http://www.biocloud.net/). The abundance of transcripts was estimated by RPKM. Genes with RPKM value greater than one in at least a tissue was considered to be expressed. To draw heat maps, the value of RPKM for each gene was normalized between −1.0 and 1.0 using the R package version 3.4.1 and analyzed for differential expression by the Fisher’s exact test. p < 0.05 was considered as differentially expressed.Expression patterns of ® Premix Ex Taq™ II  (TaKaRa) as described previously [SmPPRs were listed in SmUBQ10 was used as an internal control. qRT-PCRs were performed in three biological replicates with three technical replicates each. The expression level in stems was set to one and the levels in other tissues were given relative to this. The relative expression levels of genes were calculated by the 2−ΔΔCt method. ANOVA  was calculated using SPSS . p < 0.05 was considered statistically significant.Gene expression was analyzed using qRT-PCR. Total RNA was extracted from prepared samples using the Plant RNA EASYspin Plus . RNA integrity was analyzed on a 1.2% agarose gel and its quantity was determined using a NanoDrop 2000C Spectrophotometer . Reverse transcription was carried out using the PrimeScript™ RT reagent kit with gDNA Eraser  . PCR was performed using the SYBReviously . Gene-spPPR gene family in S. miltiorrhiza was performed. A total of 562 PPR protein genes were identified in S. miltiorrhiza genome. Gene structure and classification showed important features for this family. Tissue-specific expression analysis indicated that SmPPRs have more ubiquitous roles in S. miltiorrhiza. The expression pattern in response to different elicitors revealed SmPPRs might be involved in stress defense and secondary metabolites. Furthermore, some SmPPRs co-expressed with phenolic acid and tanshinone biosynthetic genes were shown. The results provide valuable information for future studies on characterizing the biological functions of PPR protein genes in S. miltiorrhiza.In this study, bioinformatic analysis for


Small cell lung cancer (SCLC) is a highly malignant cancer, and over 70% of patients with SCLC present with the metastatic disease. We aimed to explore some novel differentially expressed genes (DEGs) or microRNAs (miRNAs) associated with the lymph node metastasis of SCLC.The DEGs between the metastasis and cancer groups were identified, and GO functional and KEGG pathway enrichment analyses for these DEGs were implemented. Subsequently, the protein–protein interaction network and subnetwork of module were constructed. Then the regulatory networks based on miRNAs, transcription factors (TFs) and target DEGs were constructed. Ultimately, the survival analysis for DEGs was performed to obtain the DEGs related to the survival of SCLC.Here, 186 upregulated  and 144 downregulated DEGs  were identified between the SCLC patients with lymph node metastasis and without lymph node metastasis. GRM8 was attracted to the G-protein coupled receptor signaling pathway. Besides, miR-126 was identified in the miRNAs-TFs-target regulatory network. GRM8 and DACH1 were all regulated by miR-126. In particular, GSR and HCP5 were correlated with survival of SCLC patients.MiR-126, DACH1, GRM8, MET, GSR, and HCP5 were implicated in the lymph node metastasis process of SCLC. Lung cancer (LC) is a malignant lung tumor characterized by unbounded cell growth in the lung tissues . It is eLymph nodes, the central trafficking hubs for recirculating immune cells, are widely present throughout the body . ConceivThe bioinformatics analysis methods were carried out for screening DEGs correlated with the lymph node metastasis process of SCLC. Firstly, the DEGs between the metastasis and cancer groups were screened. Afterwards, Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for the DEGs were implemented to obtain the potential functions of DEGs. Afterwards, the protein–protein interaction (PPI) network and subnetwork of module were established. Then the regulatory networks based on microRNA (miRNAs), TFs and target DEGs were constructed. Ultimately, the survival analysis for DEGs was performed to obtain the DEGs related to the survival of SCLC.http://www.ncbi.nlm.nih.gov/geo/) [The gene expression profiling GSE40275 was obtained from the Gene Expression Omnibus (GEO) database (ov/geo/) , which ihttp://bioconductor.org/help/search/index.html?q=oligo/) [http://www.bioconductor.org/packages/2.9/bioc/html/limma.html) [We downloaded the raw CEL data and used the Oligo package (ver.1.38.0) (=oligo/)  in R lanma.html) . The sethttps://david-d.ncifcrf.gov/) [GO  and KEGGrf.gov/)  tool. Thhttp://www.string-db.org/) [http://www.cytoscape.org/) software was applied to construct the PPI network. The significant clustering modules were analyzed using Molecular Complex Detection (MCODE)  [The Search Tool for the Retrieval of Interacting Genes (STRING)   databases/MCODE)  plugin. http://apps.cytoscape.org/apps/iRegulon) [http://www.webgestalt.org/option.php) using the Overrepresentation Enrichment Analysis (ORA) method. The setting of threshold was count number ≥ 2 and p value < 0.05. Ultimately, the miRNAs-TFs-target regulatory network was constructed utilizing the Cytoscape software.The iRegulon   plugin ihttp://www.ncbi.nlm.nih.gov/geo/). The common samples between the SCLC samples and clinical data were screened and removed the samples with survival time less than 1 month. Here, a total of 14 samples were enrolled in the present study.GSE29016 gene expression profiling data including 20 SCLC samples and clinical data were obtained from the GEO database  in R language and median grouping method were used to conduct the survival analysis. Finally, DEGs under p value < 0.05 were selected to generate the survival curve.We obtained 330 DEGs between the metastasis and cancer groups, of these, 186 were upregulated and 144 were downregulated. The volcano map and heatmap of DEGs were presented in Fig. −4), extracellular region , and calcium ion binding . The enriched functions for downregulated DEGs were presented in Table −6), integral component of plasma membrane , and calcium ion binding . Here, the upregulated DEGs were not enriched in any pathway. However, downregulated DEGs were attracted to 5 pathways (Table −3), axon guidance , and alcoholism . Moreover, a total 6 DEGs were attracted to the pathway of transcriptional misregulation in cancer, such as MET.The enriched functions for upregulated DEGs were listed in Table −5), regulation of phosphorylation , and regulation of phosphate metabolic process . Meanwhile, the enriched functions for module a DEGs were listed in Table −3), cell surface receptor linked signal transduction , and regulation of inflammatory response . Here, our results showed that GRM8 was attracted to the G-protein coupled receptor signaling pathway.There were 178 nodes and 237 interaction pairs in the PPI network  to the sulfhydryl form glutathione (GSH). Whereas, GSH played complex roles in cancer, including the protective and pathogenic roles . In a prIn addition, HCP5 was significantly down-regulated in patients with ovarian cancer . SimilarIn summary, our results suggested that miR-126 and its target gene DACH1 may implicate in the lymph node metastasis process of SCLC. Additionally, GRM8, MET, RSD and HCP5 were implicated in the lymph node metastasis process of SCLC.


Helicobacter pylori - (H. pylori) play a role in the pathogenesis of gastritis, gastric and duodenal ulcers as well as gastric cancer. A possible involvement of outer membrane vesicles (OMVs) produced by H. pylori in the distribution of bacterial antigens through the gastric epithelial barrier and their role in the development of local and systemic host inflammatory and immune responses has been suggested. OMVs contain various biologically active compounds, which internalize into host cells affecting signaling pathways and promoting apoptosis of gastric epithelial and immunocompetent cells. OMVs-associated H. pylori virulence factors may strengthen or downregulate the immune responses leading to disease development. This review describes the biological importance of H. pylori OMVs and their role in the course of H. pylori infections, as well as H. pylori related local and systemic effects. Helicobacter pylori (H. pylori) are Gram-negative microaerophilic, spiral-shaped bacteria that belong to the ɛ subdivision of the Proteobacteria, order Campylobacterales, family Helicobacteriaceae. Due to ability to adhere to gastric epithelium or internalize inside the cells they colonize human gastric mucosa, and cause gastritis, gastric ulcers and even cancers, or even recently considered development of extragastric diseases [H. pylori and the ability to cause various diseases depend on the high genomic variability of these bacteria [diseases –5. The pbacteria .H. pylori infections is associated with a geographical region or living conditions. Moreover the demographic flux of human populations induce the occurrence of new H. pylori genetic variants [The prevalence of variants –14.x and Ley) expression, which determines molecular mimicry, and immune evasion [H. pylori resist the oxidative stress caused by excessive inflammatory response and in the chronic phase of infection, these bacteria downregulate the host immune responses driving the immunity towards tolerance rather than to a protective response [H. pylori replicate in murine bone marrow derived dendritic cells and on this way can affect immune responses mediated by these population of antigen presenting cells [These bacteria are well adapted to the harsh environment of the gastric niche due to motility, urease production, variability of adhesins, different effector molecules, and the Lewis (Le) blood group antigens  such as cytotoxin-associated gene A (CagA) protein [H. pylori also produce outer membrane vesicles (OMVs) that contain various virulence factors and this strategy supports the survival of these bacteria in the gastric mucosa [H. pylori compounds delivered by OMVs and on their role in the pathogenicity of these bacteria are ongoing. protein , 24–26. c mucosa , 28. TheEscherichia coli (ETEC) and aminopeptidase of Pseudomonas aeruginosa are elevated in vesicles, when compared to their expression in whole bacteria [tol-pal cluster [Outer membrane vesicles are defined as 20–300 nm blebs, which are naturally secreted by Gram-negative bacteria mainly during their logarithmic phase of growth although, Gram-positive bacteria also form the extracellular particles –34. The bacteria . The gen cluster .Pseudomonas fragi observed within the damaged muscles of pigs exposed to these bacteria [The process of vesiculation is influenced by stress conditions such as increased temperature or the lack of nutrients. This phenomenon is genetically regulated and determines the interaction of the pathogen with host tissues and proteolytic enzymes, which was proved by showing the presence of bleb-like evaginations on the surface of bacteria –44.Previously, it was thought that OMVs promote survival of nonpathogenic species, while in pathogenic species enhance their virulence, the ability to downregulate or stimulate host immune defense, and raise the pathogenic potential , 45–47. Bacteroides fragilis OMVs exhibited anti-inflammatory properties in patients with colitis due to upregulation of regulatory T cells. On the contrary, pro-inflammatory potential of Moraxella catarrhalis OMVs was shown in patients with chronic obstructive pulmonary disease as well as meningococcal OMVs in sepsis [Proteomic analysis of Gram-negative bacteria showed that bacterial structures present in OMVs play a role in intercellular bacteria-bacteria and bacteria-host interactions, whereas secreted OMVs are capable of interacting with and adhering to host cells . They con sepsis –59. Sincn sepsis , 60–63.H. pylori OMVs crossing the cellular barrier are considered as the effective way of H. pylori antigens translocation through the gastric mucosa to immune cells in tissues or even to circulation. This mechanism of antigen transport seem to be an alternative for the TSS4 secretion system or to direct secretion of soluble compounds to the surrounding environment [ironment , 25, 64.et al., using H. pylori OMVs separated by density gradient centrifugation and probed for inner membrane (IM) and outer membrane (OM) markers demonstrated that vesicles of these bacteria contain OM, but not IM compounds [H. pylori OM was detected by electron microscopy both in gastric biopsies of infected individuals and in cultures of these bacteria especially during the late stationary growth phase, and during biofilm formation [Olofsson ompounds . Blebbinormation , 46, 65.H. pylori strains as they are encoded by various genes. In H. pylori OMVs the following phospholipids were found: phosphatidylglycerol (PG), phosphatidylethanolamine (PE), lyso PE (LPE), phosphatidylcholine (PC), lyso PC (LPC) and cardiolipin [H. pylori OMVs. The presence of LewisXY antigens in an O-specific region of H. pylori LPS depends on iron-reach conditions, whereas the presence of LewisY determinants on iron-limited conditions [The content of OMVs might vary among particular diolipin . Lipopolnditions –69.H. pylori OMVs are divided into five families, containing Hop, Hor, Hof, Hom proteins as well as iron-regulated OMPs and efflux pump OMPs [H. pylori neutrophil activating protein (HP-NAP), adherence associated lipoprotein (AlpA), and urease [H. pylori OMVs proteins play a role in adherence, corresponding with the elevated gastric epithelial cell damage and possibly with the gastric cancer development [H. pylori isolates from various geographic areas, which may be reflected in the incidence of gastric cancer. There is a large intercountry variation in incidence of gastric cancer and H. pylori seroprevalence among Asian countries. In Japan, and Taiwan, the infection rate is 39.3%. By contrast, the prevalence of H. pylori infection is high in Vietnam (74.6%) and Bangladesh (92%), but in these regions low gastric cancer rates have been reported. This has been termed the ‘Asian enigma’. Interestingly the majority of H. pylori strains isolated from Asian population are babA2 gene positive [Proteins of ump OMPs . The fold urease , 71. Moselopment –81. The positive –84.H. pylori infection with BabA-L strains was associated with highest risk of gastric cancer, followed by BabA-H and BabA-negative strains. In Western countries, infection with BabA-L and BabA-H strains are associated with 54.8-fold and 19.8-fold risk of gastric cancer compared to BabA-negative infectors. Moreover, infection with H. pylori BabA-L-positive strain is related with higher colonization rate, neutrophil infiltration, and mucosal atrophy. The involvement of these OipA proteins is not so clear since all H. pylori isolates from East Asia are either BabA-H or BabA-L [babA2-genonegative strain was associated with lowest gastric cancer risk in babA2-genopositive strain, a lower BabA expression level seemed to be associated with higher gastric cancer risk [H. pylori markers for gastric cancer development include CagL Y58E59 vs non-Y58E59, vacA s1, vacA i1, m1 and EPIYA-D motif of CagA [Fujimoto and colleagues showed that r BabA-L . Furthercer risk , 86. In  of CagA –89.H. pylori increases the risk for gastric cancer [H. pylori DNA in gastric biopsies and the severity of precancerous lesions that is specific to CagA-positive strains [Apart from these virulence factors (released to the gastric milieu or transported via OMVs) several host-dependent determinants were shown to be involved in higher incidence of gastric cancer. There is a strong association between genetic polymorphisms in the interleukin (IL-)1β and IL-1 receptor gene cluster and in tumor necrosis factor (TNF)-α and the risk of gastric cancer , 91. Intc cancer . Similar strains . Conside strains .H. pylori OMVs contain different enzymes such as high temperature requirement (HtrA) serine protease, which induces E-cadherin cleavage resulting in the invasion of the gastric epithelium by H. pylori, and gamma-glutamyl transpeptidase (GGT), which participates in increasing gastric pH and glutamine synthesis [H. pylori to a high concentration of reactive oxygen intermediates [Besides CagA, VacA and OipA proteins, ynthesis , 97. Themediates .H. pylori OMVs were found under electron microscope in contact with the plasma membrane of human gastric mucosa cells as well as inside their endosomes, after the incubation with H. pylori broth culture filtrates [H. pylori OMVs by gastric epithelial cells can occur via clatrin-dependent endocytosis or clatrin independent (lipid raft mediated mechanism), and strongly depends on adhesins exposed on OMVs. It was shown that the presence of VacA toxin may facilitate the OMVs uptake by host cells [H. pylori in a free-soluble form, while the remaining 25% was OMV-associated and that vacuolization in cell cultures was due to free-soluble VacA activity of a H. pylori broth culture filtrate [in vitro studies.iltrates , 99. Thest cells . Once OMfiltrate . This obH. pylori OMVs can stimulate the production of both pro- and anti-inflammatory cytokines such as IL-6 and IL-10, respectively, and activate an oxidative burst [H. pylori OMVs components such as HP-NAP, which upregulates β2 integrin expression and attracts leukocytes to epithelial cells [H. pylori OMVs were also shown to induce human eosinophil degranulation and the release of eosinophil cationic protein (ECP), which enhance the expression of the intracellular adhesion molecule (ICAM)-1 on epithelial cells as well as CD11b integrin on eosinophils [H. pylori virulence factors delivered by OMVs can target transcription regulators such as a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) of the host. On this way OMVs modulate the expression of genes, which products are involved in the inflammatory response, such as the secretion of IL-8, TNF-α or IL-1β or proliferation and carcinogenesis [H. pylori OMVs independently of VacA, what suggests that vesicles without this exotoxin can cause cell damage due to induction of inflammatory response [In response to vesicle-associated virulence factors, gastric and immune inflammatory cascade is induced Fig. . Componeve burst , 103. Nial cells , 105. H.inophils , 106. H.ogenesis , 108. Itresponse .Fig. 1VaH. pylori OMVs may strengthen the immune response which lead to diseases development or, on the contrary, promote downregulation of immune reactivity which support infection persistence. Still, the effects of H. pylori OMVs on the immune cells are poorly understood. Recently, the influence of H. pylori OMVs with dendritic cells (DC) was described. These interaction resulted in NF-κB dependent upregulation of heme oxigenase-1 (Ho-1), expression and potentially modulation of DCs function [These examples show that function .H. pylori are non-invasive or low invasive and due to this secreted products of these bacteria or those introduced directly into the host cells play a role in the H. pylori induced diseases. H. pylori – related malignancy is strongly associated with the downregulation of host immunity and induction of structural and functional cell alterations that occur through H. pylori virulence factors [H. pylori may amplify its virulence. OMVs of H. pylori carrying the CagA affect cell-junctions and associated regulatory proteins, leading to chromatin changes, inappropriate transcription, and promotion of cancer development [H. pylori OMVs containing CagA increases the affinity of ATP to H1 histone, which alters the binding of DNA and histones within the nucleosome [cagA gene, treated with a colitis inducer-dextran sulfate sodium (DSS), it was shown that OMVs carrying CagA and VacA can promote inflammation [H. pylori OMVs, which were detected in gastric juice of patients with gastric cancer is suspected to participate in the process of carcinogenesis [H. pylori OMVs on the pathogenesis of gastric malignancy. H. pylori OMVs from gastric juice samples obtained from healthy controls (HC) and patients with duodenal ulcer (DU), gastric ulcer (GU) or gastric cancer (GC) patients were analysed in the context of gastric cancer incidence. The analysis showed that both H. pylori cells and H. pylori-derived OMVs were present at a significantly higher level in the gastric juices of gastric cancer patients than in normal controls and that the H. pylori-derived OMVs induce inflammation in mice model, which is associated with gastric cancer development [H. pylori OMV isolated from patients with different gastric outcomes. These results showed the association between OMVs presence and gastric lesions which should be further explored in more appropriate animal model . Chitcholtan and colleagues observed increased micronuclei formation, alteration in iron metabolism and gluthatione loss in AGS human gastric epithelial cells treated with H. pylori OMV, however this effect was observed only in OMV isolated from a toxigenic H. pylori strain [ factors , 111. Nuelopment . H. pylocleosome . Using tammation . Macrophammation , 115. Moammation . Due to ogenesis , 115. Reelopment . TransmiH. pylori OMVs are rich in membrane proteins, porins, adhesins and several molecules known to modulate chemokine secretion, cell proliferation and other host cellular processes and constitute a vehicles for the CagA and VacA [H. pylori demonstrated that not all strains express the same proteins or at least not all the proteins were detected in the analysis of the different strains. Several membrane proteins, including Omp11 and BabA, were found to be expressed by all strains. Omp26 was expressed by all disease-related strains but was only present in one out of five strains from asymptomatic carriers, which makes Omp26 a potential target for further investigation in the search for proteins unique to disease-related H. pylori strains. The authors suggest that the presence of Omp30 and absence of Omp6 within H. pylori OMVs seemed to be associated with H. pylori strains causing duodenal ulcer [H. pylori OMVs determines its content. The proteomic analysis revealed that larger OMVs contain significantly more adhesins  compared to smaller OMVs, and that most of the H. pylori proteins related with metabolism are associated within smaller OMVs. Interestingly, proteins associated with H. pylori survival or virulence were common to both small and large OMVs, including: urease A and B subunits, neutrophil activating protein, VacA, and the porin HopA [and VacA . Furtheral ulcer . It was rin HopA .H. pylori [H. pylori, mostly of toxigenic strains, induce apoptosis by the receptor-ligand pathway, which involves the CD95 ligand (CD95L) and the CD95 receptor (Fas) [H. pylori-infected patients, and increase local Fas expression [H. pylori may also induce apoptosis via the interaction of soluble or OMVs bound urease to class II major histocompatibility complex (MHC II) molecules, which are expressed in epithelial cells and play a key role in pathogen recognition [H. pylori components associated with OMVs to induce cell apoptosis may potentially contribute to the development of H. pylori-related diseases. Enhanced loss of epithelial cells due to H. pylori-induced apoptosis promote the development of gastric atrophy, which predisposes to neoplasia  . T-cellspression . H. pyloognition . The abisia Fig. . Alternaesponses , 130.FigH. pylori components present in OMVs such as LPS may induce opposite effects. The endotoxic activity of H. pylori LPS is lower than the activity of LPS of other Gram-negative bacteria due to distinct structure of lipid A. Thus OMVs decorated with H. pylori LPS may downregulate the inflammatory response or lead to it’s upregulation by the induction of fibrinolytic components [H. pylori LPS due to presence of Lewis antigens in O-specific polysaccharide chains, which share a common structure with blood group-related components of the host may lead to generation of autoimmunity due to the stimulation of autoantibody production [H. pylori infected individuals indicate that OMVs may potentially play a role in autoimmune processes related to H. pylori infection [H. pylori OMVs may strengthen the immune response leading to diseases development or promote reduction of immune reactivity and thus support the infection persistence.The mponents , 132. H.oduction . Lewis anfection . The aboH. pylori OMVs due to the induction of gastric barrier instability and dysbiosis can be translocated to deeper layers of gastric mucosa and gastrointestinal tract. OMVs have a proper size (20–200 nm) to enable their entry by circulation into lymph vessels and uptake by antigen presenting cells. Smaller OMVs ranging from 20 to 100 nm in size preferentially entered host cells via caveolin-mediated endocytosis. Whereas larger OMVs ranging between 90 and 450 nm in size entered host epithelial cells via macropinocytosis and endocytosis [H.pylori therapy studied on animal model [cagA-positive strains [H. pylori OMV localizes in a close vicinity of the tight junction protein ZO-1 and induce changes connected with cell barrier instability [H. pylori virulence factors can elucidate the involvement of H. pylori compounds in the development of local as well as systemic inflammation - crucial for the initiation or the maintenance of gastric and extragastric inflammatory diseases [ocytosis . Severalocytosis . This naal model . Consideal model . One cla strains . These mtability . This wadiseases , 140.H. pylori from the surface of these bacteria during infection. The composition of H. pylori OMVs and their ability to translocate via gastric barrier to intestines and into circulation suggests that they may play a central role in H. pylori pathogenesis by acting as delivery vehicles to transport virulence factors. Locally, OMVs induce direct effects such as tight junction disruption, cytotoxicity or apoptosis or indirectly affect the immune cells to release cytokines, proteases or tissue damaging factors such as MMP or ROS. Their translocation from gastric niche through digestive system into intestines may possibly lead to a capture and presentation by M cells to other immune cells and result with the immunomodulation of immune mechanisms leading to spread of OMVs into circulation. The studies concerning the involvement of OMVs produced by H. pylori in the pathogenesis of extra-gastric diseases such as coronary heart disease or food allergies might explain their importance and mechanisms by which they promote inflammation or tolerance.In conclusion, outer membrane vesicles are continuously shed by


Fusarium circinatum, and its close relatives in the Fusarium fujikuroi species complex (FFSC). Our results showed that the examined fungi all exhibited hallmarks of RIP, but that they differed in terms of the extent to which their genomes were affected by this pathway. RIP mutations constituted a large proportion of all the FFSC genomes, including both core and dispensable chromosomes, although the latter were generally more extensively affected by RIP. Large RIP-affected genomic regions were also much more gene sparse than the rest of the genome. Our data further showed that RIP-directed sequence diversification increased the variability between homologous regions of related species, and that RIP-affected regions can interfere with homologous recombination during meiosis, thereby contributing to post-mating segregation distortion. Taken together, these findings suggest that RIP can drive the independent divergence of chromosomes, alter chromosome architecture, and contribute to the divergence among F. circinatum and other members of this economically important group of fungi.The Repeat-Induced Point (RIP) mutation pathway is a fungal-specific genome defense mechanism that counteracts the deleterious effects of transposable elements. This pathway permanently mutates its target sequences by introducing cytosine to thymine transitions. We investigated the genome-wide occurrence of RIP in the pitch canker pathogen,  Fusarium includes a diverse assemblage of fungi—many of which are plant, animal and human pathogens  of F. temperatum markers)  and 2.4% (ca. 74 Kb) of the total RIP-affected proportion of the respective genomes. Conversely, of the 30 Mendelian markers, 26 were mapped to the genome of F. temperatum and 29 to that of F. circinatum, where they were distributed across all eleven of the core chromosomes in both species  for both species. Of the 37 TRD markers identified previously , we werercinatum . They wemarkers) . A total species . Of thesectively . Based oF. circinatum, all LRARs  were analysed. Many more putative pseudogenes were detected within and around LRARs than in the rest of the F. circinatum genome (F. circinatum. Most of these lacked start (59 pseudogenes) and/or stop codons (27), or encoded short polypeptides consisting of fewer than 100 amino acids (49). A small number of the putative pseudogenes also contained multiple stop codons within a single sequence (3) or encoded peptides with less than 20 (3) or 40 (9) amino acids. To investigate possible RIP-associated pseudogenization in m genome . On averF. circinatum and F. temperatum, changes in nucleotide identity across the length of the alignment of 14 homologous regions surrounding the TRD and Mendelian markers showing opposite RIP statuses (occurring in a region that is either RIP-positive or RIP-negative) were examined in order to determine whether RIP could bring about changes in homology and synteny. Although two of these regions  were fully homologous and mostly aligned well, the remaining 12 alignments showed extensive differences in nucleotide identity values, as well as changes in synteny and homology , or mostly internal rearrangements (marker AA/AC408bh).To investigate possible RIP-associated loss of homology, we analysed genomic regions with opposite RIP-statuses by specifically focusing on the Mendelian and TRD markers identified above. For homology . For sevF. circinatum, the location of TEs and repeated regions generally coincided with the location and frequencies of RIP mutations. These patterns thus suggest that F. circinatum and the other FFSC species examined are indeed RIP capable or were RIP capable until relatively recently. The FFSC genomes examined here harbored the known hallmarks of RIP. We observed bimodal distributions of GC content frequencies for all six genomes, with a minor peak in the low-GC content ranges (10%–30%) and a major peak at approximately 50%. Such bimodal GC content frequency distributions are common genomic features of RIP-capable fungi . Our datN. crassa [F. circinatum, we also found evidence of expression for the canonical RID and DIM-2 cytosine methyl-transferase genes, as well as those for the RIP-associated DIM-3, -5, -7, -9 and HP-1 proteins. In F. fujikuroi, we further found evidence of co-localization of DIM-5-directed H3k9me3 markers and RIP mutations, suggesting that DIM-2/DIM-5-mediated RIP occurs in this fungus and possibly also in the others examined. In the F. fujikuroi genome, the distribution of H3K9me3 markers has been associated with regions that are silenced by heterochromatin [N. crassa [F. circinatum and its relatives within the FFSC. All the FFSC genomes examined in this study contain genes that putatively encode for proteins and co-factors known to be associated with RIP and RIP-directed methylation similar to that described in . crassa ,31,38,39. crassa , followe. crassa  that rec. crassa . The lat. crassa . Our stuF. verticillioides genome to as much as 6.39% for the F. circinatum genome. In many cases, these RIP-affected regions occurred in long stretches ranging from 4 Kb up to as much as 79 Kb. Similar findings have been reported for fungi such as Neurospora tetrasperma and Leptosphaeria maculans [N. crassa genome [Metarhizium robertsii has little to no RIP products, particularly in their TE sequences [The six genomes examined differed substantially in terms of their total genomic proportions affected by RIP. These ranged from 0.89% for the maculans ,47. Furta genome , while oequences .One possible explanation for the variation in the total proportions of RIP-affected regions among FFSC genomes may relate to the reproductive strategies employed by these fungi. Although the FFSC can reproduce mitotically , many species in this complex, especially those examined here, are also known to have sexual stages in nature or under laboratory conditions . BecauseF. fujikuroi, F. nygamai and F. temperatum were all extensively targeted by RIP and contained numerous non-homologous LRARs, compared to the F. circinatum dispensable chromosome that is much less affected by RIP and had a lower LRAR content. Such large differences in the density and distribution of RIP mutations suggest the RIP pathway may play an important role in the evolution and divergence of both core and dispensable chromosomes. For example, RIP on chromosome 12 of F. temperatum is considerably higher than on any of its core chromosomes. The higher levels of RIP on dispensable chromosomes may be due to their genetic composition, particularly with respect to repeat and TE content and the presence of duplicated genes that are prime targets for RIP [L. maculans [F. poae that completely lack RIP activity or RIP mutations in the TE-rich regions of its dispensable chromosomes [Analysis of RIP substrates and products on homologous chromosomes revealed that RIP mutations are acquired in an independent or unique manner in each of the respective FFSC genomes examined. For instance, locations and proportions of the core chromosomes affected by RIP differed greatly between the American clade species despite these fungi sharing a recent common ancestor. A similar trend of apparent lineage-specificity was also observed for the dispensable chromosomes of the FFSC fungi. Our data showed that those of  for RIP ,52. Simimaculans . Howeveromosomes . NeverthF. circinatum where RIP-affected regions were gene-sparse with LRARs and their flanking regions containing numerous putative pseudogenes. Therefore, as has been shown in N. crassa, N. tetrasperma and L. maculans [RIP-affected regions differ substantially in their coding content from the rest of the genome. We showed that these regions of the FFSC genomes were particularly enriched for dinucleotides TpA and ApT associated with RIP products, as well as trinucleotide sequences associated with the so-called “amber” (TAG) and “ochre” (TAA) stop codons, which is typical for RIP-capable fungi ,56. Accomaculans ,25,47, RL. maculans, RIP-driven pseudogenization of effector and avirulence genes has contributed to the proliferation of virulent strains [Apart from promoting pseudogenization, RIP activity may also affect the overall biology of a fungus. In  strains . These s strains . Further strains ,25. RIP- strains . In thisN. crassa [F. verticillioides, where experimentally introduced duplicated regions were not inherited in a Mendelian fashion and displayed TRD [F circinatum and F. temperatum were located within or in the vicinity of RIP-affected regions, suggesting that RIP may hinder recombination during meiosis, thus causing deviation from the expected Mendelian ratios of segregation in hybrid progeny of a cross between these species. As initially observed in the late 1980s for . crassa . In the ayed TRD . In the F. verticillioides were not apparent at the time [N. tetrasperma, suppressed recombination at the mating-type (MAT) locus has affected a large proportion of the chromosome harboring it [N. tetrasperma’s MAT-containing chromosome [Although clear explanations for the TRD observed in the time , our resthe time ,57,58. Ioring it . In factromosome . This chromosome ,60,61. IN. tetrasperma MAT-chromosome, these regions in the FFSC genomes are thus likely the product of processes associated with RIP. In other words, these lineage-specific regions are formed when RIP decreases sequence similarity in the targeted regions, while RIP-directed methylation and limited sequence homology leads to the suppression of recombination that, under normal circumstances, would have eliminated or reduced sequence variability. The accumulation of such independently evolving regions may ultimately drive divergence of entire genomes. Overall, these processes may also be crucial for the formation and maintenance of species boundaries [To address the notion that RIP-associated processes may drive divergence among the genomes examined, we compared the sequences of loci with opposite RIP profiles. In most cases, these analyses revealed loss of both homology and synteny in genomic regions displaying opposite RIP statuses. Similar to what has been shown for the undaries . The whoundaries , while gundaries . Other lundaries ,14,65, lIn summary, the availability of whole-genome sequences assembled into chromosome-sized scaffolds have allowed for greater insight into the role RIP plays on a genome-wide scale in the FFSC. Our data suggest that these fungi are indeed RIP capable and that the process has affected large parts of their genomes. We also showed that RIP mutations are acquired uniquely in a lineage-specific fashion and that such regions can represent a considerable proportion of both core and dispensable chromosomes. RIP also drives the independent divergence of chromosomes and alters chromosome architecture, while RIP and the processes associated with it likely play key roles in the evolution of this important group of fungi.F. circinatum and F. temperatum from the “American Clade”, F. fujikuroi and F. mangiferae from the “Asian Clade”, and F. nygamai and F. verticillioides from the “African Clade”. The completeness of the six FFSC genome assemblies was verified with the Benchmarking Universal Single-Copy Orthologs (BUSCO) v. 3.0.2 tool using the “Sordariomyceta” dataset [N. crassa and Candida albicans, which are, respectively, RIP capable and RIP incapable [The genome sequences for eight fungi were used in this study . These i dataset  . Where needed for the respective AUGUSTUS  by usingT v. 2.5 .−5) to those described in N. crassa were identified using the CLC Genomics Workbench v. 8.0 . Their functional annotations, predicted functional domains, protein family (PFAM) membership and Gene Ontology (GO) were determined using InterProScan v. 5 [F. circinatum, their sequences were compared to the RNA-seq data. RNA was extracted from one-week old F. circinatum (FSP34) isolate grown on half-strength potato dextrose broth at 25 °C, using a RNeasy Plant mini kit  according to the manufacturer’s specifications. RNA-seq libraries  were sequenced  at the University of Stellenbosch, South Africa . RNA-seq data were analyzed using the CLC Genomics Workbench. A gene was considered expressed if it had a reads per kilobase per million mapped reads (RPKM) value ≥ 0.2 and at least 3 unique gene reads mapping to it [Nucleotide and protein BLAST searches were used to identify the genomic position of genes encoding proteins involved in the RIP pathway. These included genes encoding HP-1, RID and DIM--2, -3, -5, -7, -8 and -9 ,30,31. Fcan v. 5 . Similarcan v. 5 . To deteng to it .F. fujikuroi [N. crassa due to the actions of, amongst others, HP-1, the DNA methyltransferase DIM-2 and lysine methyltransferase DIM-5 [F. fujikuroi genome assembly and corresponding H3K9me3 marker positions determined previously [In order to gain further insight on the functionality of RIP-associated gene products, we also utilized the previously determined genomic positions of H3k9me3 markers  in ujikuroi . These mse DIM-5 . The F. eviously  and compF. circinatum chromosome assembly was used as a reference. Homologous chromosome sets were then subjected to RIP analysis using The RIPper [Homologous chromosome-sized scaffolds of the various FFSC genome assemblies were identified using LASTZ alignments v. 1.02.00  in Geneie RIPper . RIP inde RIPper .C. albicans and N. crassa.Changes in gene and GC content, and mono-, di-, and tri-nucleotide frequencies were further determined for each FFSC genome assembly investigated. Gene density and GC content were determined with the CLC Genomics Workbench. Mono-, di- and tri-nucleotide frequencies were calculated using the Jensen–Shannon divergence statistic implemented in OcculterCut (v. 1.1) . OcculteF. circinatum were determined using Blast2GO [To investigate the potential changes brought about by RIP to the genomic landscape of each species, large RIP-affected regions (LRARs) were identified using the ‘calculate LRAR’ tool implemented in The RIPper . DefaultBlast2GO . The ideBlast2GO .F. circinatum and F. temperatum [F. circinatum and F. temperatum by making use of an in silico approach that was described previously [F. circinatum and F. temperatum are haploid organisms) were investigated in the same way. These were randomly selected from those reported [P < 0.05), where the null expectation was that the frequency of RIP among the TRD markers is the same as among those inherited in a Mendelian fashion. A set of amplified fragment length polymorphism (AFLP) markers, previously demonstrated to experience TRD, were used in this study . These mmperatum . Of the mperatum . In the eviously . The seqreported  using Rareported . A chi-sF. circinatum, all LRARs and their proximal genomic regions  were investigated for the presence of putative pseudogenes. This was achieved by reannotating the genome with WebAUGUSTUS as described above, to retrieve annotations for complete, as well as partial or truncated genes. Within these sequences, the CLC Genomics Workbench was then used to identify putative pseudogenes based on the presence of premature stop codons or multiple in-frame stop codons, as well as the loss of a start codon that may cause non- or partially functional proteins.For −5). Where homologous regions could not be identified, the search included neighboring genomic regions as described before. The results were visualized with Kablamo v. 1.0 [Loss of homology due to RIP was investigated in genomic regions that harbored markers with opposite RIP statuses. For this purpose, the identified Mendelian and TRD markers were used. Homologous regions were identified with BLAST, implemented in CLC genomics and aligned using NCBI BLASTn (Expect-value < 1 × 10o v. 1.0  using th


Scientific Data 10.1038/sdata.2018.238, published online 30 October 2018Correction to: During typesetting, three paragraphs of text were erroneously removed from the Methods section of this Data Descriptor. These paragraphs have been re-inserted into both the HTML and PDF versions.


Populus euphratica Oliv. heteromorphic leaves (P. hl). To study the role of circRNAs related to transcription factors (TFs) in the morphogenesis of P. hl, the expression profiles of circRNAs in linear, lanceolate, ovate, and broad-ovate leaves of P. euphratica were elucidated by strand-specific sequencing. We identified and characterized 22 circRNAs related to TFs in P. hl at the four developmental stages. Using the competing endogenous RNAs hypothesis as a guide, we constructed circRNA–miRNA–TF mRNA regulatory networks, which indicated that circRNAs antagonized microRNAs (miRNAs), thereby influencing the expression of the miRNA target genes and playing a significant role in transcriptional regulation. Gene ontology annotation of the target TF genes predicted that these circRNAs were associated mainly with the regulation of leaf development, leaf morphogenesis, signal transduction, and response to abiotic stress. These findings implied that the circRNAs affected the size and number of cells in P. hl by regulating the expression of TF mRNAs. Our results provide a basis for further studies of leaf development in poplar trees.Circular RNAs (circRNAs) are a novel class of non-coding RNAs that are characterized by a covalently closed circular structure. They have been widely found in  Populus euphratica Oliv. is a typical stress-resistant tree that grows in desert areas of China. Its stress-resistant characteristics include tolerance to salt, alkali, drought, and cold [P. euphratica are linear and have a leaf index  ≥5. As the trees age, the leaves gradually become lanceolate , ovate , then broad-ovate . Therefore, P. euphratica has typical heteromorphic leaves, which make it an ideal model for studying leaf morphogenesis in plants [Arabidopsis endosperm [Arabidopsis [and cold . The youlate La, ≤LI<5, ovn plants . For exandosperm  and thatOryza sativa L. [http://ibi.zju.edu.cn/plantcircbase), which has allowed the regulatory roles of predicted circRNA–miRNA–TF mRNA networks to be further investigated. Salmena et al. proposed a competing endogenous RNA (ceRNA) hypothesis that ceRNAs interact using miRNA response elements (MREs) to form transcriptome-wide regulatory networks [Arabidopsis [Populus euphratica Oliv. heteromorphic leaves (P. hl), and Wang et al. [Phyllostachys edulis. However, there are few studies about the regulation of TFs by circRNAs at the transcriptional or post-transcriptional level in P. hl.Non-coding RNAs can indirectly regulate the development of plants . Circulaativa L. . Databasativa L.  and Plannetworks . Some cinetworks . For exabidopsis . It has bidopsis . Li et abidopsis  identifig et al.  found thP. hl at four developmental stages  by strand-specific sequencing, small RNA sequencing (RNA-seq), and bioinformatic tools [P. hl, 167 were known annotated miRNAs and 91 were novel miRNAs. Among them, 52 miRNAs were differentially expressed (P <0.05). The RNA-seq data analysis detected 8942 mRNAs that were differentially expressed among the P. hl at different developmental stages. A total of 1149 circRNAs were predicted from the RNA-seq data [P. hl.In the present study, we analyzed the expression profiles of circRNAs in ic tools . We idenseq data . CombineP. euphratica leaves at four developmental stages from trees growing in the Tarim Basin, Xinjiang . Li, La, Ov, and Bo leaves were selected as materials when there are 7–13 leaves on a bud. Each sample was biologically repeated three times. The Li, La, Ov, and Bo samples were named Li1, Li2, and Li3; La1, La2, and La3; Ov1, Ov2, and Ov3; and Bo1, Bo2, and Bo3. The samples were frozen immediately in liquid nitrogen and stored at −80°C before processing. Total RNA was extracted using a mirVanamiRNA isolation kit (Ambion) according to the manufacturer’s instructions, and total RNA concentration and quality were measured using an Agilent 2100 Bioanalyzer . The strand-specific sequencing process was performed as described by Levin et al. [We collected http://plntfdb.bio.uni-potsdam.de/v3.0/) to identify candidate TFs and the relative abundance of the transcripts was calculated using Bowtie2 [P < 0.05 were considered to be differentially expressed.The clean reads were aligned with sequences in the plant transcription factor database , differential expression analysis and identification of circRNA were the same as for the TFs.According to the data obtained from 12 samples sequenced, circRNAs were identified using CIRI software , circRNAhttp://www.mirbase.org/) [The small RNA was isolated and purified from the total RNA and used as sequencing sample. The miRNAs were sequenced by Illumina high-throughput sequencing, and small RNA reads were obtained by Illumina analysis  and compared with sequences in miRBase v.21 (se.org/)  to identse.org/)  to detecP. hl were compared with the Arabidopsis transcriptome sequence  to obtain the homologous genes of these TFs in Arabidopsis. The Gene Ontology (GO) annotations [https://www.omicshare.com/tools/Home/Report/goenrich). The functions of the miRNAs and circRNAs were predicted from the functions of their target TFs.The sequences of the identified TFs in the otations  of the hhttp://plantgrn.noble.org/psRNATarget/) to predict the target mRNAs of the identified miRNAs and circRNAs [https://www.omicshare.com/tools/Home/Soft/heatmap).We used the psRNATarget server (circRNAs . Then cacircRNAs  was usedwww.ncbi.nlm.nih.gov/tools/primer-blast/), and 18S RNA was used as the internal reference. All the PCR primers are listed in Supplementary Table S1. Quantification of RNA expression  was performed using the comparative Ct method. Fold changes (FCs) in the expression levels of RNAs were normalized as the ratio with 18S. Three technical replicates and three biological replicates were performed for each RNA sample.To validate the reliability of the sequencing results, a line chart was built using EXCEL software. Total RNA was extracted from the Li, La, Ov, and Bo samples using Trizol reagent . The qPCRs were performed using a qPCR kit and TB Green™ Premix Ex Taq™ . The expression profiles of nine randomly selected RNAs, namely three TFs , three circRNAs , and three miRNAs , were verified by qPCR. The TF mRNAs and circRNAs were reverse transcribed using random primers, and the miRNAs were reverse transcribed using specific primers. A Tiangen kit was used for miRNA detection, and a Takara kit was used for circRNA and mRNA detection. Primers were designed by primer-blast in NCBI  were combined in liquid nitrogen. Total RNA (pooled sample) was isolated using Trizol reagent (Invitrogen) and treated with DNase I (Takara) and RNase R (Geneseed Biotech Co.). Genomic DNA (gDNA) was extracted from the pooled sample using a MiniBEST Plant Genomic DNA Extraction Kit (Takara). Divergent primers were designed to cross the head-to-tail splicing junction using PRAPI [P. hl. The RNA was reverse-transcribed to cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara) with 1 μg total RNA and random 6-mers. The selected circRNAs were amplified and PCR products with the predicted sizes were visualized on a 2% agarose gel stained with ethidium bromide. All the primers are listed in Supplementary Table S2.Sixteen ng PRAPI , and conP. hl. The expression profile of the RNAs with possible circRNA–miRNA–TF mRNA regulatory relationships was verified by qPCR (the method referred to 2.7 section). All the PCR primers are listed in Supplementary Table S3. The correlation coefficients between the circRNA, miRNA, and TF mRNA expression profiles were calculated using Pearson coefficients with a web tool from Omicshare (https://www.omicshare.com/tools/Home/Soft/ica).We identified miRNAs and TF mRNAs that may have regulatory relationships with six randomly selected circRNAs in the 16 samples of P. hl by strand-specific RNA sequencing and small RNA sequencing. Among them, 22 (1.91%) circRNAs related to TFs were predicted from the relationships in the circRNA–miRNA–TF mRNA regulatory networks using psRNATarget (http://plantgrn.noble.org/psRNATarget/)  A. NotablTarget/) B.P. hl by analyzing the RNA-seq transcriptome data. A heat map of the circRNA expression profiles  among the four leaf shapes  showed that the 22 circRNAs related to TFs were widely expressed in P. hl  that regulated the expression of the genes encoding calcium-dependent protein kinase SK5 (XM-011001374.1). The GO annotations indicated that circRNA-0974 targeted TFs associated with developmental process (GO: 0005524) and cell differentiation (GO: 0030154)  A.In the Ov/Li comparison, 11 circRNAs, 19 miRNAs, and 23 TF mRNAs were included in the regulatory network. The 11 differentially expressed circRNAs regulated 42 downstream target genes B that weIn the Bo/Li comparison, 9 circRNAs, 24 miRNAs, and 21 TF mRNAs were included in the regulatory network, and the 21 TFs regulated 37 downstream target genes C. For exIn the Bo/La comparison, up-regulated circRNA-0227 down-regulated the expression of ptc-miR156a, ptc-miR156g, and ptc-miR156l, which regulated the expression of three TFs . This affected the expression of 10 downstream target genes D that weIn the Bo/Ov comparison, 8 circRNAs, 17 miRNAs, and 10 TFs were included in the regulatory network. The 10 TFs regulated 21 downstream target genes E. For exP. hl and analyzed their expression by qPCR. The trends of the expression levels of these RNAs determined by qPCR were basically consistent with the expression levels obtained by transcriptome sequencing. This confirmed the reliability of the strand-specific sequencing results.To verify the reliability of the strand-specific transcriptome sequencing results , we randP. hl, we used gDNA and cDNA (RNase R+) as templates with convergent and divergent primers. Five of six selected circRNAs related to TFs were amplified successfully in the cDNA sample . The results for circRNA_1102 showed that the divergent primers successfully amplified only the cDNA, whereas the convergent primers successfully amplified gDNA and cDNA (r) among circRNAs, miRNAs, and the linear TF mRNAs in 16 P. hl samples. In total, 17 of 21 regulatory relationships had negative Pearson correlation coefficients between the expression levels of the miRNAs and circRNAs, and between the expression levels of the miRNAs and TF mRNAs as determined by qPCR; however, the remaining regulatory relationships showed positive correlation (Supplementary Tables S4 and S5). For example, the Pearson correlations between circRNA_1102 and ptc-miR156g and between ptc-miR156g and XM_011034944.1, XM_011031202.1, or XM_011049364.1 were negatively correlated  indicated there was significant spatio-temporal specificity of circRNAs in the morphological development of P. hl (Previous studies found the expression of circRNAs were tissue and organ-specific ,36. We fof P. hl , implyinP. hl (P. euphratica CircRNA-0974, circRNA-0719, circRNA-0934; circRNA-0227 and circRNA-0617 were predicted to play important roles in leaf shape changes. Among them, circRNA-0719 was differentially expressed in all the pairwise comparisons and the highest expression among the four leaf types was in the Li leaves (P. hl, indicating that miR156 might play a vital role in the development of P. hl. SPL was found to be regulated by miR156 and was expressed mainly in juvenile shoots and leaves in rice [P. hl, implying that circRNA_0617 might participate in the abiotic stress response to promote the development of the medial–lateral axis or inhibit the apical–basal axis.From the RNA-Seq results and qPCR results, it was found that the expression patterns of most miRNAs were opposite to those of the circRNAs and TF mRNAs, suggesting that circular RNAs might act as miRNA sponges and could also be degraded by miRNAs in the i leaves . The resi leaves B. MacAlii leaves C. Morcili leaves D. Furthe in rice . The up-P. hl at four developmental stages. We identified 22 differentially expressed circRNAs related to TFs and established circRNA–miRNA–TF mRNA regulatory networks. Using the ceRNA hypothesis as a guide, we predicted that these circRNAs regulated the expression of 59 TFs by antagonizing 33 miRNAs. These interactions may play significant roles in regulating leaf development, leaf morphology, signal transduction, and response to abiotic stress in P. hl. Therefore, we propose that circRNAs may be involved in changing the shape and size of P. hl by regulating the expression of TF genes. Our results provide a foundation for further studies of the molecular mechanisms of leaf morphogenesis in P. euphratica.The circRNA family of regulatory RNAs participates in a variety of biological functions. In the present study, we analyzed the expression profile of circRNAs related to TFs by strand-specific RNA sequencing of Click here for additional data file.


Propionibacterium acnes, a Gram-positive commensal of the human skin. Although pain associated with infection is typically ascribed to the body’s inflammatory response, the Gram-positive bacterium Staphylococcus aureus was recently observed to directly activate nociceptors by secreting pore-forming α-hemolysins that disrupt neuronal cell membranes. The hemolytic activity of P. acnes in cultured disc specimens obtained during routine therapeutic discectomies was assessed through incubation on sheep-blood agar. The β-hemolysis pattern displayed by P. acnes on sheep-blood agar was variable and phylogroup-dependent. Their molecular phylogroups were correlated with their hemolytic patterns. Our findings raise the possibility that pore-forming proteins contribute to the pathogenesis and/or symptomology of chronic P. acnes disc infections and CLBP, at least in a subset of cases.Most patients with chronic lower back pain (CLBP) exhibit degenerative disc disease. Disc specimens obtained during initial therapeutic discectomies are often infected/colonized with  CLBP has been ranked the number one cause of years lived with disability in the United States , and subet al.  has demoet al. [Propionibacterium acnes at the time of initial therapeutic discectomies . P. acnes is a prominent Gram-positive bacterial commensal of the human skin and pilosebaceous unit. In multiple independent studies from 2001 to 2017, P. acnes was isolated from 26% of resected specimens [P. acnes as the predominant species. In a recent 368-patient study, Capoor et al. [P. acnes biofilms in situ within resected discs. This result is most consistent with chronic infection.In 2001, Stirling et al.  first pupecimens . Two 201pecimens  and 36.2pecimens , with P.r et al.  confirmeet al. [Staphylococcus aureus can directly activate nociceptor neurons through elaboration of a pore-forming toxin (α-hemolysin) that can generate action potentials in these cells through pathological ion influx. Beyond S. aureus, bacterial pathogens secrete diverse pore-forming proteins that facilitate cytotoxicity by targeting host-cell membranes [P. acnes, including isolates from orthopedic joint infections [Chronic infections are often associated with pain symptoms. This type of nociception is classically attributed to body’s inflammatory cascade, a cytokine-induced phenomenon that involves leukocyte infiltration and vascular dilatation. Nevertheless, more direct mechanisms of pathogen-induced pain have also been suggested. For instance, Chiu et al.  recentlyembranes . They arembranes ). In thifections .P. acnes also expresses pore-forming proteins during chronic intervertebral disc infection/colonization, directly interacting with sensory neurons and contributing to symptoms of CLBP. The current study explores this hypothesis by assessing the hemolytic activity of P. acnes strains obtained from three independent studies. In the first study, Capoor et al. [P. acnes in degenerated disc tissue obtained from 368 patients who underwent microdiscectomies. Of these, 119 (32.3%) were positive for P. acnes by culture (>1 colony-forming unit (CFU)/g). Only the 38 P. acnes-positive disc tissue samples with >1000 CFU/g were examined in this study. In the second study, Rollason et al. [P. acnes in degenerated disc tissue obtained from 64 patients diagnosed with lumbar disc herniation who underwent discectomy surgery. Disc samples obtained from 24 (38%) of these patients tested positive for P. acnes by culture (1–150 CFU per sample). We were able to obtain 67 separate P. acnes isolates recovered from these tissues for use in this study. In the third study, Albert et al.[P. acnes in degenerated disc tissue obtained from 61 patients who underwent primary surgery at a single spinal level for disc herniation. Anaerobic cultures were positive in 26 (43%) of these patients. We were able to obtain P. acnes-positive disc tissue samples for this study.Accordingly, we hypothesized that r et al.  assessedrt et al. assessedet al. [et al. [et al. [At the time of the three independent studies, Capoor et al. , Rollaso [et al.  and Albe [et al. , each reet al.[Homogenates  from disc tissue samples obtained during a previous study by Capoor et al., along wet al.[Frozen intervertebral disc tissue samples excised from lumbar disc herniations by Albert et al. were useP. acnes-positive disc tissue homogenates studied by Capoor et al. [Fifty-one colonies were selected visually (depending upon the hemolytic characteristics observed) from the 38 r et al. . Hemolytr et al. , 22. WheP. acnes strain from the Collection of Microorganisms  isolated phylotype IB from clinical material was used as a positive control to evaluate hemolytic activity. For phylotyping of our isolates, we utilized Nagy et al.’s methodology [P. acnes isolates from the Collection of Microorganisms.A hemolytic hodology , which wP. acnes isolates obtained from Rollason et al. [recA sequencing and monoclonal antibody typing. Hemolytic activity was assessed using Columbia blood agar supplemented with 7% (v/v) defibrinated sheep’s blood. These plates were incubated as described above. Positive hemolysis was recorded if complete zones of clearance were observed on agar plates.The sixty-seven n et al.  had alreet al. utilized matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) for identification of P. acnes, Rollason et al. utilized 16S rRNA-based PCR, and Albert et al. utilized (API) biochemical analysis using the Rapid ID 32A kit and by 16S rDNA PCR.Capoor et al. [et al. [Homogenates from the 38 disc tissue samples available from the study by Capoor et al.  were use [et al.  were simP. acnes) derived from disc tissue fragments showed β-hemolysis, while others showed either no hemolysis or a brown color likely caused by porphyrins produced by P. acnes. Of the 26 hemolytic samples, 21 had P. acnes as the only bacteria isolated from the disc, and 5 samples had additional bacteria cultured . Of the 12 non-hemolytic samples, 10 had P. acnes as the only bacteria isolated form the disc, and 2 had additional bacteria cultured (1 P. granulosum and one 1 S. epidermidus).After anaerobic culture, 26 (68%) of the 38 plates inoculated with homogenates contained colonies that showed β-hemolysis (clearing of the blood agar) as well as 12 (32%) plates with colonies that displayed no hemolytic activity. Some of the bacterial colonies  displayed β-hemolytic activity in culture. This is higher than the percentage observed by Wright et al. [P. acnes strains obtained from sterile body sites exhibited hemolytic activity with sheep blood. However, it is a lower percentage than that observed by Nodzo et al. [P. acnes strains isolated from patients who had undergone shoulder arthroplasty exhibited hemolytic activity. Follow-up studies by Boyle et al. [et al. [P. acnes isolates have exhibited hemolytic activity, suggesting that the results of this study are typical of P. acnes strains isolated from samples obtained in surgical settings.A substantial portion of the t et al. , who note et al.  and Mahy [et al.  found thet al. [P. acnes would place the infecting bacteria in proximity of these ingrown nociceptive nerves, raising the potential for an interaction between P. acnes and nociceptive neurons that results in pain  might correlate with low back pain. A dense nociceptive innervation of the vertebral endplate and the histochemical finding of Substance P in the related afferences –29 clearP. acnes isolates obtained from resected disc tissues showed that isolates from phylogroups II and IA were dominant, while isolates from phylogroups IB, IC and III were present in much smaller numbers. Although these results are somewhat different from the initial observations of McDowell et al. [et al. [P. acnes and their phylogroup distributions.Phylotyping of the l et al. , who obs [et al.  and undeP. acnes toxin (even within a disc) is not complete evidence that it is performing the physiologic function we hypothesize. In vitro experiments, in which P. acnes culture supernatant (or recombinant pore-forming exotoxin) could be applied to a neuronal tissue culture model, are needed to examine the effect of P. acnes proteins on depolarization. Identifying the genes responsible for the ability of P. acnes to form pores in neuronal membranes would require knock-in/knock-out experiments with putative hemolysin genes. Mutagenesis models of P. acnes CAMP genes have been developed [P. acnes hemolysin knockouts, which could be evaluated using designs analogous to those used in the previous animal model studies [P. acnes discitis [On a true etiologic level, the presence of a eveloped , and a s studies . Ultimatdiscitis . Such a in situ conditions. In addition, clinical studies could be proposed in patients undergoing microdiscectomy for disc herniation. Validated microbiological detection of P. acnes in the extracted disc tissue would be pursued with the two principal aims being: (i) evaluation of the association between P. acnes hemolytic activity in the infected disc tissue and pre-operative pain scores, and (ii) determination of the pore-forming factors expressed in the disc tissue specimens and evaluation of their association with pre-operative pain scores.To investigate when and how hemolysin genes are secreted, the elaboration of pore-forming exotoxins should be studied under diverse growth conditions . Protein expression within a disc may be different from all growth conditions tested, and resected disc tissue would need to be evaluated to represent P. acnes genomes, as well as the hitherto not well-appreciated formation of hemolytic zones on microbiological culture plates growing P. acnes from human discs, should make spinal researchers think deeper and with renewed curiosity. It will be of great importance to test disc material that is obtained during surgery for hemolytic activity in a larger, prospective series. With regard to the suggested use of fresh tissue, a possible limitation of the current study is that it utilized frozen intervertebral disc tissue. If the initial observations presented here were to hold true in these series, such observations would support the pathogenicity of (low-grade infected) nucleus material. It would help explain why in many patients, nucleus fragments that do not exert a significant mechanical compression on the affected nerve root on imaging can still cause such intense radiculopathy.The presence of genetic code for putative hemolysins in virtually all known Future studies could employ hemolytic assays directly on disc tissue material, proteomics using mass spectrometry on the crude disc tissue homogenate to assess whether hemolysins are expressed, mass spectrometry on liquid culture supernatants for secretome analysis, and qRT-PCR or RNA-seq for expression analysis. Hemolysin gene secretion, including assessment of the type I secretion system that does not require signal peptide, could also be studied.