The transcription factor NF-E2 p45-related factor 2 (Nrf2) is an established master regulator of the anti-oxidative and detoxifying cellular response. Thus, a role in inflammatory diseases associated with the generation of large amounts of reactive oxygen species (ROS) seems obvious. In line with this, data obtained in cell culture experiments and preclinical settings have shown that Nrf2 is important in regulating target genes that are necessary to ensure cellular redox balance. Additionally, Nrf2 is involved in the induction of phase II drug metabolizing enzymes, which are important both in degrading and converting drugs into active forms, and into putative carcinogens. Therefore, Nrf2 has also been implicated in tumorigenesis. This must be kept in mind when new therapy approaches are planned for the treatment of sepsis. Therefore, this review highlights the function of Nrf2 in sepsis with a special focus on the translation of rodent-based results into sepsis patients in the intensive care unit (ICU). Based on its role as the master regulator of the cellular antioxidant response, NF-E2 p45-related factor 2 (Nrf2) is a physiologically important transcription factor which belongs to the Cap ‘n’ collar transcription factor family ,2. It isSmall musculo-aponeurotic fibrosarcoma (sMaf) proteins function as Nrf2 heterodimerization partners to improve binding of Nrf2 to DNA . In addiConsequently, the role of Nrf2 is most significant in diseases associated with the generation of large amounts of reactive oxygen species (ROS), leading to tissue and organ damage. One of these is sepsis. It is a major cause of fatal casualties in intensive care units (ICUs) worldwide  and is cThe transcription factor Nrf2 is the master transcriptional regulator of genes involved in cellular detoxification and antioxidant defense ,28. Nrf2de-novo synthesis of GSH, via the glutamate cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit [2−), via superoxide-dismutase (SOD) [2O2) by catalase (CAT) activity [Nrf2-induced genes are involved in the detoxification of electrophile compounds and xenobiotics. Genes encoding enzymes for detoxification include UDP-glucuronosyltransferases (UGTs) , glutath subunit . Moreove subunit  are alsose (SOD)  or hydroactivity , are alsAlthough very important in maintaining the cellular redox balance to protect cells against oxidative stress, Nrf2 can also be involved in carcinogenesis  [HO-1 [Besides cell culture-based experiments, in vivo animal approaches are most important to studying, clarifying, and understanding the role of Nrf2. Due to the availability of gene knock-out animals, the role of Nrf2-dependent regulation of specific genes, which also can be used as read outs for functional Nrf2 expression, can be identified. Thus, mice have been generated with global A or tissfl/fl) . In sepsfl/fl) . Dependifl/fl) . Thus, gfl/fl) . Accordifl/fl) , necessafl/fl) . This isfl/fl) . In linel) [HO-1 ,57,58,59As outlined above, endotoxemia is also linked to organ damage. However, these disease-associated consequences are most pronounced in the more advanced mouse sepsis models, e.g., cecal ligation and puncture (CLP). In this setting, primarily peritonitis is induced, leading to multi-microbial sepsis ,61. Nrf2Organs which are often affected during sepsis progression are the kidney, liver, heart, and lung. Polymicrobial sepsis can initiate acute kidney injury (AKI), characterized by the release of kidney damage markers, such as creatinine and lipocalin-2 (Lcn-2), into the serum ,71. Cons−/−) [−/−) [In Nrf2 wild type animals A, the im−/−)  or in he−) [−/−) ,95. Mice−) [−/−) . In mice−) [−/−) , and tra−) [−/−) . In light of these differences in the immune response when Nrf2 is deleted or activated, it is important to determine the impacts of Nrf2 on different immune cell subpopulations. Thus, various studies have analyzed the roles of Nrf2 in lymphocytes, granulocytes, dendritic cells (DCs), and monocytes/macrophages . 2O2 production. It has been shown that B-cell receptor (BCR) signaling is fostered by H2O2 production in B cells, occurring in parallel [2, a Michael acceptor agent, reacts in Michael additions with cysteine residues of proteins, including Keap1 [2 provokes Nrf2 activation and was used to analyze the expression of HO-1 in murine and human normal and neoplastic B cells [2 stimulation was higher than in normal B cells. Nrf2-dependency was verified by Nrf2 siRNA experiments and the use of B cells derived from Nrf2 knock-out mice [Terminal differentiation of murine B lymphocytes to antibody-producing plasma cells is associated with endogenous Hparallel . Moreoveparallel . These oparallel . Using Lng Keap1 . Thus, 1 B cells . In thisout mice . Recentlout mice ,129. In out mice . Nrf2 is also expressed in dendritic cells (DCs), where its expression is responsible for redox balance . In DCs In keeping with their role in the immune response, granulocytes produce large amounts of ROS to fight against pathogens. Due to the detrimental nature of ROS also towards the cells that generate them, granulocytes need a system to protect themselves against ROS. Therefore, Nrf2 is highly expressed in these cells . To dete+ T cell functions are impaired [Together with granulocytes, monocytes/macrophages are innate immune cells representing the first line of defense towards pathogens. Although recent evidence supports the notion that monocytes/macrophages can acquire a kind of trained immunity, allowing improved host defense through metabolic and epigenetic changes (summarized in ), as celimpaired . Mechaniimpaired . Althougimpaired . Itaconaimpaired . Type I impaired ,138. Apaimpaired ,139. In impaired . An Nrf2impaired . Moreoveimpaired ,140. + T cell specific deletion of Keap1 revealed disturbed development of NKT cells, causing a reduced number in mice. This is associated with higher rates of NKT cell proliferation and apoptosis, and increased mitochondrial activity and glucose uptake [Finally, for the second subpopulation of lymphocytes, the T cells, several studies have shown the involvement of Nrf2 in T cell differentiation and activation. A number of different T cell subpopulations exist. One of these belongs to the innate immune system, the so called invariant natural killer T cells (NKT). Although less is known about a Nrf2-dependency in these cells, experiments in mice with a CD4e uptake . All funregs), as a T cell subpopulation belonging to the adaptive T cell group, specifically express the transcription factor FoxP3 and are important in maintaining T cell tolerance, thereby preventing autoimmunity. It could be demonstrated that in old female Nrf2 knock-out mice a multi organ autoimmune syndrome occurred . To specifically analyze the role of Nrf2 in Tregs, Klemm et al. used FoxP3-driven Keap1 knock-out mice [regs but also in epithelial cells, which probably accounts for death when Keap1 is deleted [regs, consequently associated with reduced peripheral tolerance [regs in draining lymph nodes and spleens. Mechanistically, the differentiation of Th17 cells was attenuated by a Nrf2/HO-1 dependent mechanism. As a corollary, the blockade was revered by the HO-1 specific inhibitor tin protoporphyrin IX (SnPP). Regulatory T cells  and ameliorated AKI [+ Foxp3+ Tregs was increased. Enhanced Nrf2 reduced TNF-α, IFN-γ, and IL-17 expression in these cells. Additionally, adoptive transfer of T cells with a stabilized Nrf2, reduced AKI following IR also in the recipient animal [Th17 cells are an important T cell subpopulation linked with asthma and airway inflammation. These cells are known to secrete, among others, IL-17 . In a stype mice . This wal et al.  used theated AKI . In thest animal . This st+ or CD4+ T cells derived from spleens and lymph nodes of Nrf2 knock-out and wild type mice were compared. The Nrf2 activating compound was added to freshly isolated splenocytes 30 min before classical T cell activation by αCD3/αCD28 antibodies. tBHQ concentration dose-dependently inhibited the expression of the Th1 cytokine IFN-γ in Nrf2 wild type mice, whereas it was significantly less effective in Nrf2-deficient cells. Induction of Nrf2 target genes  was only observed in Nrf2 wild type T cells in response to tBHQ. Interestingly, tBHQ in Nrf2 wild type T cells upregulated the Th2 marker IL-4 as well. A similar increase was found for IL-5 and IL-13, which are also Th2 cytokines. In Nrf2-deficient T cells, tBHQ did not induce the expression of Th2 markers. These last two studies suggested a role of Nrf2 in mediating a Th2 shift. Finally, Morzadec et al. [Early after T cell activation, Nrf2 is important to increase IL-2 expression and enhance c-Jun activation. In contrast, the expression of IFN-γ and TNF-α was reduced. This was shown in splenocytes derived from Nrf2 knock-out compared to wild type mice, using the two Nrf2 activators tBHQ and CDDO-Me . Nrf2 acc et al.  providedTaken together, in mouse models, ex-vivo or in cell culture, Nrf2 activation, when appropriately performed, is an important tool for the development of therapeutic strategies for sepsis patients, since the function of Nrf2 is generally protective by downregulating pro-inflammatory signaling. ClinicalTrials.gov”, 21 clinical trials are registered which involve Keap-1-dependent and Keap-1-independent approaches to Nrf2 activation and treatment of inflammation in patients. Furthermore, searching “ClinicalTrials.gov” for Nrf2-activating compounds identified 31 clinical trials for the synthetic triterpenoid CDDO-Me, 13 for RTA-408, 114 for di- and mono-methyl fumarate, 6 for oltipraz, 160 for ursodiol, 73 for SFN, 4 for sulforadex (SFX-01), 240 for the stilbene curcumin, 173 for the stilbene-derivate resveratrol, and finally, 11 for the nitro-fatty acid CXA-10 [® for multiple sclerosis) is being applied in rheumatoid arthritis and psoriasis, oltipraz to treat nonalcoholic steatohepatitis, the biliary acid ursodiol in chronic hepatitis C, SFN in COPD and asthma patients, curcumin in patients with CAD, AKI, and CKD, resveratrol for COPD and chronic subclinical inflammation, and CXA-10 to treat AKI and pulmonary arterial hypertension (PAH). As exemplified here, the use of Nrf2-activating compounds with respect to specific tissue or organ damage is already in clinical trials, which will, undoubtedly, end up with a licensed therapy concept, which putatively can be transferred to the sepsis patient. However, no clinical trials addressing the role of Nrf2 in sepsis patients are enrolled, as also outlined in a recent review [In “d CXA-10 . Summingt review . Apart from Nrf2 activation, tools mimicking the function of Nrf2 are also already in clinical trials. Considering glutathione (GST) as the most widely available intracellular antioxidant peptide, the ratio of its oxidized form GSSG compared to the reduced GSH and its total amount are important markers for cellular redox potential. Due to this major role of GSH in fighting reactive oxygen species-dependent modifications, induction of its generator systems in response to Nrf2 activation, as described above, might be a therapeutic concept. Restoration of the intracellular glutathione pool by the glutathione precursor molecule N-acetyl cysteine (NAC) has already been described in animal experiments to reduce pro-inflammatory cytokine expression and to improve disease outcome ,145 and From these data, it is obvious that Nrf2 is a putative target, whose activation or inhibition can interfere with the hyper- or hypo-inflammatory response observed during sepsis progression. Therefore, its activation or inhibition should be monitored in initial clinical trials. Due to the likelihood of only using Nrf2 activating compounds acutely in sepsis treatment, the carcinogenic potential of constitutive Nrf2 activity is probably negligible.Interestingly, recent progress in the understanding of COVID-19 infection and antioxidant strategies involving Nrf2 to prevent viral uptake, RNA- or DNA replication, and cytokine expression are the topics of current research ,144,155,


Several recent studies have revealed previously unknown complexity of the amphibian interferon (IFN) system. Being unique in vertebrate animals, amphibians not only conserve and multiply the fish-like intron-containing IFN genes, but also rapidly evolve amniote-like intronless IFN genes in each tested species. We postulate that the amphibian IFN system confers an essential model to study vertebrate immune evolution in molecular and functional diversity to cope with unprecedented pathophysiological requirement during terrestrial adaption. Studies so far have ascribed a potential role of these IFNs in immune regulation against intracellular pathogens, particularly viruses; however, many knowledge gaps remain elusive. Based on recent reports about IFN’s multifunctional properties in regulation of animal physiological and defense responses, we interpret that amphibian IFNs may evolve novel function pertinent to their superior molecular diversity. Such new function revealed by the emerging studies about antifungal and developmental regulation of amphibian IFNs will certainly promote our understanding of immune evolution in vertebrates to address current pathogenic threats causing amphibian decline. The animal immune system relies on a series of specified immune molecules, collectively known as immunome, to detect and repel pathogenic invaders at the molecular, cellular, and organismal levels. Species-specific or cross-species characterization of the immunome lays a cornerstone in studying animal health and immune evolution ,2,3. IntXenopus species . A . A Xenopphibians . Howeverphibians ,13,16. Aphibians ,16. Thesphibians ,12,13,16phibians ,19,20.Amphibians are an irreplaceable link in animal evolution toward terrestrial tetrapods. They reside in aquatic, terrestrial, and arboreal habitats, and are sensitive indicators for monitoring the environmental and habitat changes in our bio-ecosystem. In basic science research, amphibians have long been utilized as prominent models for studying physiological and developmental processes ,22,23,24Two types of fish IFN genes, generally one to several IFN-γ and several IFN-ϕ  genes in each species have been previously only found in teleosts ,11,12. AXenopus genome assemblies identified 6–9 intron-containing IFN genes in both IFN-I and IFN-III types, which indicated a moderate gene expansion to compare to that identified in most fish species , each species may harbor several to near 40 intronless IFN genes, and the majority of them belonging to IFN-I types [Xenopus IFN-I genes were grouped into four groups based on their intermolecular sequence similarity, and further defined into three clades based on their phylogenic relationship to other tetrapod species [Xenopus IFN-I genes were located within the mammal and reptile IFN-I clade, next to a clade containing only reptile sequences [Xenopus IFN-I genes were found to contain a single short intron and were assumed to be intermediate molecules recoding the intron loss process [Xenopus IFN-I genes located sister to the group of cartilaginous fish IFN-I genes that were next to the intronless amniote IFN-Is in a recent phylogenic analysis [Amniotic IFN-I genes are almost all intronless, which have been classified into multiple subtypes, including IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ, IFN-ω, and IFN-ζ, commonly  or species-specifically  expressed in mammalians ,14,15,16-I types ,51. Intr species ,13. For equences ,13. Thisequences ,13. The equences . Intriguanalysis . Thus, tanalysis ,13.In addition, amphibians are among the few tetrapod species having intronless IFN-III genes ,13,51. SXenopus [Xenopus but not obviously in Nanorana [In Xenopus ,13,16,51Nanorana ,15. CompNanorana ,55,56,57Xenopus IFN-III genes were ubiquitously detected in multiple tissues from healthy adult frogs [Xenopus tissues or cells treated with poly (I:C) or infected with Ranavirus (FV3) robustly stimulated the expression of both types of IFN genes and their corresponding receptors. In Xenopus kidney cells and tadpole models, administration of recombinant IFN peptides of either Xenopus IFN1 or an IFNλ induced the expression of ISGs, including IFIT5, PKR, and MX1 [Xenopus IFN1 peptides protected the kidney-derived A6 cell line and tadpoles against FV3 infection, showing the decrease of the infectious viral burdens in both cases [X. laevis frogs demonstrated significantly higher FV3-elicited IFN gene expression than tadpoles, indicating that IFN response at least partly determined the antiviral consequence [Described first for “interfering” with influenza virus in cultured chicken cells , IFNs arlt frogs ,59,60. Xsequence ,62.Xenopus and Nanorana species allows characterizing amphibian IFNs more extensively, in particular for the intronless subgroups [X. laevis was demonstrated in most tested tissues, with a particular high expression in lung and heart [X. tropicalis genome were also heavily mapped by RNA-Seq reads deposited at the public domains of NCBI, indicating functional expression of these less-studied amphibian IFNX genes  after treatment of the cells with representative microbial mimics [Similarly, in the tutively . More thtutively . More exl mimics . For exal mimics .Xenopus tadpoles and adult frogs were more conclusive toward biological significance [The results from ificance ,62. Usinificance ,62. Not ificance . The fluificance . In summificance ,60,61,62Xenopus IFN1 peptide using an insect cell-expression system [N. parkeri intronless IFNi1 and IFNi2 by direct transfection of the IFN genes in a mammalian-expression vector into Xenopus kidney cells (A6 cells) [Xenopus kidney cells only if the IFNAR receptor genes of N. parkeri were co-transfected [Xenopus IFN peptides and determined their different antiviral activity against an influenza virus [Xenopus type III IFNs were less active in antiviral activity, except an IFNL4 that showed stronger virus-suppressive activity than most type I IFNs tested. Another significant IFN example was IFNX4, which demonstrated remarkable virus-suppressive and cell protective activity similar to most intron-containing IFNs tested. Clearly, all of these antiviral analyses validate that the Xenopus IFN complex comprises a profound antiviral diversity considering their antiviral activity against the influenza and FV3 infections [The antiviral activity of amphibian IFN peptides has been tested in both cells and animals ,60,61,62n system ,60. The n system ,60. Gan 6 cells) . They densfected . This innsfected ,14,15,16za virus ,14,15. Aza virus ,14. Compfections ,60,61,62Listeria monocytogenes represents a prevalent intracellular bacterial pathogen such as in food safety and public health. We detected that in the bacteria-infected A6 cells, most amphibian IFNs were highly stimulated when the cells were intracellularly infected with the bacteria. In all tests where cells were with different bacterial dosages, the intronless IFNX3 group was highly stimulated. This indicated that this intracellular IFN subtype  might have evolved to regulate antibacterial activity inside cells. In cell cultures with a gel-overlay to restrict the bacterial intracellular growth, most IFN-expressing constructs showed antibacterial to some extent. However, most IFNs lost their antibacterial activity without a gel-overlay to limit the bacterial extracellular spreading. We also noted that IFNX2, the intracellular IFN isoform, developed significant antibacterial activity in both test conditions (with or without gel-overlay). Indeed, both intronless IFNX2 and IFNX3 are among the IFNX3 group, which showed the most antibacterial activity and were among the most inductive IFN genes in cells infected with L. monocytogenes, or treated with the ligands for either TLR2 (Pam2CSK4) or TLR4 (LPS) [Aspergillus fumigatus, type I IFNs produced by monocytes signaled the epithelial production of type III IFNs, which in turn played a critical role in regulating the antifungal neutrophil responses [The immune role of amphibian IFNs against other intracellular pathogens has also been recently tested . ListeriR4 (LPS) . ConsideR4 (LPS) ,14,63. IR4 (LPS) ,64. Curresponses ,66,67. Gesponses ,60,61,62esponses ,69,70,71Xenopus is proposed as an alternative model for cancer studies due to its evolutionary tumor immunity [Xenopus tumor immunity model can contribute to the development of IFN-based cancer immunotherapies [Several recent studies have revealed a previously unknown complexity of the amphibian IFN system ,14,15,16immunity . Antitumherapies .


GPAT1 is highly expressed in flowers, siliques, and seeds. A loss of function mutation in GPAT1 was shown to decrease seed yield but increase plant height through enhanced cell length. Transcriptomic and qRT-PCR data revealed that the expression levels of genes related to gibberellin (GA) biosynthesis and signaling, as well as those of cell wall organization and biogenesis, were significantly upregulated. These led to cell length elongation, and thus, an increase in plant height. Together, our data suggest that knockout of GPAT1 impairs glycerolipid metabolism in Arabidopsis, leading to reduced seed yield, but promotes the biosynthesis of GA, which ultimately enhances plant height. This study provides new evidence on the interplay between lipid and hormone metabolism in the regulation of plant height.Glycerol-3-phosphate acyltransferases (GPATs) play an important role in glycerolipid biosynthesis, and are mainly involved in oil production, flower development, and stress response. However, their roles in regulating plant height remain unreported. Here, we report that Arabidopsis GPAT1 is involved in the regulation of plant height. GUS assay and qRT-PCR analysis in Arabidopsis showed that  Plant height is closely correlated with yield trait owing to its crucial role in plant architecture, pod bearing number, and lodging resistance ,2,3. PlaBioactive GAs are plant diterpene hormones that are essential for multiple aspects of plant growth and development, including seed germination, leaf expansion, trichome development, stem elongation, flowering, male fertility, and fruit set ,8,9. GA GPAT5 plays a role in the production of suberin and suberin-associated root waxes, which contribute to the tolerance of plants to salt stress [GPAT7 may only contribute to suberin biosynthesis under stress, such as wounding [While the implications of lipid metabolism in the regulation of plant height remain unclarified, a few genes involved in lipid transport  and hydrt stress ,39. Lastwounding . StudiesGPAT1 was shown to act as an essential factor for tapetum and pollen development [GPAT1 in regulating Arabidopsis plant height. Our results revealed that knockout of GPAT1 increased plant height by activating GA metabolism and signaling, which then stimulated cell wall organization and biogenesis. This study provides evidence on how GPAT-mediated glycerolipid metabolism influences plant height through GA.In a previous report, Arabidopsis elopment . In thisGPAT1 was shown to be mainly expressed in developing siliques and flower buds [GPAT1, we made a construct where the GUS reporter was driven by the GPAT1 promoter, and transfected it into WT Arabidopsis plants. One transgenic GPAT1-GUSPro line was selected to analyze the expression of GPAT1 gene. GUS activity was detected in young seedlings, roots, leaves, inflorescence stems, flowers, and mature seeds by histochemical staining  siliques, as well as in mature seeds. To determine the relative expression levels of GPAT1 in different tissues of WT Arabidopsis plants, we performed qRT-PCR experiments. qRT-PCR indicated that the highest expression level of GPAT1 was in the seed, followed by a descending order of siliques, flowers, stems and leaves, roots, and seedlings  of GPAT1 (gpat1) [gpat1-c1) to further target the first exon  , which hrst exon B,C. Throodon TAA D.GPAT1. Seed oil contents of gpat1 and gpat1-c1 mutants were reduced by 11.7% and 13.7% compared with that of WT, respectively  content was 7.1% higher in gpat1 seeds than in WT seeds. Similar results were found in the gpat1-c1 line. We also analyzed the contents of 16-, 18-, 20-, and 22-carbon FAs. The levels of 16-carbon FAs were markedly lower in seeds of mutant plants, while levels of 18-, 20-, and 22-carbon FAs were similar to those of WT seeds , and measured the mRNA levels of GPAT1 in WT, gpat1, and COM by RT-PCR. The expression of GPAT1 in COM and WT was comparable while that in the gpat1 and gpat1-c1 was undetectable  terms and KEGG (Kyoto encyclopedia of genes and genomes) pathways. A total of 1585 DEGs were identified between WT and gpat1 plants, with 872 genes upregulated and 713 genes downregulated  that were significantly upregulated  gene family was most enriched, followed by genes involved in pectin modification such as PME (pectin methylesterase), PMEI (pectin methylesterase inhibitor), and PAE (pectin acetylesterase), and other polysaccharide-related genes such as polygalacturonase, galacturonosyltransferase, glycosyltransferase, cellulose synthase, endo-1,4-β-xylanase, O-fucosyltransferase, and endochitinase. In addition, other cell wall-associated genes such as expansin, extensin, and oxidase  were all included in the upregulated DEGs. Out of the 26 downregulated DEGs, there were 16 pectin-associated genes, 4 expansin genes, and 2 cellulose synthase genes. Furthermore, cell wall-related genes such as BXL2 (beta-D-xylosidase 2), TBL15 (trichome birefringence-like 15), CASP2 (casparian strip membrane domain protein 2), COBL10 (COBRA-like 10), PER2 (peroxidase 2), and FLS2 (flagellin-sensitive 2) also belonged to the downregulated DEGs.GAs are important plant hormones that function in the promotion of cell elongation. Plant cell walls determine cell wall sizes and shapes, and are thus essential for plant growth and development ,48,49. Ggpat1 mutant plants.Overall, transcriptomic analysis demonstrated that the upregulated genes in various pathways, including isoprenoid biosynthesis, GA biosynthesis and signaling, and cell wall organization or biogenesis, might be related to enhanced cell elongation, and thus, plant height in GGPPS genes, the transcript levels of GGPPS2 and GGPPS4 were increased by about 1.3-fold and 1.4-fold in the gpat1 mutant lines, respectively , gibberellin 2 oxidase (GA2ox), and CYP714A catabolic enzymes [gpat1 mutants, the mRNA levels of GA2ox1 and GA2ox4 were evidently reduced while that of GA2ox2 increased significantly  in Arabidopsis and GID2 in rice are regarded as key components [gpat1 mutants, the expression of DELLA protein GAI, SLY1, and SLY2 were not altered, while one of the three GID1 genes, GID1B, was more highly expressed than in WT. Among the three GID1 genes, GID1A contributed most to GA responses [gpat1 mutants compared with WT  and CRISPR-generated gpat1-c1 mutants, and COM (transgenic complementation lines of gpat1) A. thaliana seeds were used in this study. Seeds were sown on Murashige and Skoog (MS) medium , imbibed at 4 °C for 2–3 days in the dark, and transferred to a growth chamber with a light intensity of 200 μmol m−2 s−1 (16/8 h of light/dark at 22 °C). Ten-day-old seedlings grown on plates were transferred to potting soil under controlled growth conditions. The mutant SALK_052352 was bought from ABRC.WT (ecotype Columbia-0), GPAT1 gene was cloned into the binary vector pCAMBIA1301 carrying the GUS gene downstream of the inserted promoter [ProGPAT1::GUS was transformed into WT Arabidopsis (Col-0) plants via the floral dip method [For the GUS staining assay, a 2045-bp sequence upstream of the first ATG in the promoter . The conp method . Transgep method . Images ® Premix Ex Taq™ (Tli RNaseH Plus) and the CFX96 Real-Time PCR Detection System , as specified by the manufacturer. The primers used for RT-PCR and qRT-PCR are listed in Total RNA was extracted from various tissues of WT Arabidopsis plants using RNAiso plus reagent  following the manufacturer’s instructions. cDNA obtained using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa) was used for quantitative real-time PCR (qRT-PCR) and reverse transcription PCR (RT-PCR). qRT-PCR was performed using SYBRGPAT1 gene with the CRISPR/Cas9 gene editing system, we designed an sgRNA target sequence using the online tool CRISPR-P2.0 (http://crispr.hzau.edu.cn/CRISPR2/) [pAtU6-26:sgRNA-23p35S:Cas9 pBlunt vector [GPAT1 gene were amplified from WT and transgenic lines, after which PCR products were directly sequenced for analysis. The mutated sequences of each GPAT1 gene in the transgenic lines were revealed by aligning the sequences between WT and the transgenic lines. T-DNA insertional line SALK_052352 for GPAT1 was verified using the procedures provided by the Salk Institute Genomic Analysis Laboratory . For complementation, the full-length GPAT1 fragment was amplified from WT Arabidopsis, cloned into the pCambia1300 vector, and then introduced into gpat1 homozygote mutants via the floral dip method. The primers used above are outlined in To create a mutant of the RISPR2/) . The selt vector . The cont vector . For gengpat1, gpat1-c1, and complementation transgenic lines and fixed in FAA  for 24 h, dehydrated with a graded series of ethanol , followed by an immersion in 100% xylene and embedding in paraffin. Samples were cut into 8-µm thick transverse serial sections with a Leica RM2016 microtome and mounted on glass slides. The sections were stained with toluidine blue for 5 min, washed three times with water, and sealed with resin when dried. Samples were photographed with a Leica DM1000. The images obtained were used for cell length measurement with Image J software.For histological analysis, the first basal node of stems above the rosette leaves were collected from WT, w/v) sulfuric acid, 0.01% (w/v) BHT , 20% (v/v) toluene, and TAG-17:0 internal standard  were added to the combined lipid extracts and incubated for 2 h at 90 °C, followed by addition of 1.5 mL NaCl (0.9% w/v) to quench the transmethylation. The seed TAG was extracted with 1.5 mL of hexane and subjected to gas chromatography analysis. The total TAG content in seeds was calculated as described [TAG extraction from Arabidopsis seeds was performed as described previously ,83. Apprescribed . FA compp-value (FDR) < 0.05 and |log2FC| ≥ 1 between WT and gpat1 mutants. An enrichment analysis was performed to predict the biological processes and KEGG pathways of the DEGs via the online tool Metascape [Total RNA of tender inflorescence stems from WT and gpat1 plants was extracted for RNA-sequencing. cDNA library construction and Illumina sequencing were performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. . The RNA-seq data were aligned to TAIR10 reference genome using hisat2  with defetascape .


FXR and BRD4 was significantly reduced. In conclusion, BRD4 is a potentially novel cofactor of FXR for maintaining BA homeostasis and hepatoprotection. Although BRD4 promotes hepatic inflammation and fibrosis in cholestasis, paradoxically, BRD4 is required for the antiinflammatory, antifibrotic actions of OCA-activated FXR. Cotreatment with OCA and JQ1, individually beneficial, may be antagonistic in treatment of liver disease patients with inflammation and fibrosis complications.Activation of farnesoid X receptor (FXR) by obeticholic acid (OCA) reduces hepatic inflammation and fibrosis in patients with primary biliary cholangitis (PBC), a life-threatening cholestatic liver failure. Inhibition of bromodomain-containing protein 4 (BRD4) also has antiinflammatory, antifibrotic effects in mice. We determined the role of BRD4 in FXR function in bile acid (BA) regulation and examined whether the known beneficial effects of OCA are enhanced by inhibiting BRD4 in cholestatic mice. Liver-specific downregulation of BRD4 disrupted BA homeostasis in mice, and FXR-mediated regulation of BA-related genes, including small heterodimer partner and cholesterol 7 alpha-hydroxylase, was BRD4 dependent. In cholestatic mice, JQ1 or OCA treatment ameliorated hepatotoxicity, inflammation, and fibrosis, but surprisingly, was antagonistic in combination. Mechanistically, OCA increased binding of FXR, and the corepressor silencing mediator of retinoid and thyroid hormone receptor (SMRT) decreased NF-κB binding at inflammatory genes and repressed the genes in a BRD4-dependent manner. In patients with PBC, hepatic expression of  Treatment with obeticholic acid, a farnesoid X receptor agonist, or JQ1, a bromodomain-containing protein 4 inhibitor, has antiinflammatory, antifibrotic effects in cholestatic mice, but beneficial effects are lost in combination. Bile acids (BAs) aid in digestion of lipid nutrients but also function as signaling molecules that profoundly influence metabolism and energy balance , 2. BecaFarnesoid X receptor FXR/NR1H4) plays a central role in maintaining normal BA levels and protecting against cholestatic liver injury by transcriptional regulation of genes involved in BA synthesis, transport, and metabolism –7. The t plays a Epigenetic regulation by coordinated actions of epigenetic writer, reader, and eraser proteins plays a crucial role in maintaining homeostasis in response to environmental cues. Bromodomain-containing protein 4 (BRD4) is an epigenetic reader protein and, with BRD2, BRD3, and BRDT, belongs to the bromodomain extra-terminal (BET) family . BRD4 biShp) promoter by P300 acetyltransferase is required for FXR-mediated induction of Shp, a key regulator of BA metabolism  by infection of BRD4-floxed mice with hepatocyte-targeting AAV-TBG-Cre refs. . The galectively . Liver-synthesis , and of ynthesis , Osta, Ong, Cck1 . These rFxr were not changed in BRD4-LKD mice, hepatic mRNA levels of direct FXR target genes important for repression of BA synthesis, such as Shp, Mafg, Lsd1 . These results suggest that activation of FXR induces an indirect interaction with BRD4 in a protein complex.To test whether BRD4 coactivates FXR function, mice were briefly treated with an FXR agonist, GW4064, and the interaction of FXR with the BET proteins, BRD2, BRD3, and BRD4, in liver extracts was examined by co-IP assays. Treatment with GW4064 dramatically increased the interaction of FXR with BRD4 but not with BRD2 or BRD3 . In glutctivator , was obsShp as a model gene. BRD4 function was blocked by either siRNA-mediated downregulation or treatment with JQ1, a small molecule inhibitor of BET proteins, particularly BRD4 , a direct SHP target and key BA synthetic gene, in primary mouse hepatocytes (PMHs). As expected, treatment with GW4064 resulted in substantial increases in Shp mRNA levels and decreases in Cyp7a1 mRNA levels, and these GW4064-mediated effects were lost by downregulation or inhibition of BRD4 , an indicator of active transcription , 23, wasreatment , left. Idetected , middle.reatment , right. reatment , suggestShp promoter as expected  and TNF ligand superfamily member 4 (Tnfsf4); antagonizes NF-κB function; and represses expression of these genes, in part by recruiting the corepressors, silencing mediator of retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor 1 (NCoR1)  , 15.Il6ra and Tnfsf4, that are direct targets of both FXR and NF-κB  modified at K277, and this SUMO2 modification is critical for transrepression of NF-κB target inflammatory genes , 15. ConWe evaluated therapeutic potential of OCA and JQ1 in amelioration of cholestatic liver pathologies. Cholestatic liver injury was established first by ANIT treatment of mice for 2 days, followed by treatment for 7 days with OCA or JQ1 alone or both drugs .Shp, Bsep, and Klb, and intestinal Fgf15, and decreased those of BA synthetic genes, Cyp7a1 and Cyp8b1, and of inflammatory and fibrotic genes, but all these OCA-mediated beneficial effects were largely abolished by cotreatment with JQ1  is a toxic secondary BA that induces liver cholestasis , 4, 45. FXR and BRD4 in liver samples of 15 unidentifiable patients with PBC. Hepatic mRNA levels of FXR, as shown previously  and Ep300 (L-003486) were purchased from Dharmacon; AAV8-TBG-Cre (VB1724) and AAV8-TBG-GFP (VB1743) from Vector Biosystems; GW4064 (HY-50108), OCA (HY-12222), and JQ1 (HY-13030) from MedChemExpress; ANIT (N4525) from MilliporeSigma; and TNF-α (ab9642) from Abcam.Information on antibodies is provided in Brd4-floxed mice have been described previously and were generated and maintained in-house  for 72 hours or treated with JQ1 (500 nM) for 24 hours, respectively. To activate FXR signaling, the cells were treated with 1 μM GW4064 in corn oil or vehicle or 1 μM OCA in 1% methylcellulose or vehicle for 3–5 hours. To induce inflammation, PMHs were treated with a proinflammatory cytokine, TNF-α (50 ng/mL), or vehicle (PBS) as a control for 4 hours.PMHs were isolated by collagenase  perfusion as described , 50 and Total RNA was isolated using TRIzol  and levels of mRNA were measured. Primers used are listed in Co-IP assays were done as described previously , 50. BriShp promoter (Shp-luc) or containing 3 copies of a NF-κB site [3-luc(NF-κB)], respectively, and a plasmid for β-galactosidase (200 ng). For Shp promoter studies, cells were infected with Ad-FXR and transfected with 50 nM siBrd4 and 72 hours later were treated with 1 μM GW4064 for 3 hours. Alternatively, cells were infected with Ad-FXR, and after 48 hours, were treated with 500 nM JQ1 followed 24 hours later by treatment with 1 μM GW4064 for 3 hours. For FXR-SUMO studies, PMHs were transfected with an expression vector for BRD4 or infected with Ad-FXR-WT or Ad-FXR-K277R expressing a SUMO2-defective mutant  or PMHs were transfected with 200 ng of a luciferase promoter-reporter plasmid containing the e mutant , and 48 ChIP assays were done as described previously , 50. BriParaffin-embedded liver sections were incubated with F4/80 antibody, and the antibody was detected by IHC staining using peroxidase-based detection . Liver collagen was detected with a Trichrome Stain Kit . Liver sections were imaged with a NanoZoomer Scanner, and ImageJ software was used for image analysis and quantification.Liver specimens from 15 unidentifiable PBC patients or control individuals (organ donors) were obtained from the Liver Tissue Cell Distribution System, and mRNA levels of selected genes were quantified by RT-qPCR.t test or 1- or 2-way ANOVA as appropriate. Differences with P< 0.05 were considered statistically significant.GraphPad Prism 8 (GraphPad Software Inc) was utilized for data analysis. Statistical analysis of different groups was performed using the Student’s unpaired 1-tailed All animal studies and biosafety protocols were approved by the Institutional Animal Use and Care and Biosafety Committees of the University of Illinois at Urbana-Champaign and were in accordance with NIH guidelines. Human liver samples were not collected specifically for this study, and no one on our study team has access to the subject identifiers linked to the specimens or data. Thus this study is not considered human subjects research, and ethical approval was not required .HJ, JC, LFC, and JK designed research; HJ, JC, XH, and HS performed experiments; SYW and CMC provided critical reagents; HJ, JC, XH, HS, BK, LFC, and JKK analyzed data; and HJ, BK, LFC, and JKK wrote the paper.


Glioblastoma multiforme (GBM) is the most common primary central nervous (CNS) system malignancy with a poor prognosis. The standard treatment for GBM is neurosurgical resection, followed by radiochemotherapy and adjuvant temozolomide chemotherapy. Predictive biomarkers, such as methylation of the promoter region of the O6-methylguanine DNA methyltransferase (MGMT) gene, can successfully distinguish subgroups with different prognosis after temozolomide chemotherapy. Based on multiomics studies, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), BRAF V600E mutation, neurotrophic tyrosine receptor kinase (NTRK) fusions and other potential therapy targets have been found.We have reviewed the preclinical and clinical evidence for NTRK fusions and TRK inhibitors therapy in cancers with NTRK fusions in pan-cancer and gliomas.Several NTRK1/2/3 fusions have been reported in GBM and preclinical studies have proven that NTRK fusions are potential driver mutations in some high-grade gliomas. Tropomyosin receptor kinase (TRK) inhibitors have shown efficacy as targeted therapies for extracranial tumors with NTRK fusions in recent clinical trials, with potential CNS tolerability and activity. However, whether NTRK gene fusions can affect survival status, the efficacy and resistance of TRK inhibitors in GBMs are lacking high-level evidences.For GBM patients, NTRK fusions and TRK inhibitors are potential target therapy strategy but remain biological mechanism and clinical significance unclarified. More clinical data and future clinical trials are needed to provide more evidence that supports targeted therapy for GBM with NTRK fusions. Glioblastoma multiforme (GBM) is the most common primary malignant tumor of the central nervous system (CNS) and accounts for 48% of CNS malignancies, with a morbidity of 3.22 per 100,000 people. The morbidity of GBM increases positively with age, and the median age of patients is 65 years old  radiochemotherapy and adjuvant TMZ chemotherapy, with or without a tumor treatment field (TTF) therapy; the median overall survival (mOS) of patients with or without TTF therapy is 21 months (m) or 14.6 m, respectively . MethylaNTRK genes, including NTRK1/2/3, encode the Tropomyosin receptor kinase A/B/C (TrkA/B/C). During the period of embryo development, Trks regulate neurological homeostasis. TrkA/B/C have roles in pain responses, movement, body temperature and weight regulation, memory, and proprioception . Over-acNTRK fusions are detected at a relatively low rate in most types of cancer, with an average rate of 0.31% in adults and children. However, NTRK fusions can be detected in over 80% of cases of infantile fibrosarcoma and secretory breast carcinoma . NTRK fuBased on The Cancer Genome Atlas (TCGA) database and previous studies, NTRK fusions are detected in approximately 0.56–1.69% of adult GBM patients; this detection rate is similar to that in adult low-grade glioma but is much lower than that in pediatric high-grade glioma. For children younger than 3 years old who are diagnosed with high-grade glioma, the detection rate of NTRK fusions ranges from 5.3 to 40% . In otheThe NTRK fusions that have been reported in GBM are shown in An ETV6-NTRK3 fusion has been reported in many types of solid cancers, including secretory breast carcinoma, carcinoma of salivary glands and gastrointestinal system, and acute myelocytic leukemia (AML) . ETV6-NTNTRK1/2/3 encode TrkA/B/C of the TRK family, respectively. Trk proteins contain an extracellular ligand-combining domain, a transmembrane domain and an intracellular tyrosine kinase domain. TrkA/B/C are regulated by nerve growth factor (NGF), brain-derived growth factor (BDGF) and neurotrophin-4/5 (NT-4/5) and NT-3, respectively, and promote cell proliferation and survival through downstream mitogen-activated protein kinase/extracellular regulated protein kinases (MAPK/ERK), phospholipase C gamma/protein kinase C (PLCγ/PKC), and phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling Figure 1NTRK1 is located on 1q21-22, while NTRK2 is located on 9q21.1. Both NTRK1/2 genes have 17 exons with exons 13–17 encoding the tyrosine kinase domain. Similarly, the NTRK3 gene is located on 15q25.3 and has 18 exons, with exons 13-18 encoding the tyrosine kinase domain. Generally, NTRK fusions occur in introns, with the 5’-region of the fusion partner gene connected with the 3’-end of the NTRK gene. For NTRK1/2/3, the gene breakpoint is located at the 5’ region to exons 13–17 (and exons 13–18 for NTRK3), leaving a complete sequence that encodes the tyrosine kinase domain , 30. AppThe formation of gene fusions is a complex process, including balanced rearrangements and unbalanced rearrangements in chromosomal level, generally. Balanced chromosomal rearrangements, including translocation, insertion and inversion, result in no loss or gain of genes. And unbalanced chromosomal rearrangement, such as gene deletion, can lead to copy number variations. In tumor cell, structural variation maybe more complex and usually involved more than two genes, such as local-distant cluster, chromoplexy or chromothripsis, which may cause gene fusions. Also, transcripts can fusion together by trans-splicing and cis-splicing and produce a functional fusion protein without chromosomal rearrangement –33. ThroMost functional NTRK fusion proteins lack the extracellular ligand-combining domain of Trk; instead, the partner gene is expressed in a homogenous dimer, which induces ligand-independent activation of the tyrosine kinase domain and upregulates downstream cancer-associated pathways. However, some NTRK fusions cannot be expressed into functional proteins, and the mechanisms remain unclear , 30. NTRNTRK genes are rarely expressed in GBM, but a significantly higher level of expression of NTRK1 and increased activation of the NGF/TrkA pathway were found in GBM with NTRK1 fusions , 19. HowFor tumors with NTRK fusions, the coactivation of Trk and other signaling pathways, such as TP53, tyrosine kinases, and cell cycle pathways, has been reported . The coeAside from gene fusion, other alterations of the NTRK gene have been reported, such as somatic mutations, insertions, deletions and amplifications. Whether these mutations can act as driver mutations of cancer is still unknown. Several NTRK gene mutations can cause resistance to TRK inhibitors, including resolvent-front alteration of TRKA G595R and TRKC G623 in the ATP binding pocket of the kinase domain; TRKA G667C, TRKB G709C, and TRKC G696A alteration in the xDFG site; and gatekeeper mutations such as TRKA F589L, TRKB F633L, and TRKC F617L .in situ hybridization (FISH), RT-PCR and next-generation sequencing (NGS). The detection of gene fusions by IHC occurs at the protein level; thus, nonexpressing NTRK fusions cannot be detected. IHC can present false-negative results in samples from both adults and children, and the sensitivity and specificity of detection of NTRK3 fusions are much lower than those for NTRK1/2 (The detection of NTRK fusions mainly relies on immunohistochemistry (IHC), fluorescence  NTRK1/2 , 39. Nat NTRK1/2 , which c NTRK1/2 .FISH is the main technique for the detection of chromosomal rearrangement, but it cannot identify the 5’-ending partner gene and expression of NTRK fusions . Break-aNucleoside-based methods for detection of gene fusions, such as RT-PCR, RNA sequencing (RNA-seq) and DNA sequencing (DNA-seq), can screen known fusions and unknown NTRK fusions with a clear identification of 5’-ending partner genes. RT-PCR and Sanger sequencing can help confirm NTRK fusions that are screened by RNA-seq; this process was described in a study by Frattini et al., in which GBMs with FASC-NTRK1 fusion were detected . BecauseRecently, molecular pathology is routinely performed to help treatment decisions. As adult GBM lead to a worse survival probability, detection of NTRK fusions shown a new chance for them, especially those with poor diagnosis under standard therapy. It is a worthy practice point to detecting NTRK fusions for GBM patients who do not have typical therapy targets and driver mutations like methelytion of MGMT promoter. The advantages and disadvantages of NTRK fusions detection assays were summarized in TRK inhibitors that can target TrkA/B/C have potential efficacy in tumors with functional NTRK fusions, namely, those generating proteins with complete kinase domains. These inhibitors include the selective TRK inhibitor larotrectinib, the multikinase inhibitor entrectinib and others that have entered into preclinical or clinical research Table 3.In 2018, larotrectinib, or LOXO-101, became the first Food and Drug Administration (FDA)-approved pan-TRK inhibitor for adult and pediatric solid tumors with NTRK fusions in the United States . The effin situ high-grade glioma mouse model with BCAN-NTRK1 fusions   is a multitarget inhibitor and has been researched in a phase II single-armed trial in relapsed glioblastoma. For patients without a history of anti-angiogenesis therapy, high/low dose cabozantinib (140 or 100 mg/d) showed ORRs of 17.6 and 14.5% and mOS rates of 7.7 and 10.4 m, respectively . For GBMFor GBM, therapies targeting NTRK fusions need further study. Rosen et al. reported a single-center cohort analysis of TRK inhibitors in multiple cancers, including salivary carcinoma and thyroid carcinoma and sarcoma, and showed an ORR of 67.6% (n = 23/34). Four patients with GBM were involved in this study, with NTRK1-AFAP1, NTRK1-TPM3, NTRK2-BCR and NTRK4-EML4 fusions, and all patients had a history of neurosurgery, radiotherapy, and adjuvant chemotherapy. However, the two evaluable patients showed no response to TRK inhibitors .With the confirmation of diagnostic and predictive biomarkers, such as IDH1/2 mutation and MGMT gene promoter methylation, molecular pathology is routinely performed to diagnose adult GBM and guides prognosis evaluation and treatment decisions. However, patients with GBMs are still suffering from a poor prognosis. For patients that accord with including criteria, clinical trials are recommended by National Comprehensive Cancer Network (NCCN) guideline in central nervous system cancers , which iCoactivation of NTRK fusions and tyrosine kinase family members, cell cycle or other cancer-associated pathways, and driver mutations indicates a new landscape of combined target therapy in GBM . AdditioStudy design: YW and WM. Manuscript writing: YKW and PL. Papers searching: YKW and PL. Manuscript formatting and revising: YKW, PL, YW and WM. All authors contributed to the article and approved the submitted version.1. Beijing Municipal Science and Technology Commission (7202150), 2. Beijing Municipal Science and Technology Commission [19JCZDJC64200(Z)], 3. Chinese Academy of Medical Sciences (2016–I2M–2–001), and 4. Peking Union Medical College Hospital (2019ZLH101).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.


The volatile components of milks fermented using traditional starter cultures (Streptococcus thermophilus and Lactobacillus bulgaricus) and probiotics  were investigated by means of gas chromatography-mass spectrometry (GC-MS) combined with simultaneous distillation extraction (SDE). A total of 53 volatile compounds were detected, being 10 aldehydes, 11 ketones, 10 acids, 11 hydrocarbons, 7 benzene derivatives, and 4 other compounds. The starter culture was found to significantly affect the composition of volatile components in the fermented milks. Ketones and hydrocarbons were the dominant compounds in milk before fermentation, while acids were dominant compounds in the fermented samples. Compared with probiotics, there was greater abundance of volatile components in fermented milks with traditional strains. The importance of each volatile compound was assessed on the basis of odor, thresholds, and odor activity values (OAVs). Of the volatile compounds, 31 of them were found to be odor-active compounds (OAV > 1). The component with the highest OAVs in most samples was -2,4-decadienal. Heatmap analysis and principal component analysis were employed to characterize the volatile profiles of milks fermented by different starter cultures. The results could help to better understand the influence of starter cultures on the odor quality of milks. Fermentation is a common method of producing new and palatable dairy products from milk . FermentStreptococcus thermophilus and Lactobacillus bulgaricus, are widely used for the industrial fermentation of yogurt. Many studies have been performed to investigate the factors affecting the volatile composition of yogurt, such as extraction techniques [The flavor of fermented milk is formed by the action of starter bacteria and originated from biochemical changes of carbohydrates, lipids, and proteins . Starterchniques , source chniques ,10, packchniques , processchniques , and stochniques .Bifidobacterium fermentation increased low-molecular volatile compounds  in milk [Lactobacillus pentosus and confirmed that butanoic acid, methyl ester, octanoic acid, and decanoic acid were the main volatile compounds in the sample [In recent years, along with the increase of consumers’ demand for nutritious and healthy yogurts, more and more probiotics have been used in milks fermentation. It has been well-reported that probiotics are associated with such health benefits as prevention/treatment of hypercholesterolemia , certain in milk . Pan et e sample . HoweverLactobacillus lantarum strains (1-33 and 1-34) could improve the odors of acetaldehyde, diacetyl, and acetoin based on OAVs in yogurt [In addition, not all difference of the volatile components found in fermented milks are of sensory importance, as these components differ in concentration. Only when a volatile compound concentration exceeds an odor threshold can the components can be perceived . MeasuriChemometric techniques are of extreme importance and widely relevant in the resolution of problems in flavor chemistry. Among them, hierarchical cluster analysis (HCA) and principal component analysis (PCA) have often been applied in interpreting the chromatographic data related to an extensive number of compounds and a large number of variables involved. PCA and HCA are exploratory multivariate data analysis techniques. PCA is a projection technique, the goal of which is to reduce large amounts of data and represent them efficiently in a graphical form. HCA is a clustering technique complementary to PCA, which groups a dataset by similarities or dissimilarities .Streptococcus thermophilus s2 (STs2), Lactobacillus bulgaricus 8 (LB8), Lactobacillus lactis 28 (LL28), Lactobacillus bifidus 531 (LB531), Lactobacillus casei 475 (LC475), and Lactobacillus plantarum 45 (LP45). Gas chromatography-mass spectrometry (GC-MS) combined with simultaneous distillation extraction (SDE) was applied to analyze the composition of volatiles in samples. Chemometrics was performed by heatmap analysis and PCA to screen characteristic volatiles of different fermented milks. This study could be important for fostering a better understanding of typical volatile compounds in fermented milks to improve the quality of product.The objective of this study was to characterize the volatile profiles of milks fermented by different starter cultures including Relative concentration and compound amount of different type volatile compounds identified in milk before fermentation (BFM); traditional starter cultures (STs2 and LB8); and probiotics  samples are shown in −1, from low to high in BFM, LB8, LP45, LL28, STs2, LB531, and LC475 samples, respectively. The total concentrations of volatile compounds in STs2 and probiotics were significantly higher than BFM and LB8 samples (p < 0.05). Of these volatiles, 16 were commonly detected in all investigated samples, and 21 of them were found to be simultaneously present in the milks fermented by different starter cultures. The volatile compounds identified in BFM, fermented samples are shown in n-alkanals, and one aldehyde belonged to 2,4-alkadienals. Eight, four, eight, four, four, three, and five aldehydes were detected in BFM, STs2, LB8, LL28, LB531, LC475, and LP45 samples at different concentrations of 36.64, 9.46, 35.60, 11.00, 13.07, 12.57, and 14.52 mg·kg−1, respectively (p < 0.05). Aldehyde concentrations were relatively low, but the number and content of aldehydes were significantly different. Benzaldehyde, nonanal, and -2,4-decadienal were detected in all samples. Octanal was only detected in BFM and STs2. Benzeneacetaldehyde was detected in LB8 and LL28 samples. Decanal, 2,4-dimethyl-benzaldehyde, and dodecanal were only in BFM and LB8 samples, and undecanal was only in LB8, LB531, and LP45. Tetradecanal was detected in just BFM samples. Aldehydes can be derived from amino acid catabolism [Ten aldehyde compounds were detected in the volatile fraction of milks fermented by different starter cultures. Among the detected aldehydes, three compounds belonged to aldehydes containing benzene, six aldehydes belonged to ectively . Among otabolism . In additabolism . Therefo−1 were in BFM, STs2, LB8, LL28, LB531, LC475, and LP45 samples, respectively  were the lowest in the LB8 sample. As shown in Ten acids were detected in all samples. In the BFM sample, only two acids (dodecanoic acid and tetradecanoic acid) with a concentration of 11.90 mg·kgectively . After f−1 were in BFM, STs2, LB8, LL28, LB531, LC475, and LP45 samples, respectively . Among detected benzene derivatives, 1,2,4,5- tetramethyl-benzene, naphthalene, 1-methyl-naphthalene, and 2,3-dimethyl-naphthalene could not be detected in fermented samples. Naphthalene has only been detected in LB8 fermented milk. Benzene derivatives usually come from the milk of cows fed on pasture grass [Seven benzene derivatives were detected in low concentrations in samples by different starter cultures. Seven, three, four, three, three, three, and three benzene derivatives were detected in BFM, STs2, LB8, LL28, LB531, LC475, and LP45 samples at different concentrations of 36.56, 38.98, 35.6, 30.59, 26.02, 22.0, and 28.49 mg·kgectively . Fermentre grass . The abire grass .p < 0.05) reduced the concentration of 2,4-bis-phenol. The reason for this concentration decrease is unknown and needs further study.In addition, there were four other compounds detected, including 2-pentyl-furan, limonene, benzothiazole, and 2,4-bis-phenol in fermented milks from different starter cultures. Most of them are common volatile compounds in yogurt. The compound 2-pentyl-furan only presented in BFM samples, which is a secondary oxidation product of linoleic acids . LimonenBecause different volatile compounds have different thresholds, not all components have a large impact on the overall aroma character of fermented milks from different starter cultures. The OAV is an important method to evaluate the contribution of various volatile compounds on the olfactory impression. Compounds with OAV > 1 were identified as odor-active compounds. The aroma characteristics of 32 volatile compounds were described in previous works ,34,44. A−1, “fatty and green”), trace changes had a huge impact on odor intensity of samples. Octanal contributed fat, soap, lemon, and green odor and presented extremely high OAVs in BFM (1608), STs2 (1035), and LP45 (1302). Dodecanal contributed “soapy, waxy, and citrus” odor, and presented 5836 and 7324 OAVs in BEM and LB8 samples, respectively. Benzene acetaldehyde (LB8 and LL28); decanal ; and undecanal (LB8 and LB531) showed high OAVs and contributed “sweet and floral”, “fatty”, and “fatty” odors to samples, respectively. Nonanal  and decanal (fatty) were identified as medium OAVs in STs2, LB531, and BFM LB8 samples. Benzaldehyde belonged to low OAVs and was present in almost all samples except LC475 samples. Among detected odor-active aldehydes, nonanal and decanal were detected in milk fermented by Lactobacillus plantarum, but they did not contribute odor to a sample’s OAV [As shown in le’s OAV . Nonanaln BFM 160, STs2 (1−1). “Fruity and floral” are pleasant odors for people. The LB8 sample had extremely high OAVs (33047), which are about 10 times higher than other starters. This may be why it is often selected as an industrial strain and used together with Streptococcus thermophilus. The ketone 2-heptanone contributed a “fresh cream” odor with high OAVs between 386 and 815. The ketones 2-nonanone and 2-undecanone, which had extremely high OAVs or high OAVs in some samples, contributed “grassy, fruity, and floral” and “orange, grass, and fresh” to samples. Ketones 2-decanone (sweet and waxy) and 2-dedecanone  with medium OAVs only contributed odor in the BFM sample. Among the odor-active ketones, 2-heptone, 2-nonanone, and 2-undecanone contributed medium OAVs of “fresh cream”, “hot milk and foam”, and “foam and grass” to yogurt fermented by Lactobacillus plantarum [Six ketones were found to affect aroma characteristics of samples from different starter cultures. Odor-active ketones belonged to second-odor-strength compounds, which were inferior to aldehydes. The ketone 2-none-4-one, which contributed “fruity and floral” odors, showed extremely high OAVs in STs2 (3391), LB8 (33047), LL28 (1915), LB531 (3939), and LP45 (1917) samples due to their low threshold , which had a slight contribution to sample odor. Octanoic acid, decanoic acid, dodecanoic acid, and tetradecanoic acid, which provide “chess”, “sweaty”, and “rancid” odors, were detected as odor-active compounds and were classified as high OAVs or medium OAVs. However, compared to other fermented samples, milk, which was fermented by LB8, showed lower OAVs. Hexanoic acid ; nonanoic acid (rancid); and undecanoic acid (rancid) with medium OAVs were detected in fermented samples expected for LB8 samples. Heptanoic acid and pentadecanoic acid with low OAVs only were detected in STs2-fermented milk. Acid compounds are important characteristic odor components in fermented milks. These detected acids also are odor active components in dairy products. Tian et al. (2019) reported that hexanoic acid and octanoic acid with “spicy, rancidity, and floral” and “chess” characteristic odors were detected as odor-active compounds in yogurt which were fermented by ium OAVs . In addiium OAVs .p-xylene, and styrene showed medium odor intensity and high odor intensity in all samples, respectively. Naphthalene and 1-methyl-naphthalene presented high odor intensity in BFM samples. Among all fermented samples, only naphthalene was detected in LB8 samples as an odor-active compound with high OAVs. Benzene derivatives such as ethylbenzene and p-xylene were reported as providing “phenol, spice” and “leathery” odors to yogurt [Five odor-active benzene derivatives, including ethylbenzene, p-xylene, styrene, naphthalene, and 1-methyl-naphthalene, gave a “phenolic” odor to samples. OAVs of ethylbenzene, o yogurt . Other cp-xylene, 1-heptadecene -2,4-decadienal, 2-nonen-4-one, and benzothiazole. This group showed high concentrations in STs2 and LB8, both of which are lactic acid bacteria commonly used in industry. These volatile compounds may be the typical volatile compounds in daily yogurt and may reflect the main differences between industrial strains and probiotics. C group, which showed high content in BFM samples, included six aldehydes, five ketones, seven hydrocarbons, five benzene derivatives, and three other compounds. Most hydrocarbons, benzene derivatives, and other compounds, and parts of aldehydes and ketones, were concentrated in the C group. These compounds represent volatile compounds in raw milk. For example, benzene derivatives are representative compounds that come from raw milk [A heatmap was generated to show the variations in the contents of volatile compounds in milks fermented from different starter cultures . Green craw milk . Hydrocaraw milk .p-xylene, which showed high odor intensity in LB8 and STs2 samples. Acids and benzene derivatives provided “rancid” and “phenolic”, which expressed odor characteristics of two industrial strains [ strains ,44. Ther strains ,34,44. TPCA was applied to the concentrations of 53 volatile compounds to analyze differences among milks fermented from different starter cultures . In FiguS. thermophilus is a multifunctional lactic acid bacteria also used as a probiotic in some studies. As shown in heatmap and PCA, S. thermophilus behaved more like other probiotic strains. The different distribution of odor components may be caused by the probiotic’s activity. The inherent relationship between probiotic activity and volatile components needs further study.PCA also was applied to the concentrations of 31 odor activity characteristics compounds (OAV > 1) to analyze differences among milks fermented from different starter cultures . The PC1S. thermophilus is a multifunctional lactic acid bacteria also used as a probiotic in some studies [S. thermophilus behaved more like other probiotic strains. The different distribution of odor components may be caused by the probiotic’s activity. The inherent relationship between probiotic activity and volatile components needs further study.The heatmap and PCA showed a highly consistent distribution of volatile profiles based on the content and the OAVs in this research. Samples collected from different starter culture fermentations were clustered into three groups. STs2 and LL28, LB531, LC475, and LP45 samples belonged in one kind. LB8 and BFM belonged to the second and third groups and presented a closer distribution trend. LB8 and STs2 presented a further distribution; however, they can synergistically enhance the odor quality of fermented milk. All of these reasons may explain why they have been selected as industrial fermentation strains. However, synergistic action in further research is necessary to study yogurt odor mechanisms.  studies . As showPasteurized full-fat milk from a local retailer  was fortified with 6% sugar. Homogenization was performed at 5 pa, divided into 500 mL glass bottles, and then heated at 98 °C for 15 min. Milk was then immediately cooled to about 35 °C in a water bath and inoculated with different starter cultures . −1) was added as an internal standard. Then, an U-tube apparatus was filled with water and 50 mL diethyl ether. An additional 10 mL diethyl ether was poured into a finger-shaped-bottom flask, and both flasks (one containing diethyl ether and another containing the sample) were heated to boiling. The extraction time was 2 h. After extraction, the distillate in the 100 mL flask was dried over anhydrous sodium sulfate (5 g), concentrated using vacuum rotary evaporation, and stored in headspace vials. The volatile compounds of samples were extracted using SDE in a Likens-Nickerson apparatus. A mixture of 200 g of sample and 2 L of deionized water was placed in a 5 L flask and connected to a Likens-Nickerson apparatus. Aqueous 2, 4, 6-trimethylpyridine  equipped with a mass spectrometer  and fitted with a DB-5 capillary column . Helium at 1 mL·minn-alkanes  that elute before and after the target compound (i) for the same chromatographic conditions.These volatiles were further confirmed by matching their linear retention indices (LRIs) and odor descriptions in the literature ,34,44. T−1) was used as internal standard for quantitative analysis. The quantitative calculation (mg·kg−1) was based on:Aqueous 2, 4, 6-trimethylpyridine  followed by Turkey and Dunnet’s multiple comparison test was adopted to evaluate the differences in volatile concentrations among yogurt samples. Heatmap and PCA were performed using MultiExp Simc-P 11.5  to assess differences in volatile compounds and odor-active compounds, respectively. Unless specified, In the present study, the influence of starter cultures on the volatile profiles of milks were investigated. A total of 53 volatile compounds and 31 odor-active compounds were detected in all samples. Ketones and hydrocarbons were the predominant volatile components in the milk before fermentation, while acids became the predominant volatiles in all fermented samples. -2,4-decadienal, 2-nonanone,2-heptanone, and 2-undecanone with high OAVs were characteristic odor compounds in all samples. Fermentation increased OAVs of acids in samples. Compared with probiotics, there were greater abundance volatile compounds and odor-active compounds detected in fermented milks with STs2 and LB8. The heatmap and PCA showed a highly consistent distribution of volatile profiles based on the content and the OAVs. By means of heatmap and PCA, samples were divided into three kinds. STs2 and probiotics  belonged to one kind, and LB8 and BFM separately belonged to the other two kinds. The obtained data could help to better understand the influence of starter cultures on the odor quality of fermented milk and significant values for strain utilization and dairy development in dairy industry.


Actinidia chinensis) is a rich nutritious fruit crop owing to a markedly higher content of vitamin C and minerals. To promote fruit set and to increase the yield of kiwifruit, forchlorfenuron (CPPU) has been widely applied. However, the molecular details regarding CPPU controlling kiwifruit development, especially at the fastest fruit growth stage, remain unknown. In the present study, we measured the effect of CPPU on developmental regulation in red-fleshed kiwifruit (Actinidia chinensis ‘Hongyang’). Additionally, a cytological analysis was performed to clarify the precise changes in the cell structure of the CPPU-treated kiwifruits. Moreover, the concentration of endogenous phytohormones, including indoleacetic acid (IAA), zeatin (ZT), gibberellic acid 3 (GA3), and abscisic acid (ABA), were measured by Enzyme-linked Immunosorbent Assay (ELISA). Furthermore, RNA-Seq was performed to dissect the complicated molecular mechanisms, with a focus on biosynthesis, metabolism, and signaling compounds, such as endogenous hormones, sugars, and L-ascorbic acid. Our results demonstrated that CPPU treatment not only regulates the size and weight of a single fruit but also improves the quality in ‘Hongyang’ kiwifruit through the accumulation of both soluble sugar and vitamin C. It was also seen that CPPU regulates kiwifruit development by enhancing cell expansion of epidermal cells and parenchyma cells, while, promoting cell division of subepidermal cells. Additionally, CPPU significantly increased the gibberellin and cytokinin biosynthetic pathway and signaling, while repressing auxin and ABA biosynthetic pathway; thus, signaling plays an essential role in CPPU controlling kiwifruit development. Notably, transcriptomic analysis revealed that a total of 2244 genes, including 352 unannotated genes, were differentially expressed in kiwifruits because of CPPU treatment, including 127 transcription factors. These genes are mainly enriched in plant hormone signal transduction, photosynthesis, MAPK signaling pathway, starch and sucrose metabolism, and phenylpropanoid biosynthesis. Overall, our results highlight that CPPU regulation of kiwifruit development is mainly associated with an antagonistic and/or synergistic regulatory role of endogenous phytohormones, and enhancing the energy supply. This provides new insights into the molecular details of CPPU controlling kiwifruit development at the fastest fruit growth stage, which is of agricultural importance for kiwifruit breeding and crop improvement.Kiwifruit ( Arabidopsis thaliana and Solanum lycopersicum (tomato) [SlPIN4), Transport Inhibitor Response 1 (TIR1), GA 20-oxidases (GA20ox) and 9-cis epoxycarotenoid dioxygenase1 (NCED1) [Fw2.2 gene encoding plant-specific and fruit-specific protein, functions in regulating fruit size in tomato [TOMATO AGAMOUS (TAG)-LIKE1 (TAGL1), resulted in a reduction in pericarp thickness from 25 to 15 layers and an absent inner pericarp [After fertilization, the main contributors to fruit development are cell division and cell expansion, which are tightly regulated by genotypes and environmental conditions –4. Recen(tomato) –7. Among(tomato) , 9. Inte(tomato) . It has  (NCED1) –14, playn tomato . Loss ofpericarp . Notablypericarp .Actinidia) is native to China, and is a rich nutritious fruit crop because of its high concentration of vitamins, minerals, and amino acids [Actinidia species [A. arguta, A. melanandra, A. macrosperma, and A. polygama [A. deliciosa ‘Hayward’ [Actinidia arguta ‘Mitsuko’ [Kiwifruit  [A. eriantha ‘White’ [SPS), sucrose-6F-phosphate phosphohydrolase (SPP), and sucrose synthase (SuSy), were gradually increased during fruit development, which might explain the observation that the accumulation of soluble sugars increases in kiwifruits from 1.8% of fresh weight at the S2 stage (70 days after anthesis) to 7.12% of fresh weight (FW) at the S7 stage (140 days after anthesis) [Alase), nonheme hydroxylase (CHY), chlorophyll b reductase (CBR), and 1-aminocyclopropane-1-carboxylate oxidase (ACO)) [The draft genome of the kiwifruit  in 2013 . Afterwangyang’) , 20, and ‘White’ . Additio ‘White’ . Among tnthesis) . Furthere (ACO)) . Additioe (ACO)) .These data from the genome and transcriptome of the kiwifruit provide a valuable resource not only for biological discovery but also crop improvement. However, our understanding of the molecular mechanism underlying CPPU controlling kiwifruit development (required at the fastest growth stage) remains quite limited. In the present study, we disassembled this complicated mechanism through (1) analyzing the effects of CPPU on kiwifruit characteristics, including the size and weight of a single fruit and the soluble sugar and vitamin C contents, (2) illustrating the change at the cellular level of the kiwifruit after CPPU treatment, (3) monitoring the concentration of endogenous plant hormones in CPPU-treated kiwifruits, and (4) obtaining a deeper understanding of molecular mechanisms of CPPU treatment in kiwifruit.A. chinensis ‘Hongyang’ were used for the present study and were grown in the orchard of Chongqing University of Arts and Sciences, Chongqing, China. The CPPU treatments were performed as previously described [-1 of CPPU  for 10 s. Double-distilled water (ddH2O) was used for the control treatment. Kiwifruit samples were collected after 0 d, 20 d, 40 d, 60 d, 80 d, and 120 d of CPPU treatments. For each concentration of CPPU treatment, 10 different kiwifruits at each time point were harvested. All samples were immediately frozen in liquid nitrogen and stored at -80°C.Three-year-old plants of escribed . BrieflyThe phenotypes of kiwifruits at each time point were recorded. Moreover, the weight, the transverse diameter, and longitudinal diameter of single fruit were also measured. In addition, to assess the soluble sugar and vitamin C contents in kiwifruits, the plant soluble sugar content test kit and vitamin C content test kit  were used according to the manufacturer’s protocol, respectively. To test the dry matter content of kiwifruits, the weight of kiwifruits and aluminum cans were measured before drying treatment and were recorded as (A) and (B), respectively. These kiwifruits were placed into the aluminum cans and were desiccated until they reached a constant weight at 70°C; finally, the weights were recorded as (C). Therefore, dry matter content was calculated according to the formula (C—B)/A × 100%. For all results, three technical replicates were performed, and all data represent the mean with standard deviations (n = 10).-1 CPPU treatment, those kiwifruits were sectioned in the transverse plane, and then photographed. These kiwifruits after 20 d of ddH2O treatment were used as control. Briefly, samples were taken from the outer pericarp (OP) and related tissues at the middle of the fruits and were immediately immersed in fixative. Then, these samples were cut into 4 μm sections and fixed with FAA [To unravel how CPPU controls kiwifruit development at the cellular level, after 20 d of 20 mg Lwith FAA . All sec3), cytokinin , and abscisic acid (ABA), enzyme-linked immunosorbent assays were performed. Briefly, fresh flesh (0.2 g) from 0 d, 20 d, 40 d, 60 d, 80 d, 100 d, and 120 d samples of both 20 mg L-1 CPPU-treated and ddH2O-treated kiwifruits were ground in liquid nitrogen. Then, the powders were mixed with 1.8 mL of phosphate-buffered saline  and were centrifuged at 3000 r/min for 20 min. Subsequently, the content of IAA, GA3, ZT, as well as ABA were measured using the IAA Elisa Kit, GA3 Elisa Kit, ZT Elisa Kit and ABA Elisa Kit , respectively [To evaluate the concentration of auxin , gibberellin  and NanoDrop 2000 spectrophotometer (Thermo) [™ RNA Library Prep Kit from Illumina  according to the manufacturer’s protocol, which was performed by ABOROAD . To obtain high-quality reads, the adaptor and low-quality bases were trimmed. Clean reads were aligned to the kiwifruit reference genome database (http://bioinfo.bti.cornell.edu/cgi-bin/kiwi/home.cgi) [-5 [2Ratio ≥ 1 and q  < 0.05 [http://mapman.gabipd.org) [Total RNA was isolated from kiwifruit samples using EasyPure Plant RNA kit . Briefly, total RNA was extracted from 3 kiwifruits after 20 d of 20 mg L(Thermo) , 33. Theome.cgi) . Furthercgi) [-5 . The gen) < 0.05 . Furtheripd.org) . The tra-1 CPPU treatment. The kiwifruits treated with ddH2O were used for the control. At each time point, 3 CPPU-treated and 3 ddH2O-treated kiwifruit samples were used. Briefly, 0.5 μg of total RNA was using for cDNA synthesis via HiScript® II Q Select RT Supermix  [https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) (-ΔΔCt method. AcActin (GI:149938963) was used as an internal reference gene [To test the validation of our RNA-seq databases, the expression levels of 11 randomly selected genes were measured by qRT-PCR using the same RNA samples for the transcriptome analysis. Additionally, expression patterns of 8 candidate genes were monitored in kiwifruits after 0 d, 20 d, 40 d, 60 d, 80 d, 100 d, and 120 d of 20 mg L, China) . The priastHome) . The lennce gene , 38.The significant differences of all data were evaluated by SPSS version 16.0  according to Duncan’s multiple range test (P < 0.05). For all results, three technical replicates were performed, and the results were represented as the mean with standard deviations , 37.A. chinensis ‘Hongyang’, the fresh weight, transverse diameter, and longitudinal diameter of every single fruit (n = 10) was measured every 20 days after CPPU treatments. Compared to the control, we found that 5 mg L-1 of CPPU treatment strongly increased both fresh weight and fruit size of single fruit , abscisic acid (ABA) and auxin . The concentration of ZT was significantly higher in CPPU-treated kiwifruits than in the control throughout entire fruit developmental phases   , indicatCompared with that of the control, a total of 2244 genes were differentially expressed (DEGs) in the kiwifruits after 20 days of CPPU treatment. Among these genes, 923 and 1321 genes were upregulated and downregulated, respectively . InteresTo further dissect how the upregulation and downregulation of DEGs affect kiwifruit development, we grouped them based on their individual metabolic pathways through gene ontology (GO) enrichment analysis . After C3, ZT and ABA were dramatically changed in the kiwifruits in response to CPPU treatment (3 (LOG3) and GA 20-oxidase 1 (GA20ox1), and strongly induced expression of cytokinin signaling pathway positive regulators, e.g., ARR3, ARR5, and ARR9. In contrast, dramatically repressed transcripts of the gibberellin signaling negative regulator DELLA was also found . Consistent with a significantly decreased IAA content in CPPU-treated kiwifruits  were also strongly decreased (GH3 genes (GH3.1 and GH3.6) that encode IAA-amido synthase functioning to conjugate amino acids to auxin , which encode auxin efflux carrier involved in the maintenance auxin gradients, were dramatically repressed , which act as repressor of auxin-inducible gene expression, were also evidently reduced  encoding a critical ethylene biosynthetic enzyme , and ERF017, were significantly induced  and 1 myoinisitol oxygenase (AcMIOX1) (SUS1), tonoplast monosaccharide transporter 2 (TMT2), hexose carrier protein 6 (HEX6), and sugars will eventually be exported transporter 17 (SWEET17)  . AdditioSWEET17) . TherefoAcARR3, AcGA20ox1, AcDELLA, AcPP2C, AcETR2, AcSAUR50, AcSUS1, and AcMIOX1, respectively. Compared with the control, and consistent with the content changes of plant hormones, sugar and vitamin C in kiwifruits in response to CPPU treatment; the expression levels of AcARR3, AcGA20ox1, AcSUS1, and AcMIOX1 were significantly induced  DEGs are unannotated . The reaongyang’ . TherefoA. deliciosa ‘Hayward’ [GULLO3, GULLO6, MIOX1, SUS1, TMT2, HEX6, and SWEET17  to AsA [SUS gene, which encoded a protein with sucrose synthase activity, gradually increased during kiwifruit development [Furthermore, we observed that the soluble sugar and vitamin C contents were strongly increased in CPPU-treated ‘Hongyang’ kiwifruits, suggesting that CPPU treatment improved not only the yield but also the quality of ‘Hongyang’ kiwifruits . SimilarHayward’ . Consist SWEET17 . In addial phase . The trareatment . These r) to AsA . Furtherelopment .GA 20-oxidase 1 (GA20ox1) and cytokinin phosphoribohydrolase ‘Lonely guy’ (LOG3)  and nine-cis-epoxycarotenoid dioxygenase (NCED3)  . These rn tomato , 48. Theanthesis . We also (NCED3) . Similarth stage . Strikinth stage , suggestARR3, ARR5, and ARR9, and PP2C were found in CPPU-treated kiwifruits Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


A higher CE score was a predictor of mortality; coronary artery disease (CAD) and elevated CE scores were associated with an increase in mortality . Kaplan–Meier (KM) analysis of the entire cohort (n = 51) demonstrated a decrease survival in the CE group, p = 0.026. KM survival analysis of hydrocortisone, ascorbic acid, and thiamine (HAT) therapy and control patients not receiving steroids (n = 45) showed CE and IE was associated with a decrease in survival (p = 0.003); of interest, there was no difference in survival in CE patients after stratifying by HAT therapy (p = 0.18). These findings suggest a possible treatment effect of corticosteroids, HAT therapy, endotype, and outcome. Conclusion: This subset of patients from the ORANGES trial confirmed previous retrospective findings that a 33-mRNA classifier can group patients into IE, AE, and CE endotypes having prognostic significance. A novel finding of this study identifying an association between endotype and corticosteroid therapy warrants further study in support of future diagnostic use of the endotyping classifier.Background: Retrospective analysis of the transcriptomic host response in sepsis has demonstrated that sepsis can be separated into three endotypes—inflammatory (IE), adaptive (AE), and coagulopathic (CE), which have demonstrated prognostic significance. We undertook a prospective transcriptomic host response analysis in a subgroup of patients enrolled in the Outcomes of Metabolic Resuscitation Using Ascorbic Acid, Thiamine, and Glucocorticoids in the Early Treatment of Sepsis (ORANGES) trial. Methods: Blood was obtained from 51 patients and profiled using a pre-established 33-mRNA classifier to determine sepsis endotypes. Endotypes were compared to therapy subgroups and clinical outcomes. Results: We redemonstrated a statistically significant difference in mortality between IE, AE, and CE patients, with CE patients demonstrating the highest mortality (40%), and AE patients the lowest mortality (5%,  Sepsis, defined as a dysregulated immune response to an acute infection, confers an extremely high mortality and utilization of health care resources ,2. A comRecently, based on pre-clinical and clinical experience there has been great interest in employing the combination of hydrocortisone, intravenous ascorbic acid (AA) and thiamine (known as HAT therapy) in the management of patients with sepsis ,7. HowevOver the last decade microarray analysis, genomics and transcriptomics studies have demonstrated that the host response in sepsis explains some of the heterogeneity of the syndrome ,4. A proSweeney et al. previously reported the discovery and validation of three sepsis endotypes across 1300 patients with bacterial sepsis at hospital or ICU admission . Whole bWe here prospectively studied whether a previously defined 33-mRNA classifier for these transcriptomic endotypes holds predictive validity in septic patients in a substudy of the ORANGES trial .ClinicalTrials.gov Identifier: NCT03422159.Study population: Outcomes of Metabolic Resuscitation Using Ascorbic Acid, Thiamine, and Glucocorticoids in the Early Treatment of Sepsis (O.R.A.N.G.E.S.) The ORANGES trial was a randomized, double-blinded, placebo-controlled trial assessing the utilization of an ascorbic acid, thiamine, and hydrocortisone treatment bundle for the management of septic and septic shock patients admitted to an ICU and is described in detail elsewhere . This stSample collection: Whole blood was drawn in PAXgene Blood RNA tubes at enrollment along with other standard laboratory parameters. Data collection included demographic information, clinical scores , laboratory results, length of stay, and clinical outcomes. Patients were followed up daily until time of discharge. PAXgene Blood RNA samples were shipped to Inflammatix , where RNA was extracted and the 33 mRNAs were quantitated using NanoString nCounter, as described ,16.ARG1, LCN2, LTF, OLFM4, HLA-DMB; Adaptive (AE), YKT6, PDE4B, TWISTNB, BTN2A2, ZBTB33, PSMB9, CAMK4, TMEM19, SLC12A7, TP53BP1, PLEKHO1, SLC25A22, FRS2, GADD45A, CD24, S100A12, STX1A; Coagulopathic (CE), KCNMB4, CRISP2, HTRA1, PPL, RHBDF2, ZCCHC4, YKT6, DDX6, SENP5, RAPGEF1, DTX2, RELB. We then applied the previously defined multi-class logistic regression model to these three input gene expression scores, which yields a probability of endotype assignment  + p(Adaptive) + p(Coagulopathic)] sums to 1). Each sample is assigned an endotype according to the highest probability , were on mechanical ventilation , and had higher APACHE IV scores, p = 0.017  was used to designate every patient’s host response endotype as one of IE (34%), AE (39%), or CE (27%) . Their cp = 0.032 [p = 0.026) (p = 0.023), and showed negative correlation with AE scores  and positive correlation with CE scores   when compared to both the IE (10%) and the CE (40%),  = 0.032 . Kaplan–= 0.026) . Mortali= 0.005) p = 0.007).Unlike previous studies, no difference in age was observed among endotypes in our cohort. The current study shows procalcitonin (not previously studied with sepsis host response endotypes) was highly induced in IE patients, but not in the AE patients . Similarp = 0.020 & HR 15.5, 95% CI 1.14–211, p = 0.039, respectively).In this cohort, several factors were significantly associated with mortality, including age, clinical severity (APACHE IV score), mechanical ventilation, the presence of coronary artery disease and the CE score. We employed Cox proportional hazards analysis and showed both coronary artery disease and CE score to be significant predictors of mortality (Hazard ratio (HR) 12.3, 95% CI 1.4–101, n = 23), a comparator group who received steroids off protocol (n = 6), and a comparator group who did not receive steroids per protocol (n = 22). In general, there were few differences between the groups    and validated preset clinical findings previously shown to be associated with the endotypes, lending credence to the findings.The 33-mRNA classifier is one possible way to reduce the clinical heterogeneity and inform therapeutic decisions. We suggest that future studies of immunomodulatory therapy in sepsis should at a minimum draw RNA-stabilized blood at the time of enrollment so that a prospective blinded endotypes analysis (using ours or other classifiers) can be performed. It is also important that future studies include specific markers of coagulopathy in order to have a more robust analysis of CE. It is only through such further blinded prospective endotype-based analysis that we will gain the confidence to apply the endotypes in interventional treatment randomization.More work is needed to identify and confirm a companion-diagnostic approach to immunomodulatory therapy and sepsis. We emphasize that transcriptomic evaluation of septic patients is leading to a paradigm shift in risk stratification and may enable the identification of patients responding to different therapeutic approaches, thereby leading to a personalized approach to individual patients.