Cerebral ischemia-reperfusion injury involves multiple independently fatal terminal pathways in the mitochondria. These pathways include the reactive oxygen species (ROS) generation caused by changes in mitochondrial membrane potential and calcium overload, resulting in apoptosis via cytochrome c (Cyt c) release. In addition, numerous microRNAs are associated with the overall process. In this review, we first briefly summarize the mitochondrial changes in cerebral ischemia-reperfusion and then describe the possible molecular mechanism of miRNA-regulated mitochondrial function, which likely includes oxidative stress and energy metabolism, as well as apoptosis. On the basis of the preceding analysis, we conclude that studies of microRNAs that regulate mitochondrial function will expedite the development of treatments for cerebral ischemia-reperfusion injury. With substantial incidence, prevalence, and mortality , stroke Mitochondria maintain homeostasis through energy production, synthesis of many compounds, and participation in cell signaling networks . DetaileEmerging evidence suggests that micro-RNAs (miRNAs) are associated and localized to mitochondria, indicating that the intricacy of the regulation of mitochondrial function is mediated by proteins which encoded by the nuclear and mitochondrial genomes ,18,19. BThe pathogenesis of cerebral ischemia-reperfusion injury involves miRNAs that alter the mitochondrial response and regulate the expression of key elements that mediate neuronal survival and apoptosis ,26,27,28Mitochondria are found in almost all eukaryotic cells and function as “power houses” . The mit2−), hydrogen peroxide (H2O2), and hydroxide radical (·OH−) [Mitochondria are potential major intracellular sources of ROS in almost all cells. The electron transport chain localized to the mitochondrial inner membrane is the main site for ROS generation . Specifil (·OH−) . Furtherl (·OH−) . ROS reml (·OH−) ,43. Duril (·OH−) ,45.et al. (2013) considered that mitochondrial changes during brain ischemia-reperfusion injury involve the following steps: ischemic starvation, reperfusion-induced hyperactivation, mitochondrial dysfunction, and delayed neuronal death [Sanderson al death . During al death .2+ maintains homeostasis through its uptake and release via the mitochondrial calcium uniporter [+/Ca2+ exchanger, mitochondrial permeability transition pore (MPTP), and pathways including the Na+-independent pathway of Ca2+ efflux and H+/Ca2+ antiporter pathway, etc. [2+ overload is detected, the sustained opening of the MPTP is stimulated [+ is triggered, all of which inhibits ROS production. When blood supply is reestablished, as it occurs due to membrane instability during early reperfusion, the reintroduction of oxygen enhances electron leakage, which markedly increases ROS production  [2+ inhibits complex I, which increases ROS generation in vitro and in vivo [Furthermore, intracellular Caniporter ,49, mitoay, etc. . Under pay, etc. . During ay, etc. . Insuffiimulated , and the“burst”) . Ca2+ in in vivo .Mitochondria generate ATP for most cells through oxidative phosphorylation to produce more than 95% of a cell’s energy under physiological conditions via complexes I–V . In almoUnder pathological conditions, most cells in the penumbra remain viable after 2 h of ischemia; however, glucose and ATP contents decrease considerably and the phosphocreatine content decreases to approximately 70% of non-ischemic values . A portiCyt c, which plays a major role in apoptosis, is located in the inner membrane of mitochondria, is the first proapoptotic protein discovered in the mitochondria . Its funThe Bcl-2 family and its regulation of mitochondrial permeability were the focus of extensive investigations. The members of the Bcl-2 family can be categorized according to their specific functions during apoptosis  as folloThe mitochondria involved in brain ischemia-reperfusion injury trigger apoptosis . The promiRNAs (20–22 nucleotides) regulate the expression of genes by binding to the 3′ untranslated regions of mRNAs, causing downregulation of gene expression or mRNA degradation ,78. The The expression of more than 30% of genes for coding proteins are regulated by miRNAs . Moreove2−, H2O2, and ·OH−. Moreover, miRNAs regulate ROS generation to a certain extent under conditions of mitochondrial oxidative stress. H2O2-induced upregulation of MDH is likely mediated by the downregulation of miR-743a [2+ overload [miRNAs have been detected within or in association with mitochondria from various tissues and cell types involved in a variety of biological processes ,92,93. TmiR-743a . miR-128miR-743a . miR-145overload . Similaroverload .miRNAs influence the balance of ROS. For instance, NOX4 and NOX2 belong to the NOX family of NADPH oxidases that produce large amounts of ROS . NOX4 wa2− to H2O2 is inhibited by miR-21, which occurs when miR-21 directly decreases SOD3 levels or indirectly reduces SOD2 levels, and ROS levels are affected by miR-21 [2O2-induced ROS activity is partly protected by the overexpression of miR-21 [Under conditions of pathological reperfusion, three antioxidant enzymes, MnSOD, GPX2, and Txnrd2, present in mitochondria, are negatively regulated by miR-17* . Moreovey miR-21 . Furtherf miR-21 . ROS is f miR-21 .2−, H2O2, and hydroxyl radicals (OH·), are produced in the mitochondria during brain ischemia-reperfusion injury. The nuclear transcription factors HIF-1α and NF-κB contribute to the generation of ROS during cerebral ischemia-reperfusion. SOD detoxifies O2− by converting it to H2O2, which is converted to H2O by CATs or GSHPx/Gpx1. NOX4 and NOX2 generate ·O2−-generating enzymes. miRNAs targeting mitochondrial-related proteins and antioxidative enzymes as well as nuclear transcription factors under mitochondrial oxidative stress are shown in Excessive amounts of ROS including ·OMitochondrial energy metabolism during brain ischemia-reperfusion injury is regulated by miRNAs . Table .During hypoxia, glucose, ATP, and phosphocreatine levels are reduced, and miRNAs regulate ATP generation and conversion to ADP. For instance, miR-15b modulates the concentration of ATP by targeting Arl2, and miR-15b overexpression reduces Arl2 expression, and therefore inhibits ADP/ATP exchange and ATP synthesis . FurthermiR-378 inhibits PGC-1β expression , whereasDuring cerebral ischemia, oxidative metabolism and glucose content decrease considerably, but glycolysis is sustained, causing impaired oxidative metabolism of pyruvate by mitochondria. The process of generating energy by glucose oxidative metabolism mainly includes the generation of pyruvate, pyruvate metabolism, and TCA cycle. Lipid peroxidation occurs during reperfusion. The miRNAs involved in this process are indicated on the right.Mitochondria-induced neuronal apoptosis during ischemia-reperfusion involves reduction of the mitochondrial membrane potential and opening of the MPTP that is associated with Bcl-2 family proteins . Cyt c, in vitro and in vivo and reduces ischemic brain injury by attenuating neuronal vulnerability [The mitochondrial apoptotic pathway is inhibited by miR-221/222 through the regulation of the BH3-only protein PUMA . Bnip3, rability . Moreoverability ,150. miRrability  and in irability .Moreover, Bcl-2 is a target of miR-134, miR-497, and miR-302 ,128,152.Cerebral ischemia-reperfusion injury triggers the mitochondrial release of Cyt c, AIF, Diablo, and EndoG via Bak. Cyt c then binds to procaspase-9 and Apaf-1 to form the apoptosome. The procaspase-9 complex is transactivated to activate caspase-9. Caspase-9 cleaves and activates downstream caspases such as caspase-3 to induce apoptosis. XIAP simultaneously inhibits caspases-9 and caspase-3. Bcl-2 and Bax prevent Cyt c release and interfere with this pathway. p53 and PUMA are involved in this process. miRNAs that target the mitochondrial apoptotic pathways are indicated on the right in Stroke is a multi-factor disease, with limited therapeutic strategies. Therefore, numerous clinical trials to treat this disease have failed . The ear


Pelosinus fermentans JBW45, isolated from a chromium-contaminated site in Hanford, Washington, USA, has been completed with PacBio sequencing. Nine copies of the rRNA gene operon and multiple transposase genes with identical sequences resulted in breaks in the original draft genome and may suggest genomic instability of JBW45.The genome of  Pelosinus fermentans JBW45 was isolated from groundwater stimulated for hexavalent-chromium reduction by injection of a polylactate compound  and the type strain R7 from Russian kaolin clays are similar to each other but show little synteny with JBW45 or the completed genome of Pelosinus sp. UFO1, isolated from Oak Ridge, Tennessee, USA  RSII instrument with P4-C2 chemistry as described previously . Two sinPelosinus spp. with a competitive advantage upon nutrient stimulation.The final assembly was circularized, resulting in a 5,380,816-bp chromosome with a G+C content of 39.5% and 250-fold sequence coverage. A total of 4,743 protein-coding, 77 tRNA, and 28 rRNA  genes were identified. Four of the 16S rRNA genes contained a 100-bp insertion, which is consistent with previous findings of intragenomic 16S rRNA gene heterogeneity in UFO1 , 27. TheCP010978. The version described in this paper is the first version, CP010978.1.This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number


It turned out those fibrosis indicators were typically inhibited by r-sHSA, suggesting its capacity in HSCs inactivation. The antifibrotic activity of r-sHSA was further investigated in vivo on CCl4-induced hepatic fibrosis, in view of significant improvement of the biochemical and histological indicators. More specifically, CCl4-intoxication induced a significant increase in serological biomarkers, e.g., transaminase , and alkaline phosphatase (ALP), as well as disturbed hepatic antioxidative status; most of the parameters were spontaneously ameliorated to a large extent by withdrawal of CCl4, although the fibrotic lesion was observed histologically. In contrast, r-sHSA treatment markedly eliminated fibrous deposits and restored architecture of the liver in a dose dependent manner, concomitantly with the phenomena of inflammation relief and HSCs deactivation. To sum up, these findings suggest a therapeutic potential for r-sHSA in hepatic fibrosis, though further studies are required.Hepatic fibrosis is an effusive wound healing process, characterized by an excessive deposition of extracellular matrix (ECM), as the consequence of chronic liver injury of any etiology. Current therapeutic repertoire for hepatic fibrosis is limited to withdrawal of the noxious agent, which is not always feasible. Hence, in this article, the antifibrotic effects and possible mechanisms of r-sHSA, a recombinant protein with hepatoprotection potential, were investigated. Using NIH/3T3 (mouse embro-fibroblast cell line), skin fibroblasts  and HSC-T6 (rat hepatic stellate cell line), the  In animals, the liver is a vital organ characterized by its crucial roles in maintaining metabolic homeostasis. However, the unique position of the liver also confers vulnerability to a wide variety of insults and injury , which ietc.  and pathological changes, characterized by hepatocellular necrosis, inflammation, and steatosis ,18. A se4 treatment led to a noticeable rise of hepatic MDA (a marker of lipid peroxidation levels) and depletion of GSH and TAOC, suggesting the impairment of hepatic antioxidant capabilities [4 control group. Even more apparently, r-sHSA treatment restored hepatic GSH and TAOC levels, as well as ameliorated hepatic MDA contents. , glutathione (GSH), and total antioxidant capacity (TAOC). The data indicated, as compared to normal mice, CClbilities . After tontents. 4-induced fibrogenesis and r-sHSA effects were evaluated through histopathology . The next two-week spontaneous resolution significantly decreased the levels of these markers (P < 0.05). However, r-sHSA treatment further accelerated the recovery, evidently performing the antifibrotic effects at higher dosage (60–120 μg/kg), as compared with the CCl4 control group (P < 0.05).The events were also confirmed via biochemical analyses. Serological or histological biomarkers, such as hyaluronic acid (HA), tissue inhibitor of metalloproteinases-1 (TIMP-1), hydroxyproline (Hyp) and collagen III, were engaged to the estimation of fibrogenesis and reversibility . Those fin vivo influence on HSCs activation of r-sHSA was assessed , the staining pattern shrank, even close to normal.With regard to the distribution of α-SMA-positive fibrogenic cells, assessed A. In nor4 substantially initiated tissue inflammation, characterized by wide immunopositivity in hepatic sinusoid, portal tracts, fibrous septa, and necrotic hepatocytes. In the CCl4 control group, immunopositivity persisted with some degree of improvement, followed by the partial resolution of fibrous structure. However, treated by r-sHSA, immunopositive deposits were sporadically distributed in inflammatory cells infiltration in the portal area and hepatic sinusoidal cytoplasm, with a significant decrease at higher dose.In addition, the inflammatory response during hepatic fibrogenesis was estimated by changes of proinflammatory factors, TNF-α B and TGF4 markedly caused hepatocellular apoptosis, featured by wide distribution of positive staining for TUNEL and Fas protein, round fiber hyperplasia, pseudolobuli and portal inflammation. The case was partially improved but continued, after spontaneous recovery. However, apoptotic cells decreased significantly after r-sHSA treatment, which showed a dose-effect relationship.The apoptosis of parenchymal and nonparenchymal cells were represented by positive staining of TUNEL D and Fasin vitro and in vivo respectively. Consistent to previous hypothesis, the results showed that r-sHSA could inhibit different fibroblasts, demonstrated by the descending collagen and α-SMA. Furthermore, in vivo study also suggested that r-sHSA could significantly protect liver against fibrosis induced by CCl4, on account of the histopathologic examination and improvement on fibrotic indexes.In this work, the antifibrotic effects of r-sHSA, a recombinant protein with hepatoprotective potential, were investigated 4 is proposed to cause fatty infiltration and centrilobular necrosis in liver, as well as destruction of microsomal cytochrome P-450 [4 hepatoxicity, derives from its active metabolites generated by a cytochrome P-450 dependent step, which can bind covalently with membrane structures, thereby inducing lipid peroxides [4, a dramatic ascent was observed in a series of biomarkers  in model mice; oxidative stress also was demonstrated by alteration of relevant parameters, such as MDA, GSH, and TAOC, which indicated the status of oxidative attack on hepatocytes and tissue [As an important experimental hepatotoxicant, CClme P-450 . The majeroxides ,22. Increroxides ,24. In od tissue . Howeverd tissue .4 for two weeks, the above-mentioned biochemical parameters in CCl4 control mice indicated essential melioration of oxidative stress and hepatocellular damage, albeit slightly less effective than the groups treated with r-sHSA. Additionally, previous studies stated that the classical biomarkers, such as serum aminotransferase levels, do not necessarily reflect the degree and progression of chronic liver damage [4-intoxicated group, besides an accumulation of ECM, which characterized the presence of fibrosis [4 control group, with a shrinkage in α-SMA immunopositive area and collagen deposition, after spontaneous recovery for two weeks, yet inferior to the therapeutic effects of r-sHSA, which also manifested the potency of r-sHSA on fibrosis reversal.Being a highly dynamic process, liver fibrosis is now known as reversible in human and animal models, following control of the noxious stimulus ,28,29, ir damage ,32, sincr damage ,34. Hencfibrosis . In contLiver fibrosis represents a common response to chronic hepatic damage in various diseases, which results in ECM remodeling and scar tissue formation, characterized by the excessive deposition of collagen . For dec4, increased α-SMA perisinusoidal immunopositivity, as well as the deposition of collagen in damaged hepatic areas, indicated activated HSCs are responsible for the fibrosis seen in the CCl4-intoxicated mice. Nevertheless, α-SMA positive expression, was observed to decrease after r-sHSA treatment, accompanied by the descending collagen content. Similarly, a marked fall was observed in the mRNA expressions of α-SMA and collagen I, in two kinds of fibroblasts and HSC-T6 cells, as witness to that case in vitro. Those results demonstrated that r-sHSA could inhibit HSCs activation, which paves the way for fibrosis recovery.In fact, the antifibrotic activity of r-sHSA likely involves such crucial mechanisms. As the principal collagen-producing cells , HSCs ac4, apparent deposits of TGF-β1 present in necrotic areas, suggests an active producing of this profibrotic cytokine, which predictably lead to consequent HSCs activation and ECM accumulation. Concomitantly with the resolution of fibrosis, r-sHSA treatment diminished TGF-β1 expression by comparison.From another aspect, the crucial role of HSCs activation in fibrotic process leads to extensive concerns on numerous signals, of which HSCs serve as both the principal source and the target . Hithert4 group suggested a high levels of TNF-α, accompanied by obvious steatosis and oxidative lesion, which was sustained in CCl4 control group with moderate improvement. The therapeutic activity of r-sHSA was contrastively associated with substantial decrease in TNF-α production, concomitantly with the histological improvement in the livers.As a pleiotropic cytokine, TNF-α participates in a variety of physiological and pathophysiological processes . It has 4 intoxication was associated with a high proportion of hepatocyte apoptosis, characterized by ubiquitous immunopositivity of TUNEL and Fas protein, which coincidently matched the enhancement of TNF-α. By contrast, r-sHSA decreased hepatocyte apoptosis, along with an amelioration of tissue injury, which might suggest its involvement in protection of hepatic parenchyma from inflammatory and fibrotic disorders.Increasingly as a nexus, apoptosis is associated with liver disease through which many key pathways converge. As to hepatic fibrogenesis, a pervasive but complex interplay takes place among those cellular and molecular bases, such as HSCs activation, peroxidation, inflammatory mediators, as well as apoptosis . In ordeEscherichia coli BL21(DE3) with the recombinant vector pET-28a-sHSA and purified as previously described [r-sHSA was expressed in escribed . Brieflyescribed . Induced2 atmosphere at 37 °C in DMEM medium, 10% fetal bovine serum (FBS), 1% vitamins, 2mM l-glutamine and 1% penicillin/streptomycin . For all experiments, SFBs were studied between passages 4 and 8 [Primary cultures of human skin fibroblasts (SFBs) were kindly provided by John Varga ,which were established from neonatal foreskin biopsies by previously described explant techniques . SFBs we 4 and 8 .2 in DMEM growth medium, supplemented with 10% FBS, 2mM l-glutamine and standard antibiotics  [Another two kinds of cell line, mouse embryo fibroblast NIH/3T3 and rat hepatic stellate cell HSC-T6 were obtained from Type Culture Collection of the Chinese Academy of Sciences . Cells were cultured at 37 °C in 5% COUT, USA) .in vitro effects of r-sHSA on the fibrosis development were investigated on multiple cell types. For each type, a total of 6 × 105 cells were seeded in a 12-well plate and allowed to 90% confluency, and then replaced with fresh medium containing 0.1% BSA. After serum-starvation for 24 h, cells were treated with TGF-β1  in the absence or presence of r-sHSA at indicated various concentration for 24 h. Following exposure to TGF-β1 for 6 h, cells were harvested for measuring the expressions of endogenous TGF-β1, α-SMA and alpha-1 type I collagen (Col1A1) by quantitative RT-PCR analysis (qRT-PCR) [The qRT-PCR) .® plus kit . First strand cDNA was synthesized from 2 μg of total RNA using the M-MLV Reverse Transcriptase  and used as a template for qRT-PCR. PCR amplifications were performed using the primer pairs for qRT-PCR  were seeded onto eight-well Lab-Tek II chamber glass slides  to confluence, and then subjected to serum-starvation for 24 h. Fresh media with r-sHSA (20 ug/mL) were added and the incubations continued for 24 h, then treated with TGF-β1 (4 ng/mL) for another 24 h. At the end of the experiments, cells were fixed, permeabilized, and incubated with primary antibodies to Type I collagen  or to α-SMA  at 1:500 dilution. Cells were then washed with PBS and incubated with secondary antibodies at 1:500 dilution  and viewed under a Nikon A1R Cell Imaging Facility  [SFBs  .Male C57BL/6 mice of clean grade (body weight 18–22 g) were purchased from Comparative medicine center of Yangzhou University , with certification number of SCXK (Su) 2012-0004. The mice were maintained at 12 h light/dark cycle, at constant temperature (20 ± 1 °C) and humidity (50% ± 5%). All animal care and research protocols were performed in compliance with the appropriate laws and animal welfare guidelines, and were approved by the Animal Ethical Committee of China Pharmaceutical University .n = 12): normal, CCl4, CCl4 control (observed for spontaneous resolution), r-sHSA groups . Liver fibrosis was induced by subcutaneous administration of CCl4 (dissolved in peanut oil) at a dose of 1mL/kg body weight twice-weekly for four consecutive weeks, while normal group received an equal volume of peanut oil. The CCl4 group was examined with histopathology, by killing the animals 72 h after the last CCl4 injection. For the next two weeks, the CCl4 control group was observed for spontaneous resolution. As in the protein-treated groups, r-sHSA was dissolved in saline and administered intraperitoneally (i.p.) at a dose of 30, 60, or 120 μg/kg daily, respectively. The dosages were based on preliminary studies [4 control groups received vehicle instead. At 24 h after the last administration, whole blood was collected from the orbit of ether anesthetized mice. Then all animals were sacrificed by cervical dislocation, and liver tissue was harvested for biochemical analysis or histopathology examination [After five days of acclimation, mice were randomly divided into several groups, with 12 mice in each group . As biomarkers of liver function, serum enzymes, including alanine aminotransferase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP), were measured by Liver function tests kits . Levels of serum hyaluronic acid (HA) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were assessed by ELISA kits .g for 10 min, to collect the supernatant for protein quantitation and multiple analysis on fibrosis indexes. A series of biochemical indicators of inflammation and fibrosis, such as malonaldehyde (MDA), total antioxidant capacity (TAOC), glutathione (GSH), inducible nitric oxide synthase (i-NOS), hydroxyproline (Hyp) and collagen III, were measured using commercially available kits, correspondingly calibrated by the protein concentration . All the procedures were performed at 4 °C [Hepatic tissues were homogenized in saline and centrifuged at 3500  at 4 °C .Liver specimens were fixed in 10% formalin, embedded in paraffin, and cut into 4 μm thick sections. Sections for histopathological examination were stained with hematoxylin and eosin (H & E) and Mallory trichrome stain, according to standard procedure.The extent of fibrosis was examined blindly by a pathologist, numerically graded in the light of the formula of Scheuer , with slin situ cell death detection kit , according to the manufacturer’s instructions. TUNEL-positive nuclei were identified by the BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) solution as blue/violet staining.Apoptotic hepatocytes in sections were detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, using the For immunohistochemistry, sections were deparaffinized, rehydrated, and stained using the MaxVision™ IHC kit  as described . Brieflyt-test for single, or analysis of variance for multiple, group comparison. In all tests, P < 0.05 was considered statistically significant.Data are presented as means ± SD. Statistical analysis was performed using an unpaired Student’s in vitro. In vivo, r-sHSA therapy could result in a complete reversion of progressive liver fibrosis by meliorating fibrotic pathology in the light of indicant and histological levels. As far as the current data is concerned, the antifibrogenic potential of r-sHSA, might involve inactivation of HSC and expression down-regulation of profibtic cytokines . The results obtained from present study suggest the therapeutic application of r-sHSA in hepatic fibrosis, although further studies are required.In summary, r-sHSA could inhibit HSCs activation and ECM composition by reducing the expression of α-SMA and collagen


Escherichia coli serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing E. coli (STEC) serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of E. coli O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 E. coli strains belonging to the 21 covered serogroups and 18 E. coli strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections. Escherichia coli (STEC) are important food-borne zoonotic pathogens responsible for a broad spectrum of clinical symptoms in humans, ranging from mild diarrhea to hemorrhagic colitis (HC) and the life-threatening hemolytic uremic syndrome (HUS) [Shiga toxin (Stx)-producing me (HUS) . Althougme (HUS) ,3, whichme (HUS) .E. coli clones including both pathogenic and commensal ones are currently identified by the combination of their O (lipopolysaccharide) and H (flagellum protein) antigens [E. coli (EHEC). This term was originally coined to denote strains that cause HC and HUS, produce Stx and attaching and effacing (A/E) lesions, and possess the 60-MDa virulence plasmid [antigens . To dateantigens . However plasmid ,7. Disea plasmid ,7,8, as  plasmid ,9,10. Fu plasmid –13.E. coli serotyping is typically performed by agglutination reactions using antisera raised in rabbits against the different O and H standard reference strains [E. coli serotypes are strongly needed. strains . HoweverE. coli is a consequence of the extensive genetic diversity within the rfb (O-antigen) gene cluster, which encodes many of the enzymes involved in O-antigen biosynthesis and assembly [rfb region maps flanked by the two housekeeping genes galF and gnd on the E. coli chromosome. Indeed, the JUMPstart sequence, which is a 39-bp conserved element located in the intergenic region between galF and the O-antigen gene cluster, and the gnd sequence, which is present downstream the cluster, have been used to successfully amplify the entire O157 O-antigen gene cluster by PCR [wzx (enconding the O-antigen transporter or flippase) and wzy (enconding the O-antigen polymerase) [wzx and wzy, show relatively low similarity among different E. coli serogroups, and therefore primers targeting wzx and wzy are generally used to develop serogroup-specific PCR assays [Much of the O-antigen variation in assembly . The rfbr by PCR . The cluymerase) . SeveralR assays –21.E. coli O5 and O76 reference strains were fully sequenced, their associated genes were identified on the basis of homology, and specific primers targeting wzx were designed for each serogroup. Specific primers targeting wzx or wzy were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences from each serogroup. Previously published specific primers targeting wzx of serogroups O26, O103, O111, O121, and O145 were added to the designed primers to further develop three serogroup-specific multiplex PCR assays able to detect these 21 serogroups. These PCR assays were shown to be highly specific and sensitive, and suitable for the detection of the most clinically relevant STEC serogroups.In this study, the O-antigen gene clusters of E. coli O5 and E. coli O76 from Statens Serum Institute  were used for nucleotide sequence analysis of their O-antigen gene clusters. A collection of 10 E. coli O5 and 15 E. coli O76 strains isolated from feces of humans and animals and from food at different time periods in Spain [E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella was used for testing of primer specificity and validation of the serogroup-specific multiplex PCR assays developed . Published oligonucleotides 482 (5′-CAC TGC CAT ACC GAC GAC GCC GAT CTG TTG CTT GG-3′) and 412 (5′-ATT GGT AGC TGT AAG CCA AGG GCG GTA GCG T-3′) complementary to the JUMPstart sequence and to the proximal end of gnd [http://www.ncbi.nlm.nih.gov/gorf/). BLAST and PSI-BLAST were used for searching databases, including GenBank, COG, and Pfam (http://www.ncbi.nlm.nih.gov/blast/).Chromosomal DNA from d of gnd , respectwzx sequences determined before from E. coli O5 and E. coli O76 reference strains , according to the manufacturer’s instructions, in an ABI 2720 thermal cycler (Applied Biosystems) as follows: denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min, repeated 25 times. Each reaction contained 400–800 nM of each primer  . In ordeh primer . AdditioE. coli O5 and O76 O-antigen gene clusters have been deposited in GenBank under the accession numbers KM881565 and KM881564, respectively.The DNA sequences of the gnd was obtained from E. coli O5, and eleven open reading frames (ORFs) were found , with the same organization and 78% DNA identity. E. coli and Salmonella are closely related, and several cases in which the O-antigen structures are identical or highly similar in the two species have been documented [Salmonella and E. coli O-antigen gene clusters that express identical O-antigen backbones is close to the lower end of the range for their housekeeping genes (between 76% and 100% DNA identity), indicating that O-antigen gene clusters for each structure originate from a common ancestor [A sequence of 9,915 bases between the JUMPstart sequence and re found . All there found . A sequere found . Likewisre found . As expee Tables  and 3 [2cumented ,28. The wzx genes of E. coli O5 and O76 were designed . Such a result was not surprising, since pool 1 contained an E. coli O9 strain and it is well known that the gene cluster encoding for serogroup O104 has the same genes in the same order as the K9 gene cluster [E. coli strains belonging to serogroups O8, O9, and O9a [E. coli O9 strain included in pool 1. Likewise, pool 15 gave a positive result when tested with the primer pair specific for wzx gene of E. coli O118 (409 bp). This result was also expected, since pool 15 contained an E. coli O151 strain and the O-antigen gene clusters of both E. coli O118 and E. coli O151 have been shown to be organized in the same manner and to share high level identity (> 99% DNA identity) [wzx genes of E. coli O118 and E. coli O151 are identical [E. coli type strains representing 169 different serogroups and therefore suitable for being used in PCR assays for serogroup identification.Primer pairs specific for and O177  were useShigella  in orderShigella , and no  cluster , and the and O9a , which wE. coli O9 strain gave a positive PCR result (272 bp) when tested with multiplex 3, due to the presence of the primer pair specific for wzx gene of E. coli O104 on this multiplex PCR assay, as discussed before. To test the sensitivity of the three multiplex PCR assays, they were carried out to amplify serially diluted chromosomal DNA  prepared from strains belonging to the 21 covered serogroups, and positive PCR results were obtained from as little as 1 ng of DNA for each of the strains.In order to develop a less laborious PCR method, the 21 serogroup-specific primer pairs were combined in three multiplex 5′-nuclease PCR assays (multiplex 1 to 3)  aiming tE. coli. The combination of these three multiplex PCR assays enables the reliable detection of genes encoding the O antigen in E. coli strains belonging to the most clinically relevant STEC serotypes, including typical, atypical, and emerging EHEC serotypes. This method of molecular serotyping is a faster, simpler, and less expensive technique than traditional serotyping, also enabling the detection of E. coli O antigens even when they cannot be expressed by the bacteria. As a consequence, these PCR assays could be an efficient and convenient strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, especially in those where PCR is already a routine tool, and so their development could benefit clinical diagnosis, epidemiological investigation, surveillance, and control of STEC infections.In conclusion, the three serogroup-specific multiplex PCR assays developed in this study were found to be highly specific and sensitive, and suitable for serogroup identification in S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Evolutionary analysis indicated that MebZIPs could be divided into 10 subfamilies, which was further supported by conserved motif and gene structure analyses. Global expression analysis suggested that MebZIPs showed similar or distinct expression patterns in different tissues between cultivated variety and wild subspecies. Transcriptome analysis of three cassava genotypes revealed that many MebZIP genes were activated by drought in the root of W14 subspecies, indicating the involvement of these genes in the strong resistance of cassava to drought. Expression analysis of selected MebZIP genes in response to osmotic, salt, cold, ABA, and H2O2 suggested that they might participate in distinct signaling pathways. Our systematic analysis of MebZIPs reveals constitutive, tissue-specific and abiotic stress-responsive candidate MebZIP genes for further functional characterization in planta, yields new insights into transcriptional regulation of MebZIP genes, and lays a foundation for understanding of bZIP-mediated abiotic stress response.The basic leucine zipper (bZIP) transcription factor family plays crucial roles in various aspects of biological processes. Currently, no information is available regarding the bZIP family in the important tropical crop cassava. Herein, 77  Due to their sessile nature, plants cannot move to avoid unfavorable conditions, thus they have to cope with various harsh environmental factors334cis-element, resulting in ABA response or environmental adaptation. In Arabidopsis, accumulated evidence has suggested the roles of group A bZIPs, such as, AtbZIP39, AtbZIP36, AtbZIP38, AtbZIP66, AtbZIP40, AtbZIP35 and AtbZIP37, in abiotic stress response or ABA signaling567In plants, there is considerable evidence showing that bZIP TFs play crucial roles in various aspects of biological processes, including organ differentiation, embryogenesis, seed maturation, flower and vascular development35To date, numerous bZIP family members have been identified by genome-wide analyses in various species4101114bZIP genes, we use BLAST and Hidden Markov Model searches to search the cassava genome database with bZIP sequences from Arabidopsis and rice as queries. After these programs, 77 putative bZIP members were characterized from cassava, and further conserved domain detection confirmed that all the identified bZIPs harbor the conserved bZIP domain that is the basic characteristics of bZIP family. The 77 predicted full-length cassava bZIP proteins varied from 129 (MebZIP69) to 766 (MebZIP1) amino acid residues and the relative molecular mass ranged from 15.0 (MebZIP69) to 83.2 (MebZIP1) kDa, with isoelectric points in the range of 5.23–10.18  and wild subspecies (W14) for transcriptome analysis. Transcriptome analysis showed a transcript abundance of 55 MebZIP genes in different tissues, while the rest of the 22 MebZIP genes were not covered in the RNA-seq libraries (To seek insights into the roles of ibraries .MebZIP genes (100%) showed transcripts in different tissues, in which 28 (50.9%), 26 (47.3%), and 27 (49.1%) genes exhibited high transcriptional abundance  in stem, leaf, and storage root tissues, respectively. Moreover, there were 21 MebZIP genes  that had high expression levels  in all the tested tissues. Additionally, MebZIP72 did not show transcripts in stem; MebZIP-72, -53, -48, -71 did not show transcripts in leaf; and MebZIP-64, -49, -48, and -71 did not show transcripts in storage root.For Arg7 variety, all the 55 MebZIP genes, except for MebZIP58, had transcripts in different tissues, in which 23 (42.6%), 27 (50%), 25 (46.3%) genes had high transcriptional abundance  in stem, leaf, and storage root tissues, respectively. Moreover, 19 MebZIP genes  showed high transcriptional levels  in all the tested tissues. Additionally, MebZIP-71 and -58 did not show transcripts in stem; MebZIP-53, -72, -49, -48, and -58 did not show transcripts in leaf; and MebZIP-59, -64, -71, -48, and -58 did not show transcripts in storage root.For W14 subspecies, all the 55 MebZIP genes in different tissues between Arg7 and W14, 49 genes (89.1%) had transcripts accumulation in all tissues of Arg7, while 47 genes (85.5%) in W14, suggesting the constitutive expression patterns for these genes. On the contrary, the absence of detection might represent the distinct temporal or spatial expression patterns for the remaining MebZIP genes. Notably, 18 MebZIP genes  showed high transcript abundance  in all tested tissues of Arg7 and W14, indicating key roles for these genes in tissue development. Overall, MebZIP genes had similar expression profiles in different tissues of Arg7 and W14, indicating that most of bZIP genes played similar roles in tissue development of the two genotypes. However, there were some genes that exhibited differential expression profiles. MebZIP-18, -10, and -8 had high transcript abundance  in stem of W14, whereas low in stem of Arg7. On the contrary, MebZIP-33, -52, -54, -74, -64, -16, -47, and -57 had high transcriptional abundance  in stem of Arg7, while low in stem of W14. This phenomenon was also observed in leaf and storage root of Arg7 and W14. These findings indicated differential roles for these genes in tissue development in different genotypes.For comparing the expression profiles of MebZIP genes responding to drought stress, cassava seedlings of three genotypes were subjected to water withholding and then the leaves and roots tissues were sampled to extract RNA for subsequent RNA-seq analysis  and 23 (30.3%) MebZIP genes were induced by drought in leaves and roots, respectively. In South China 124 (SC124) variety, 28 (36.8%) and 26 (34.2%) MebZIP genes showed upregulation after drought in leaves and roots, respectively. In W14 subspecies, 21 (27.6%) and 40 (52.6%) MebZIP genes were upregulated by drought in leaves and roots, respectively. These results suggest that the number of MebZIP genes upregulated by drought was greater in W14 than those in Arg7 and SC124, suggesting the comprehensive response of bZIP genes to drought at transcriptional levels in W14 subspecies. Based on the transcriptome data, we also found that the number of bZIP genes up-regulated by drought was significantly more in roots than those in leaves in W14, whereas less in Arg7 and SC124.To get some clues on the roles of analysis . AccordiMebZIP genes in response to drought were similar between Arg7 and SC124, which differs from W14. Seven genes  showed induction in leaves of W14, whereas downregulation or no response in leaves of Arg7 and SC124 after drought treatment. Seventeen genes  were upregulated in roots of W14, whereas downregulated or no response in roots of Arg7 and SC124 after drought treatment. This differential expression profile suggested that bZIP-mediated response underlying cassava tolerant to drought stress might be different between cultivated varieties and wild subspecies. In addition, some bZIP genes had close evolutionary relationships; however, they displayed different responses to drought stress, such as, MebZIP-5/-14, MebZIP-65/-67, MebZIP-66/-34, MebZIP-48/-49, MebZIP-13/-16, MebZIP-36/-44, MebZIP-55/-63, MebZIP-68/-77, MebZIP-18/-19, MebZIP-1/-2, and MebZIP-39/-40. Together, the expression profiles of bZIP genes responding to drought in cultivated varieties and wild subspecies will be benefit for further investigation of the mechanisms underlying strong drought resistance in cassava.Generally, the expression patterns of MebZIP genes to various environmental stresses and related signaling at transcriptional levels, the transcripts of MebZIP genes under these treatments were determined by quantitative real-time PCR. Eight MebZIP genes  induced by drought based on RNA-seq data in different cassava genotypes were chosen for further examination of their expression patterns after osmotic, salt, cold, ABA and H2O2 treatments  in ABA response or environmental adaptation through regulating expression of ABA-dependent genes. However, the identity and function of cassava bZIPs have remained unknown.bZIP family members from cassava. Previous studies have identified 75 bZIPs in Arabidopsis, 89 in rice, 92 in Sorghum, 64 in cucumber, 125 in maize, 55 in grapevine, 131 in soybean, and 96 in Brachypodium distachyon410bZIP in cassava had expanded compared to that in Arabidopsis, cucumber, and grapevine, whereas had shrunk compared to that in rice, Sorghum, maize, soybean, and Brachypodium distachyon. Evolutionary analysis indicated that the cassava bZIPs could be clustered into 10 subfamilies, which is in line with previous evolutionary classification of bZIPs in Arabidopsis, grapevine and Sorghum510MebZIPs varied from 0 to 12  upregulated by drought based on RNA-seq data in different cassava genotypes were chosen for further examination of their transcriptional response various treatments , leaves (90-days-old) and storage roots (150-days-old) were collected from Arg7 and W14 under standard conditions. To examine the transcriptional changes of cassava bZIP genes to drought stress, leaf samples (90-days-old) were collected from and W14, Arg7 and SC124 under standard conditions or drought treatments for 12 days. For various stimuli, 60-days-old seedlings of Arg7 were subjected to 300 mM NaCl for 14 d, 200 mM mannitol for 14 d, 100 μM abscisic acid (ABA) for 24 h, 10% H2O2 for 24 h, and low temperature (4 °C) for 48 h, respectively.The characteristics of cassava genotypes W14, SC124 and Arg7 have been described in previous studies30Arabidopsis and rice was obtained from the Phytozome (http://www.phytozome.net/cassava.php), UniPort (http://www.uniprot.org/) and RGAP (http://rice.plantbiology.msu.edu/) databases, respectively3233bZIP family genes, the local Hidden Markov Model-based searches (http://hmmer.wustl.edu/) was firstly built from known bZIPs to search the cassava genome databaseArabidopsis and rice bZIPs as queries. Finally, all candidate protein sequences were further examined by the CDD (http://www.ncbi.nlm.nih.gov/cdd/) and PFAM (http://pfam.sanger.ac.uk/) databases36Arabidopsis and rice were performed by Clustal X 2.0. The bootstrap neighbor-joining evolutionary tree was created by MEGA 5.0 software with 1000 bootstrap replicates based on the sequence alignments38The whole bZIP protein sequence of cassava, http://expasy.org/) was employed to detect the molecular weight and isoelectric points of predicted MebZIP proteinshttp://meme.nbcr.net/meme/cgi-bin/meme.cgi), the conserved motifs in full-length cassava bZIP proteins were identified with the following parameters: maximum number of motifs was 20 and the optimum width of motifs was set between 10 and 50http://www.ebi.ac.uk/Tools/pfa/iprscan/)http://gsds.cbi.pku.edu.cn/) was employed to identify gene structures of cassava bZIPsThe ExPASy proteomics server  for transcriptome analysis. Three μg total RNA of each sample were used to construct the cDNA libraries following the Illumina instructions, and subsequently sequenced by Illumina GAII. Data preprocessing and analyses were performed according to our previous studyMebZIP genes responding to various stimuli, including osmotic, salt, cold, ABA and H2O2 were determined by quantitative real-time PCR analysis on Stratagene Mx3000P Real-Time PCR system using SYBR® Premix Ex Taq™ . The PCR amplification conditions used for all reactions were implemented as follows: 10 min at 95 °C, and followed by 40 cycles of 10 s at 95 °C, 15 s at 50 °C and 30 s at 72 °C. The relative expression levels of the target genes were calculated by the 2–ΔΔCt methodMebZIP genes in each treated time point were compared with that in each time point at normal conditions.Transcriptional changes of How to cite this article: Hu, W. et al. Genome-wide characterization and analysis of bZIP transcription factor gene family related to abiotic stress in cassava. Sci. Rep.6, 22783; doi: 10.1038/srep22783 (2016).


Streptococcus pneumoniae strain isolated in Monterrey, Mexico, MTY1662SN214, from a man with purpura fulminans. The strain belongs to the invasive and multidrug-resistant serogroup 19A, sequence type 320 (ST320). The draft genome sequence consists of 60 large contigs, a total of 2,069,474 bp, and has a G+C content of 39.7%.We report here the draft genome sequence of a  Streptococcus pneumoniae serotypes not included in PCV7  in the United States and other western countries, there was a significant reduction in the incidence of invasive pneumococcal disease (IPD) , 2. Howe in PCV7 , increas in PCV7 –6.S. pneumoniae 19A ST320 has been implicated in both IPD and noninvasive pneumococcal disease , in Northeast Mexico. The patient was a 43-year-old male with purpura fulminans. The strain belonged to serotype 19A and ST320. Strain MTY1662SN214 was multidrug resistant, with resistance to penicillin, erythromycin, trimethoprim-sulfamethoxazole, tetracycline, quinolone, clindamycin, and cefotaxime  with an average length of 250 × 2 nucleotides (nt), providing about 25-fold genome coverage. software . The uncIn order to obtain a functional annotation of the genome, all the assembled contigs were annotated by NCBI Prokaryotic Genome Annotation Pipeline . The genLKAA00000000. The version described in this paper is version LKAA01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Juglans regia L.) has been less well studied compared with other species. We discovered 25 JrMAPK members in the Juglans genome in this study. The JrMAPK gene family was separated into four subfamilies based on phylogenetic analysis, and members of the same subgroup had similar motifs and exons/introns. A variety of cis-acting elements, mainly related to the light response, growth and development, stress response, and hormone responses, were detected in the JrMAPK gene promoters. Collinearity analysis showed that purification selection was the main driving force in JrMAPK gene evolution, and segmental and tandem duplications played key roles in the expansion of the JrMAPK gene family. The RNA-Seq (RNA Sequencing) results indicated that many of the JrMAPK genes were expressed in response to different levels of Colletotrichum gloeosporioides infection. JrMAPK1, JrMAPK3, JrMAPK4, JrMAPK5, JrMAPK6, JrMAPK7, JrMAPK9, JrMAPK11, JrMAPK12, JrMAPK13, JrMAPK17, JrMAPK19, JrMAPK20, and JrMAPK21 were upregulated at the transcriptional level in response to the drought stress treatment. The results of this study will help in further investigations of the evolutionary history and biological functions of the MAPK gene family in walnut.Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr (serine/threonine) protein kinases that play very important roles in plant responses to biotic and abiotic stressors. However, the MAPK gene family in the important crop walnut ( Biotic and abiotic stressors occur frequently during plant growth and development, in which drought stress and infection by pathogens seriously affect crop yield and quality . MitogenAtMPK3, AtMPK4, and AtMPK6 have been extensively researched. For instance, the transcription levels of AtMPK3, AtMPK4, and AtMPK6 are induced by osmotic stress, salt, cold stress, and a variety of pathogens [AtMPK3, AtMPK4, and AtMPK6 play critical roles in both abiotic and biotic stress reactions [AtMPK3 and AtMPK6 also play important roles in stomatal development, embryonic development, anther and ovule development, and inflorescence formation [The Arabidopsis MAPK gene family can be divided into TEY and TDY subtypes based on the characteristics of the conserved domains. The common feature of the TEY subtype of MAPK members is that they contain the T-E-Y (Thr-Glu-Tyr) motif, which can be further divided into groups A, B, and C. The TDY subtype is also called group D, which is characterized by the T-D-Y (Thr-Asp-Tyr) domain . The rolathogens ,6,7. Muteactions ,6,7. AtMormation ,9,10,11.Juglans regia L.) has rich nutritional value and is popular with people all over the world. It is widely planted in temperate regions and is one of the most widely distributed, commercially important tree species. The genomic database of the ‘Chandler’ cultivar has been sequenced, providing a good platform for mining and identifying walnut MAPK family members at the genomic level [JrMAPK genes under drought stress and Colletotrichum gloeosporioides infection. The results will provide new insight and the basis for further investigation on the biological functions of MAPK gene family members in walnut.Several MAPK transcription factors of diverse species have been excavated and identified with the release of the genomic databases of various species, enabling researchers to have a deep understanding of the MAPK family members ,15,16,17ic level ,19. In tJrMAPK genes were identified from the walnut genomic database and named JrMAPK1–JrMAPK25 (JrMAPK genes were 179 (JrMAPK16) to 623 (JrMAPK2) amino acids in length. The molecular weights were from 37.52 (JrMAPK24) to 70.76 (JrMAPK2) kDa. The isoelectric points were from 4.79 (JrMAPK16) to 9.63 (JrMAPK1). The predicted total average hydrophilicity (GRAVY) score ranged from −0.579 (JrMAPK22) to −0.07 (JrMAPK16). The instability index ranged from 33.38 (JrMAPK14) to 46.83 (JrMAPK5), and 14 JrMAPK proteins were unstable. According to subcellular localization prediction, 25 JrMAPK proteins were found in the nucleus. In addition, none of the JrMAPK genes had signal peptides  to reveal the exon/intron arrangement of each gene  to 10   and two genes in group D (JrMAPK22 and JrMAPK23) had the same intron-exon distribution pattern.We also analyzed the gene structure of the ach gene B. The dirMAPK23) B, which JrMAPK genes on the chromosomes. As shown in JrMAPK25) located on the scaffold, 24 JrMAPK genes were unevenly distributed on 11 chromosomes. The number of JrMAPK genes on chromosome 11 was the largest, with 4 JrMAPK genes. Next were Chr7, Chr12, and Chr15, which had five JrMAPK genes each. Chr1, Chr2, Chr5, and Chr6 each had two JrMAPK genes. On chromosomes 8, 9, and 14, only one JrMAPK gene was discovered each. We studied gene duplication events to elucidate the potential expansion mechanism of the JrMAPK gene family, including tandem duplication and segmental duplication  existed as 2 or more copies. A total of 10 JrMAPK genes underwent tandem replication, and 14 JrMAPK genes underwent segmental replication. In order to determine the driving force of the evolution of the JrMAPK gene family, we also studied the nonsynonymous and synonymous substitution ratio (Ka and Ks) of duplicate genes , abscisic acid (ABA), and other hormone response elements. Additionally, the discovery of stress-response-related elements, such as MBS (MYB binding site implicated in the stress response), ARE (anaerobic induction of cis-elements), and the LTR (low-temperature response element), showed that JrMAPK genes are engaged in the stress response. We also detected CAT box, circular elements, and RY elements, etc., which are cis-acting elements associated with growth and development.We investigated the cis-acting elements in the elements . The G-bJrMAPK genes in the defense reaction of walnuts, we analyzed the transcriptional regulation of 14 JrMAPK proteins after infection with C. gloeosporioides (JrMAPK proteins in F26 (anthracnose-resistant) and F423 (anthracnose-susceptible)were induced by C. gloeosporioides infection. Among them, the expression levels of JrMAPK3 in F26 and F423 were 4.25 and 3.14 times higher, respectively, at 72 hpi (hours post inoculation) than those at 0 hpi, and the transcription levels of JrMAPK6 in F26 and F423 were 2.20 and 1.57 times higher, respectively, than those at 0 hpi. The transcription levels of JrMAPK1 and JrMAPK13 in F26 were 1.94 and 1.93 times higher at 48 hpi than those at 0 hpi, and 1.17 and 1.45 times higher in F423 than at 0 hpi. Moreover, the transcription levels of JrMAPK4, JrMAPK7, and JrMAPK9 were downregulated by C. gloeosporioides infection in F26 and F423. JrMAPK5 expression was upregulated in F423 and downregulated in F426. The expression levels of JrMAPK11, JrMAPK12, and JrMAPK19 were downregulated in F423 and upregulated in F26. The transcription pattern of the other JrMAPK proteins changed not significantly.To explore the function of the orioides . AccordiJrMAPKs under drought stress was detected by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). As indicate in JrMAPK genes were significantly up-regulated after drought stress and were many times higher than that of the control, but the peak times varied between the genes. Among them, JrMAPK1, JrMAPK3, JrMAPK4, JrMAPK5, JrMAPK6, JrMAPK7, JrMAPK9, JrMAPK11, JrMAPK12, JrMAPK17, JrMAPK19, and JrMAPK21 reached their highest expression levels 20 days after the drought stress, and their gene expression levels were 3.33, 5.07, 10.89, 1.53, 8.55, 6.21, 6.87, 4.93, 10.01, 13.21, 1.60, and 7.94 times higher, respectively, than those of the control. In contrast, the expression of JrMAPK13 and JRMAPK20 were highest 30 days after the drought stress, and their gene expression levels were 6.80 and 34.57 times higher, respectively, than those of the control.Next, the expression profile of AtMAPK genes, we predicted several important interactions and preliminarily speculated the possible functions of some JrMAPK genes (AtMAPK4 (homologue of JrMAPK3/13/18/25) and MKS1 genes. MKS1 encodes nuclear localized members of the plant-specific gene family and participates in mediating the response to pathogens. MEK1 and AtMAPK4/6 plays a role in a signal pathway that regulates gene expression in response to biotic and abiotic stresses and plays an important role in pathogen defense through negative regulation of innate immunity. The MKK1-AtMPK6 module responds to drought and salt stress by mediating ABA-dependent CAT1 expression. During seed germination, the MKK1-AtMPK6 module is also implicated in sugar signaling. MPK3 and MPK6 activate upstream MKK4 by phosphorylation. Stomatal cell fate is regulated by the MKK4-MPK3/MPK6 module before the guard mother cell (GMC) is determined. In addition, AtMAPK3 (homologue of JrMPK6/21), AtMAPK4 (JrMAPK3/13), and AtMAPK6 (homologue of JrMAPK11/20) are predicted to interact with WRKY33, and WRKY33 is involved in plant defense response to fungal pathogens. AtMAPK4 (homologue of JrMPK3/13/18/25) and AtMAPK6 (homologue of JrMAPK11/20) interact with AT2G30020. AT2G30020 is a protein phosphatase that negatively regulates the defense response. Inactivated MPK4 and MPK6 MAP kinases are involved in stress and defense signals.In order to analyze the function of JrMAPK proteins, the interaction network map was constructed based on the homologous protein family in Arabidopsis. Using well-studied PK genes . InteresC. gloeosporioides fungus has had a tremendous impact on the growth and development of walnut and fruit quality [Solanum lycopersicum [JrMAPK genes were discovered in the Juglans genome.Walnut trees are often exposed to abiotic and biotic stressors during growth and development. In particular, the  quality . Various quality . Therefopersicum , maize [persicum , apple [persicum , tobaccopersicum , Arabidopersicum , and potpersicum , with 16JrMAPK transcription factors were divided into groups A, B, and C, containing the TEY motif, and group D, containing the TDY motif. In addition, all MAPK members had a CD domain, except for JrMAPK24 in group D, which has been described as LHDXXDEP. This is consistent with the descriptions for Brassica napus and Solanum tuberosum. The members of groups C and D also have CD domains in these two species [It is now generally believed that the MAPK gene family can be categorized into groups A, B, C, and D ,29,30. T species ,32.JrMAPK gene family members were systematically and comprehensively analyzed in this study. The JrMAPK gene family members were composed of 1–10 exons. Groups A and B had 5 introns, Group C contained 1–3 introns, and Group D contained 3–10 introns. The JrMAPK genes in the same subgroup had comparable intron numbers and exon lengths. Similar structural patterns have been observed in other plants, which are highly conserved within the group, but large differences are observed between groups [JrMAPK genes contained motif 4, except JrMAPK16, which is different from other species [JrMAPK16 lacks some amino acids before TEY in motif 4. The reliability of our subfamily classification is strongly supported by the similar conserved motif and gene structure analysis of MAPK genes in the same branch.The positions of the exons and introns in the genome generally offer crucial evidence for phylogenetic analysis. The structural distribution and length of exons and introns of n groups ,34. Our  species ,30,35. IJrMAPK genes underwent tandem duplication  is crucial for genome expansion and rearrangement, as well as in in the diversification of gene function and the generation of a large number of gene families . In thislication , and at lication . These fdistance . As a refamilies ,39,40.JrMAPK gene promoter region contained a large number of cis-acting elements. For example, the existence of MYB elements, ABRE (ABA response elements), MYC elements, CGTCA motifs, TGACG motifs , and variable light response elements indicates that the JrMAPK genes may be involved in various stress signal transductions. Similar cis-acting elements of MAPK genes have been also identified in other species [The analysis of cis-acting elements revealed that the  species ,35,41.AtMPK3, AtMPK4, and AtMPK6, which are enabled by different triggers, such as pathogenic bacteria, abiotic stress, and oxidative stress [JrMPK6 and JrMPK11, which are homologous to AtMPK3 and AtMPK6 in anthracnose-resistant F26, was higher than that in anthracnose-susceptible F423 after being infected by C. gloeosporioides. JrMPK3 and JrMPK13, which are homologous to AtMPK4, were also induced by C. gloeosporioides. This result confirms that the same group of MAPK proteins may have similar functions in different species [JrMPK1 and JrMAPK12 in group C were upregulated in anthracnose-resistant F26, which is consistent with the results of research on group C in rice and alfalfa [JrMAPK3, JrMAPK6, JrMAPK11, JrMAPK13, and JrMAPK20 interact with MKS1, MEK1, and WRKY33, which are involved in mediating pathogen responses. We speculate that these genes have a significant role in walnut biological stress.Many studies have shown that the plant MAPK cascade signaling pathway is widely implicated in various abiotic and biotic stress responses ,43. The e stress . In our  species ,44. In a alfalfa ,46. ProtAtMPK6 in subgroup A and AtMPK4 in subgroup B participate in the abiotic stress reactions to cold and drought [JrMAPK genes were significantly induced after drought stress. The gene expression levels of JrMAPK20 in subgroup A and JrMAPK13 in subgroup B were 34.57 and 6.80 times higher than those of their respective controls 30 days after the drought stress. In addition, some studies have reported that genes in groups C and D participate in the environmental stress response [JrMAPK12 and JrMAPK17 in group C and of JrMAPK4 in group D were more than 10 times higher than their respective controls 20 days after the drought stress. In the upstream regions, cis-acting elements relevant to the stress response were discovered, indicating that these genes are important in regulating drought conditions. Moreover, from the results of the interaction network, there is a close relationship between JrMAPK3, JrMAPK6, JrMAPK11, JrMAPK13, JrMAPK20, JrMAPK21 and abiotic-related MEK1. This finding further emphasizes the specific function of these genes in regulating abiotic stress. In addition, most of the selected JrMAPK genes responded to two different stress conditions. For example, JrMAPK1, JrMAPK3, JrMAPK5, JrMAPK6, JrMAPK11, JrMAPK12, JrMAPK13, and JrMAPK19 were not only induced by C. gloeosporioides, but also responded to drought stress, indicating that they were the main factors involved in crosstalk between different signal transduction pathways to cope with the response to the biotic and abiotic stressors. However, the specific biological functions of JrMAPKs under biological and abiotic stress need further experimental verification.Drought stress is the main limiting factor in global crop yield . Studies drought . In the response ,50. In thttp://plants.ensembl.org/index.html, accessed on 1 April 2022) was used to find the Juglans regia genome_v2.0 data. The serial/threshold protein kinase domain (PF00069) Hidden Markov Model file was obtained from the pfam database . The walnut genome database was searched for MAPK genes using HMMER 3.0 . The default settings were used, with a 0.01 cut-off value. To confirm that the predicted MAPK genes contained the conserved domains, they were submitted to the SMART  and NCBI CDD  databases. The physical and chemical characteristics of the MAPK proteins, such as their isoelectric point, relative molecular weight, and instability index, were predicted using the online program ExPASy . The SignalP5.0 server  was used to predict signal peptides. Subcellular localization was predicted using PlantmPLoc .The Ensembl database  and its default settings. The full-length amino acid sequences of the MAPK proteins in Arabidopsis and walnut were utilized to create the evolutionary tree using the neighbor-joining (NJ) method and MEGA 5.0 software . The following parameters were used with the neighbor-joining (NJ) method: Complete deletion, amino, and bootstrap (1000 replications) p-distance [The 25 distance .JrMAPK gene family members using Meme v 5.0.2  software [JrMAPKs gene structure was shown using the gene structure display server (GSDS) program  [JrMAPK genes’ upstream 1.5 kb genomic sequences were uploaded to the PlantCARE database  [JrMAPK protein sequences as query conditions, homologous Arabidopsis genes were selected as reference. Protein interaction is predicted using String 10.0 .We identified the conserved motifs of the software . The folil 2022) . In ordeil 2022) . Using 2http://plants.ensembl.org/index.html, accessed on 1 April 2022), chromosomal location images were obtained for the JrMAPK genes using Mapchart 2.32 software [Based on the chromosomal positional information provided by the Ensembl database  in a greenhouse. The drought treatment was started when the plants reached a height of about 1500 mm. The designated control plants always received the normal irrigation plan, while the treatment group plants were suspended from irrigation for 30 days, and on days 0, 10, 20, and 30 of the water deficit, leaves were collected. The leaf tissues were instantly submerged in liquid nitrogen and kept at −80 °C.JrMAPK response to C. gloeosporioides were taken from Feng et al. [C. gloeosporioides fungus, which had been grown on PDA media for 5–7 days in the dark at 28 °C. Samples were collected 0, 24, 48, and 72 hpi (hours post inoculation), and 0 hpi was used as the control.The RNA-Seq (RNA Sequencing) data for the g et al.   were applied to create a heatmap.The Foregene plant total RNA isolation kit  was used to extract total RNA. The cDNA was synthesized using the M5 sprint qPCR RT kit with the gDNA remover , and primers were designed using Primer Premier 6.0 software  . Gene exp-value < 0.05 was deemed significant.SPSS Statistics 23  was used to analyze all data, and the results of each treatment were compared using Duncan’s test and one-way analysis of variance. JrMAPK genes in the walnut genome. Based on bioinformatics methods, JrMAPKs were extensively analyzed for chemical and physical qualities, gene structure, conservative motifs, chromosomal location and evolution, and cis-acting elements. We discussed the response of the JrMAPK genes to drought stress and C. gloeosporioides infection. Fourteen selected genes were induced by drought stress. In addition, JrMAPK1, JrMAPK3, JrMAPK5, JrMAPK6, JrMAPK11, JrMAPK12, JrMAPK13, and JrMAPK19 were highly expressed after C. gloeosporioides infection. Moreover, we constructed a protein interaction network, indicating that multiple MAPK genes are involved in the expression regulation of stress. These conclusions set a theoretical foundation for comprehending the stress resistance mechanism of walnut and establish a groundwork for future research on the evolution of the JrMAPK gene family and its function under biotic and abiotic stress.In this study, we systematically analyzed the walnut MAPK gene family and identified 25


NFE2L3, also known as NFE2L3, is a nuclear transcription factor associated with the pathogenesis and progression of human tumors. To systematically and comprehensively investigate the role of NFE2L3 in tumors, a pan-cancer analysis was performed using multi-omics data, including gene expression analysis, diagnostic and prognostic analysis, epigenetic methylation analysis, gene alteration analysis, immune feature analysis, functional enrichment analysis, and tumor cell functional status analysis. Furthermore, the molecular mechanism of NFE2L3 in liver hepatocellular carcinoma (LIHC) was explored. The relationship between NFE2L3 expression and survival prognosis of patients with LIHC was analyzed and a nomogram prediction model was constructed. Our study showed that NFE2L3 expression was upregulated in most cancers, suggesting that NFE2L3 may play an important role in promoting cancer progression. NFE2L3 expression is closely related to DNA methylation, genetic alteration, immune signature, and tumor cell functional status in pan-cancers. Furthermore, NFE2L3 was demonstrated to be an independent risk factor for LIHC, and the nomogram model based on NFE2L3 expression had good prediction efficiency for the overall survival of patients with LIHC. In summary, our study indicated that NFE2L3 may be an important molecular biomarker for the diagnosis and prognosis of pan-cancer. NFE2L3 is expected to be a potential molecular target for the treatment of tumors. Globally, cancer is one of the most severe diseases threatening human health and poses a heavy health and economic burden to society as a result of its high morbidity and mortality . Most maNFE2L3 is a member of the Cap“n”Collar (CNC) family, which belongs to the basic region-leucine zipper (bZIP) transcription factor . The NFEOur study comprehensively analyzed NFE2L3 expression in pan-cancers using different databases. The diagnostic and prognostic value of NFE2L3 in pan-cancers was also evaluated. The correlations between NFE2L3 expression and DNA methylation, genetic alterations, immune features, and tumor cell functional status in pan-cancers was also investigated. A gene function enrichment analysis of NFE2L3 in pan-cancer was performed and the molecular mechanism of NFE2L3 in LIHC was evaluated. NFE2L3 was identified as an independent risk factor for OS in LIHC. In conclusion, the study revealed the essential biological function of NFE2L3 in pan-cancers and showed that NFE2L3 has the potential to be used as an effective therapeutic target and tumor diagnostic marker.https://www.proteinatlas.org/) (https://sites.broadinstitute.org/ccle) (http://timer.comp-genomics.org/) (https://www.cancer.gov/). The subcellular localization of NFE2L3 was investigated using the HPA database and the differential expression of NFE2L3 protein in various tumor and normal tissues was analyzed. R software v3.6.3 and the ggplot2 package v3.3.3 were used for statistical analysis and visualization. P-values were calculated with the Wilcoxon signed rank test and Wilcoxon rank sum test, and p < 0.05 was accepted as statistically significant   database (as.org/) . RNA-seqrg/ccle) . The difcs.org/) . Analyti 0.0001) .http://gepia2.cancer-pku.cn/#index) (The RNA-seq data (level3) and related clinical information were obtained from TCGA cohort. The relationship between NFE2L3 expression and survival prognosis in patients with pan-cancer was analyzed using univariate Cox regression and the results were visualized using the “forest plot” R package. The differential expression Wilcoxon signed rank analysis of NFE2L3 among tumor pathological stages was performed on Gene Expression Profiling Interactive Analysis v2.0 (GEPIA2) (/#index) . P-valueReceiving operating characteristic (ROC) curves were constructed to evaluate the diagnostic value of NFE2L3 in multiple tumors. The area under the ROC curve (AUC) was rated as outstanding discrimination (AUC ≥ 0.90), excellent discrimination (0.8 ≤ AUC < 0.9), acceptable discrimination (0.7 ≤ AUC < 0.8), and poor discrimination (AUC < 0.70). The pROC package v1.17.0.1, and the ggplot2 package v3.3.3 were used for analysis and visualization, respectively.http://www.bioinfo-zs.com/smartapp/) (http://xena.ucsc.edu/) .Methylation data were obtained from the Illumina Infinium DNA methylation platform arrays HumanMethylation450. DNA methylation levels of NFE2L3 in TCGA pan-cancer were analyzed using the SMART database (artapp/) . Comparisc.edu/) . The corhttps://wlab.ethz.ch/protter/start/) is a web-based application tool used for visualizing the sequences, topologies, and annotations of individual proteins   .http://cis.hku.hk/TISIDB/) (http://tide.dfci.harvard.edu/). In addition, the correlation between NFE2L3 expression and immune checkpoint blockade (ICB) overall survival outcome was analyzed in the immunotherapy dataset and the ability of NFE2L3 knockdown to regulate lymphocyte-mediated tumor killing efficacy in the CRISPR Screen dataset was evaluated  . Based oTISIDB/) . The potvaluated . All corhttp://biocc.hrbmu.edu.cn/CancerSEA/)  .p-value < 0.05 was considered to be statistically significant. In addition, tumor patients in TCGA cohort were divided into high and low expression groups according to the median expression level of NFE2L3 mRNA, including BRCA, CHOL, ESCA, HNSC, KIRC, PRAD, UCEC, and THCA. The differentially expressed genes in the high and low expression groups in the multiple tumors described above were analyzed using the DESeq2 package (version 1.26.0). GSEA analysis of the differentially expressed genes was performed in these tumors using the clusterProfiler package (version 3.14.3).GeneMANIA was used to identify the top 20 genes related to NFE2L3 . STRING p-values < 0.05. GO and KEGG enrichment analyses were performed for the DEGs that met the screening requirements. In addition, all the differential genes between the high and low expression groups of NFE2L3 were included in the GSEA analysis. The top 10 signaling pathways from the analysis were presented as merged plots of GSEA. R (version 3.6.3) was used for statistical analysis and visualization, and the main R package that was used included the ggplot2 package v3.3.3 and the cluster Profiler package v3.14.3.The top five genes positively associated with NFE2L3 expression and the top five negatively associated genes in LIHC were evaluated. The results were visualized using a heat map with Spearman’s correlation. Furthermore, TCGA-LIHC cohort was divided into a high and low expression group according to the expression level of NFE2L3 mRNA, and the differentially expressed genes (DEGs) between the high and low expression group of NFE2L3 in LIHC were analyzed . The resp ≥ 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).The correlation between NFE2L3 expression and multiple clinical characteristics of LIHC were analyzed using logistic regression. The factors associated with the overall survival (OS) of patients with LIHC were analyzed using univariate and multifactorial Cox regression. In addition, a nomogram integrating NFE2L3 expression and the prognostic factors of the multivariable model for OS in LIHC from TCGA data was created using the nomogram function from the RMS package v6.2-0 and survival package v3.2-10. The accuracy of the nomogram model for predicting overall survival of patients with LIHC was evaluated using calibration curves. The related RNA-seq and clinical data were accessed from TCGA database, normalized as transcripts per million (TPM), and then log2 transformed. The Wilcoxon signed rank test was used to determine statistical significance  of eight tumors, including ACC, CESC, KIRC, OV, PAAD, THCA, LIHC, and BRCA .To further explore the relationship between NFE2L3 expression and prognosis, overall survival (OS), progression-free survival (PFS), and disease-free survival (DSS) analyses with median group cutoff by univariate Cox regression analysis for pan-cancers were performed. This study showed that higher expression of NFE2L3 had poorer prognosis for OS in six tumor types, including KIRC, KIRP, brain lower grade glioma (LGG), LIHC, mesothelioma (MESO), and pancreatic adenocarcinoma (PAAD). In contrast, higher expression of NFE2L3 was associated with a better prognosis for OS in BLCA, skin cutaneous melanoma (SKCM), and STAD. For PFS, higher expression of NFE2L3 had a poor prognosis in seven tumor types, including GBM, KIRC, LGG, LIHC, MESO, PAAD, and THYM. However, higher NFE2L3 expression was associated with a better prognosis for PFS in patients with STAD . FurtherThe diagnostic value of NFE2L3 expression in pan-cancer was evaluated by using the ROC curve. As shown in Tumorigenesis and epigenetic modifications of genes are closely related, and DNA methylation is one of the most widely studied types of epigenetic modification. Thus, DNA methylation levels of NFE2L3 in normal and primary tumor tissues in pan-cancer were assessed. The DNA methylation levels of NFE2L3 were lower in BLCA, CESC, CHOL, COAD, ESCA, HNSC, KIRC, KIRP, PRAD, LUSD, LUSC, PAAD, READ, THCA, and UCEC than in normal tissues. However, the DNA methylation levels of NFE2L3 were higher in BRCA, LIHC, and PRAD tissues than in normal tissues . In addiAccumulation of gene mutations is one of the causes of tumorigenesis; therefore, the landscape of genetic alterations of NFE2L3 in pan-cancer were analyzed. Transmembrane protein topology of NFE2L3 revealed a natural missense variant of Valine441 in membrane localization . The genThe immune response of tumors is an important process in tumor development, thus the role of NFE2L3 in tumor immune regulation was investigated by analyzing the immune features and immunotherapeutic responses. The correlations between the expression of NFE2L3 and different immune signatures in TCGA pan-cancer cohort were evaluated, including immune cell infiltration, immune checkpoints, immunosuppressive cell infiltration, and immune cell markers. A significant positive correlation between the expression of NFE2L3 and the level of immune cell infiltration in TCGA pan-cancer cohort was demonstrated, except for COAD, OV, UCEC, and UCS . HoweverThe effect of NFE2L3 expression on immunotherapy was evaluated by analyzing the correlation between NFE2L3 expression and TMB or MSI. The results showed that NFE2L3 expression was significantly positively correlated with MSI in STAD, KICH, and THYM, however NFE2L3 expression was negatively correlated with DLBC and CHOL . NFE2L3 The expression level and functional status of NFE2L3 in single tumor cells using the CancerSEA database was determined. NFE2L3 expression was shown to be closely related to the cellular functional status of a variety of tumors, including acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), breast cancer (BRCA), astrocytoma (AST), glioblastoma (GBM), glioma, oligodendroglioma (ODG), head and neck cancer (HNSCC), renal cell carcinoma (RCC), melanoma (MEL), retinoblastoma (RB), and uveal melanoma (UM). In most tumor cells, NFE2L3 expression was positively correlated with differentiation, angiogenesis, apoptosis, inflammation, and tumor cell stemness however, NFE2L3 expression negatively correlated with DNA damage, DNA repair, and invasion . NFE2L3 The molecular functions of NFE2L3 was evaluated in different tumors by performing GO and KEGG pathway enrichment analyses. The top 20 genes that interacted with NFE2L3 were evaluated using the GeneMANIA database . Then thGSEA analysis in BRCA, CHOL, ESCA, HNSC, KIRC, PRAD, UCEC, and THCA was performed. p-value < 0.05. We identified 749 DEGs, including 668 upregulated and 81 downregulated genes   . In addi= 0.032)  The cali= 0.032) .Cancer is one of the greatest threats to human health worldwide, with a high incidence and mortality rate . Early dHowever, no studies to our knowledge have evaluated the significance of NFE2L3 expression in pan-cancer analyses. The molecular mechanisms of NFE2L3 in cancer was evaluated by performing a comprehensive analysis of NFE2L3 in 33 types of human tumors based on data from the TCGA, CCLE, and HPA databases, including gene expression, epigenetic methylation, genetic alteration, functional enrichment, immune features, and survival prognosis. Our results showed that NFE2L3 was significantly upregulated in most human tumors including BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PRAD, READ, STAD, THCA, and UCEC. Furthermore, NFE2L3 protein was shown to be upregulated in OV, TGCT, LUSC, and CESC. NFE2L3 expression was significantly correlated with different pathological stages in ACC, CESC, KIRC, OV, PAAD, THCA, LIHC, and BRCA. In addition, upregulated expression of NFE2L3 was significantly associated with poor OS, DFS, and DSS in most tumors, including KIRC, KIRP, LGG, LIHC, MESO, PAAD, GBM, and THYM. NFE2L3 also had promising efficacy in tumor diagnosis, especially in the diagnosis of CHOL, COAD, KICH, READ, and STAD. Thus, our study demonstrated that NFE2L3 expression was upregulated in a variety of cancers and was associated with poor prognosis, consistent with previous reports . The aboDNA methylation is a common epigenetic modification that is closely related to tumorigenesis, regulating gene transcription and expression, and has become the focus in epigenetics . HypermePrevious studies have shown that tumor development is closely related to genetic alterations, especially mutations in oncogenes and tumor suppressor genes . Our resWith the continuous development of biomedical technology, tumor immunotherapy has become the fourth most effective treatment method after surgery, chemotherapy, and radiotherapy . The potRecently, single-cell sequencing technology has developed rapidly, and its most important feature is the ability to sequence the genome at the individual cell level compared to traditional sequencing technologies, thus enabling further exploration of patterns of tumor heterogeneity . CancerSThe molecular function of NFE2L3 in pan-cancer was investigated through gene function enrichment analysis. Our study showed that NFE2L3 was mainly involved in cell cycle regulation by KEGG pathway enrichment analysis, which was consistent with the study performed by The function of NFE2L3 was investigated by analyzing the co-expression of genes of NFE2L3 and DEGs between the NFE2L3 high and low expression groups in LIHC. Subsequently, GO and KEGG pathway enrichment analyses and GSEA on these DEGs were conducted and it was shown that they were mainly enriched in immune and metabolic processes. Our study also investigated the relationship between the expression level of NFE2L3 and clinicopathological features using logistic regression analysis and the clinical characteristics related to OS of LIHC using univariate and multivariate Cox regression analyses. NFE2L3 expression level correlated with sex, age, T stage, pathologic stage, histologic grade, and AFP in LIHC, and NFE2L3 expression was an independent risk factor for OS in patients with LIHC, which was consistent with previous studies . These rThere are some limitations to the present study. Only online public databases were used for analysis, which may cause systematic bias. The study lacked actual clinical data and further experimental validation using cells and animal models to prove the results observed .Recently, with the rapid development of bioinformatics, an increasing number of computing methods have been developed and used in tumor research to promote tumor diagnosis and treatment technology . To inve


Morus alba) trees have become the best food source for silkworms (Bombyx mori). Nevertheless, the molecular and genomic basis of defense response remains largely unexplored. In the present study, we assessed changes in the transcriptome changes of mulberry in response to silkworm larval feeding at 0, 3, and 6 h. A total of 4709  and 3009  unigenes were identified after 3 and 6 h of silkworm infestation, respectively. MapMan enrichment analysis results show structural traits such as leaf surface wax, cell wall thickness and lignification form the first physical barrier to feeding by the silkworms. Cluster analysis revealed six unique temporal patterns of transcriptome changes. We predicted that mulberry promoted rapid changes in signaling and other regulatory processes to deal with mechanical damage, photosynthesis impairment, and other injury caused by herbivores within 3–6 h. LRR-RK coding genes  was predicted participated in perception of cell wall perturbation in mulberry responding to silkworm feeding. Ca2+ signal sensors (CMLs), ROS , RBOHD/F, CDPKs, and ABA were part of the regulatory network after silkworm feeding. Jasmonic acid (JA) signal transduction was predicted to act in silkworm feeding response, 10 JA signaling genes  and 21 JA synthesis genes  were upregulated after silkworm feeding for 3 h. Besides, genes of “alpha-Linolenic acid metabolism” and “phenylpropanoid biosynthesis” were activated in 3 h to reprogram secondary metabolism. Collectively, these findings provided valuable insights into silkworm herbivory-induced regulatory and metabolic processes in mulberry, which might help improve the coevolution of silkworm and mulberry.As a consequence of long-term coevolution and natural selection, the leaves of mulberry ( Morus alba) displays tolerance to many metals, including copper, cadmium, and zinc [Bombyx mori), mulberry leaves possess various nutrients, such as proteins, soluble sugars, and fat, which play an essential role in the growth and development of silkworms [Mulberry  generation of reactive oxygen species (ROS), and (iv) mitogen-activated protein kinase (MAPK) activity [Plants are continuously exposed to attacks from different herbivores. Plants have evolved complex defense mechanisms after the million years of selection pressure exerted by insect herbivores ,9. Speciressalis ,9,10,11.thaliana ,13. Chewactivity ,11.2+ signaling, ROS signaling, and downstream MAPK signaling are activated, triggering the biosynthesis of Jasmonoyl-l-isoleucine (JA-Ile), the bioactive form of jasmonic acid (JA) [2O2 accumulation can be stimulated by Ca2+-activated NADPH oxidases (RBOHD and RBOHF) or by GOX in oral secretions [PRRs recognize DAMPs from injured cells at the plasma membrane . Membrancid (JA) ,11,15. Ccid (JA) ,16. Suchcretions ,18. Prevcretions .A. thaliana, N. attenuate, cotton and tea plant [Plant structural traits such as leaf surface wax, thorns or trichomes, and cell wall thickness/and lignification form the first physical barrier to feeding by the insects; the secondary metabolites act as toxins and also affect growth and development; and digestibility reducers form the next barriers that defend the plant from subsequent attack ,21. The ea plant ,24,25,26Although many studies have shown the regulatory mechanism underlying silkworm metabolism and silk protein synthesis, few studies have focused on how mulberry trees respond to silkworm feeding. In the present work, we assessed the dynamic transcriptome of mulberry plants in response to silkworm feeding. Moreover, we explored the target genes and critical signaling implicated in activating herbivory-induced indirect defense.The leaf samples were sequenced for gene expression at 0, 3, 6 h after silkworm feeding A–D. Aftehttps://www.ncbi.nlm.nih.gov/, accessed on 1 June 2021), NT (NCBI non-redundant protein sequences) and PFAM  databases with an E-value threshold of 1 × 10−5  unigenes were annotated in at least one database. A total of 24,632 (64.69%), 23,936 (62.86%), and 17,375 (45.63%) unigenes showed similarity to sequences in NR , 3009  unigenes were filtered as dysregulated genes in comparison of EL_3h vs. CK, EL_6h vs. CK with the cutoff of padj < 0.05 and |log2(foldchange)| > 1 A,B. OverTo further provide insights into the functional transitions towards insect response in mulberry, we clustered the 5708 DEGs into eight clusters using the Euclidean distance clustering algorithm . The genp-value < 0.05  of EL_3h v CK, EL_6h v CK = 3.977, 3.320), was also detected significantly overexpressed by qRT-PCR method . In the present study, DEGs in cluster C6 were remarkably downregulated at 3–6 h  insect feeding causes damage to the leaves of trees, and the response of trees can be quickly started within 6 h. For example, Wang et al. reported profound transcriptomic changes in tea plants . In mulberry, the overexpression of cell wall precursor synthesis, cellulose and lignin genes after silkworm feeding  were upregulated after 3 h of silkworm feeding, and one LRR-RK coding gene  was significantly overexpressed after 6 h. Chewing herbivores cause mechanical damage, and such damage significantly alters the extracellular space by releasing cell wall fragments and intracellular components. THESEUS1 is a receptor kinase in plants that senses cellulose-related cell wall integrity [FERONIA can monitor cell wall integrity in response to salt stress by binding to pectin [THE1  and FER  homologous genes were upregulated after 3 h of silkworm infestation , calmodulin-like proteins (CMLs), and calcium-dependent protein kinases (CDPKs) [2fc = 2.096) was overexpressed in mulberry after 3 h of silkworm feeding , OST1 , RBOHD , and RBOHF  suggested that Ca2+ signal sensors, ROS, CDPKs, and ABA were part of the regulatory network after silkworm feeding in mulberry  was upregulated after 3 h of silkworm feeding, indicating that this gene positively regulated resistance to the silkworm.Upon insect perception, early signaling is reflected by increased cytosolic Ca (CDPKs) . We show feeding . In Arabolerance . Calciumolerance ,33. The mulberry . Since RLOX2 , AOS , ACX1 , OPR3 , JAR1 , JAZ1 , JAZ3 , and JAZ10   A. This fMYC2, MYC3, MYC4, and MYC5 exhibit increased susceptibility to S. littoralis and S. exigua feeding [MYC2 , MYC3 , and MYC4  coding genes were significantly over-expressed after silkworm feeding , MYB62 , and MYB111 . Members of the MYB gene family have experienced many evolutionary events, such as gene duplication, during plant evolution [WRKY40 , WRKY51 , and WRKY19   and WRKY51 were overexpressed after 3 h of silkworm feeding. We speculated that these genes of mulberry regulated responding with silkworm. TFs play a fundamental role in mediating defense signaling pathways. Basic helix–loop–helix (bHLH) MYCs regulate defense against herbivores in Arabidopsis by forming a transcriptional complex. Single and higher-order mutants of  feeding ,37. In t feeding . This fivolution . We spec 2.0514) . A previS. exigua and Helicoverpa zea, leads to the release of complex volatile blends, such as β-myrcene, (E)-β-ocimene, DMNT, and (E)-β-caryophyllene, resulting in the increased foraging efficiency of predators and parasitoids [ACAT , HMGCS , HMGCR , MVK , and FDPS   B. Howeve2+, JA, TFs, and secondary metabolisms in response to silkworms. However, several questions remain unanswered. For example, how potential PRRs are linked to these early signaling events and how regulatory steps are modulated. Therefore, comprehending the topology of signaling networks of plants in response to herbivory is necessary.It is reasonable to point out the limitation of this study. The temporal expression trend of genes provided clues for the involvement of CaM. alba) wildtype were cultivated at the Key Lab of Plant Stress Research, Shandong Normal University, Shandong Province, China. The mulberry seedlings were maintained in a multipurpose and pest-free greenhouse at a temperature of 25 ± 2 °C with a 14/10-h light/dark photoperiod. In addition, 2-month-old mulberry seedling clones grown in pots were used as the experimental materials. In addition, nine healthy mulberry seedlings with consistent growth were chosen for the experiment.Mulberry plants . The quality and integrity of purified RNA were evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system  and the NanoDrop 2000 spectrophotometer . ® UltraTM RNA Library Prep Kit from Illumina®  was adopted to generate sequencing libraries. Index codes were supplemented to attribute sequences of each sample. Briefly, poly-T oligo-attached magnetic beads were employed to purify mRNA from total RNA, followed by synthesis of the first- and second-strands of cDNA. Subsequently, the AMPure XP system  was used to purify 150~200-bp cDNA fragments. Finally, the size-selected, adaptor-ligated fragments were isolated and enriched by PCR amplification. The PCR amplicons were subjected to high-throughput sequencing on an Illumina HiSeq X platform . All genetic data were submitted to the NCBI Sequence Read Archive (SRA) database , SRA accession: PRJNA862319. The NEBNexthttp://www.novogene.com/, accessed on 5 March 2021). Clean reads were acquired by removing adapter-containing reads, ploy-N-containing reads, and low-quality reads. The remaining high-quality reads were used for transcriptome assembly using the Trinity software pipeline with default parameters [−5.Reference sequence libraries for mulberry leaves were generated through RNA sequencing and de novo transcriptome assembly. The RNA sample of every accession was sequenced separately. Next, cDNA library construction and Illumina pair-end 150-bp sequencing (PE150) were conducted at Novogene Co., Ltd.  | > 1.A total of 9 independent transcript libraries were created for mulberry control/insect feeding leaves. The expressions of genes were assessed by RSEM . The cleA. thaliana gene IDs by sequence similarity searching against the genome of A. thaliana with an E-value cutoff of 1 × 10−5. The differentially expressed genes (DEGs) in mulberry samples were subjected to the GO enrichment analysis using the topGO package of R. The DEGs of mulberry unigene IDs were converted to the Arabidopsis TAIR locus IDs. The software KOBAS was adopted to validate the statistical enrichment of DEGs in KEGG pathways in mulberry [A. thaliana genome database. The obtained KOs were submitted to iPath tools  for the phytoalexin biosynthesis pathway analyses. MapMan (version 3.5.1 R2) was chosen for metabolic pathways enrichment for DEGs in leaf samples.The unigenes of mulberry were mapped to mulberry . KEGG OrA. thaliana alpha subunit of the elongation factor-1 complex in mulberry was adopted as the housekeeping gene. Premier 5.0 software was employed to design gene-specific primers (21–24 bp) ((ΔΔCt) method.The expression patterns identified by the RNA-seq analysis were confirmed by qRT-PCR. The isolated RNA specimens were subjected to DNaseI digestion and reversely transcribed to cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser . Six upregulated genes  and six downregulated genes  were randomly chosen for the qRT-PCR assay. Ortholog (Cluster-14363.8722) of 1–24 bp) . qRT-PCR2+ signal sensors, ROS, RBOHD/F, CDPKs, and ABA were part of the regulatory network after silkworm feeding. Jasmonic acid signal transduction might act in mulberry responding silkworm were upregulated after silkworm feeding for 3 h. Additionally, genes of “alpha-Linolenic acid metabolism” and “phenylpropanoid biosynthesis” were activated in 3 h to reprogram secondary metabolism. Collectively, these findings provided valuable insights into silkworm herbivory-induced regulatory and metabolic processes in mulberry, which might help improve the coevolution of silkworms and mulberry.In the present study, we assessed changes in the transcriptome changes of mulberry in response to silkworm larval feeding at 0, 3, and 6 h. A total of 4709 and 3009 unigenes were identified after 3 and 6 h of silkworm infestation, respectively. The structural traits such as leaf surface wax, cell wall thickness and lignification were predicted to form the first physical barrier to feeding by the silkworms. We predicted that mulberry promoted rapid changes in signaling and other regulatory processes to deal with mechanical damage, photosynthesis impairment, and other injury caused by herbivores within 3–6 h. Ca