However, some IBD patients are non-responders to these drugs, which are also associated with serious side effects. Thus, it has been hypothesized that therapies aimed at restoring anti-inflammatory signals, by exploiting the tolerogenic potential of cytokines , immune cells , or mesenchymal stem cells, might offer promising results in terms of clinical efficacy with fewer side effects. In this review, we provide new insights into putative novel treatments aimed at restoring anti-inflammatory signaling pathways in IBD.Inflammatory bowel disease (IBD) is caused by a dysregulated immune response against normal components of the intestinal microflora combined with defective functioning of anti-inflammatory pathways. Currently, all therapies approved for IBD manipulate the immune system by inhibiting pro-inflammatory mechanisms, such as tumor necrosis factor-α, gut-homing α Hyperac kinases . Thus, t kinases . In thisLactobacillus lactis (LL-Thy12) for direct mucosal delivery proved to have a significantly better safety profile, reducing the frequency of the side effects commonly registered in other trials [IL-10 is a key immunoregulatory cytokine that can be expressed by virtually any innate and adaptive immune cell, including macrophages, natural killer cells, eosinophils, neutrophils, and B- and T-lymphocytes ,25. Althr trials . Up to dr trials . Further+ and IL-17A+ T cells expressing the gut-homing protein CCR9 [The expression of the anti-inflammatory cytokine TGF-β is unexpectedly high in the inflamed mucosa of IBD patients in comparison to control gut . Howeverein CCR9 ,42, patiein CCR9 . Moreoveein CCR9 , which hein CCR9 . A furthein CCR9 . In a phein CCR9 . The priein CCR9 . The safein CCR9 . A post ein CCR9 . A phaseein CCR9 . This noein CCR9 . A phaseein CCR9 .p = 0.01) and achieved remission , compared to the placebo arm. In addition, the rate of response was significantly higher at all the considered time points, including 30-day follow-up (p = 0.03), as well as the rate of remission at the end of the 30-day follow-up (p = 0.02), and a significant improvement of the quality of life was demonstrated comparing the Sargramostim group to the placebo one at all time points, but especially at 30-day follow-up (p = 0.006) [The GM-CSF is a glycoprotein that enhances the function of mature white cells, including neutrophils, monocytes, and macrophages, and stimulates the expansion and differentiation of hemopoietic progenitors, acting as a potent stimulant of the innate immunity ,47,48. T= 0.006) . Due to 6, 107, 108, 109 autologous ova-Tregs) and were followed-up for 12 weeks. The results showed that 40% of patients responded at Weeks 5 and 8 with a CDAI score reduction of 100 points or more and a significantly higher reduction of the same score in those receiving doses of 106 cells at both time points. Nevertheless, the benefit did not last over time, as 12 weeks after infusion, the CDAI score was comparable to baseline [+ resting Tregs and showed that the latter have an epigenetically-stable FOXP3 locus and do not switch into a Th17 phenotype in vitro [+ Tregs express a high level of α4β7 integrin, CD62L, and CC motif receptor 7 and have been shown to home to the human small bowel in a xenotransplant model of severe combined immunodeficiency [+ Tregs [Tregs encompass a heterogeneous group of T cells that are involved in maintaining the peripheral tolerance to both self- and external antigens, thus downregulating unnecessary inflammatory processes ,65. Regabaseline . In addiin vitro . Furtherficiency . Another[+ Tregs . At pres4β7-integrin+ and CCR9+ Tregs through TGF-β and retinoic acid [+ DCs display low production of TGF-β and reduced activity of the ALDH1A2 gene, one of the enzymes involved in retinoic acid synthesis together with ALDH1A1 and ALDH1A3, thus leading to a limited amount of Foxp3+ Tregs and an increase in IFN-γ-producing T cells [+ DCs isolated from patients with CD do not express the ALDH1A1 enzyme [DCs migrate from peripheral tissues to T cell areas of secondary lymphoid organs, including the gut, where they act as antigen-presenting cells . Upon stoic acid ,75,76,77 T cells . Likewis1 enzyme .6 DCs/mL, 5 × 106 DCs/mL, 10 × 106 DCs/mL) and the number of doses (one or three every two weeks) [A phase 1, single-center, sequential-cohort, dose-range study investigated the safety and tolerability of autologous tolerogenic DCs, derived from monocytes by leukapheresis and administered by ultrasound-guided intraperitoneal injection in nine patients with refractory moderate-to-severe CD ,80. The o weeks) . Amongsto weeks) . In parao weeks) . No adveo weeks) . The pero weeks) . In addio weeks) . These fo weeks) .Amongst stem cells, MSCs are the best candidate as therapy in IBD due to their migration to inflamed intestinal tract, where they exert a regenerative function and the absence of immunogenicity . In addiImmunotherapy represents an intriguing therapeutic option that has the aim of restoring the physiological T cell tolerance towards specific antigens, while leaving the rest of the immune system unaffected. Antigen- and epitope-based immunotherapy has rapidly developed over the last decade, especially for oncologic and allergic disorders, in which immunodominant disease triggers are increasingly recognized, and therefore can be targeted . The devOtelixizumab is a chimeric/humanized Fc-engineered monoclonal antibody against CD3/ε showing encouraging results in patients with new or recent onset of diabetes mellitus type 1 ,93. HoweIn addition to the current therapeutic strategies blocking pro-inflammatory cytokines, lymphocyte gut homing, and Janus kinases, several novel treatments aimed at bolstering the immunoregulatory pathways have been proposed for the treatment of IBD patients . Cytokin


Viruses are intracellular parasites that require a permissive host cell to express the viral genome and to produce new progeny virus particles. However, not all viral infections are productive and some viruses can induce carcinogenesis. Irrespective of the type of infection (productive or neoplastic), viruses hijack the host cell machinery to permit optimal viral replication or to transform the infected cell into a tumor cell. One mechanism viruses employ to reprogram the host cell is through interference with signaling pathways. Polyomaviruses are naked, double-stranded DNA viruses whose genome encodes the regulatory proteins large T-antigen and small t-antigen, and structural proteins that form the capsid. The large T-antigens and small t-antigens can interfere with several host signaling pathways. In this case, we review the interplay between the large T-antigens and small t-antigens with host signaling pathways and the biological consequences of these interactions. Polyomaviridae family consists of naked viruses with an icosahedral capsid structure. Although originally isolated in mammals, polyomaviruses (PyV) also infect birds and, recently, PyV sequences have also been detected in insects, fish, amphibians, and reptiles. However, it remains to be established whether PyV can actually infect these species [The  species ,2. The cSo far, 14 different human polyomaviruses (HPyV) have been described. BKPyV and JCPyV were the first HPyV to be isolated in 1971 and they were named after the initials of the patient ,4. In thViruses, including polyomaviruses, recruit the host cell machinery to favour their replication, and, in the case of oncoviruses, to cause carcinogenesis. One way to take control or perturb cellular processes is by interfering with signaling pathways regulating processes such as DNA replication, the cell cycle, the immune response, transcription, metabolism, DNA repair, cell survival, cell motility, and angiogenesis ,35,36,37One way to explore the impact of HPyV on signaling pathways is to identify which cellular proteins can bind to LT and sT. Several methods such as co-immunoprecipitation, tandem affinity purification coupled to mass spectrometry, GST pull down of in vitro translated proteins, stable isotope labeling by amino acids in cell culture (SILAC)-based pull down, and yeast two-hybrid have been used to identify cellular interaction partners of HPyV LT and sT to understand the function of these proteins . A list The human phosphatome consists of 264 enzymes that, depending on their substrate, include non-protein phosphatases , and protein phosphatases. The latter group contains protein tyrosine phosphatases, serine/threonine phosphatases, and dual specificity phosphatases . HPyV viMCPyV sT was found to bind the catalytic subunit of PP1 . The bioPP2A is a phosphoserine/threonine phosphatase that exists as a heterotrimer composed of a structural subunit A, a regulatory subunit B, and a catalytic C subunit . SeveralThe effect of HPyV sT: PP2A interaction on signaling pathways was investigated for HPyV6 and TSPyV sT. Both sT were shown to bind PP2A-A and PP2A-C subunits when overexpressed in human embryonal kidney HEK293 cells ,54. WildMCPyV sT was reported to interact with PP4 ,49,58,592) into phosphatidylinositol-3,4,5-triphosphate (PIP3), which then recruits the protein serine/threonine kinase-3′-phosphoinositide-dependent kinase 1 (PDK1). PDK1 phosphorylates and activates AKT. AKT signaling promotes mTOR activity through inhibitory phosphorylation of tuberous sclerosis complex (TSC) proteins 1/2, which act as mTOR inhibitors. mTOR is a complex that consists of mTORC1 and mTORC2 [A central signaling cascade that regulates cellular processes such as growth, motility, survival, metabolism, and angiogenesis is the phosphatidyl-3-kinase/AKT/mammalian target of the rapamycin (PI3K/AKT/mTOR) pathway . Perturbd mTORC2 ,71.H1047R mutation [Earlier studies had shown that SV40 LT, through an interaction with insulin receptor substrate 1 (IRS-1) and sT, and via inhibition of protein phosphatase 2A (PP2A) could activate the AKT pathway ,73. It wmutation , which lmutation . HoweverPIK3CA gene) and AKT gene, as well as the phosphorylation levels of AKT at Thr308 in Merkel cell carcinoma (MCC) tumor samples and cell lines were investigated [PI3KCA gene, whereas none of the eight cell lines had mutations. The most common mutation that activates AKT, glutamine residue 17 into lysine [PIK3CA and in AKT, and of the presence of MCPyV. The latter indicates that neither LT nor sT are responsible for AKT phosphorylation in MCC. Another study found that the mRNA levels of TSC1, TSC2, and mTOR but not the protein levels, were significantly higher in virus-negative samples compared with virus-positive MCC tumors. AKT phosphorylation at Thr308 was also significantly higher, whereas phosphorylation of Ser473, which is another activation event of AKT, was not statistically significantly different between virus-negative and virus-positive MCCs [The presence of mutations in the PI3K encoding gene . 4E-BP1 belongs to a family that contains 4E-BP1, -2, and -3 and exists in one unphosphorylated (4E-BP1α) and three phosphorylated isoforms  with increasing degrees of phosphorylation . In its The Wnt signaling cascade is a major pathway in cells and aberrant expression is tightly associated with cancer . The Wnthttps://web.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes) including genes encoding proteins involved in migration and invasion , anti-apoptosis , and angiogenesis  were upregulated by LT. The mechanism by which LT interferes with the β-catenin cascade is not known, but LT may stabilize β-catenin. LT increases β-catenin levels by inhibiting its proteasomal degradation. However, this LT-mediated stabilization of β-catenin is independent of GSK3α [It was demonstrated that JCPyV LT can stimulate transcription of β-catenin target genes and can interact with β-catenin and co-localize in the nuclei ,92,93. Tof GSK3α . The autProtein kinase C (PKC) is a family of serine/threonine kinases that consists of the members α, βI, βII, γ, δ, ε, η, θ, ζ and ι . PerturbThe MAPK pathways are involved in processes controlling gene expression, cell division, cell survival, cell death, metabolism, differentiation, and motility. The conventional MAPK pathways consist of a cascade of three consecutive phosphorylation events executed by a MAPK kinase kinase, a MAPK kinase, and a MAPK. There are four different subfamilies of MAPK: extracellular-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), p38 MAPK, and big MAPK. The atypical MAPK are not organized in the classical three tier cascade, and include ERK3/4, ERK7/8, and Nemo-like kinase .Infection studies with JCPyV in the human glial cell line SVG-A demonstrated rapid activation of ERK1/2. Increased ERK1/2 phosphorylation could be observed 15 minutes after infection and was sustained for at least 6 hours. At 9 hours post infection, ERK1/2 phosphorylation returned to baseline levels ,105,106.HPyV may also interfere with other MAPK pathways. One of the substrates of JNK is c-Jun, which is a transcription factor that is part of the dimeric activating protein 1 (AP-1) complex . AP-1 cop = 0.04) higher in MCPyV-positive non-small cell lung cancer samples (n = 6) compared with MCPyV-negative non-small cell lung cancer samples (n = 10) and an adjacent benign tissue. Moreover, phosphoSer-445 BRAF levels were also significantly higher in the virus-positive specimens than in virus-negative tumours [The interaction between MCPyV and MAPK pathways has not been intensively studied. Transcript and protein levels of RAF were significantly ( tumours . A largeCBF1/RBPjκ/Su(H)/Lag-1), which results in transcriptional activation of target genes [The Notch signaling pathway mediates cell-cell communication because Notch ligands are membrane bound and will interact with the Notch receptors, which are transmembrane proteins. There are four human Notch receptors (Notch1–4), while there are several groups of ligands, including Jagged 1 and 2, and Delta-like proteins. Binding of the ligand to the Notch receptor results in conformational change that exposes Notch to a proteolytic cleavage to release the Notch intracellular domain (NICD). NICD enters the nucleus and will bind to and displace the transcriptional receptor CSL  in MCPyV-positive MCCs (n = 29) than in MCPyV-negative MCCs (n = 21) [SHH-1B gene and one silent point mutation in exon 5 of the GLI1 gene in 14 samples [The hedgehog signaling pathway owes its name to the polypeptide ligand, because fruit flies lacking the gene encoding this protein had a phenotype that resembled hedgehogs. In humans, there are three hedgehog ligands: sonic (SHH), Indian (IHH), and desert (DHH) hedgehog. The Patched 1 (PTCH1) receptor forms a heterodimeric complex with the transmembrane protein Smoothened (Smo). In the absence of HH, PTCH1 suppresses Smo by preventing its localization to the cell surface. Binding of HH to the receptor releases this inhibition and allows translocation of Smo to the cell surface. This triggers a signaling cascade resulting in the activation of the DNA binding proteins Gli-1, Gli-2, and Gli-3. In the presence of HH, Gli-1 acts as a transcriptional activator, while Gli-3 is a transcriptional repressor. Depending on the post-translational modifications, Gli-2 can act as a repressor or an activator ,124. Exp(n = 21) . Brunner(n = 21) . However samples . MCPyV-i samples .DNA damage repair (DDR) pathways will delay or arrest cell cycle progression in cells with damaged DNA. This pathway is controlled by the kinases ataxia telangiectasia mutated (ATM), ATM-related and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) ,129. ATMP99A mutant had no effect on NER activity, which indicates that JCPyV sT can induce chromosome instability in a PP2A-independent manner [Several studies have shown induction of the DDR pathway by JCPyV. JCPyV infection, LT, or sT expression in primary human astrocytes, human neuroblastoma IMR-32, human lung carcinoma H1299 cells, and human glial cell line SVG-A caused genomic instability and DNA damage as shown by aneuploidy/hyperploidy of infected cells, and inhibited nucleotide excision repair (NER) activity. Furthermore, increased phosphorylation of H2AX at Ser-139  and the ATM and ATR substrates Cdc2, Chk1, Chk2, replication protein A32, and p53 was measured. Elevated levels of DNA repair proteins, including RAD51, p53, Ku70, and phosphorylated Artemis were observed ,134,135.t manner .Elegant work by the group of Reiss has unveiled the mechanism by which JCPyV LT affects RAD51. They showed that JCPyV LT perturbs homologous DNA repair by interfering with the IGF-1/IGF-IR/insulin receptor substrate 1 (IRS-1) signaling axis and this requires RAD51. The receptor of insulin-like growth factor I (IGF-IR) is a receptor tyrosine kinase that becomes activated by the insulin-like growth factor 1 (IGF-1) and IGF-2. Besides its role in metabolism, this signal transduction pathway is also involved in normal cell growth, DNA repair, regulation of cell-cell adhesion, and cell survival. Compromised activity of the IGF-IR signaling pathway is implicated in cancer ,147,148.Cip1/Waf1 [Cip1/Waf1, and DNA damage. In accordance with elevate p21 levels, cell cycle arrest and inhibition of cell proliferation were observed in LT expressing cells. Full-length LT, but not C-truncated LT, which is typically found in MCPyV positive Merkel cell carcinoma (MCC) tumours, induced the ATR/Chk1/p53 pathway. Full-length had a decreased potential to stimulate cell proliferation and anchorage-independent cell growth compared to truncated LT. These observations may explain why LT is C-terminally truncated in MCC. The authors also showed that MCPyV sT or 57kT failed to increase phosphorylation of Chk1 and Chk2. In another study, the same group showed that MCPyV utilizes host DDR factors for replication of its genome [The effect of MCPyV on the DDR pathway has also been studied. Infection of osteosarcoma U2OS cells with MCPyV induced the DDR pathway, as shown by enhanced phosphorylation of ATM/Chk1 and ATR/Chk2 . Expressip1/Waf1 ,153. Thes genome . Hence, The retinoblastoma family or pocket proteins is a family of tumour suppressors that consist of the three members pRb (RB1), p107, and p130 (RB2). They have a pivotal role in controlling cell cycle progression from the G1 to the S phase. During the G1 to the S phase transition, RB1 is converted from its hypophosphorylated form to its hyperphosphorylated form, while p107 and p130 become hyperphosphorylated during the late G1 to S phase ,156,157.The LT of BKPyV, JCPyV, WUPyV, MCPyV, and HPyV7 have been shown to interact with the pocket proteins ,169,170.A direct interaction between the sT of MCPyV and TSPyV and the pocket proteins has not been demonstrated, but induced expression of TSPyV sT, but not MCPyV sT, which enhanced phosphorylation of pRb in HEK293 cells . Hence, DNA methyltransferease 1 (DNMT1) gene and this activation depends on the E2F binding sites in the promoter and on LT’s ability to interact with the pocket proteins [The biological consequences of the interaction with the pocket proteins was not always investigated. The interaction of JCPyV LT and the three T’ variants inhibited phosphorylation of the pocket proteins and promoted their degradation, whereas association of sT with p107/p130 drove the cells into the S phase and promoted replication of the viral genome ,178. BKPproteins . Infectiproteins ,181. Thuproteins ,183,184.proteins . MCPyV fproteins . The bioTP53 gene are observed in most cancers [The tumour suppressor p53 is a transcription factor that binds as a tetramer to DNA in a sequence-specific manner and activates or represses the expression of several hundred target genes . p53 is  cancers . Further cancers . It is,  cancers ,192,193. cancers . BKPyV-i cancers ,196. p53 cancers .The C-terminal truncated LT that is expressed in MCPyV-positive MCCs lacks the conserved p53-binding domain present in LT of other HPyV and, hence, cannot bind p53 . SurprisProgrammed cell death or apoptosis plays an important role in development, aging, tissue homeostasis, and in the defense mechanisms against DNA damage and infections. Failure of apoptosis results in pathological conditions, including developmental defects, autoimmune diseases, neurodegeneration, and cancer ,203. VirBAG3 gene. JCPyV LT may compete with AP-2 for the same site because the JCPyV LT binding motif 5′-GRGGC-3′ (R = A or G) and the AP-2 consensus site 5′-CCSCRGGC-3′  are sim C or G; . JCPyV-m C or G; . Hence,  C or G; .p = 0.05) downregulated in MCPyV-positive non-small cell lung cancer samples compared to virus-negative tumours and healthy adjacent tissue, while there were no significant differences in mRNA levels of the pro-apoptotic Bax gene between the different specimens [Bcl-2 gene was not investigated. However, in another study on a larger cohort of MCC patients (n = 116), no statistically significant correlation was found between the presence of the Bcl-2 protein (levels not quantitated) and MCPyV DNA in the tumour samples [Bax and Bcl-2 levels [Expression of Bcl-2, which is an anti-apoptotic protein that promotes cell survival , was sigpecimens . Few sam samples . Transcr2 levels . The exaAnother inhibitor of cell death is survivin, and its expression is upregulated in most cancers . By compUbiquitination involves the covalent attachment of a 76 amino acid peptide to target proteins. This labels the proteins for proteolytic degradation by a multiprotein complex known as the 26 S proteasome. The proteasome function is essential for protein homeostasis and influences the regulation of most cellular processes, including cell survival, cell signaling, and cell cycle progression ,219. ConFbw7  [JCPyV LT binds β-transducin-repeat containing proteins 1 and 2 (βTrCP1 and βTrCP2), which are F-box proteins that are part of the E3 ubiquitin ligase complex SCFining 7) . This inining 7) . This suining 7) . Moreoveining 7) , which mFbw7. This resulted in stabilization of MCPyV LT, which is a substrate for the E3 ligases SCFFbw7, β-TrCP, and Skp2 [Fbw7 targets such as the oncoproteins c-MYC and cyclin E [Fbw7-dependent [Fbw7 or β-TrCP, as well as between LT and SCFFbw7. Furthermore, sT was demonstrated to stabilize LT independently of SCFFbw7 [MCPyV sT was found to bind and inhibit the E3 ubiquitin ligase complex SCFand Skp2 . sT-inducyclin E . Furtherependent . Moreoveependent  and β-Trependent , and by ependent . All the SCFFbw7 . The autMCPyV and BKPyV sT can form a complex with STIP1 homology and U-box containing protein 1 (STUB1), which is also known as carboxy terminus of Hsc70 interacting protein (CHIP) . This prNFκB is a family of transcription factors that consists of five members that can form homodimers and heterodimers. NFκB is kept in an inactive state in the cytoplasm through interaction with members of the inhibitor of κB (IκB) proteins. Activation of the canonical NFκB pathway is mediated by the IκB kinase (IKK) complex, a heterotrimer of the protein kinases IKKα and IKKβ, and the regulatory subunit IKKγ, which is also referred to as an NFκB essential modulator (NEMO). Activation of IKK depends on phosphorylation of IKKα and IKKβ. The activated IKK complex will then phosphorylate IκB, which results in its degradation. Hence, NFκB is released and translocates to the nucleus, where it affects transcription of NFκB-responsive genes . The traCCL20, CXC-9, IL-8, and TANK in HEK293 and the virus-negative MCC cell line MCC13 [p = 0.034) expression of pSer-536 RelA/p65 subunit of NFκB was observed in MCPyV-positive (n = 24) compared to virus-negative (n = 17) MCCs. This phosphorylated form resided exclusively in the nucleus [Ectopically expressed MCPyV sT was found to inhibit the NFκB pathway and downregulate expression of NFκB target genes such as ne MCC13 . The autne MCC13 . Althougne MCC13 . Taken t nucleus . Neither nucleus , which sp.i. most NEMO was relocated to the cytoplasm. At three days p.i., however, little or no phosphorylated ATM was detected in JCPyV-infected cells. Because inhibition of ATM suppressed viral replication, the authors speculated that stress induced upon JCPyV infection activates ATM, which, in a NEMO-dependent manner, activates NFκB. This transcription factor has previously been shown to stimulate transcription of the early and late viral genes and viral DNA replication [The DDR can activate the NFκB pathway also referred to as the “inside-out” or “nuclear to cytoplasm” NFκB signaling . This nulication .p-TEFb, and chromatin remodeling proteins. Moreover, it can interfere with the NFκB pathway by interacting with IκB [Bromodomain protein 4 (Brd4) is a member of the bromodomain and extra terminal domain family of proteins that recognized acetylated lysine. It acts as a transcriptional and epigenetic regulator by activating transcription factors, transcription elongation factor with IκB ,239. Itswith IκB ,241,242.with IκB . It is nwith IκB .The innate immune system forms an important defense against viruses. Specific pattern recognition receptors (PRRs) serve to identify pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) ,246. OneISG15 and IFIT3 in human endothelial cells [p.i. [Infection of primary human foetal glial cells and U87MG glioblastoma cells with JCPyV induced the expression of interferon-(IFN) stimulated genes . Anotheral cells . BKPyV ial cells . The traal cells . A recenls [p.i. . It is uls [p.i. . Alternals [p.i. ,252.The primary function of cytokines, including chemokines, is the induction of inflammation and immune responses during viral infection ,256,257.Nuclear receptors are a diverse family of intracellular receptors that also act as transcription factors. Their ligands can pass the plasma membrane and include steroid hormones, thyroid hormone, vitamin D3, retinoic acid, and fatty acid metabolites. Ligand binding, posttranslational modifications, and recruitment of co-activators result in activation of nuclear receptors. They bind as monomers, homodimers, or heterodimers to specific DNA sequences, which directs transcription of their target genes. Nuclear receptors regulate cellular processes such as cell proliferation, development, metabolism, inflammation, tissue homeostasis, apoptosis, and reproduction ,266. TheCo-expression of BKPyV LT and sT stimulated oestrogen receptor-mediated transcription. However, when expressed separately, only LT induced oestrogen receptor-mediated transcription . The mecSPHK1 and SPHK2 genes can be excluded. SK1 becomes activated by Ser-225 phosphorylation and this site is dephosphorylated by PP2A [Sphingolipids are bioactive (phospho)lipids that act as important regulators of cellular processes such as proliferation, cell survival, differentiation, migration, autophagy, and immune responses . These m by PP2A . Therefo by PP2A , so it mViruses can alter the host metabolism for their own benefit to promote optimal viral replication conditions . Human tMost human polyomaviruses establish a life-long persistent, but harmless infection in healthy people. However, they may cause diseases in immunocompromised individuals. Only one HPyV and MCPyV is recognized as a human oncovirus, whereas BKPyV and JCPyV are possibly oncogenic viruses . Current


Grain size and weight are crucial components of barley yield and quality and are the target characteristics of domestication and modern breeding. Despite this, little is known about the genetic and molecular mechanisms of grain size and weight in barley. Here, we evaluated nine traits determining grain size and weight, including thousand grain weight (Tgw), grain length (Gl), grain width (Gw), grain length-width ratio (Lwr), grain area (Ga), grain perimeter (Gp), grain diameter (Gd), grain roundness (Gr), and factor form density (Ffd), in a double haploid (DH) population for three consecutive years. Using five mapping methods, we successfully identified 60 reliable QTLs and 27 hotspot regions that distributed on all chromosomes except 6H which controls the nine traits of grain size and weight. Moreover, we also identified 164 barley orthologs of 112 grain size/weight genes from rice, maize, wheat and 38 barley genes that affect grain yield. A total of 45 barley genes or orthologs were identified as potential candidate genes for barley grain size and weight, including 12, 20, 9, and 4 genes or orthologs for barley, rice, maize, and wheat, respectively. Importantly, 20 of them were located in the 14 QTL hotspot regions on chromosome 1H, 2H, 3H, 5H, and 7H, which controls barley grain size and weight. These results indicated that grain size/weight genes of other cereal species might have the same or similar functions in barley. Our findings provide new insights into the understanding of the genetic basis of grain size and weight in barley, and new information to facilitate high-yield breeding in barley. The function of these potential candidate genes identified in this study are worth exploring and studying in detail. Hordeum vulgare L.) has become one of the most important cereal crops cultivated around the world, and is widely used as animal feed, potential healthy food products and is a major raw material for malting and brewing industries  and the degree of grain filling, are two crucial components that affect barley yield and malt quality . In the Nud  population containing 122 lines derived from Huaai 11 (six-rowed and naked) and Huadamai 6 (two-rowed and hulled) was employed to identify QTLs that control barley grain size and weight. Details of the DH population and their parents can be found in our earlier studies . The DH 2), grain perimeter , grain diameter  and grain roundness (Gr) using a camera-assisted phenotyping system  (2) according to the following formula: Ffd = At maturity, kernels of six uniform plants in the middle of each plot were bulk-harvested and sun-dried for phenotypic evaluation. Then, 200 to 300 fully filled grains of each line were used to measure thousand grain weight , grain length , grain width , grain length-width ratio (Lwr), grain area  . Ga and 00×Gl×Gw .t-test, normality test (Shapiro–Wilk), correlation analysis and analysis of variance (ANOVA) were performed using SPSS v24.0 . Broad sense heritability (H2) estimates were calculated from ANOVA using the following formula: H2 = 1 − MS2/MS1, where MS1 and MS2 are the mean squares of genotype and genotype × environment, respectively (The best linear unbiased prediction (BLUP) value of the three replicate measurements for each year were used for statistical analysis and QTL mapping. The calculations of descriptive statistics, Student’s ectively . Frequen2 = PVE(A) and PVE(E) are the additive genetic (A) and environmental (E) components of the multi-environment variance. Moreover, to overcome the interference of row type (Rt) and caryopsis type (Ct), we used Rt and Ct as a covariate, respectively, and conducted QTL mapping with a covariance analysis. The covariate QTL analysis was performed using software QTL.gCIMapping from the R website  on all investigated grain size and weight traits  in all years. The phenotypic variation coefficient ranged from 10.11 to 33.25% . VariancAdditionally, we also calculated the correlation coefficient among the nine grain size and weight traits in the DH population based on the mean value of the three-year data . The resUsing the ICIM BIP module for single-environment QTL analysis, we identified 168 QTLs distributed on all chromosomes of barley for nine grain size and weight traits, including 45, 63 and 60 QTLs in 2016, 2017 and 2018, respectively . Out of We also performed MET analysis using the ICIM MET module. A total of 12, 7, 17, 10, 17, 11, 17, 12, and 10 MET QTLs were identified for Tgw, Gl, Gw, Lwr, Ga, Gp, Gd, Gr, and Ffd, respectively . Of thesFurthermore, to eliminate the potential confounding effect of row type (Rt) and caryopsis type (Ct) on grain size and weight traits, we used these two traits as a covariate, and performed covariate QTL analysis using genome-wide composite interval mapping (GCIM) methods. A total of 312 covariate QTLs distributed on all seven chromosomes were successfully identified, including 108, 117, and 87 covariate QTLs for using Rt, Ct, and Rt+Ct as a covariate, respectively . Among tIn this study, we found a considerable number of overlapping QTLs for different traits. By integrating all the QTLs identified using the five mapping methods, we found 27 hotspots on six chromosomes involving 421 QTLs, including 3, 2, 10, 1, 2, and 9 hotspots for 1H, 2H, 3H, 4H, 5H, and 7H, respectively . For insTo identify candidate genes for QTLs and their correspondence to grain size/weight related genes in other cereal species, we collected 38 barley genes and 148 other cereal crops genes  associated with grain size/weight or yield , S8. BasImproving barley yield has always been an important objective of barley genetic research and breeding programs. Grain size is one of the key factors determining barley yield. Although some QTLs related to grain size and weight have been identified, and several genes affecting grain size or weight have been characterized, our knowledge on the genetic and molecular mechanisms that regulate grain size in barley remain largely unknown. In this study, we have identified 593 QTLs for nine barley grain size and weight traits using five mapping methods. A total of 45 potential candidate genes were identified, providing important insight into the genetic basis of barley grain size and weight.QTL analysis of thousand grain weight (Tgw) has been performed previously , but somGs3 gene on chromosome 7H (HORVU7Hr1G001910), which may be the same to the QTL hotspot 7_9 (7H: 4.97–7.56 Mb) identified in our study. The QTL hotspot 3_3 consisting of 19 QTLs for Tgw, Gl, Gw, Ga, Gp, Gd, and Ffd on chromosome 3H at 29.5–33.5 cM (631.86–646.92 Mb) identified here may share the same location with the QTL LEN-3H for Gl described by QTGW. MC3H.1 for Tgw detected by QTL-3H-9 for Tgw found by In the present study, we successfully identified 60 reliable QTLs for the nine traits of grain size and weight and found 27 hotspot regions that distributed on chromosome 1H, 2H, 3H, 4H, 5H, and 7H , 4. CompIn addition, some novel QTLs were also identified in our study. For example, QTL hotspot 4_1 for Tgw, Gw, Ga, and Gd on chromosome 4H at 529.57–532.00 Mb identified here are different from the QTLs or QTL hotspots for grain size and weight described on chromosome 4H in previous reports . Similarsdw1/denso controls plant height, the number of tillers, grain yield, and grain size  was also mapped to this region, which has previously been reported to affect row type, grain size and Tgw (nud gene (HORVU7Hr1G089930) that determines the hulled/naked caryopsis phenotype  contained 61 QTLs for all grain size and weight traits. The  and Tgw . Anotherhenotype . Severalnud gene . Since onud gene . Althougvrs1/int-d and nud genes, the vernalization gene Vrn-H3/Sgh3/HvFT1 (HORVU3Hr1G087100) and flowering gene HvCO9/HvCMF11 (HORVU1Hr1G056120), which are associated with barley yield, were physically close to QTL hotspot regions 1_1 (415.25–423.42 Mb) and 3_4 (623.15–624.57 Mb), respectively (sdw1/denso (HORVU3Hr1G090980) and dwarf gene btwd1 [close to SNP marker 7HL_6335336 (7H: 345673515–345673602 bp)] were located in the QTL hotspots region of 3_3 (631.86–646.92 Mb) and 7_5 (345.67–381.62 Mb), respectively , which encodes the leucine-rich repeat receptor-like protein kinase family protein and is nearby the QTL hotspot 3_1 (661.19–670.02 Mb) identified in this study  for Tgw, Ga, and Gd, encoding a grain length protein  domain, is a negative regulator of grain size and organ size , 9 and 4 genes from rice, maize, and wheat, respectively . The baris study . The ric in rice . Hence,  protein . The ricgan size . Many pr of rice . Moreovein wheat . TherefoIn summary, in this study, we identified 60 reliable QTLs and 27 QTL hotspots for the traits of grain size and weight in barley, using a single-environment QTL analysis, MET analysis, and covariate QTL analysis. Moreover, we also systematically explored the genetic correspondence between the QTLs identified in this study and known yield-related genes in barley and grain size/weight related genes in other cereal species. A total of 45 barley genes or orthologs were identified as promising candidate genes for barley grain size and weight, 20 of which were located in the QTL hotspot region underlying barley grain size and weight. These potential candidate genes are worth exploring and studying in detail. Our findings will enhance our understanding of the genetic basis of barley grain size and weight and may provide new information to facilitate high-yield breeding in barley.DS and GS conceived this study and designed the experiments. QW, BD, YC, and YW conducted the experiments and phenotyping measurements. QW performed the statistical analysis and wrote the manuscript. XR coordinated the experiments and oversaw the data analysis. DS and GS modified the manuscript. CL produced the Huaai 11 and Huadamai 6 DH population. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antibiotic resistance mediated by bacterial enzymes constitutes an unmet clinical challenge for public health, particularly for those currently used antibiotics that are recognized as “last-resort” defense against multidrug-resistant (MDR) bacteria. Inhibitors of resistance enzymes offer an alternative strategy to counter this threat. The combination of inhibitors and antibiotics could effectively prolong the lifespan of clinically relevant antibiotics and minimize the impact and emergence of resistance. In this review, we first provide a brief overview of antibiotic resistance mechanism by bacterial secreted enzymes. Furthermore, we summarize the potential inhibitors that sabotage these resistance pathways and restore the bactericidal activity of inactive antibiotics. Finally, the faced challenges and an outlook for the development of more effective and safer resistance enzyme inhibitors are discussed. The emergence, prevalence, and rapid spread of antibiotic resistance in pathogens represent an urgent threat to our medical system for the treatment of bacterial infectious diseases ,2,3. AccThese compounds that directly inhibit antibiotic resistance could be explored as antibiotic adjuvants. Thus, elucidation of antibiotic resistance mechanisms would contribute to novel antibiotic adjuvants’ discovery. To date, the molecular mechanisms of antibiotic resistance involve reduced cell membrane permeability, chemical structure changes of targets, active efflux of the intracellular antibiotics, and inactivation of antibiotics by resistance enzymes ,28,29. TDifferent from conventional antibiotics that directly target the components or progress that are essential for bacterial growth, inhibitors of resistance enzyme always have no or little direct antibacterial activity. However, these inhibitors could decrease the activity of resistance enzymes by specifically binding to the active site or inhibiting the transcription and translation of resistance genes. For example, clavulanic acid, a β-lactam-containing natural product, is a valuable inhibitor of β-lactamase . Most noMost antibiotics act by specifically binding to their targets, thereby preventing the physiological function of these targets. However, in the long-term competition with antibiotics, some bacteria have evolved multi-detoxifying enzymes to inactivate clinically relevant antibiotics such as β-lactams, carbapenems, and aminoglycosides . Based oKlebsiella pneumoniae carbapenemase (KPC) . Mo. Momcr-1bacteria . Meanwhitability ,113,114.tability ,116. Suctability ,118. Nottability ,120.An alternative strategy to counter the antibiotic resistance mediated by resistance enzyme is the use of inhibitors that specifically bind to the active site of enzymes and restore the activity of antibiotics. During the past decades, many enzyme inhibitors have been identified and introduced into the clinical setting, particularly for β-lactamases. In the following, we will briefly summarize the development of hydrolytic enzyme inhibitors and modifying enzyme inhibitors occurred in the past decades and highlight the novel, recently identified enzyme inhibitors.1)), a β-lactam-containing natural product, was the first identified β-lactamases inhibitor in the 1970s, isolated from a β-lactam antibiotic-producing strain of Streptomyces clavuligerus [2)) and sulbactam (3)) that are paired with piperacillin and ampicillin, respectively [4)) maintains the capacity to covalently acylate its β-lactamase targets [K. pneumoniae carbapenemase (KPC) [5)) paired with imipenem or cilastatin are also being investigated [A. baumannii infections, owing to a wide variety of class D β-lactamases expressed by this micoorganism [6)) was optimized as the best candidate with high inhibition activity towards clinically relevant class A, C, and D β-lactamases and penicillin-binding proteins (PBPs). Importantly, the combination of sulbactam and ETX2514 shows high efficacy in MDR A. baumannii mouse infection models [7)) based on a cyclic boronic acid pharmacophore was discovered, which is used paired with meropenem against Gram-negative bacteria, particularly for KPC-producing carbapenem-resistant Enterobacteriaceae (CRE) [Clavulanic acid  ,135. Avyse (KPC) . Besidesstigated ,138. Notorganism . By modin models . In addiae (CRE)  ), a fungus-derived natural product [K. pneumoniae [Ki = 0.4 ± 0.1 µM, Kd ≈ 0.2 µM), phosphonoacetate, and acetylphosphonate (9/10/11)), were shown to be potent inhibitors of the fosfomycin-resistance protein FosA [Except for Ser-β-lactamase inhibitors, many efforts are being made to identify potential metallo-β-lactamases inhibitors because of the increasing concern for MDR Gram-negative bacterial infections. There are two different strategies to identify clinically useful metallo-β-lactamases inhibitors, namely, Znin vitro ,143. Besin vitro . By meanin vitro . Notablyin vitro . Aspergi product ,148 disceumoniae . Besidesein FosA .12/13)), restored gentamicin activity against ANT-(2″)-Ia-expressing bacteria [14)) was designed based on the 1,3-diamine pharmacophore found in aminoglycoside structures and were shown to be competitive inhibitors of ANT(2″)-Ia and APH(3′)-IIIa [Aminoglycoside antibiotics are the foremost bactericidal drugs that specifically target the ribosome , which sbacteria . Besides3′)-IIIa . A detai3′)-IIIa .15/16/17)) [18)) also displays inhibitory activity towards APH(2´´)-Ia [19)) binds to and inhibits APH(3´)-I (a dominant resistance mechanism in Gram-negative pathogens) by a pathway that is distinct from the binding mode of human kinases [Similarly, several potent inhibitors of APHs were identified . Because/16/17)) . Besides(2´´)-Ia , owing t kinases , offerin20)) was found. It inhibits the acetylation of aminoglycosides in vitro with IC50 values of 39.7 and 34.9 μM using kanamycin A or amikacin as substrates [Mycobacterium tuberculosis, Keith et al. tested 23,000 molecules, of which 300 (1.3%) showed a reasonable degree of inhibition against AACs, and one compound (21)) showed IC50 values of 0.364 μM [22)), a natural product isolated from Gymnascella aurantiaca, circumvents aminoglycoside antibiotics resistance by inhibiting AME AAC(6′)-Ie/APH(2′′)-Ia [The development of computer-assisted technology undoubtedly facilitates a versatile screening approach for novel resistance enzyme inhibitors, particularly for high-throughput screening (HTS). For instance, in silico molecular docking simulation provides a viable strategy to identify enzyme inhibitors ,162,163.bstrates . To scre0.364 μM . Aranoro(2′′)-Ia  , trans-3,5-dimethoxy-4′-hydroxystilbene), isolated from fresh leaves or fruit, has been previously reported owing to its potent anticancer [E. coli of both human and animal origins was observed. However, more mechanistic studies are required to elucidate the potentiation activity of pterostilene. Transcription inhibition of enzyme-coding genes also provides a potential approach to rescue antibiotic activity, such as specifically designed antisense oligonucleotides [24)) was designed to target mcr-1 mRNA, preventing translation and modulating colistin resistance [Two successful strategies were applied for screening MCR-1 inhibitors, including directly looking for molecules interacting with MCR-1 protein and targeting its mRNA . Pterostticancer ,177, antticancer , and antticancer . Recentlticancer , which rleotides ,182,183,leotides . Besidesleotides . Therefoleotides , cell-peleotides , polymerleotides , and nanleotides  would fasistance . Excitinsistance ,192,193,sistance ,196,197,sistance ,198, andsistance ,201,202.Although numerous compounds that inhibit different bacterial resistance enzymes have been found, instances of their clinical use are limited to β-lactamases inhibitors. There are two major concerns, that is, low efficacy and unspecific toxicity of these compounds in vivo. It is noteworthy that some candidates failed to demonstrate significant efficacy in vivo due to their liability to degradation by proteases. Thus, activity screening in vitro alone is not enough to show evidence of their therapeutic potential. Alternatively, some animal infection models could be straightforwardly utilized to evaluate the therapeutic effectiveness of identified leads, such as the silkworm infection model, which has conserved immune response and comparable pharmacokinetics with mammals . In addiAntibiotics resistance, caused by bacterial resistance enzymes that have hydrolytic and modifying effects, poses a foremost and serious threat to public health worldwide. Despite the challenges faced in the screening of novel resistance enzyme inhibitors and in bringing them into the market, the discovery of enzyme inhibitors provides a complementary strategy to new antibiotic discovery. Importantly, these inhibitors can potentiate or restore the antibacterial activity of some promising antibiotics that are vulnerable and can be hydrolyzed or modified. The wide application of β-lactamase inhibitors in the clinic over the past decades is the most robust proof of concept. In addition, the pairing of inhibitors with antibiotics also contributes to minimizing the burden and emergence of antibiotic resistance. Overall, resistance enzyme inhibitors show great promise. Nevertheless, other novel strategies should be continuously explored to preserve the efficacy of antibiotics in this resistance era.


Chelidonium majus is a well-known plant, the seeds of which are dispersed by ants. Previous studies have identified the presence of primary fatty acids in its elaiosomes and seeds. However, the molecular mechanisms underlying fatty acid biosynthesis in elaiosomes remain unknown.Elaiosomes are specialized fleshy and edible seed appendages dispersed by ants. Lipids are the primary components of elaiosomes. C. majus, and understand the expression patterns of genes associated with fatty acid biosynthesis, four different developmental stages, including the flower-bud (Ch01), flowering (Ch02), young seed (Ch03), and mature seed (Ch04) stages, were chosen to perform whole-transcriptome profiling through the RNA-seq technology (Illumina NGS sequencing).In order to gain a comprehensive transcriptional profile of the elaiosomes and seeds of DESA1), and malonyl CoA-ACP transacylase were involved in palmitic acid synthesis; stearoyl-CoA desaturase and DESA1 were involved in oleic acid synthesis, and acyl-lipid omega-6 desaturase was involved in linoleic acid synthesis.A total of 63,064 unigenes were generated from 12 libraries. Of these, 7,323, 258, and 11,540 unigenes were annotated with 25 Cluster of Orthologous Groups, 43 Gene Ontology terms, and 373 Kyoto Encyclopedia of Genes and Genomes pathways, respectively. In addition, 322 genes were involved in lipid transport and metabolism, and 508 genes were involved in the lipid metabolism pathways. A total of 41 significantly differentially expressed genes (DEGs) involved in the lipid metabolism pathways were identified, most of which were upregulated in Ch03 compared to Ch02, indicating that fatty acid biosynthesis primarily occurs during the flowering to the young seed stages. Of the DEGs, acyl-ACP thioesterases, acyl carrier protein desaturase ( Elaiosomes are highly specialized nutrient-rich structures that are attached to the seeds of many plant species. The term elaiosome was introduced for the first time by Myrmecochory is regarded as a mutualistic relationship that is beneficial for both plants and ants. Ants typically collect and transfer whole diaspores (seed + elaiosome) to their nests, wherein the elaiosomes are consumed and the seeds are discarded unharmed within or outside the nests, which are in turn generally regarded to provide optimal germinating conditions . The elaMany studies have described several factors that likely determine the efficiency of myrmecochory. The sizes of seeds and elaiosomes, the elaiosome size/seed size ratio, and their physical surfaces influence the ability of ants to disperse the diaspores . In addiChelidonium majus L., commonly known as greater celandine, is the only species of the genus Chelidonium in the Papaveraceae family. C. majus is an herbaceous perennial plant  with conspicuous coronal elaiosomes  conducted a detailed study on the complete ontogeny of C. majus elaiosomes and seeds using standard techniques for plant anatomy and histochemistry ; after flowering stage (post-pollination) mitotic activity ceases, and the elaiosome develops solely through very active cell growth. The mature elaiosome of C. majus is composed of two cell types, small basal cells and very elongated outer cells. Both cell types, especially the outer cells, contain a large amount of lipids , also known as high-throughput sequencing, has emerged as a powerful tool for profiling the whole transcriptome from non-model plants that do not have available reference genomes , fertilized ovaries, young seeds (testa white to brown), and mature seeds (testa black and hard) were collected as RNA-seq materials. They were frozen in liquid nitrogen and stored at −80 °C until further use. Three samples were collected as biological repeats for each developmental stage.The sampled plants of nditions . To gain defined . In the  defined , floweri defined , young s defined , and mat defined  for compTo obtain transcriptome expression profiles, RNA-seq transcriptome libraries were prepared using the TruSeqTM RNA sample preparation Kit from Illumina  and five μg of total RNA. First, messenger RNA was isolated through the polyA selection method using oligo (dT) beads and fragmented in a fragmentation buffer. Next, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit  and random hexamer primers . Then, the synthesized cDNA was subjected to end-repair, phosphorylation, and “A” base addition according to Illumina’s library construction protocol. Libraries were size selected for cDNA target fragments of 200–300 bp on a 2% Low Range Ultra Agarose gel followed by polymerase chain reaction (PCR) amplification using Phusion DNA polymerase  for 15 cycles. After quantification using TBS380, the paired-end RNA-seq sequencing library was sequenced using Illumina HiSeq 4000 (2 × 150 bp read length). RNA samples were named Ch01a/b/c (flower-bud stage), Ch02a/b/c (flowering stage), Ch03a/b/c (young seed stage), and Ch04a/b/c (mature seed stage). A total of 12 cDNA libraries were then constructed and sequenced using the Illumina deep sequencing platform.E-value of less than 1.0 × 10−5 was set. The BLAST2GO program was used to get Gene Ontology (GO) annotations of uniquely assembled transcripts for describing biological processes, molecular functions, and cellular components . ChamQ SYBR Color qPCR Master Mix (2X)  was used for Quantitative real-time PCR (qRT-PCR). qRT-PCR was performed on Line gene 9600 plus Fluorescence quantitative PCR instrument . Each sample contains three independent biological replicates.https://doi.org/10.6084/m9.figshare.8044838.v1, File 1). Some reads comprise of a contig, and some contigs comprise of a unigene. We obtained a total of 63,064 unigenes  for all studied samples; these unigenes had an average length and N50 value of 1,114.56 bp and 1,565 bp, respectively. After sorting the assembled unigenes from large to small, the N50 value was calculated as half of the total length of the unigenes. The size distribution of the assembled transcripts is shown in C. majus elaisomes and seeds were reliable.In this study, a total of 12 libraries were sequenced. After removing the low-quality reads and adaptor sequences, the remaining clean data comprised of approximately 61 Gb of sequence data (171 million reads from the flower-bud stage (Ch01); 141 million reads from flowering stage (Ch02); 148 million reads from young seed stage (Ch03); and 154 million reads from mature seed stage (Ch04)). The results are shown in E-values < 1e−5. After alignment, gene annotation, functional classification, and metabolic pathway assignment, a total of 30,143 (47.80%) of the unigenes were successfully annotated , which indicated that their functions were relatively conserved and diverse.To verify the potential functions of the 63,064 assembled unigenes, we performed sequence similarity BLASTx searches against the NR (NCBI protein nonredundant), COG, GO, and KEGG protein databases with E-values of the hits in the NR database indicated that 20,378 unigenes, representing more than half of the mapped sequences (69.43%), showed significant homology , while 8,973 unigenes (about 30.57%) had E-values ranging from 1.0 × 10−30 to 1.0 × 10−5 . Smaller E-values distribution , Phoenix dactylifera , Populus trichocarpa , Prunus persica , and Jatropha curcas   were mapped to the NR database. In addition, the distribution of ribution  indicateribution . To iden, 3.63%) . A totalC. majus are closely associated with enzyme-catalyzed reactions, cell structure formation, and metabolic processes.By conducting GO functional classification, a total of 258 unigenes were classified into one or more terms and divided into 43 functional groups, belonging to three major categories . In the C. majus. Notably, 322 unigenes (4.40%) were classified under the “lipid transport and metabolism” category, which could provide useful information for further studies on lipid metabolism in C. majus elaisomes and seeds.To predict the potential function of genes and evaluate the completeness of the transcriptome libraries, all assembled unigenes were searched against the COG database. A total of 7,323 mapped unique sequences were clustered into 25 functional categories . Among tC. majus elaisomes and seeds, all annotated unigenes were mapped to the reference pathways in the KEGG. The analysis identified a total of 11,540 unigenes that were assigned to five clusters and distributed across 373 KEGG pathways , glycerolipid metabolism , fatty acid degradation , biosynthesis of unsaturated fatty acids , fatty acid elongation , fatty acid biosynthesis , ether lipid metabolism , sphingolipid metabolism , linoleic acid metabolism , steroid biosynthesis , arachidonic acid metabolism , steroid hormone biosynthesis , synthesis and degradation of ketone bodies , and primary bile acid biosynthesis  pathways were involved in these pathways  (77.9%), followed by the monounsaturated fatty acid, oleic acid (C18:1) (16%) (C. majus was formed during the flower-bud stage (Ch01), while elaiosome cell growth and enlargement primarily occurred from the flowering stage (Ch02) to the young seed stage (Ch03); the maturation of the seed and elaiosome occurred from the young seed stage to the mature seed stage (Ch04). Therefore, to obtain insight into the regulation of the biosynthesis of saturated fatty acids and unsaturated fatty acids in elaiosomes and seeds, we analyzed the DEGs of the lipid metabolism pathways in Ch02 compared to Ch01, Ch03 compared to Ch02, and Ch04 compared to Ch03.Analysis of the fatty acid compositions of 1) (16%) . Further1) (16%)  indicateC. majus elaisomes and seeds (fabG) (ACOX1) (DESA1) (fabF) (Accase) (ACSL)   (fabZ) (FAD2) (FatA/B) (SCD)  and the biosynthesis pathway of unsaturated fatty acids (Path: ko01040) at the four developmental stages of nd seeds , we idens (fabG) , acyl-Co (ACOX1) , acyl ca (DESA1) , and β-k) (fabF)  were fou(Accase) , acyl co) (ACSL) , DESA1, e (fabD) , fabF, fe (fabI) , β-hydro) (fabZ) , acyl-li) (FAD2) , acyl-AC(FatA/B) , and steB) (SCD)  were cleelopment , the remACSL, DESA1, fabD, fabF, fabG, fabI, fabZ, and FatA/B, were significantly upregulated in Ch03. On the other hand, a gene expression analysis in Ch04 compared to Ch03  but primarily occurs from the flowering stage to the young seed stage. C. majus was not included in Hughes’s study, whether the high palmitic acid content in C. majus elaiosomes is similar to that in the ants that disperse the C. majus seeds remains unknown and needs further study.The enzymes esis see , are parfabG. The second process is the conversion of stearic acid (C18:0) to oleic acid , which involves genes encoding the enzymes, SCD (Delta-9 desaturase) and DESA1. The third process is the conversion of oleic acid  to linoleic acid , which involves genes encoding the enzyme, FAD2 (Delta-12 desaturase). The above findings showed that some palmitic acid was converted into stearic acid (saturated fatty acid), and the latter was subsequently desaturated to form oleic acid and linoleic acid.C. majus indicated that the whole diaspores (seed + elaiosome) were probably used to attract ants that contain high levels of  nutrients. In addition, oleic acid was shown to promote ant removal behavior  represents significantly regulated genes.Click here for additional data file.10.7717/peerj.6871/supp-3Supplemental Information 3ACCase, FatB, FabA, FatA/B, DesA1/2, ACSL.Red represents up-regulated gene, green represents down-regulated gene. Overview of de novo fatty acid biosynthesis pathways: (A) Different genes expression between Ch01 and Ch02; (B) Different genes expression between Ch02 and Ch03; (C) Different genes expression between Ch03 and Ch04. Indentified enzymes include: Click here for additional data file.10.7717/peerj.6871/supp-4Supplemental Information 4Click here for additional data file.


Phytophthora infestans (Mont.) de Bary, a hemibiotrophic oomycete, has caused severe epidemics of late blight in tomato and potato crops around the world since the Irish Potato Famine in the 1840s. Breeding of late blight resistant cultivars is one of the most effective strategies to overcome this disruptive disease. However, P. infestans is able to break down host resistance and acquire resistance to various fungicides, possibly because of the existence of high genetic variability among P. infestans isolates via sexual and asexual reproduction. Therefore, to manage this disease, it is important to understand the genetic divergence of P. infestans isolates. In this study, we analyzed the genomes of P. infestans isolates collected from Egypt and Japan using various molecular approaches including the mating type assay and genotyping simple sequence repeats, mitochondria DNA, and effector genes. We also analyzed genome-wide single nucleotide polymorphisms using double-digest restriction-site associated DNA sequencing and whole genome resequencing (WGRS). The isolates were classified adequately using high-resolution genome-wide approaches. Moreover, these analyses revealed new clusters of P. infestans isolates in the Egyptian population. Monitoring the genetic divergence of P. infestans isolates as well as breeding of resistant cultivars would facilitate the elimination of the late blight disease. Phytophthora infestans (Mont.) de Bary is a hemibiotrophic oomycete that causes late blight disease in tomato (Solanum lycopersicum) and potato (Solanum tuberosum). Globally, many epidemics of tomato and potato late blight have been reported since the end of the 19th century, among which the Irish Potato Famine in the 1840s was probably the worst. P. infestans is reported to have originated either in South America [P. infestans remains a major threat to global food security [P. infestans is a heterothallic pathogen with two mating types (A1 and A2), which reproduce sexually through oospores [P. infestans also exhibits asexual reproduction, with high genetic variability. Since 1980, the A1 mating type was dominant globally, outside the region of its origin [P. infestans is able to break down major resistance genes in host plant species and develop resistance against various fungicides [ America  or in ce America , 4. Alth America , P. infesecurity . P. infeoospores . P. infes origin . This was origin . This rengicides .Solanum pimpinellifolium, in breeding programs [P. infestans is complicated and has evolved over time, probably because of sequence variations in virulence genes, also known as effector genes, which encode proteins that suppress the plant immune system and alter the host response to accelerate the infection process. Therefore, knowledge of the genetic divergence of P. infestans isolates is important to control the late blight disease.Breeding of late blight resistant cultivars is one of the most effective strategies to overcome this disruptive disease. Resistance genes and/or DNA markers linked to resistance loci are powerful tools for the selection of resistant lines via marker-assisted selection. To the best of our knowledge, genetic loci have been identified in late blight resistant germplasms of wild tomato, programs –15. MoreP. infestans populations [P. infestans reference genome sequence [P. infestans populations on a genome-wide level. The genome-wide genotyping methods have been applied to P. infestans populations to reveal their phylogenetic relationship [P. infestans isolates tested has been limited and insufficient to cover the P. infestans populations spread across the world.Over the last several decades, PCR assays for assessing nucleotide sequence variations in mitochondrial DNA (mtDNA), simple sequence repeats , and effector genes have been employed to investigate the genetic diversity of ulations –20. Advasequence , reducedsequence  and restsequence , have betionship , 19; howP. infestans clonal linages, based on DNA analysis, epidemiology, and population dynamics of this destructive plant pathogen [P. infestans populations collected from Egypt and Japan; neither of these regions has been represented in the genome-wide genotyping analysis of P. infestans isolates to date. We investigated the mating types, mtDNA haplotypes, and SSR marker genotypes of the isolates as well as genome-wide genotypes, based on double-digest restriction-site associated DNA sequencing (ddRAD-Seq) and WGRS approaches, to gain insights into the diversity of P. infestans populations at the genome-wide level.A decision support system in agricultural domains would be essential for successful plant disease management. Knowledge of the evolution of pathogen , would bP. infestans isolates were used in this study; these included 62 isolates collected from seven counties in Egypt, and 18 isolates obtained from the National Institute of Agrobiological Sciences (NIAS) in Japan, which were collected from three prefectures  markers to detect sequence variations in P1, P2, P3, and P4 regions of the mitochondrial genome  Tables. AVR1, AVR2, AVR2-like, AVR3a, and AVR4, across all 80 isolates. DNA amplicons were obtained for four genes  (NitRed), but not for AVR1. The AVR4 and NitRed genes were amplified from all 80 isolates. However, the remaining three genes showed polymorphism; AVR3a, AVR2, and AVR2-like were not detected in 4, 12, and 19 isolates, respectively. All of these three genes  were not detected in the Egyptian isolate EG_36, while AVR2-like was not amplified in all of the Japanese isolates.We tested the PCR assay to detect five effector genes, nd AVR4)  Tables aAVR2 amplicon. The other two clusters contained only Japanese isolates, with 13 isolates in JPN1 and five in JPN2. Isolates in the JPN1 cluster were collected from 1987–1992, while those in the JPN2 cluster were collected in 2013.We tested 16 SSR (or microsatellite) markers in the 80 isolates. A total of 54 peaks were detected, ranging from two peaks in five markers  to seven peaks in SSR4  Tables. P. infestans strain T30-4 (version ASM14294v1) at an average mapping rate of 92.4%, and 996 loci were detected as high-confidence SNPs based on the read alignments.The ddRAD-Seq approach was used to analyze genome-wide SNPs. A total of 23 Egyptian isolates were removed from subsequent analysis because of low-quality data, probably because the DNA available for analysis was of poor quality and insufficient quantity. An average of 430,489 high-quality ddRAD-Seq reads were obtained per sample from 57 isolates . Reads wClustering analysis, based on SNPs, grouped the tested isolates into four clusters, EGY1, EGY2, JPN1, and JPN2, containing 32, 7, 13, and 5 isolates, respectively, in accordance with the results of the SSR marker analysis . FurtherP. infestans at an average mapping rate of 94.5%, and 760,928 high-confidence SNPs, including 479,784 transitions and 281,144 transversions (Ts/Tv ratio = 1.7), were detected. The Egyptian isolates EG_18 and EG_73 contained the lowest and highest number of SNPs, respectively. The average density of SNPs was 3.3 per kb. Analysis of SNP annotations using SnpEff indicated that intergenic variants were the most common , followed by synonymous variants  and missense variants   was obtained . The rea8; 7.4%) . A totalAmong the Egyptian isolates, CNVs were detected between EG_18, as a reference, and each of the remaining 16 isolates. Between any two isolates, an average of 5,402 CNVs were detected in each isolate, ranging from 533 CNVs in EG_22 to 6,735 CNVs in K92-61. The average size of a CNV was 4,068 bp, with the longest of 79 kb in EG_75.P. infestans isolates, we used the SNP data and data publicly available on 11 isolates [To determine the lineage of isolates . On the P. infestans T30-4 reference genome sequence, 594 and 194 genes have been annotated as encoding RxLR- and Crinkler-type effector proteins, respectively. Among these, four genes were identified in the P. infestans T30-4 genome sequence: Avr1 (PITG_16663) in the contig sequence of supercont1.51, Avr2 (PITG_08943 and PITG_22870) on supercont1.16, Avr3a (PITG_14371) on supercont1.34, and Avr4 (PITG_07387) on supercont1.11. A total of six non-synonymous SNPs causing missense mutations were identified. Four of the six missense mutations caused amino acid substitutions at positions 19, 80, 103, and 124 of PITG_14371 (Avr3a). While the first three mutations were observed in the ten isolates belonging to clusters EGY1-1 and EGY1-2, the fourth mutation was identified in all 12 isolates belonging to the EGY2 cluster, in addition to the isolates in EGY1-1 and EGY1-2 clusters. Among the two remaining missense mutations, one was detected at amino acid position 31 of PITG_08943 (Avr2) in the two isolates belonging to the JPN2 cluster and one isolate (GBK 14) belonging to JPN1, and the other was detected at amino acid position 139 of PITG_07387 (Avr4) in seven isolates belonging to clusters EGY2, JPN1, and JPN2. No SNPs were detected in PITG_16663 (Avr1).In the Avr1, Avr2, Avr3a, and Avr4. CNVs of Avr1 and Avr4 were observed in 12 isolates of EGY1-1, EGY1-2, and JPN2, while CNVs of Avr2 were detected in three isolates belonging to the cluster JPN1 and 12 isolates belonging to EGY1-1, EGY1-2, and JPN2. The CNV of Avr3a was identified in only one isolate, EG_75, belonging to the sub-cluster EGY1-1.CNVs were detected in P. infestans isolates collected from Egypt and Japan using a series of thorough genotyping assays at different levels, including the target gene level (SSRs and RFLPs), genome-wide level (ddRAD-Seq and WGRS), and reproduction biology level (mating type assay). While A1 and A2 mating types were observed among Japanese isolates, the A1 mating type was predominant among Egyptian isolates tested. This suggests that the A1 mating type might be adapted to the Egyptian environment [P. infestans genome may be one of the possible drivers of genetic diversity [P. infestans. However, effector genes do not diverge among the Egyptian isolates, possibly because most of the tomato and potato cultivars in Egypt are susceptible to late blight, leading to minimal selection pressure and low mutation rates. Nonetheless, the population structure of P. infestans isolates, regardless of their reproduction mode, was broader than that suggested by previous studies on American and European isolates ; these isolates were collected from the leaves and fruits of tomato plants and leaves of potato plants growing in three prefectures, Hokkaido, Ibaraki, and Shizuoka, from 1987–2013  . Additio987–2013 . P. infeeviously . The DNAP. infestans isolates and those of EG_136 [Mycelium plugs of f EG_136  and K92-f EG_136 .AVR1, AVR2, AVR2-like, AVR3a, and AVR4, in P. infestans isolates was tested in the current populations (The mtDNA of isolates was amplified from the genomic DNA using primers listed in ulations . The PCRP. infestans isolates using sequence-specific primers (Sixteen SSR markers –42 were  primers . DNA ampPstI and MspI restriction enzymes and used to construct ddRAD-Seq libraries. The libraries were sequenced using MiSeq (Illumina) in paired-end 251 bp mode, as described previously [Genomic DNA of isolates was double-digested with eviously , 44.http://hannonlab.cshl.edu/fastx_toolkit), respectively. The cleaned reads were mapped onto the reference genome sequence of P. infestans strain T30-4  [Data processing was performed as described previously , 44. Low0000000)  using Bo0000000) . To obta0000000)  and the 0000000) . High-co0000000) , based oP. infestans, as described previously [Sequencing libraries with an insert size of 600 bp were prepared for 17 isolates of eviously , 49, andeviously . CNVs weeviously .Dendrograms were constructed using the unweighted pair-group method with average linkage (UPGMA). Additionally, PCA was performed to determine the relationship among samples using the TASSEL software , with thAccession numbers: Double digest restriction-site associated DNA sequencing (ddRAD-Seq) and whole genome resequencing (WGRS) data are available at the DDBJ Sequence Read Archive database under the accession number DRA007610.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Fagopyrum esculentum Moench) is a widely cultivated minor crop with excellent economic and nutritional value in temperate zones. However, little is known about the molecular mechanisms of seed development in common buckwheat (Fagopyrum esculentum). In this study, we performed RNA-Seq to investigate the transcriptional dynamics and identify the key genes involved in common buckwheat seed development at three different developmental stages. A total of 4619 differentially expressed genes (DEGs) were identified. Based on the results of Gene Ontology (GO) and KEGG analysis of DEGs, many key genes involved in the seed development, including the Ca2+ signal transduction pathway, the hormone signal transduction pathways, transcription factors (TFs), and starch biosynthesis-related genes, were identified. More importantly, 18 DEGs were identified as the key candidate genes for seed size through homologous query using the known seed size-related genes from different seed plants. Furthermore, 15 DEGs from these identified as the key genes of seed development were selected to confirm the validity of the data by using quantitative real-time PCR (qRT-PCR), and the results show high consistency with the RNA-Seq results. Taken together, our results revealed the underlying molecular mechanisms of common buckwheat seed development and could provide valuable information for further studies, especially for common buckwheat seed improvement.Seed development is an essential and complex process, which is involved in seed size change and various nutrients accumulation, and determines crop yield and quality. Common buckwheat ( Arabidopsis and rice through analysis of mutants, transcriptomes, and quantitative trait locus (QTLs) [Brassica napus [Seed or grain development is one of the most crucial processes in plant development. It not only determines the successful racial continuation of seed plants, but also affects the final seed yield and seed quality ,2. Geners (QTLs) ,6,7,8,9.s (QTLs) ,11, wheas (QTLs) ,13, soybs (QTLs) ,15,16, bs (QTLs) , Tartarys (QTLs) ,18, chics (QTLs) , and Braca napus ,21 by trFagopyrum esculentum) is an annual minor crop which belongs to the eudicot family Polygonaceae, mainly cultivated in temperate regions of Asia, Europe, and North America [Fagopyrum tataricum). Therefore, it is of considerable interest to identify genes and dissect the molecular mechanisms of seed development in common buckwheat, as this will contribute to common buckwheat and tartary buckwheat seed improvement, including seed formation, morphogenesis, size, and nutrient accumulation. To date, several studies have investigated the transcriptional profile of the developing seeds of common buckwheat though RNA-Seq [Common buckwheat , filling stage (S2), and initial maturity stage (S3)) with the purpose of investigating its transcriptional dynamics and identifying candidate genes involved in seed development. In addition, some vital seed-development candidate genes were verified by quantitative real-time PCR (qRT-PCR). The results obtained in this study will enhance our understanding of the molecular mechanisms of common buckwheat seed development and provide candidate genes for future common buckwheat seed improvement.To gain insight into the transcriptional dynamics of seed development in common buckwheat, nine libraries from three samples  were constructed and subjected to high-throughput RNA-Seq. An overview of the sequencing data is shown in To identify the DEGs during the common buckwheat seed development, the gene expression profile clustering was subjected to first analysis. All 54,582 genes were assigned to seven different clusters by the K-means method, which led to the identification of genes with similar expression during all stages of seed development, and indicated that a large number of genes were differentially expressed between different samples . Then, gene expression levels among the three developmental stages were subjected to a pairwise comparison. A total of 4619 DEGs were identified, of which 745 (492 up- and 253 down-regulated), 2449 (1506 up- and 943 down- regulated), and 3680 (2162 up- and 1518 down-regulated) were found between S1 and S2, S2 and S3, and S1 and S3, respectively B. Of theTo further explore the function of these DEGs, Gene Ontology (GO) enrichment analysis was carried out. As a result, 395 (S1 vs. S2), 1244 (S2 vs. S3), and 1878 (S1 vs. S3) DEGs were divided into biological process, cellular component, and molecular function categories . As showTo investigate the metabolic pathways involved by the DEGs, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. A total of 237 (S1 vs. S2), 789 (S2 vs. S3), and 1244 (S1 vs. S3) DEGs were assigned to 69, 99, and 113 KEGG pathways, respectively . All of 2+ signaling pathway regulate many plant development processes, including seed development [2+ signaling pathway were identified (2+ exchangers (CCXs), Ca2+-dependent protein kinases (CDPKs), CBL-interacting protein kinases (CIPKs), Ca2+-transporting ATPase, and Cation/H+ antiporter (CHX). As shown in CML), up-regulated at S2 and down-regulated at S3 , only down-regulated at S2 (1 CDPK), only up-regulated at S3 (2 CMLs), and only down-regulated at S3 .Genes involved in Caelopment ,30. Accoentified . These gIAA) was up-regulated at S2, five IAA genes were up-regulated at S3, and one ortholog of AFR encoding for auxin response factor was up-regulated both at S2 and S3  and one AHP (histidine-containing phosphotransfer protein) were increased at S2 and S3, respectively (PIF3 (phytochrome-interacting factor 3) showed up-regulated trends during seed development, and one ortholog of bHLH127 was increased only at S3 (PP2C (protein phosphatase 2C) and ABF (encoding for ABA responsive element binding factor), respectively. In addition, one PYR/PYL (abscisic acid receptor), one PP2C, and one SnRK2 (serine/threonine protein kinase) were down-regulated at S3 (EIN3 (Ethylene-Insensitive Protein 3) were up-regulated at S2 and S3, respectively. Furthermore, two ETR (Ethylene Receptor), one EIN4 (Ethylene-Insensitive Protein 4), and one CTR1 (serine/threonine-protein kinase) were down-regulated at S3 (BRI1 (Brassinosteroid Insensitive 1) was down-regulated at S3, and other BRI1 displayed up-regulated at S2 and down-regulated at S3 (JAZ) at S3 and down-regulated (TGA) at S2 , including the auxin, CK, GA, BR, ABA, ET, JA, and SA signaling pathways . In the 2 and S3 . Moreoveectively . In the ly at S3 . In the ed at S3 . In the ed at S3 . In the ed at S3 . In addiA) at S2 , respectAP2 (9), bHLH (11), and AP2 (22) for these three comparison groups, respectively  (DWF4 and CYP724B1) and one gene (GIF1) showed up-regulation only at S2 or at S3, respectively; five genes  were up-regulated during seed development stages; four genes , with high expression at S1 and S2, were down-regulated at S3; two genes (CYP78A13 and ANT) were up-regulated at S1 compared to S2, and followed by down-regulation at S3; however, three genes  displayed the opposite expression pattern with CYP78A13 and ANT, which were down-regulated at S1 compared to S2, and followed by up-regulation at S3  . Of theson at S3 . SUS), 3 UDP glucose pyrophosphorylase (UGPase), 14 ADP glucose pyrophosphorylase (AGPase), 8 granule bound starch synthase (GBSS), 24 starch synthase (SS), 4 starch-branching enzyme (SBE), and 3 debranching enzyme (DBE) genes were identified by using the starch biosynthesis-related genes in Arabidopsis to perform a homologous comparative search in the common buckwheat genome database  for the three replicates of each sample. These results indicated that the RNA-Seq had higher quality. Through comparison of gene expression patterns between different samples, 4619 DEGs were identified, and the DEGs involved in the Ca2+ signaling pathway, hormone signaling pathway, TFs, seed size, and starch biosynthesis were the main focus of this study. In total, 242.86 million clean reads were obtained from nine sequencing libraries, and 63.32 to 69.46% clean reads for each library were mapped to the reference genome. The map rate was relatively low, which might have been caused by the draft assembly of the common buckwheat genome . The Q302+ signal transduction pathways participate in many developmental processes in plants, including pollen tube growth, stem elongation, vascular development, embryogenesis, and seed germination [2+ signal transduction pathways play an important role in seed development with a focus on CDPK. For example, several CDPK genes were differential or special expression during seed development in rice, maize, and peanut, some of which expressed dominant in the early stage, while others expressed prominently in the latter stage [CDPK genes might be involved in different development process of seeds. Furthermore, OsCDPK2, OsCDPK11, and OsCDPK23 in rice and RcCDPK1 in castor have been functionally verified to be essential for seed development [CDPK genes were identified as DEGs during common buckwheat seed development. Among them, two were up-regulated at S2 and down-regulated at S3, and one was only decreased at S3, indicating that they might have an important role in the early stages of common buckwheat seed development. In contrast, the other one was down-regulated at S2, suggesting that it might have an opposed function during common buckwheat seed development with the previous three CDPK genes. In addition, we also identified another 18 DEGs involved in the Ca2+ signaling pathway, including 3 CML, 2 CBL, 2 CCX, 5 CIPK, 4 2+-ATPaseCa, and 2 CHX genes, which displayed similar expression patterns with CDPK genes. These results indicate that the Ca2+ signaling pathway is conserved in regulatory seed development and that these identified DEGs could be key candidate genes for the seed development of common buckwheat. The Camination ,30. In aer stage ,34,35,36elopment ,34,35,36Fagopyrum Tararicum), a dozen of these hormone-related DEGs were identified by RNA-seq, which displayed different expression patterns during seed development [JAZ and one TAG gene, which are the key genes in the JA and SA signaling pathways, respectively, were also differentially expressed during common buckwheat seed development, implying that the JA and SA signaling pathways probably also have important roles in common buckwheat seed development. It is well known that hormone signaling, including auxin, CK, GA, BR, ABA, and ET signaling, plays a crucial role in seed development ,9. In taelopment ,18. In tArabidopsis and rice. Among these, the expression level of the orthologs of AtAP2, AtDA1, AtEOD1, and OsSLG were down-regulated during seed development. Interestingly, AtAP2 [AtDA1 [AtEOD1[OsSLG [Arabidopsis and rice, respectively. This indicates that these genes from common buckwheat may also negatively regulate the seed size. In contrast, the orthologs of several positively-regulated genes for seed size, such as AtIKU2 [OsGRF4 [OsGIF1 [OsCYP78A13 [OsCYP724B1 [AtDET2 [AtDWF4 [OsWRKY53 [AtANT [AtTTG2 [OsCYP78A13 and AtANT were up-regulated at filling stage and down-regulated at initial maturity stage, indicating that they might regulate the accumulation of seed storage materials and ultimately affect seed size. In addition, an ortholog of AtIKU2 and two orthologs of OsSRS5 (positively regulated seed size) were down-regulated at both filling stage and initial maturity stage, suggesting that they might preform function for seed size at the embryogenesis phase, mainly related to cell division and expansion. Seed size is a key factor to determine yield in crops. The identification of genes associated with seed size will support yield improvement. To date, over 90 genes have been identified and functionally validated to regulate seed size in different seed plants [y, AtAP2 ,38, AtDA2 [AtDA1 , AtEOD1[1 [AtEOD1,40, and D1[OsSLG  have bees AtIKU2 , OsGRF4  [OsGRF4 ,44, OsGI [OsGIF1 ,44, OsCYCYP78A13 ,46, OsCYCYP724B1 , AtDET2  [AtDET2 , AtDWF4  [AtDWF4 , OsWRKY5OsWRKY53 , AtANT [3 [AtANT , and AtT [AtTTG2 , were inSUS, UGPase, AGPase, GBSS, SS, SBE, and DBE, catalyze starch biosynthesis [SUS, 3 UGPase, 14 AGPase, 8 GBSS, 24 SS, 4 SBE, and 3 DBE genes. Of these, seven SUS, one AGPase, one UGPase, one GBSS, two SS, two SBE, and one DBE genes were differentially expressed during the seed development of common buckwheat. This finding suggests that these identified starch biosynthesis DEGs might be the major functional genes for starch biosynthesis in common buckwheat seed, and could accelerate further functional analysis of them as well. The seeds of common buckwheat contain abundant starch, accounting for over 70% of seed dry weight ,24,25. Hynthesis . In our 2+ signaling, hormone signaling, TFs, seed size, and starch biosynthesis-related genes, and its differential expression patterns during common buckwheat seed development were verified by qRT-PCR. The results are helpful to further dissect the molecular mechanism of buckwheat seed development and provide a basis for buckwheat seed size and quality improvement.Seed development is predominantly regulated by genetic factors and is also greatly influenced by physiological pathways ,2. UsualThe common buckwheat cultivar “Chitian No. 1” was used in this study. It was planted in the growth chamber of the Research Center of Buckwheat Industry Technology, Guizhou Normal University , China, in Spring 2018. All plants were grown under natural daylight and subjected to normal management during the growth periods. Seeds were harvested on the 8th, 14th, and 21st days after full bloom, which corresponded to the pre-filling stage (S1), filling stage (S2), and initial maturity stage (S3). For each stage, approximately 200 mixed seeds were collected from the same five plants, and three independent biological replicates were performed. All samples were immediately frozen in liquid nitrogen and stored at −80 °C for RNA-Seq and qRT-PCR validation analyses.®Ultra™ II RNA Library Prep Kit for Illumina® . Finally, the constructed cDNA libraries were sequenced using the Illumina XtenPE150 system by Biomarker Technologies Co., Ltd. .Total RNA was extracted using the EASYspin Plus Plant RNA Kit  with three replicates for each sample, according to the manufacturer’s instructions. Contaminated DNA in the total RNA samples was removed using DNase I . The quality and concentration of the total RNA were determined using 1.2% agarose gel electrophoresis and a NanoDrop 2000c spectrophotometer . Then, the cDNA libraries for Illumina sequencing were constructed using the NEBNextQ < 20), and poly-N stretches (>10%). Then, the clean reads were mapped to the common buckwheat reference genome (ftp://ftp.kazusa.or.jp/pub/buckwheat/) to obtain the unigenes by using Tophat2 software [The raw reads were processed to produce clean reads by removing the adaptor sequences, low-quality sequence reads , software , KOG [59software , and KEGsoftware . The transcript abundance of each unigene was calculated and normalized to fragments per kilobase of transcript per killion fragments mapped (FPKM) . SignifiFeActin7 was used as the internal control. RT-qPCR was conducted using the SYBR premix Ex Taq kit  on ABI 7500 Fast Real-time PCR system . Each sample was analyzed in triplicate. The relative expression change of each gene was calculated using the 2Ct−ΔΔ method [qRT-PCR analysis was performed to verify the RNA-Seq data. These genes that reflected some of the functional categories and different expression levels were selected. The reference cDNA sequences of these genes were obtained from the common buckwheat genome sequence database. The RT-qPCR primers were designed using Primer 5.0 software according to the reference cDNA sequences . FeActint method . The Pea


The renin-angiotensin system (RAS), especially the angiotensin II (Ang II)/angiotensin II type 1 receptor (AT1R) axis, plays an important role in the aging process of the kidney, through increased tissue reactive oxygen species production and progressively increased oxidative stress. In contrast, the angiotensin 1-7 (Ang 1-7)/Mas receptor (MasR) axis, which counteracts the effects of Ang II, is protective for end-organ damage. To evaluate the ability of resveratrol (RSV) to modulate the RAS in aging kidneys, eighteen-month-old male C57BL/6 mice were divided into two groups that received either normal mouse chow or chow containing resveratrol, for six months. Renal expressions of RAS components, as well as pro- and antioxidant enzymes, were measured and mouse kidneys were isolated for histopathology. Resveratrol-treated mice demonstrated better renal function and reduced albuminuria, with improved renal histologic findings. Resveratrol suppressed the Ang II/AT1R axis and enhanced the AT2R/Ang 1-7/MasR axis. Additionally, the expression of nicotinamide adenine dinucleotide phosphate oxidase 4, 8-hydroxy-2′-deoxyguanosine, 3-nitrotyrosine, collagen IV, and fibronectin was decreased, while the expression of endothelial nitric oxide synthase and superoxide dismutase 2 was increased by resveratrol treatment. These findings demonstrate that resveratrol exerts protective effects on aging kidneys by reducing oxidative stress, inflammation, and fibrosis, through Ang II suppression and MasR activation. Aging causes progressive deterioration of organs, leading to impaired tissue function, increased vulnerability to stress, and death ,2. The kVarious processes are involved in the deterioration seen in aging kidneys, including oxidative stress, mitochondrial dysfunction, inflammation, altered calcium regulation, and activation of the renin-angiotensin system (RAS) ,7. The RResveratrol is a polyphenolic phytoalexin that exists naturally in various plant parts and products, such as grapes, red wine, berries, and peanut skins, and has numerous beneficial health effects ,14. It an = 7) received normal mice chow  and the resveratrol-treated group (n = 7) received normal mice chow mixed with resveratrol . The food was changed every 24 h and a calculated regular amount of food was fed daily, for a six month period. The experimental conditions were set with reference to the article of Baur et al. [Eighteen-month-aged male C57BL/6 mice were purchased from the Korea Research Institute of Bioscience and Biotechnology . The Animal Care Committee of the Catholic University of Korea approved the experimental protocol and the experiments were performed in accordance with our institutional animal care guidelines. Mice were housed in a controlled temperature and controlled light environment with 12:12-h light-dark cycles and had free access to water. The aged male C57BL/6 mice were divided into two groups: The control group  with access to water and food for 24 h. Urine collection was done every four weeks and data from month 18 and 24 was used in this experiment. Albuminuria  and urine creatinine concentration  were measured using ELISA kits. Serum creatinine concentrations and Blood urea nitrogen were measured using i-STAT system Cartridges . Creatinine clearance was calculated using a standard formula (urine creatinine (mg/dL) × urine volume (mL/24 h)/serum creatinine (mg/dL) × 1440 (min/24 h)).Kidney tissue samples were fixed in 10% formalin. The tissues were embedded in low-temperature melting paraffin, and 4 microns, thick sections were processed and stained with a periodic acid–Schiff (PAS), and Masson’s trichrome. The glomerular volume and mesangial matrix were quantified for kidney tissue cross-sections, using PAS staining. The relative mesangial area was expressed as mesangial/glomerular surface area. A finding of tubulointerstitial fibrosis was defined as a matrix-rich expansion of the interstitium in Masson’s trichrome. Ten randomly selected fields, per section, were assessed. All of these sections were examined in a blinded manner, using a color-image analyzer  and quantified using image J .2O2, in methanol, to block the endogenous peroxidase activity. Nonspecific binding was blocked with 10% normal horse serum. After incubating with the primary antibody to Ang II, AT2R and MasR , AT1R and 3-nitrotyrosine , and 8-OHdG , at 4 °C, overnight, antibodies were visualized with a peroxidase conjugated secondary antibody, using the Vector Impress kit . Sections were then dehydrated in ethanol, cleared in xylene and mounted without counterstaining. All sections were assessed using a color-image analyzer .Immunohistochemistry was performed to determine changes of Ang II, angiotensin target receptors  and oxidative stress using 8-hydroxy-deoxyguanosine (8-OHdG) and 3-nitrotyrosine. Paraffin sections were deparaffinized in xylene and hydrated in ethanol, before staining, and treated with an antigen-unmasking solution, consisting of 10 mM sodium citrate, pH 6.0 and then washed with phosphate-buffered saline. Sections were incubated with 3% HTotal protein was extracted from the kidney tissues, using Pro-Prep Protein Extraction Solution , according to the manufacturer’s instructions. Extracted protein was subjected to SDS-polyacrylamide gel electrophoresis and transferred onto a Nitrocellulose membrane . Membranes blocked with 3% nonfat milk in Tris-buffered saline (TBS), containing 0.1% Tween-20 for 1 h, at room temperature. Then membranes were incubated overnight, at 4 °C, with primary antibodies in 3% nonfat milk or 3% BSA in Tris-buffered saline (TBS), containing 0.1%. After that, the membranes were washed in TBS containing 0.1% Tween-20 and were then incubated with peroxidase-conjugated secondary antibody, for 2 h, at room temperature. Immunoreactive bands were detected using Amersham ECL Prime Western Blotting Detection Reagent . Western blot analysis was performed using the following antibodies—transforming growth factor-β , collagen IV , fibronectin , ACE , ACE2 , AT1R , AT2R , prorenin receptor , MasR , endothelial nitric oxide synthase , phosphorylated (phospho)-Ser1177 eNOS , Nicotinamide adenine dinucleotide phosphate oxidase 2 , Nicotinamide adenine dinucleotide phosphate oxidase 4 , superoxide dismutase 1 , superoxide dismutase 2 , and β-actin .Levels of Ang II and Ang 1-7 in Serum and renal tissue homogenates were measured using competitive enzyme immunoassay , according to the manufacturer’s protocols.t-test . p values of less than 0.05 were considered significant.Data are expressed as means ± standard deviation (SD). Differences between the groups were examined for statistical significance, using ANOVA and unpaired Changes in renal function of aging mice were measured, before and after resveratrol treatment, and the results were compared with control mice. Serum creatinine was decreased in resveratrol-treated mice, compared with the control group mice  and fibronectin (control 1.00 ± 0.19 vs. resveratrol 0.64 ± 0.21-fold) were decreased significantly by the resveratrol .In this study, we investigated the beneficial effects of resveratrol on the kidneys of aging mice. A six-month treatment with resveratrol resulted in a reduced albuminuria, better renal function, and improved renal histological changes, including diminished tubulointerstitial fibrosis, glomerulosclerosis, and inflammatory cell infiltration. Positive changes of the RAS were also observed in the resveratrol-treated aging mice. Specifically, the expression of ACE, Ang II, and AT1R was suppressed, while the expression of ACE2, Ang 1-7, AT2R, and particularly, MAS was stimulated. Furthermore, the expression of oxidative stress markers  and inflammation markers  was improved in the resveratrol-treated mice. The novel finding of the present study is that resveratrol not only inhibits ACE, Ang II, and AT1R, the well-known classic axis, but also stimulates the ACE2/Ang 1-7/MasR axis in the aging kidneys. The altered expression of RAS components was confirmed by the western blot, as well as immunohistochemical assays.Aging is a complex, multifactorial process, characterized by gradual deterioration of function and progressive structural changes ,7. VarioRecently, multiple lines of evidence suggested an association between an increased ACE2 signaling and improvements in the aging-related tissue injury. First, activation of ACE2 improved metabolic profiles during aging . Second,Numerous studies have reported that aging induces changes in the RAS ,27. For Many of the molecular and cellular effects of Ang II are mediated by stimulating the production of ROS. Of the many types of ROS generated, NOX is considered as the prime producer ,49. To dOur results demonstrated that the resveratrol treatment improves kidney function, albuminuria, glomerulosclerosis, tubular interstitial fibrosis, inflammation, and oxidative stress of age-related renal injury. These changes occur through decreases in the PRR/ACE/Ang II/AT1R axis and increases in the ACE2/Ang 1-7/MasR axis. A therapeutic strategy targeting the ACE2/Ang 1-7/MasR axis with resveratrol may postpone age-related renal structural and functional deterioration, through its antioxidant and antifibrotic effects. However, additional clinical investigations of the safety and anti-aging efficacy of resveratrol, are necessary.