Dimocarpus longan Lour.). In this study, 23 genes in the CCD, D27, and SMXL family were identified in the longan genome. The phylogenetic relationships, gene structure, conserved motifs, promoter elements, and transcription factor-binding site predictions were comprehensively analysed. The expression profiles indicated that these genes may play important roles in longan organ development and abiotic stress responses, especially during early somatic embryogenesis (SE). Furthermore, GR24  and Tis108 (SL biosynthesis inhibitor) could affect longan early SE by regulating the levels of endogenous IAA (indole-3-acetic acid), JA (jasmonic acid), GA (gibberellin), and ABA (abscisic acid). Overexpression of SMXL6 resulted in inhibition of longan SE by regulating the synthesis of SLs, carotenoids, and IAA levels. This study establishes a foundation for further investigation of SL genes and provides novel insights into their biological functions.Strigolactones (SLs), a new class of plant hormones, are implicated in the regulation of various biological processes. However, the related family members and functions are not identified in longan ( D27 acts upstream of MAX1 in the SL biosynthetic pathway in Arabidopsis [D27 leads to a deficiency in strigolactone biosynthesis [ccd7 and ccd8 mutants [ccd7 and ccd8 are deficient in SL and exogenous application of GR24, a synthetic analogue to SL, and rescued the excess branching phenotype of ccd7 and ccd8 [Strigolactones (SLs) are defined as carotenoid-derived plant hormones, which are divided into oxygen-free carotenes, such as b-carotene, and xanthophylls that contain oxygen, such as lutein ,2. SLs wbidopsis . The mutynthesis ,24. In rynthesis . D27 alsynthesis . CCD1, Cynthesis ,29,30. E mutants ,6. Studiand ccd8 .SMXL6, 7 and 8 in Arabidopsis [It was demonstrated that SLs employ SMXL (SUPPRESSOR OF MAX2-LIKE) family members for signal transduction in Arabidopsis ,33, and bidopsis .Arabidopsis, as well as GR24 and Tis108 (an SL biosynthetic inhibitor) on SE correlated with changes in the expression of AUXIN RESPONSIVE FACTORs, which are required for the production of embryogenic tissue [Plant somatic embryogenesis (SE) is defined as a general cultivation technique in vitro, where embryos develop from somatic cells without gamete formation and fertilisation ,42. Recec tissue . These sThe interactions between SLs and other plant hormones were also reported. In tomato, the level of SLs decreased after treatment with specific ABA inhibitors . ConversDimocarpus longan Lour is cultivated widely for tropical/subtropical evergreen fruit trees. Since the establishment of the longan SE system by Lai [SMXL6 (DlSMXL6) inhibited the differentiation of longan somatic embryos, which may be related to the synthesis of IAA by upregulating the expression of longan YUC5 (DlYUC5) and longan YUC10 (DlYUC10). These findings provide a valuable resource for better understanding the biological roles of SL biosynthesis and response genes in longan SE.m by Lai ,58, whicm by Lai . HoweverIn this study, a total of 23 genes were identified in the SL biosynthesis and signalling pathway, including 11 members in the CCD gene family (DlCCD), eight members in the SMXL gene family (DlSMXL), and four members in the D27 gene family (DlD27). To gain insight into the biological functions of DlCCD, DlSMXL, and DlD27 family members, the protein physicochemical properties of the three gene families were further analysed . DlCCD, DlCCD6 and DlCCD7 were found in peroxisomes. DlSMXL6 was predicted to localise in the extracellular matrix. Four members were found primarily in the nucleus, including DlSMXL2, DlSMXL3, DlSMXL7, and DlD27c. In addition, no signal peptides exist for all full members of the three gene families, suggesting that none of the DlCCD, DlSMXL, or DlD27 family members were secreted proteins.Most family members were predicted to localise in the chloroplast. DlCCD1, DlCCD2, DlCCD3, DlSMXL,1 and DlSMXL2. DlSMXL family genes were located on Chr1, Chr5, Chr7, Chr12, and Chr14. The DlD27 genes were located on Chr5, Chr7, and Chr15. Additionally, Chr4, Chr10, Chr12, and Chr15 only contained one number.As shown in DlSMXL3-DlSMXL7 and DlSMXL4-DlSMXL8, which were both classified in subgroup VI of the phylogenetic tree  and DlSMXL7 (Chr12) corresponded to two genes in Arabidopsis  was searched for analysis . Among tcis-acting elements of the promoter could combine with specific transcription factors and then form a transcription initiation complex to initiate gene-specific expression in various biological processes [The rocesses . To inveDlCCD2, DlCCD3, DlCCD11, and DlSMXL2 were highly expressed in the flower. DlCCD2, DlCCD4, and DlSMXL7 were highly expressed in the leaf. DlD27a, DlSMXL6, and DlSMXL7 were highly expressed in the roots. DlCCD1, DlCCD3, DlSMXL1, DlSMXL2, DlSMXL6, DlSMXL7, DlD27a, and DlD27d were highly expressed in the seed and might be involved in longan seed dormancy and germination. DlSMXL2, DlCCD5, DlCCD6, and DlCCD7 were highly expressed in the young fruit, indicating that these genes might be involved in fruit development and maturation.We further analysed the expression patterns of DlCCD, DlSMXL, and DlD27 family genes in different tissues and treatments by transcriptome data . The resDlCCD2, DlCCD6, DlCCD7, DlCCD11, DlSMXL3, and DlSMXL6 were highly expressed under blue light. DlD27a, DlD27d, DlCCD5, DlCCD8, DlCCD10, DlSMXL5, and DlSMXL7 were highly expressed under white light , indicating that these genes might be involved in cell self-repair under high temperature  for 24 h. We first determined the endogenous SL content of longan ECs under different hormone treatments. The results show that exogenous IAA and GA significantly increased the SL content compared to the control, while ABA and JA decreased the SL content in longan ECs  and SL biosynthetic inhibitor Tis108 (3 μM) were applied in this study . The micTo further explore the interaction relationship between SLs and other hormones in longan, endogenous hormone contents were determined with the SL synthesis analogue GR24 and the synthesis inhibitor Tis108. The results show that GR24 decreased the IAA levels of longan somatic embryos compared with the control, while Tis108 increased the IAA levels at 6 and 8 days C. These DlSMXL6 expression decreased gradually during longan SE and was downregulated by IAA treatment, indicating that it might play a role in somatic embryo development. To verify this prediction, the full-length DlSMXL6 CDS was cloned into the pCAMBIA-1301-35S overexpression vector and stably transformed into longan ECs. We selected the OE-1, OE-2, and OE-3 cell lines with high expression levels for further analyses  by auxin was reported [DlCCD4, DlCCD8, DlD27a, DlSMXL2, and DlSMXL6, indicating that these genes may be involved in SL synthesis under IAA treatment to regulate longan SE. To further demonstrate the effects of SLs on ECs differentiation and the endogenous hormone content changes during longan SE, GR24 and Tis108 were applied to treat longan ECs. The results show that GR24 could promote the transformation of ECs into GEs, and Tis108 inhibited the process. In this study, GR24 decreased the IAA level of longan embryos, while Tis108 could increase the IAA level, indicating that GR24 and Tis108 could regulate the SE process by altering the endogenous IAA content [Hormones play crucial roles in plant growth and development, especially in plant SE. Recently, the interactions between SLs and other hormones were uncovered in plant development ,83. Auxiby auxin ,88,89,90 content ,91.DlCCD5, DlCCD7, and DlD27d were downregulated in longan ECs by ABA treatment. GR24 did not lead to an increased content of ABA [ABA plays an important role in plant SE, which can accelerate the synthesis and accumulation of nutrients ,94,95,96t of ABA , and negt of ABA . In chert of ABA . HoweverDlCCD5, DlCCD8, DlD27d, and DlSMXL2. GR24 could significantly increase the GA content in seeds [Jatropha curcas, SL acted antagonistically to gibberellin in the control of lateral bud outgrowth [GA was reported to be necessary for the induction of SE in spinach ,103,104 in seeds , and SL in seeds . In Jatrutgrowth ,106. HowDlSMXL6 and downregulating the expression of DlD27a. The JA content significantly increased upon GR24 treatment at 6 and 8 days and significantly decreased upon Tis108 treatment, which may play an important role in longan SE upon SL regulation.It was reported that JA inhibits embryo germination in angiosperms ,109, andTaken together, GR24 promoted longan SE by decreasing the levels of IAA and increasing the levels of JA at the early stage, while Tis108 inhibited the process by regulating the levels of IAA at the early stage and ABA and JA at the late stage A. HoweveSMXL6, 7, and 8 in Arabidopsis [DlSMXL6 was specifically highly expressed in ECs and IcpEC, and expressed at low levels in GE, indicating its role in somatic embryo development. The results show that SMXL6-OE cells could not differentiate into GE cells on the 12th day, while WT and 1301-GUS cells exhibited typical GEs.As a downstream regulatory factor of SL, SMXL6, 7, and 8 are important switches of the SL signalling pathway and play an important role in regulating the growth and development of plant branches, root architecture, and leaf shape ,36,38,39bidopsis . CompareDlSMXL6-overexpressing cell lines compared to the WT and 1301-GUS cell lines by upregulating the expression of DlYUC5 and DlYUC10. As reported, the requirement of IAA content decreased during the plant SE [DlSMXL6 in longan ECs also promoted the accumulation of SL precursor carotenoids, which may be due to the feedback regulation of the SL pathway  supplemented with 100 μM IAA, ABA, JA, or GA for 24 h. Additionally, to investigate the effects of SL on the conversion of ECs into GEs in longan, the ECs were cultured in MS liquid medium added with GR24 (1 μM stock in acetone) and Tis108 (3 μM stock in DMSO). The same volume of acetone (Mock1) and DMSO (Mock2) were as controls at 25 °C with 120 r·min−1 shaking in the dark for 10 days. The test materials were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent tests. Each assay was repeated three times.The ‘Hong He Zi’ (‘HHZ’) longan ECs were obtained as previously described . The exphttp://www.arabido-psis.org (accessed on 10 May 2021). They were used as base alignment sequences with the local BLAST of TBtools [https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 10 May 2021) and SMART (http://smart.embl-heidelberg.de/ (accessed on 15 May 2021)) tools to verify their reliability as targets [https://www.expasy.org/ (accessed on 15 May 2021). The subcellular localisation of SL biosynthesis and signalling proteins were predicted with Plant-PLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant/ (accessed on 15 May 2021) [http://www.cbs.dtu.dk/services/SignalP/ (accessed on 15 May 2021). Chromosomal localisation information was extracted from the GFF file, and the results were visualised with Tbtools. The cis-acting elements were analysed with PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 15 May 2021), which were present from 2000 bp upstream of the gene initiation codon. PlantTFDB (http://planttfdb.cbi.pku.edu.cn/ (accessed on 15 May 2021) was used to predict the transcription factor-binding sites (TFBSs) on promoters with the parameter set of p value ≤ 1 × 10−6. MEME (https://meme-suite.org/meme/tools/meme (accessed on 15 May 2021) was used to analyse the proteins’ conserved motifs. Finally, TBtools software  was used to annotate the gene structure and visualise the conserved domains and motifs. MCScanX software was used to predict the collinearity of the genes of SL biosynthesis and signalling and to mark the collinear genes [https://sourceforge.net/ (accessed on 20 May 2021) was used to estimate synonymous (Ks) and nonsynonymous (Ka) substitution rates [The genes involved in SL biosynthesis and signalling in Arabidopsis were obtained from TAIR  . The sigar genes . KaKs_Caon rates .Arabidopsis thaliana) from TAIR (http://arabidopsis.org (accessed on 20 May 2021) and rice (Oryza sativa) from hytozome v12.1 . The amino acid sequences of Zea mays, Citrus sinensis, Glycine max, Solanum lycopersicum, and Triticum aestivum were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 20 May 2021). The phylogenetic trees were constructed using MEGA 5.0 software with neighbour-joining (NJ) [To compare the phylogenetic relationship of the genes involved in SL biosynthesis and signalling in longan with their counterparts in other plant species, we obtained the amino acid sequences of Arabidopsis (ing (NJ) .The FPKM expression profile of genes involved in the SL pathway in longan was extracted from four transcriptome datasets, including three for the ‘Hong He Zi’ (HHZ) cultivar and one for the ‘Si Ji Mi’ (SJM) cultivar. RNA-seq data during early longan SE were available in the NCBI SRA repository under accession code SRR21979789, SRR21979788 and SRR21979787 (ECs), SRR21979786, SRR21979785 and SRR21979784  and SRR21979783, and SRR21979782 and SRR21979781 (GEs). RNA-seq data of different temperature treatments on ECs are available in the NCBI SRA repository under accession code SRR21921625, SRR21921624 and SRR21921623 (15 °C), SRR21921622, SRR21921621 and SRR21921620 (25 °C), SRR21921619, and SRR21921618 and SRR21921617 (35 °C). The different light qualities  on ECs are available in dataset . The fraThe levels of IAA, GA, JA, ABA, carotenoid, and SL were determined with a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The specific steps were as follows: the standard, sample extract, and horseradish peroxidase (HRP)-labelled detection antibody were added to the coating micropores, their corresponding antibodies were added, and the samples were incubated and washed thoroughly. The substrate 3,3′,5,5′-tetramethylbenzidine was used for colour development. TMB was transformed into blue under the catalysis of peroxidase and finally into yellow under the action of acid. The colour depth was positively correlated with the content of the substance to be measured in the sample. The absorbance was determined with a microplate reader  at a 450 nm wavelength to calculate the sample activity. Each assay was repeated three times.Total RNA was extracted using the TransZol Up reagent kit  and treated with RNase-free DNase I  following the manufacturer’s instructions. The RNA quality was analysed by agarose gel electrophoresis and quantified using a Nanodrop 2000 spectrophotometer . Only RNAs that met the criteria 260/280 ratio of 1.8–2.1 and 260/230 ratio ≥ 2.0 were used for further analyses and stored at −80 °C.DlFeSOD (EU330204) was used as a reference gene for the internal control [−ΔΔCt method [The RNA was used to synthesise first-strand cDNA using a PrimeScript RT Master Mix  cDNA Synthesis Kit . DNAMAN software was used to design the qRT–PCR primers . The eff control . The opet method .SMXL6 coding sequences in longan ECs were amplified by PCR and then inserted into a separate pCAMBIA-1301-35S vector at the BamH I and Sal I restriction sites using the In-Fusion HD Cloning Kit . The generated pCAMBIA1301-35S-SMXL6 recombinant plasmids were used for longan ECs stable transformation.The SMXL6 constructs were transformed into the Agrobacterium tumefaciens strain (GV3101) following the manufacturer’s instructions . The rapidly proliferating longan ECs were transferred into the bacterial liquid of Agrobacterium tumefaciens (OD600 = 0.7–0.8). After 30 min of infection, the infected ECs were transferred into coculture MS solid medium containing 30 g/L sucrose for three days and then transferred into MS solid medium containing 1.0 mg/L 2,4-D, 20 g/L sucrose and 300 mg/L cefotaxime and harvested after 20 days. The proliferating ECs were transferred into MS solid selection medium containing 1.0 mg/L 2,4-D, 20 g/L sucrose, 300 mg/L cefotaxime, and 20 mg/L hygromycin and subcultured every one month until resistant ECs formed. GUS staining  and PCR amplification were used to detect the transformed ECs. For the differentiation of early somatic embryos, longan wild-type and transformed ECs were cultured in MS solid medium containing 300 mg/L cefotaxime, and samples were harvested after 6 and 12 days. The morphological characteristics of transformed somatic embryos were observed by microscopy (Olympus).The pCAMBIA1301-35S-p < 0.05), “**” (p < 0.01), “***” (p < 0.005), or “****” (p < 0.001) using Student’s t test.Data were analysed, and graphs were drawn using GraphPad Prism 8 . In all graphs, error bars indicate standard deviation, and significant differences are indicated with “*” (


Intramuscular injection of anemoside B4 (AB4) has a superior therapeutic effect on clinical mastitis in lactating cows. Here, we explored AB4’s effect on milk whey in clinical mastitis-affected cows using proteomics. Among fifty clinical mastitis cows received AB4 administration , twelve healed cows were selected and marked as group T. Twelve clinically heathy cows received the same dose of saline for 7 days, marked as group C. Collected milk whey of group T before and after AB4 administration marked as T1 and T2, respectively. The milk whey of group C after saline injection marked as C1. Milk whey protein changes were detected using tandem mass tag-based quantitative proteomic. We identified 872 quantifiable proteins in the samples. Among them, 511 proteins between T1 and C1, and 361 proteins between T2 and T1 were significantly altered. T1 than C1 had significantly more proteins associated with inflammatory damage and trans-endothelial migration of leukocytes, whereas these proteins were reduced in T2 treated with AB4. Compared with C, proteins associated with fibrin clot degradation and complement system activation were downregulated in T1 but upregulated in T2. In summary, AB4 can exert its therapeutic effect on clinical mastitis in cows mainly by reducing inflammatory damage, activating the complement system, inhibiting trans-endothelial migration of leukocytes, and promoting degradation of milk fibrin clots. Pulsatilla chinensis (Bunge) Regel, (a synonym of Anemone chinensis Bunge) belongs to the family of Ranunculaceae, being used as a kind of medical herb for a long history. In traditional Chinese medicine (TCM), P. chinensis is used to clear heat in body and detoxify, to cool blood and stop dysentery such as bacterial dysentery, amebic dysentery, especially good at clearing damp-heat of gastrointestinal tract and blood-heat toxin, in addition, it was reported to treat malaria and relieve spasm and pain as well1. Modern pharmacological studies confirmed that the extracts or the active compounds, such as anemoside B4 (AB4), anemoside B5 (AB5), isolated from P. chinensis played important roles in anti-inflammatory, anti-microbial and antiviral3. AB4 can inhibit the increase of TNF-α and E-selectin secretion from RIMECs caused by LPS with IL-6 unchanged, inhibits both LPS and concanavalin A-induced splenic lymphocyte proliferation, and reduces LPS-induced NF-κB activation and IL-6 production5. The in vivo anti-inflammatory activities of AB4 assessed through the intraperitoneal injection of 2 mg/kg AB4 in mice showed protection against LPS-induced acute lung injury, accompanied by reduction of IL-6, neutrophil infiltration, and NF-κB activation6. AB4 (12.5–50 mg/kg) significantly suppresses xylene-induced mouse ear edema and ameliorated LPS-induced kidney and lung inflammation damage, inhibiting pro-inflammatory response by NF-κB pathway in mice4. Findings from these in vitro and in vivo studies demonstrate the anti-inflammatory and immunomodulatory effects of AB4, highlighting its utility as a potential therapy for inflammation and immune response-related conditions, which is clearly related to its’ traditional use.7. It results in considerable monetary losses to the dairy industry due to decreased milk yield and quality, reduced reproductive performance, treatment, and premature culling9. Mastitis is the inflammation of the mammary gland, typically arising due to intramammary infections, broadly classified into subclinical (SCM) and clinical mastitis (CM) based on the severity of infection10. CM displays symptoms such as fever, depression, and anorexia and is usually associated with visible local and systemic signs of inflammation, marked by changes in milk, such as the appearance of clots, flakes, or watery texture12. CM is mainly caused by infection with pathogenic microorganisms such as Escherichia coli, Streptococcus agalactiae, and Staphylococcus aureus. E. coli releases bacterial cell wall components, such as lipopolysaccharide (LPS), which induces markedly increased local inflammatory mediators with a strong systemic acute phase response13. Activation of TLR4-dependent signaling pathways by LPS results in either the production of proinflammatory factors or initiation of endothelial apoptosis. Blocking TLR4 activation may inhibit the inflammatory response caused by LPS14. The S. agalactiae infection may affect cell proliferation and milk composition by reducing immune cell migration and inhibiting ATP synthesis and pathogen recognition pathways, thereby preventing immune response7. S. aureus is a gram-positive, opportunistic, and contagious bacterium that is one of the most prevalent causes of mastitis in cattle15. S. aureus-induced mastitis is difficult to eradicate with available therapies and is prone to persistent or recurrent infections due to the mechanisms that escape the attack from the host’s immune16. The desire to eradicate pathogens and assist the immune response established the routine use of intramammary antibiotics for the treatment of CM17. However, the extensive use of antibiotics has led to the evolution of antibiotic resistance in many bacterial species18. Therefore, government regulations have encouraged reduced antibiotic usage.Mastitis, with an incidence and prevalence ranging from 20% to 60 in dairy herds, respectively is the most common disease in dairy cows19. Here, we analyzed milk whey from dairy cows intramuscularly injected with AB4 after natural infection of CM using a tandem mass tag (TMT) proteomic approach and aimed to detect target proteins and molecular pathways associated with the therapeutic effect of AB4.Previous studies indicate that intramuscular AB4 injection significantly attenuates clinical signs and manifestations in dairy cows with mastitisThis study was carried out in compliance with the ARRIVE guidelines.All experiments were performed in accordance with Ethics Statement Approval of Institute Animal Ethics (IAEC) committee guidelines and reviewed by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (DYY-S20174610).11, with Somatic cell count  above 500,000 cells/mL, positive California mastitis test results , clinical signs of mastitis (included changes in milk appearance (flakes and clots in milk) and different stages of udder inflammation , were recruited to the study from a dairy farm in the region of Southern Sichuan Province. All CM cows received an intramuscular infusion of AB4   for 7 days. During this period, clinical symptoms and milk traits were observed every day to judge the therapeutic effect. CM cows without clinical symptoms, such as change in the appearance of milk  and local clinical signs of inflammation of the udder parenchyma , were assessed as clinically cured. Among the clinical cured cows, twelve clinical cured cows, at the 2nd–3rd lactation stage, weighted 565–659 kg and aged 3–4 years, were selected and marked as group T. Before (T1) and after (T2) AB4 injection, 10 mL milk samples were collected from groups T. Twelve health cows with same lactation stage, body weight and age, received an intramuscular injection of same dose saline for 7 days, marked as group C. Collected 10 mL milk samples of group C after saline injection (C1). There are 3 groups in this study. Each group has 3 biological replicates, which consists of 4 milk samples. Sample details and TMT tag information are shown in Table Fifty natural infected CM Chinese Holstein cows, identified and confirmed by a veterinary surgeon according to the criteria of Turkg for 10 min to remove the cream layer . Then, CaCl2  was added to a final concentration of 60 mM. The skim milk was acidified at pH 4.6 with the addition of 10% hydrochloric acid  to precipitate casein. The whey protein was centrifuged at 189,000×g for 60 min. The supernatant was collected for further TMT analysis. Three sample pools of each group were prepared by mixing milk whey from the four dairy cows. Meanwhile, SCC and inflammatory factors  in whey were detected by SCC counter  and ELISA kit  to evaluate the effectiveness of AB4 treatment.Scrub teats with a pad soaked in 70% alcohol  and dried; massage the udder and discard the first three streams of milk from the teat before sampling. Collected milk samples was centrifuged at 10,000×20. Protein concentration was determined using the BCA kit . Proteins were digested using filter-aided proteome preparation (FASP) with trypsin  hydrolysis20. The peptides derived from pooled samples were labeled using a TMT kit  according the manufacturer’s instruction. The samples were labeled as 128 (C1), 129 (T1) and 130 (T2). The TMT labelling efficiency was 100%.Sample proteins were extracted using the SDT method C, Thermo Fisher Scientific, USA).A C18 fractionation column  was preequilibrated with 0.1% (v/v) trifluoroacetic acid  and eluted using an acetonitrile (containing 0.1% trifluoroacetic acid) gradient. Loaded the labeled peptides onto the analytical column , followed by the addition of deionized water. Centrifuged the mixture at a low speed to desalt . Eluted the peptide from the C18 column using a gradient of acetonitrile. Digested peptide samples were dried in vacuum freeze dryer  and reconstituted in trifluoroacetic . Detected the peptide concentration at OD280  coupled to an Easy nLC system . TMT-labeled peptides were dissolved in 0.1% formic acid (V/V) acetonitrile solution , loaded onto a C18 column , and separated on an analytical column  at a flow rate of 300 μL/min.The MS was operated in positive ion mode using Q-Exactive. Full-scan MS spectra were acquired in the range from 300 to 1800 m/z. The MS1 resolution was 70,000 at 200-m/z. The automated gain control (AGC) target was 1e6. The maximum IT was 50 ms. The dynamic exclusion was 60 s. The 20 strongest signals of the parent ions were selected for secondary fragmentation. The MS2 activation type was high-energy collision dissociation (HCD). The isolation window was 2 m/z. The MS2 resolution was 35,000 at 100 m/z (TMT 10-plex).The normalized collision energy was 30 eV.https://www.uniprot.org/)-Bos-taurus-20191014.Analyzed the RAW data of the MS results by Mascot 2.2 and Proteome Discoverer 1.4. The relevant parameters and descriptions were as follows: enzyme, trypsin; max missed cleavages, 2; fixed modifications, Carbamidomethyl (C), TMT 10plex (N-term),TMT 10 plex (K); peptide mass tolerance, ± 20 ppm; fragment mass tolerance, 0.1 Da; peptide FDR, ≤ 0.01. Proteins’ GenInfo identifier (GI) accession numbers were converted into official gene symbol by uniport (http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes 21 pathway analysis was performed using Blast2Go (https://www.blast2go.com/). Fisher’s exact test was used to analyze the GO and KEGG functional enrichments. Functional network analysis was performed using STRING (http://string-db.org/) and Cytoscape platform version 3.7.2 (https://cytoscape.org) based on bos-taurus genes.Gene ontology .P < 0.05) according to mass spectrum data. Supplemental Table We identified 1583 quantifiable proteins using a TMT-based quantitative proteomics approach according to the set criteria . Supplemental Table  P value = 0.37), stress response , organonitrogen compound metabolic process , etc. MF included nucleosomal DNA binding , chromatin DNA binding , and protein binding . CC included the Golgi membrane , nuclear chromosome , chromatin , etc. , interleukin-1 beta production , regulation of interleukin-1 production , etc. MF included serine-type endopeptidase activity , cytoskeletal protein binding , and transforming growth factor beta receptor binding . CC included the extracellular region , cytoplasm , and the cortical cytoskeleton  , cellular components (CC), and molecular functions. The top 20 enriched GO terms of T1/C1 and T2/T1 are shown in Fig. tc. Fig. . Among t55) Fig. .Figure 2KEGG enrichment analysis results are shown in Supplemental Table Among the 268 upregulated proteins in dairy cows with CM (T1/C1), 164 were downregulated after AB4 treatment  signaling pathway is activated to produce pro-inflammatory factors and APPs, and cause mastitis22. S. aureus colonizes the mammary gland, adheres to the host epithelial cells and their extracellular matrix, synthesizes and secretes factors that allow the invasion, penetration, and destruction of the mammary tissue, including several exotoxins (hemolysins and leukocidins) and various hydrolytic enzymes such as proteases, coagulase, lipases, and hyaluronidases24. Additionally, S. aureus can escape, and also modulate the host immune system by producing a range of factors such as S. aureus superantigen toxins, protein A, and polysaccharide capsule25.CM is caused by the invasion of pathogens. Gram-negative bacteria, such as 26. However, we found that APOA1, APOA4, APOE, and RBP4 were downregulated during clinical mastitis. These proteins were transferred from the blood to the pathogens causing mastitis. When the consumption of these proteins was greater than the rate of transfer from the blood, this may have caused conflicting results. This indicated that changes in the abundance levels of these proteins were not sensitive enough to act as biomarkers to predict infection of the mammary gland. Proteins involved in innate immunity and antimicrobial functions  and are associated with the immune response to pathogens  are abundantly expressed in whey from S. aureus mastitis milk27. HP, SAA1, DEFB10, and SERPINB3 are also observed to increase in milk from cows with mastitis28. Sudipa et al. Changes in the expression of HP and FN from Holstein Friesian correlated with disease progression, and angiogenin and cofilin-1 were upregulated while ubiquitin family members were downregulated during disease transition29. Proteins that stand out as logical candidates for further analyses include various APPs and vascular-derived proteins such as C3, C4, TF, ALB, TTR, FGA, and ITIH4 in mastitis whey of E. coli infected cows by 2-DE and label-free methods, respectively31. Host defense-related proteins such as HP, SERPINB1, SERPINB4, and ITIH4 were upregulated in our study, consistent with previous studies.Turk et al. (2021) found that proteins involved in host defense such as α2- macroglobulin (A2M), α1-microglobulin/bikunin protein (AMBP), proteins involved in transport such as apolipoproteins AII and F , and retinol-binding protein 4 (RBP4), are increased in milk but reduced in the serum of mastitis cowsS. agalactiae-induced mastitis are mostly enriched in complement and coagulation cascades, glycolysis/gluconeogenesis, pentose phosphate pathway, purine metabolism, NOD-like receptor signaling pathway, inflammatory mediator regulation of TRP channels, bacterial invasion of epithelial cells, chemokine signaling pathway proteasome, cell adhesion molecules (CAMs), and ribosomes32. S. aureus-infected mastitis also detected complement and coagulation cascades, pentose phosphate pathway, Fc gamma R-mediated phagocytosis pathway, leukocyte transendothelial migration pathway, acute phase response signaling, LXR/RXR activation, antigen processing and presentation pathway, and ECM–receptor interaction pathway34. E. coli-induced mastitis also found complement and coagulation cascades pathway, glycolysis/gluconeogenesis, pentose phosphate pathway, acute phase response signaling, lysosome, cytokine-cytokine receptor interaction, and cell adhesion molecules.DEPs of E. coli were complement and coagulation cascades, glycolysis/gluconeogenesis, pentose phosphate pathway, leukocyte transendothelial migration35. Most of the AB4-targeted pathways identified here were consistent with the above pathways, including glycolysis/gluconeogenesis, pentose phosphate pathway, leukocyte transendothelial migration pathway, Fc gamma R-mediated phagocytosis, regulation of actin cytoskeleton, and complement and coagulation cascades.The top canonical pathways detected in 36. Gluconeogenesis is a synthesis pathway of glucose from no carbohydrate precursors, and it is essentially a reversal of glycolysis with minor variations in alternative paths37. When mastitis developed (T1/C1), GPI, TPI1, GAPDH, PGK1, PGAM1, ENO1, and PKM in the glycolysis/gluconeogenesis pathway were upregulated, which promoted pyruvate synthesis with large amounts of ATP production. ATP is present in inflamed tissues in vivo at extracellular concentrations sufficient for P2 receptor activation, promoting leukocyte recruitment and NALP3-inflammasome activation via P2X738. Lowering extracellular ATP levels in inflamed tissues can inhibit inflammation. Following AB4 treatment, GPI, TPI1, GAPDH, PGK1, PGAM1, ENO1, and PKM were downregulated, stimulating ATP breakdown to reduce inflammatory damage. LDHA and LDHB which were downregulated promote anaerobic metabolism of pyruvate with l-lactate and NADPH production39. This results in less energy for the cow and is not conducive to fighting inflammation. LDHA and LDHB were upregulated with AB4 treatment, which enhanced the aerobic metabolism of pyruvate to provide energy for fighting against inflammation. In the meantime, GPI, G6PD, PGD, and TALDDO1 in the pentose phosphate pathway was upregulated in CM cows, leading to bulk NADPH production. Activated in nature by microbes and microbial-derived products, the phagocyte NADPH oxidase rapidly assembles and generates reactive oxidant intermediates (ROIs) in response to infectious threats40. NADPH oxidase plays a key role in modulating inflammation and injury, distinct from its antimicrobial function40. However, ROIs can directly injure cells by damaging DNA, proteins, and lipids41. GPI, G6PD, PGD, and TALDDO1 downregulation with AB4 can prevent oxidative damage by excessive NADPH.Glycolysis is the process of converting glucose into pyruvate and generating small amounts of ATP (energy) and NADH (reducing power)42. In cows with CM, Rac2 upregulation promotes leukocyte motility43; RhoA and MLC upregulation indirectly promotes tail retraction44; ERM, actin, and α-actin upregulation indirectly regulates the docking structure45, and CLDN8 downregulation promotes leukocyte transendothelial migration. This is also responsible for the high SCC in mastitis milk. After AB4 treatment, Rac2, MLC, ERM, actin, and α-actin were downregulated, and leukocyte migration was inhibited. ITGB2 binds to JAM1, JAM3, and ICAM3, promoting transendothelial migration of neutrophils and T cells, and phagocytosis of apoptotic neutrophils by macrophages, respectively47. ITGB2 was downregulated by AB4. These results indicate that AB4 has a great potential to reduce SCC.Leukocyte migration from the blood into the sites of infection is a vital immune surveillance strategy48. After opsonization with antibodies (IgG), foreign extracellular materials are recognized by Fc gamma receptors. IgG is downregulated in mastitis cows, reducing the ability to recognize antigens, be caused by a vast array of immune evasion mechanisms of S. aureus. CFL1 plays a role in the regulation of cell morphology and cytoskeletal organization in epithelial cells49. The Arp2/3 complex mediates the formation of branched actin networks in the cytoplasm, providing a force for cell motility50. CFL1, ARPC4, and ARPC3 are upregulated during cow mastitis, promoting macrophage, neutrophil, and monocyte motility to destroy infectious pathogens52. Following AB4 treatment, IgG was upregulated, whereas CFL1, ARPC4, and ARPC3 were downregulated, indicating that invasive pathogens were effectively controlled.Phagocytosis plays an essential role in host defense mechanisms through the uptake and destruction of infectious pathogenswith S. aureus34. The complement system is a proteolytic cascade in blood plasma and a mediator of innate immunity, a nonspecific defense mechanism against pathogens. The main consequences of complement activation are the opsonization of pathogens, recruitment of inflammatory and immunocompetent cells, and direct killing of pathogens53. Pathogens have a greater effect on the blood-milk barrier, and consequently, a larger transfer of blood proteins to milk occurs in mastitis54. Together with fibrinogen alpha (FGA) and fibrinogen beta (FGB), polymerize to form an insoluble fibrin matrix55. FGA and FGB are upregulated with fibrin monomer production, further transfer to fibrin clots, causing deterioration in milk quality. Plasmin, which transfers fibrin clots to fibrin degradation products via the fibrinolytic system, reduced production due to the downregulation of PLG, HCII, F2, A1AT, and α2AP. C1q is the first subcomponent of the C1 complex in the classical pathway of complement activation56. Functions in the lectin pathway of complement play a key role in innate immunity by recognizing pathogens through patterns of sugar moieties and neutralizing them. The lectin pathway is triggered upon the binding of mannan-binding lectin (MBL) and ficolin to sugar moieties, leading to activation of the associated proteases MASP1 and MASP257. Here, C1, MBL, MASP1, and MASP2 were downregulated in mastitis. The classical and lectin pathways of the complement cascade were inhibited, and AB4 treatment C1 and MBL were upregulated, activating the complement system. VTN and CLU protect cells against apoptosis and cytolysis by complement59. Bacterial invasion into the mammary gland induced VTN and CLU downregulation, promoting cell apoptosis and cytolysis. Both VTN and CLU were upregulated after AB4 treatment. The membrane attack complex (MAC) forms trans–plasma membrane channels on the surface of pathogenic bacteria, causing cell lysis and death; only five of the complement system proteins eventually formed MAC subunits: one unit each of complement C5b, C6, C7, and C8 and several units of complement C960. C8A, C8B, C8G, and C9 were downregulated in mastitis, indicating that the host's ability to eliminate the bacteria is diminished. Following AB4 treatment, all proteins were restored to normal levels.The most common pathway of mastitis infection is the complement and coagulation cascade pathway, also the main pathway for the host to deal In summary, pathogenic bacterial infection upregulated PKM, LDHB, LDHA, ALDOA, PGD, GPI, and ALDOC, increased ATP production, which promotes leukocyte recruitment and NALP3-inflammasome activation, increases NADPH production, and promotes ROIs modulating inflammation and injury. These proteins were restored by AB4, which indicates that inflammation was inhibited. Invasion of pathogenic bacteria leads to transdermal migration of leukocytes into the mammary tissue, elevating somatic cells in the milk, increasing fibrinogen precursors, and decreasing plasmin production, resulting in fibrin clot deposition, eventually causing deterioration in milk quality. Bacterial infection of mammary tissue inhibits activation of the complement system, reduces MAC production, and protects pathogenic bacteria from killing through the complement immune system, thereby promoting apoptosis of mammary epithelial cells. Intramuscular infusion of AB4 can downregulate GPI, TPI1, GAPDH, PGK1, PGAM1, ENO1, PKM, GPI, G6PD, PGD, and TALDDO1 and restore LDHB and LDHA, reducing inflammatory damage caused by ATP and NADPH; downregulating Rac2, RhoA, MLC, ERM, actin, and α-actin ITGB2, inhibiting transendothelial migration of leukocytes, thereby reducing milk SCC; restoring PLG, FGA, FGB, reducing milk fibrin clots; upregulate C1, MBL, CLU, VTN, activating the complement system and reducing MAC, and directly inhibiting the invasion of pathogenic bacteria.We investigated the differences in the milk proteomes of mastitis-affected cows following intramuscular AB4 injection. Our results suggested that AB4 may treat CM through multiple pathways by easing inflammation, reducing SCC, decreasing milk clots, and eradicating pathogenic bacteria. These results contribute to a better understanding of the AB4 mechanism in CM, providing theoretical support for its clinical application. Application of AB4 can reduce antibiotic usage, therefore, economic losses because of antibiotic residues in milk can be avoided, bacterial resistance will be controlled, and environmental pollution caused by antibiotic misuse will be reduced. Future research confirming the target proteins identified in this study of AB4 on dairy cows with CM is needed.Supplementary Information 1.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.


Alzheimer’s disease (AD) is a neurodegenerative disorder molecularly characterized by the formation of amyloid β (Aβ) plaques and type 2 microtubule-associated protein (Tau) abnormalities. Multiple studies have shown that many of the brain’s immunological cells, specifically microglia and astrocytes, are involved in AD pathogenesis. Cells of the innate immune system play an essential role in eliminating pathogens but also regulate brain homeostasis and AD. When activated, innate immune cells can cause programmed cell death through multiple pathways, including pyroptosis, apoptosis, necroptosis, and PANoptosis. The cell death often results in the release of proinflammatory cytokines that propagate the innate immune response and can eliminate Aβ plaques and aggregated Tau proteins. However, chronic neuroinflammation, which can result from cell death, has been linked to neurodegenerative diseases and can worsen AD. Therefore, the innate immune response must be tightly balanced to appropriately clear these AD-related structural abnormalities without inducing chronic neuroinflammation. In this review, we discuss neuroinflammation, innate immune responses, inflammatory cell death pathways, and cytokine secretion as they relate to AD. Therapeutic strategies targeting these innate immune cell death mechanisms will be critical to consider for future preventive or palliative treatments for AD. Alzheimer’s disease (AD) is a debilitating disease affecting approximately 44 million people worldwide . The pro1-42 is the major component of plaque formation; Aβ1-42 has a highly hydrophobic C-terminus that initiates Aβ aggregation and oligomerizes into higher order insoluble structures that diffuse throughout the brain [AD is characterized by the presence of two structural brain abnormalities caused by the proteins amyloid β (Aβ) and type 2 microtubule-associated protein (Tau). The characteristic AD-inducing plaques, caused by Aβ deposition between nerve cells in the brain, can manifest several years before clinical symptoms unfold, and plaques play a major role in cognitive decline and neuronal cell death ,4. Aβ ishe brain ,7,8. Thehe brain .Along with Aβ aggregation, the dysregulation of the protein Tau also results in AD . Tau is While Aβ plaques and NFTs in neurons are key features of AD, the disease pathogenesis is not limited to the neuronal compartment, and many of the brain’s immunological cells  are also involved. The activation of astrocytes and microglial cells releases proinflammatory cytokines, including IL-1β, TNF-α, and IL-6; these cytokines can trigger Tau hyper-phosphorylation . AdditioInnate immune signaling pathways in microglial cells are activated in response to a myriad of stimuli such as infection, injury, and chronic disease. As a result of an activated immune response, programmed cell death (PCD) can occur and has a significant role in pathogenesis in the CNS, with specific roles in brain development, homeostasis, and clearance of infected/transformed cells. There are several forms of PCD with defined molecular signatures , rangingMany questions remain about the precise mechanisms through which innate immunity and PCD pathways modulate neuronal pathogenesis. Here, we discuss the link between neuroinflammation, innate immune responses, and AD. Innate immune sensors and molecular mechanisms of PCD, as related to AD, are also described, along with proposed AD therapeutic strategies targeting fundamental regulators of PCD. Considering the innate immune components of AD pathogenesis and identifying strategies to therapeutically target these pathways will be critical for informing clinical interventions.Neuroinflammation is involved in the propagation of several neurodegenerative disorders and has been shown to be a major contributor to AD pathogenesis and progression. Inflammation in the CNS can occur as a result of cells sensing Aβ or other damage- or pathogen-associated molecular patterns (DAMPs or PAMPs). Cells contain several pattern recognition receptors (PRRs), both on the cell surface and in the cytoplasm, that are responsible for recognizing DAMPs and PAMPs; sensing can induce inflammatory signaling pathways and immune responses. The five primary PRR families include Toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), and the absent in melanoma 2 (AIM2)-like receptors (ALRs) . This PAMicroglia are self-renewing immune cells of the CNS  that ariTREM2, CD33, INPP5D, CLU, CR1, SPI1, ABCA7, EPHA7, MS4As, HLA-DRB5-DRB1, CASP7, and CASP8 . Nucleic acid-containing Aβ elicits a TLR- and RLR-derived type I IFN response for C3-dependent synapse destruction . The expThe activation of PRRs, especially inflammasome sensors, can lead to the release of IL-1β and IL-18, and these proinflammatory cytokines are also correlated with AD severity ,109,110.The CNS relies heavily on astrocytes for maintaining homeostasis and assisting with synapse formation and elimination ,118. AstEGFL7, FLT1, and VWF (angiogenic growth factors and their receptors) and B2M and HLA-E (antigen presentation machinery) [Single-nucleus transcriptome analyses from prefrontal cortical samples from patients with AD identified a variety of endothelial transcriptomic changes, specifically in angiogenesis and the immune response pathway when compared with healthy patients . The endchinery) , suggestThe major pathological hallmarks of AD include extracellular Aβ deposition, the intraneuronal aggregation of NFTs, and neuronal loss (neurodegeneration) . In mammPyroptosis is an inflammatory PCD pathway initiated by the assembly of a multimeric protein complex called the inflammasome , as desc1-42 can induce pyroptosis in cortical neurons [In pyroptosis, caspase-1-dependent GSDMD cleavage, and the subsequent release of IL-1β and IL-18, have been well characterized in immune cells such as macrophages . However neurons ,143  that recruits adaptor proteins (including TRADD and FADD) and pro-caspase-8 ,156. CasMany studies have shown that compared to healthy brains, the expression profiles of several components of apoptosis, most notably the Bcl-2 family members, are altered in AD . Bcl-2 fIn addition to the Bcl-2 family members, apoptotic caspases are also associated with AD. Stimulating microglia with inflammogens can activate caspase-8, -3, and -7 in BV2 cells and in mice, and these caspases are activated in the microglia of patients with AD . MoleculNecroptosis can be initiated in response to multiple signaling pathways, including TLR signaling, death receptor engagement, and IFN signaling . The necNecroptosis can impair cognitive function; in the APP/PS1 mouse model, mice expressing constitutively active MLKL performed worse in the Morris water maze and had fewer neurons . In addiCASP1, CASP3, CASP6, CASP7, CASP8, and CASP9 have all been found in the entorhinal cortex of patients with AD with severe dementia [PCD pathways have long been thought of as segregated pathways with little overlap. However, numerous studies have found significant crosstalk among the components of pyroptosis, apoptosis, and necroptosis. Components of noninflammatory apoptosis can undergo crosstalk with molecules involved in executing lytic, inflammatory PCD pathways ,184. Fordementia , suggestdementia ,190,191.dementia ,30,35,36Innate immune signaling pathways and cell death mechanisms often culminate in the release of cytokines and chemokines. Cytokines, which are largely released by microglia and astrocytes, are major contributors of neuroinflammation. They are involved in chemoattraction, pro- and anti-inflammatory processes, neuronal injury, and the microglial response to Aβ deposits. Since cytokines modulate microglial activation, their presence or absence can be influential in the development and progression of AD  Table 4Table 4. Inflammation must be tightly balanced in the brain. A proinflammatory environment, such as that seen in AD brains, accounts for damaging pathology. The increased caspase-1 activation in AD leads to higher levels of IL-1β release ,210; thiIn addition to cytokines, chemokines also enhance local inflammation in AD by regulating microglial migration to areas of neuroinflammation . In AD, Several AD therapeutics have attempted to target Aβ plaques and Tau. However, the clearance of Aβ plaques through anti-Aβ immunotherapy has failed to provide any cognitive benefit in patients with AD thus far . The U.Snd generation tetracycline blocking cytochrome c release and upregulating Bcl-2 expression, was ineffective in patients with mild AD [Mouse models, such as the APP/PS1 model as well as others, allow researchers to gain an understanding of relevant disease-related PCD signaling pathways and their regulation. The immediate hope is that PCD, in some capacity, can be targeted to modulate neurological disease severity and progression. However, this approach has also faced challenges. Historically, there has been an association between apoptosis and the expression of key apoptotic proteins with neurodegenerative disorders; yet in vivo targeting of apoptosis has failed to show effective therapeutic results. A recent clinical trial for minocycline, a 2 mild AD ,229. Thi mild AD , suggestA correlation has also been identified between inflammatory cytokines and their signaling pathways and AD, with microglia playing a key role in coupling inflammation with neurodegeneration ,243,244.In addition to immune, inflammatory, and cell death pathways, the meningeal lymphatic vasculature (mLV) system may also be a promising therapeutic target for AD. The mLV system moves immune cells from the brain to the peripheral immune system; it is crucial in maintaining fluid homeostasis and enabling appropriate innate immune responses ,247. MicRecent studies have significantly expanded our understanding of neuroinflammation, innate immune responses, and tightly orchestrated cell death signaling in AD pathogenesis. The brain immune cells (microglia) are integrally associated with innate immune cell death in AD pathology. Excessive neuroinflammation during Aβ and Tau clearance could be diminished by specifically regulating the innate immune cell death response. The continued elucidation of cell death pathways and the central innate immune sensor signaling pathways involved in regulating neuroinflammation and Aβ/Tau clearance will have a significant impact on the AD research field. The emerging concept of PANoptosis is likely to play a role in disease pathogenesis and understanding its relevance in AD therapeutics. Emerging AD studies are focusing on this network of inflammasomes, inflammatory cell death, and neuroinflammation. Further investigations into these processes will potentially aid in identifying novel therapeutic targets for AD management.


SnRK2 (sucrose non-fermenting 1-related protein kinase 2) is a plant-specific protein kinase that plays a crucial role in abscisic acid and abiotic stress responses in some model plant species. In apple, corn, rice, pepper, grapevine, Arabidopsis thaliana, potato, and tomato, a genome-wide study of the SnRK2 protein family was performed earlier. The genome-wide comprehensive investigation was first revealed to categorize the SnRK2 genes in the Liriodendron chinense (L. chinense). The five SnRK2 genes found in the L. chinense genome were highlighted in this study. The structural gene variants, 3D structure, chromosomal distributions, motif analysis, phylogeny, subcellular localization, cis-regulatory elements, expression profiles in dormant buds, and photoperiod and chilling responses were all investigated in this research. The five SnRK2 genes from L. chinense were grouped into groups (I–IV) based on phylogeny analysis, with three being closely related to other species. Five hormones-, six stress-, two growths and biological process-, and two metabolic-related responsive elements were discovered by studying the cis-elements in the promoters. According to the expression analyses, all five genes were up- and down-regulated in response to abscisic acid (ABA), photoperiod, chilling, and chilling, as well as photoperiod treatments. Our findings gave insight into the SnRK2 family genes in L. chinense and opened up new study options.Protein kinases play an essential role in plants’ responses to environmental stress signals.  Liriodendron, which belongs to the Magnoliaceae family. L. chinense is a native of South China, and L. tulipifera comes from eastern North America. The medications produced from the two species varied in America, China, and other countries .,8.L. chiWe used the Total RNAPlant Extraction Kit as directed by the manufacturer . The cDNA was produced using the TaKaRa PrimeScript 1st Strand cDNA Synthesis Kit , following the manufacturer’s instructions. The SYBR Green Real-time PCR Master Mix  was used to perform the qRT-PCR on an ABI7500 Real-Time PCR System  .Lchi13910, Lchi00543, Lchi25623, Lchi12999, and Lchi01348 in the dormant bud subjected to various environmental conditions were validated by qRT-PCR analysis  (accessed on 5 June 2022) [The data were analyzed by Statistix 8.1 software  using one-way ANOVA, and the data were presented as the mean ± SD (Standard deviation) of the three replicates. The differences in the mean values of the dormant buds between photoperiod, chilling, chilling, as well as photoperiod, and different treatments (ABA-10 µM and ABA-100 µM) of ABA plants were analyzed using an LSD (least significant difference) test at ne 2022) .SnRK2s from the A. thaliana genome, we investigated five SnRK2 genes in the L. chinense genome  structure, and the projected 3D structures showed that the LchiSnRK2 protein has similar conserved structures. The gene expansion of the K2 genes . To furtd motifs A–C were ectively B. The SnSnRK2 genes. Furthermore, five SnRK2 genes included 10 conserved motifs  with 50 amino acids; one motif (three) with 40 amino acids, while two motifs (four and six) with 29 amino acids, motif nine with 20, motif eight with 19, and motifs seven and 10 with nine amino acids  structures show that three subfamily proteins have similar 3D structures at their N terminals but differ at their C terminals   and cell cycle regulation (three). In addition, two sensitive metabolic elements—one each for the control of zein metabolism and anaerobic induction—were found  to analyze the PlantCARE database to determine the cis-regulatory elements and functions of the genes. In the chosen region of the elements . In SnRKtabolism B. Identitabolism . The majtabolism A–D. Theserature) . In addielements . Most ofre found .LchiSnRK2 genes were subjected to qRT-PCR-based expression profiling to look into their expression profiles under contrasting photoperiods (long day (P-LD) and short-day (P-SD)), chilling, various abscisic acid concentrations (ABA-10 µM and ABA-100 µM), and chilling as well as photoperiod  conditions (Lchi13910 gene significantly increased under ABA-10 µM and photoperiod long day (P-LD) and significantly decreased under chilling as well as photoperiod long day (C + P-LD), according to the RT-qPCR data. In other conditions, including ABA-100 µM, P-SD, and C + P-SD, Lchi13910 expression level displayed a non-significant difference. Similarly, the expression of the Lchi00543 gene significantly decreased under all environmental circumstances, including chilling, photoperiod, and various ABA concentrations. In P-SD, chilling, and C + P-LD conditions, the expression level of Lchi25623 was significantly up-regulated, whereas, in ABA-100 µM conditions, it was significantly downregulated. In the ABA-10 µM and P-LD conditions, Lchi25623 displayed non-significant differences. The Lchi12999 gene was significantly up-regulated in the chilling condition, whereas it was down-regulated in the ABA-100 µM, P-LD, and C + P-LD conditions. In the ABA-10 µM, P-SD, and C + P-SD conditions, the expression level of Lchi12999 did not display any significant differences. The Lchi01348 transcript expression was also highly up-regulated in P-SD but significantly down-regulated in all other environmental conditions  Sarg. (L. chinense), which is typically found in the mountains at altitudes between 450 and 1800 m [Liriodendron is an ideal natural resource [Liriodendron wood [Liriodendron leaves on tumor cell lines, as well as their inhibitory activity against farnesyl protein transferase (FPTase) and tumor cell development. [Liriodendron’s high economic worth and L. chinense’s threatened status, little is known about their population’s genetic makeup and geographic variation [Liriodendron twigs, photoperiod substantially impacted the budburst percentage (BB%) and interacted with chilling [Liriodendron twigs responded strongly to photoperiod. In this species, the BB% under LD conditions was consistently higher than 50%, but under SD conditions, it was lower than 20%. This was true regardless of the length of the chilling period [Liriodendron twigs, Phoebe, and Torreya seedlings), lowering the risk of frost damage brought on by false springs [Only two species of the genus al value ,46. Chind 1800 m . Researcresource . Numeroulopment.  Despite ariation . ABA appariation . In numeariation . For perariation . Diverseariation . In LiriSnRK2 genes, as well-known members of the serine-threonine protein kinase subfamily, are essential for combating environmental challenges such as drought [SnRK2s mostly regulate the development of abscisic acid-dependent plants and their responses to environmental stressors. SnRK2s are either not activated or weakly activated in plants in response to ABA regulating abiotic stress responses. Furthermore, SnRK2 genes are essential controllers of the ABA signal-transduction pathway [L. chinense whole-genome sequencing data are now accessible [There are numerous forms of unfavorable environmental conditions to which plants are subjected. Plants can mitigate the negative impacts of extreme environmental conditions by utilizing various morphological, physiological, and molecular defense mechanisms ,60. Acco drought ,63. SnRK pathway ,65,66. Fcessible , which pSnRK2 gene uniquely responds to multiple environmental conditions. The majority of SnRK2s in model plant species such as rice [SnRK2 gene was discovered from an ABA-treated wheat embryo cDNA library [SnRK2 genes. However, the SnRK2 gene family in L. chinense had not yet been identified. Five distinct SnRK2 genes were detected in the L. chinense genome by genome-wide analysis in the current study. This is fewer than the number of SnRK2 genes found in other plant species, such as 20 in cotton [N. tabacum [SnRK2 genes have been reported for several plant species.According to numerous research, each  as rice , arabido as rice , and mai as rice  have bee library . Many pln cotton , 22 in N tabacum , 10 in a tabacum , 10 in r tabacum , eight i tabacum , eight i tabacum , 11 in m tabacum , 10 in s tabacum , 10 in s tabacum , nine in tabacum , and 12  tabacum . AccordiLchiSnRK2s was analyzed and revealed two patterns. Two genes had nine exons and eight introns, while two other genes had six exons and five introns, and only one gene had two exons and one intron. The former gene structure pattern is consistent with those of cotton [N. tabacum [SnRK2 family in plants has undergone very little evolutionary change.The gene structure of f cotton , N. taba tabacum , arabido tabacum , rice [2 tabacum , mungbea tabacum , grapevi tabacum , maize [ tabacum , sugarca tabacum , sorghum tabacum , pepper  tabacum , and app tabacum . The SnRL. chinense, A. thaliana, Solanum lycopersicum, Capsicum annuum, Solanum tuberosum, Vitis vinifera, Zea mays, Oryza sativa, and Malus domestica. The phylogenetic analysis showed that the LchiSnRK2 genes were grouped into the four previously described groups  [Lchi00543 was the only member of the group I, and Lchi01348 was the only member of group II. Lchi13910 and Lchi12999 were the two members of group III, and Lchi25623 was in Group IV. These findings demonstrate a potential evolutionary pattern within the SnRK2 gene family [A thorough phylogenetic analysis was carried out by building a phylogenetic tree to explore the evolutionary relationship between  and IV) ,26,66. LLchiSnRK2 genes will help us better understand how these genes react to different environmental situations. Our findings revealed four types of cis-elements, including those that responded to hormones, stress, growth and biological processes, and metabolism (SnRK2 genes were shown to have a significant role in specific agricultural plants under various environmental conditions by others who also reported comparable findings in these same plants [LchiSnRK2 genes in diverse contexts and conditions. The possible functionalities of various LchiSnRK2 were also characterized using 3D structural modeling. The LchiSnRK2 box, which forms a single helix and is packed parallel to the -helix in the N-terminal lobe, is a notable feature of LchiSnRK2. For kinase activity, the LchiSnRK2 box-C contact has been essential [The cis-regulatory motifs and elements present in the promoter sequences of the tabolism . Light, ssential . UnderstSnRK2s have a role in various abiotic stress responses. This study looked at how LchiSnRK2 expression changed in response to ABA, chilling, and photoperiod treatments. ABA also increased the expression of numerous SnRK2 genes from maize [SnRK2 genes are differently controlled by ABA in various organs of the rice plant [TaSnRK2.4, TaSnRK2.7, and TaSnRK2.8 expression levels are increased in response to various stressors [SnRK2s, such as TaSnRK2.3, TaSnRK2.4, TaSnKR2.7, and TaSnRK2.8 from wheat [ZmSnRK2.3 and ZmSnRK2.7 from maize, have been demonstrated to Huai et al. [LchiSnRK2 genes may be effective genetic modifiers for chilling, photoperiod, and various ABA levels tolerance in plants and crops.Numerous investigations have shown that om maize . Numerouce plant . Wheat Ttressors . SnRK2s,om wheat ,70,71; ai et al. . AdditioLchiSnRK2 genes were identified during genome-wide analysis of L. chinense SnRK2 genes. The SnRK2 gene family in L. chinense was subjected to a genome-wide investigation, and the expression patterns in dormant vegetative buds under ABA foliar sprays, photoperiod, and chilling conditions were studied. Through molecular breeding, it may be possible to increase the photoperiod, chilling tolerance, and bud dormancy release of L. chinense and other crops. Our understanding of the mechanism behind L. chinense stress tolerance may be aided by the molecular elucidation of these genes.In conclusion, five


Euroxenomys minutus (von Meyer, E. minutus in Tagay is an indicator for an Orleanian European-Siberian bioprovince during the Mid-Miocene Climate Optimum, and for a continuous belt of humid, warm-temperate to subtropical forests, stretching from Europe to Siberia, and probably further to East and South-Eastern Asia. In Eurasia, beaver remains are an indicator of permanent water bodies, which is in agreement with the palaeoenvironment of the Tagay locality.The castorid dental material described in this paper derives from Miocene, fossiliferous deposits of the Baikal rift valley, exposed at Tagay Bay on Olkhon Island in the Lake Baikal, in eastern Siberia. It consists of maxillary fragments and isolated upper and lower teeth of the small trogontheriine beaver  Euroxenomys minutus, Steneofiber eseri and ?Dipoides sp., medium-sized Chalicomys jaegeri and Steneofiber depereti and large-sized Anchitheriomys suevicus have been reported , is particularly rich with many localities in Austria, Czech Republic, France, Germany, Greece, Poland, Portugal, Spain, Switzerland and Turkey Hugueney . From thHugueney . The norHugueney .Euroxenomys nanus, the medium-sized Steneofiber siamensis, ?Steneofiber changpeiensis, Hystricops mengensis and Monosaulax tungurensis, the large-sized Minocastor godai and Anchitheriomys tungurensis, and the giant-sized Youngofiber sinensis have been reported . From localities in the Junggar, Linxia and Qaidam basins, in the Tunggur area in Inner Mongolia, in the coastal areas of eastern and northern China, in the Mae Moh and Chiang Muan basins in northern Thailand and in the Kani and Mizunami basins in Central Japan the small-sized The goal of our paper is to describe the first Miocene Castoridae from eastern Siberia, which also represent the northernmost record in East Asia.The Tagay section  at Tagay bay is located on the northwestern coast of Olkhon Island in Lake Baikal , St. Petersburg, Russia.Anatomical abbreviations: dP4: upper deciduous molar, P4/p4: upper and lower premolar, M1/m1: upper and lower first molar, M2/m2: upper and lower second molar, m3: lower third molar.Institutional abbreviations: ZIN: collection of the Zoological Institute of Russian Academy of Sciences, St. Petersburg.Order Rodentia Bowdich, 1821Family Castoridae Hemprich, 1820Euroxenomys Samson and Radulesco, 1973Genus Euroxenomys minutus , Irkutsk Region; Eastern Siberia; Tagay Formation; Early/Middle Miocene transition, MN5.Material: left maxillary fragment with P4 (ZIN 106953), right maxillary fragment with P4, M2 (ZIN 106954), right maxillary fragment with P4 (ZIN 106955), right maxillary fragment with M1-2 (ZIN 106960); isolated teeth: right P4 (ZIN 106956), right P4 (ZIN 106957), right P4 (ZIN 106958), right DP4 tooth germ (ZIN 106959) , right M1/2 (ZIN 106961), right M1/2 (ZIN 106962), right p4 (ZIN 106963) , right m1/2 (ZIN 106964), left m1/2 fragment (ZIN 106965) (1978), right m3 (ZIN 106966) , seven I fragments (ZIN 106967) .Measurements (in mm): See Table Description: The upper dentition is represented by four maxillary fragments with P4, P4 and M2, and M1-M2 in situ; additionally there are isolated teeth: one DP4 tooth germ, three P4 and two M1/2; there is no M3 preserved. One of the P4 (ZIN 106958) and the two M1/2  from Layer 9 most likely represent one individual, based on preservation and tooth wear. The upper P4 is the largest of all cheek tooth positions, accordingly the upper premolars are significantly larger than M1/2. In contrast to the upper dentition, the lower dentition is represented by isolated teeth only: one p4, two m1/2, and one m3. The premolar is the largest of the lower cheek teeth. Most of the cheek teeth are considerably worn. In some flexi(-ds)/fossettes(-ids)/striae(-iids) there is little tooth cementum preserved, but it seems that there were no massive cementum fillings.DP4 tooth germ  in France  in Czech Republik  localities, and a common beaver in Middle and Late Miocene (MN 6 – MN 11) sites  of Japan, which up to now represented the only record of the genus in Asia (Mörs and Tomida Euroxenomys wilsoni from the Early Miocene (early Hemingfordian) of Colorado and Nebraska (Korth Euroxenomys inconnexus from the Middle Miocene (early Barstovian) of Montana  to the Late Miocene . The magnetostratigraphic correlation of the vertebrate-bearing, upper part of the Tagay-1 section with the subchrons C5Cn.2r – C5Cn.1r of Chron C5C, indicates a late Burdigalian, Early/early Middle Miocene age, ~16.5 to ~16.3 Ma that can be correlated with MN4/5 , and for a continuous belt of humid, warm-temperate to subtropical forests, stretching from Europe to Siberia, and probably further to East and South-Eastern Asia during the Mid-Miocene Climate Optimum.The beaver remains indicate the presence of permanent water bodies, which is in agreement with the palaeoenvironment of the Tagay locality.


GJA1), reduced GJ intercellular communication from ~85% to 5–8% after 24 h incubation with 120 μM 18α-glycyrrhetinic acid, 400 nM 12-O-tetradecanoylphorbol-13-acetate, and 90 μM dichlorodiphenyltrichloroethane, as confirmed by a dye-transfer assay. Following inhibition, NANOG, OCT3/4, and SOX2 were up-regulated 2–4.5 times more; other pluripotency-related genes, such as KLF4, CBX7, and SPRY2 were elevated; lineage-specific differentiation-related genes were down-regulated in quantitative-PCR and RNA-sequencing. Connexin43-siRNA introduction also confirmed the up-regulation of NANOG, OCT3/4, and SOX2. YAP, a co-transcriptional factor in the Hippo signaling pathway that regulates pluripotency gene expression, co-localized with GJA1  in the cell membrane and was translocated to the nucleus after GJ inhibition. Adherent culture is usually more suitable for the stable expansion of cells than is a suspension culture. GJ inhibition is suggested to be a simple method to up-regulate pluripotency in an adherent culture that involves a Cx43-YAP axis in pluripotent stem cells, such as Muse cells.Gap junctions (GJ) are suggested to support stem cell differentiation. The Muse cells that are applied in clinical trials are non-tumorigenic pluripotent-like endogenous stem cells, can be collected as stage-specific embryonic antigen 3 (SSEA-3+) positive cells from multiple tissues, and show triploblastic differentiation and self-renewability at a single cell level. They were reported to up-regulate pluripotency gene expression in suspension. We examined how GJ inhibition affected pluripotency gene expression in adherent cultured-Muse cells. Muse cells, mainly expressing gap junction alpha-1 protein ( The cells of multicellular organisms have many different ways to communicate with neighboring cells to maintain cellular and tissue homeostasis. Gap junction (GJ) channels are one of the representative systems that enables the exchange of small molecules, second messengers, and electrical signals to connect the cytoplasmic space of two adjacent cells. GJs consist of connexin (Cx) proteins that oligomerize into hexamers to form hemi-channels, namely connexons. One connexon is presented from each cell to form a GJ channel between the two cells ,2.GJ intercellular communication (GJIC) is also suggested to be important in regulating stem cell differentiation. For example, the ablation of GJIC led to the disruption of differentiation in embryoid bodies derived from mouse embryonic stem (ES) cells . MoreovePOU5F1) , NANOG, and SRY (sex determining region Y)-box 2 (SOX2), at moderate levels when compared with those in ES and iPSCs, and can differentiate, spontaneously, not only into adipogenic-, chondrogenic-, and osteogenic-lineage cells, but also into other mesodermal-lineage cells , as well as into endodermal-  and ectodermal (microtubule-associated protein 2 and neurofilament)-lineage cells from a single cell in vitro without cytokine induction . A. A26]. ANANOG, OCT3/4, and SOX2 in Muse cells after treatment with the GJ inhibitors. Compared to the untreated-Muse cells, the elevation of these factors was the most prominent in Muse cells treated with 90 μM DDT, where NANOG, OCT3/4, and SOX2 expression levels were 1.85 ± 0.2 (p < 0.001), 4.56 ± 1.26 (p < 0.001) and 3.92 ± 0.59 (p < 0.001) times higher, respectively  and a 2.3 (p < 0.05) times higher elevation of NANOG and SOX2, when compared to the untreated Muse cells, respectively, while OCT3/4 did not show significant changes  was used in the following experiments.GJA1 gene expression and to observe the gene expression levels of NANOG, OCT3/4, and SOX2 in the Muse cells.Cx43 siRNA was used to knock down the GJA1 knockdown was confirmed by a Western blot analysis, which showed that the Cx43 protein levels were down-regulated to 10–20% for up to 5 days after transfection . Effective sfection .NANOG , OCT3/4 , and SOX2  in the Muse cells 4 days after GJA1 knockdown when compared to the untreated Muse cells , brain-expressed X-linked protein 1 (BEX1), P/Q voltage-dependent calcium channel (CACNA1A), zinc finger protein 521 (ZNF521), hepatocyte growth factor (HGF), and tyrosine-protein kinase (KIT) were significantly down-regulated, while, pluripotency-related genes, such as kruppel-like factor 4 (KLF4), alkaline phosphatase (ALPL), chromobox homolog 7 (CBX7), sprouty RTK signaling antagonist 2 (SPRY2), and SPRY4 were significantly up-regulated in DDT-Muse cells when compared to the untreated-Muse cells , compared with that in the untreated Muse cells  transcription factor . Large tA laser confocal microscopic analysis of Cx43/YAP double staining showed that both signals were adjacent to each other, nearby the cell membrane, in untreated-Muse cells A. Howevep < 0.01)  B.p < 0.05). Histone H3, the nuclear loading control, was detected mainly in the nucleus, and α-tubulin, the cytoplasmic loading control, was detected in the cytoplasm . These data are consistent with previous reports suggesting that Cx43 is positively correlated with differentiation and negatively correlated with pluripotency gene expression in stem cells since GJIC plays a role in the differentiation of pluripotent stem cells and Cx43 levels are elevated during differentiation into many different cell types ,40,41,42Hippo pathway plays an evolutionarily conserved role in organ size control by inhibiting cell proliferation, promoting apoptosis, and regulating the fates of stem/progenitor cells . YAP, whYAP has another important aspect. It is a primary sensor of the cell’s physical nature, e.g., cell structure, shape, and polarity . YAP actIt is reported that YAP and TEAD2 were highly expressed in self-renewing mouse ES cells and that YAP expression is up-regulated during iPSC reprogramming ,45. IndeGJA1 siRNA-transfected Muse cells. Other studies, using iPS cells (Cx43 knockout (GJA1−/−)) iPSCs, generated using CRISPR-Cas9 gene ablation), maintained characteristics typical of iPSCs and successfully differentiated into cells of ectoderm, mesoderm, or endoderm lineages [In this study, we focused on the role of Cx43 in Muse cells treated with these three different GJIC inhibitors that work by very different biochemical mechanisms to inhibit functional GJIC at the posttranscriptional level, as well as in lineages . While tlineages ,51.OCT4 gene, as well as the extra copy of the exogenous OCT4 gene. By definition, these iPS cells, when injected into an adult animal, form teratomas or the three primary germ lines. Whereas the organ-specific adult stem cells do not form teratomas. The normal human adult stem cells, i.e., human breast stem cells, do not express GJA1, nor do they have functional GJIC [OCT4 is transcriptionally repressed, GJA1 is expressed, and functional GJIC is evident. If, however, the normal adult breast stem cells are transfected with the Large T antigen gene of SV40 virus, the cells do not express GJA1, maintain expression of OCT4, and do not differentiate. They stay in the stem cell state or remain “immortal”. Clearly, further studies must be done to clarify the exact mechanism of how YAP interacts with the promoters of pluripotency related-genes in these cells, as well as the differences between these Muse cells and iPS cells in the future.These iPS cells have both the endogenous nal GJIC . When thGJA1 is different between naïve and primed pluripotent states. Human primed pluripotent stem cells express higher GJA1 than they do in the naïve state [GJA1 and the responsiveness of pluripotency gene expression by GJ inhibition, supports the assumption that Muse cells are similar to primed pluripotent stem cells.The expression of ve state , and naïve state . Muse ceve state . The resNANOG, OCT3/4, and SOX2, when GJIC was inhibited, other systems might also be involved in this phenomenon. Furthermore, multiple steps/systems might intervene in the inhibition of GJ and pluripotency gene up-regulation. These interesting subjects need to be clarified in future studies.While we demonstrated the involvement of a Cx43/YAP system in the up-regulation of pluripotency genes,


PyK2 is a member of the proline-rich tyrosine kinase and focal adhesion kinase families and is ubiquitously expressed. PyK2 is mainly activated by stimuli, such as activated Src kinases and intracellular acidic pH. The mechanism of PyK2 activation in cancer cells has been addressed extensively. The up-regulation of PyK2 through overexpression and enhanced phosphorylation is a key feature of tumorigenesis and cancer migration. In this review, we summarized the cancer milieu, including acidification and cancer-associated molecules, such as chemical reagents, interactive proteins, chemokine-related molecules, calcium channels/transporters, and oxidative molecules that affect the fate of PyK2. The inhibition of PyK2 leads to a beneficial strategy to attenuate cancer cell development, including metastasis. Thus, we highlighted the effect of PyK2 on various cancer cell types and the distribution of molecules that affect PyK2 activation. In particular, we underlined the relationship between PyK2 and cancer metastasis and its potential to treat cancer cells. Proline-rich tyrosine kinase 2 (PyK2) is a member of the proline-rich cytoplasmic tyrosine kinase family, which is ubiquitously expressed and dominantly localized in neuronal cells, endothelial cells, and hematopoietic cells ,2,3,4,5.2+ signaling, have not been revealed. Thus, we elucidated the modulatory role of PyK2 and PyK2-associated molecular mechanisms in a cancer system.In addition to PyK2, tyrosine kinases have two other members, focal adhesion kinase (FAK) and Src . The str2+ ([Ca2+]i) increases to activate EMT in pancreatic cancer cells i) induced PyK2 phosphorylation i is increased by the activation of various Ca2+ channels and transporters that are located on intracellular organelle and plasma membranes. Intracellular Ca2+ is stored in intracellular organelles, including the nucleus, mitochondria, and endoplasmic reticulum (ER), to maintain Ca2+ homeostasis. The mitochondrial protein Lon is involved in protein quality control and maintains mitochondrial homeostasis [2+]i through the involvement of a mitochondrial Na2+/Ca2+ exchanger, and enhanced chemoresistance to cisplatin in human oral squamous carcinoma cells (OEC-M1) i in a process called store-operated Ca2+ (SOC) entry (SOCE) [2+ influx through TRPM2 induces apoptosis through goldnano-conjugated doxorubicin) [2+ signaling presents further challenges in verifying the precise mechanism for cancer therapy.The ER, another intracellular Ca (STIM1) . This Cay (SOCE) . The dowy (SOCE) . The knoy (SOCE) . Additioy (SOCE) ,130,131.y (SOCE) . The knoy (SOCE) . Althougrubicin) , enhance2O2, reactive nitrogen species (RNS), and nitric oxide (NO) [In cancer cells, oxidative modification has pathological roles in protein alterations through the involvement of second messengers, including ROS, Hide (NO) ,135,136.ide (NO) ,138. Oxiide (NO) . Hypoxicide (NO) . The migide (NO) .2O2 in ESCC [2O2 levels in ESCC cells under hypoxic conditions and decreased ESCC proliferation [2O2 enhanced the phosphorylation of PyK2 in mouse left ventricular myocytes [2O2 stimulated PyK2 phosphorylation, and the activation of PyK2 phosphorylation increased the production of ROS in CTLs [PyK2 was reported to bind with dihydronicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in KySE30 and KySE410 esophageal squamous cell carcinoma (ESCC) . Hypoxia in ESCC . NOX5 shferation . Oxidatiferation ,143,144.myocytes  and H9c2myocytes . The delmyocytes . Additio2+-related molecules, as well as oxidation-related molecules, in cancer cells. The features of the acidic microenvironment and Ca2+ signaling in PyK2-associated molecular mechanisms have been demonstrated. Thus, further investigation is required to include its multiple regulators. Cells possess various ion transporters and channels, including those discussed above, and electrolyte transporters such as potassium, sodium, and chloride are also involved in cellular systems. Thus, further experimental evidence, including the relationship between ion channels and transporters and cancer, should be determined. In addition, investigations of PyK2 as an extracellular milieu-sensing protein in cancerous processes might provide further information on the responsiveness to the cancer milieu.PyK2 has been studied as a key regulator of cancerous processes. pH-associated kinase PyK2 is regulated by various molecules such as chemical reagents and interactive proteins, including chemokine-related and Ca


C. albicans-administered 5/6 nephrectomized (Candida-5/6 Nx) mice. At 20 weeks post 5/6 Nx, Candida-5/6 Nx mice demonstrated increased 24 h proteinuria, liver enzymes, and serum cytokines , but not weight loss, systolic blood pressure, hematocrit, serum creatinine, or gut-derived uremic toxins (TMAO and indoxyl sulfate), compared to in 5/6 Nx alone. The gut leakage in Candida-5/6 Nx was more severe, as indicated by FITC-dextran assay, endotoxemia, and serum BG. The areas of fibrosis from histopathology, along with the upregulated gene expression of Toll-like receptor 4 (TLR-4) and Dectin-1, the receptors for LPS and BG, respectively, were higher in the kidney, liver, and heart. In vitro, LPS combined with BG increased the supernatant IL-6 and TNF-α, upregulated the genes of pro-inflammation and pro-fibrotic processes, Dectin-1, and TLR-4 in renal tubular (HK-2) cells and hepatocytes (HepG2), when compared with LPS or BG alone. This supported the pro-inflammation-induced fibrosis and the possible LPS–BG additive effects on kidney and liver fibrosis. In conclusion, uremia-induced leaky gut causes the translocation of gut LPS and BG into circulation, which activates the pro-inflammatory and pro-fibrotic pathways, causing internal organ fibrosis. Our results support the crosstalk among several organs in CKD through a leaky gut.Uremic toxins and gut dysbiosis in advanced chronic kidney disease (CKD) can induce gut leakage, causing the translocation of gut microbial molecules into the systemic circulation. Lipopolysaccharide (LPS) and (1→3)-β-D-glucan (BG) are the major gut microbial molecules of Gram-negative bacteria and fungi, respectively, and can induce inflammation in several organs. Here, the fibrosis in the kidney, liver, and heart was investigated in oral  Candida cell wall), from the intestinal translocation activate their receptors, it leads to the enhanced inflammatory responses that further facilitate the progression of CKD [Candida albicans are the microbes with the most and the second most abundance in the human intestine. Therefore, increases in both molecules through a leaky gut might lead to clinical adverse effects [Chronic kidney disease (CKD) has been acknowledged as a significant global burden , leadingn of CKD . Notably effects .Candida spp. to be detectable in stool cultures [Candida spp. in the gut does not directly cause disease, gut fungi alter the gut microbiota and provide a higher BG in gut content [Candida exotoxin, and endogenous alcohol) [Candida spp. possibly facilitate the growth of specific bacteria with the glucanase enzyme, which can digest BG from the fungal cell wall [Candida on gut bacteria, gut fungi alter microbial homeostasis, and the oral administration of Candida causes gut dysbiosis, with leaky gut and inflammatory reactions observed in several mouse models [However, fungi are less prominent in the mouse gut than in the human intestine, and the influence of intestinal fungi in mouse models is possibly underestimated . Contrarcultures . Althoug content , which p content ,18,19,20alcohol) ,22,23,24alcohol) . Meanwhiell wall . Despitee models ,28,29,30e models ,32.Candida gavage at 16 weeks after 5/6 nephrectomy (5/6 Nx), despite a synergistic pro-inflammatory effect of LPS plus BG [Gut translocation of LPS and BG causes systemic inflammation , and chr plus BG . However plus BG  and endo plus BG ,40, (ii) plus BG ,40,41,42 plus BG ,43, the Candida gavage were observed for 20 weeks with the exploration of fibrosis in other internal organs (liver and heart) along with in vitro experiments. To understand the pathophysiologic effects of fungi on fibrosis, BG and LPS  were tested on hepatocytes (HepG2 cells) and renal tubular cells (HK2 cells).Here, 5/6 Nx and Candida administration during the 20 weeks observation, more prominent systemic inflammation and organ injuries in Candida-administered 5/6 Nx mice, compared with 5/6 Nx alone, were demonstrated by the increased 24 h proteinuria  [Candida-administered 5/6 Nx mice than in 5/6 Nx alone, as indicated by FITC-dextran assay, endotoxemia, and increased serum BG  , liver eserum BG A–C. Notaserum BG , supportAlthough inflammatory maladaptation from uremia and LPS alone can cause fibrosis and organ dysfunction ,48,49, aLikewise, LPS and BG also enhance inflammation in hepatocytes because LPS and BG from gut translocation are directly transported to the liver through the portal vein . Here, aCandida administration in mice is used to examine the influence of gut fungi in several models, taking advantage of the lower abundance of C. albicans in mouse feces compared with humans (Candida in mouse feces is detectable only by polymerase chain reaction (PCR) [Candida-5/6 Nx mice had more severe gut leakage (FITC-dextran assay), with a higher level of serum endotoxin and BG (glucanemia) compared to non-Candida 5/6 Nx mice, perhaps due to the direct intestinal invasion of fungi, gut dysbiosis, and pro-inflammatory enterocytes [Candida-induced gut dysbiosis in uremic mice, as previously described [Candida 5/6 Nx were higher than non-Candida 5/6 Nx, and serum BG might be the main factor responsible for the hyper-inflammation in Candida uremic mice. Additionally, the inflammatory activation of LPS and BG in the blood of 5/6 Nx mice might be responsible for the increased proteinuria, elevated liver enzyme, and organ damage , as indicated by histology. Although Candida did not alter systolic blood pressure in 5/6 Nx mice, cardiac fibrosis was more prominent in Candida-5/6 Nx than 5/6 Nx alone, implying an impact of some non-blood pressure-related profibrotic factors possibly, which was due to the direct myocardial activation by LPS and BG. An upregulation of TLR-4 and Dectin-1 in these organs  supported an inflammatory synergy of LPS and BG in Candida-5/6 Nx mice. Likewise, enhanced fibrosis in the liver might be correlated with LPS–BG from a leaky gut that was directly transported to the liver through the portal vein [Candida administration is important as renal fibrosis at 20 week, but not at 16 weeks of the experiment [Candida administration (both heat-killed and viable cells) was also previously demonstrated [Candida-5/6 Nx mice, supporting the importance of gut fungi in the condition with systemically chronic inflammation. Thus, pro-fibrosis of the internal organs in other conditions with chronic inflammation from leaky gut, such as obesity, inflammatory bowel disease, and cirrhosis, would be interesting to explore further.Oral on (PCR) , but noton (PCR) , which don (PCR) . Here, Cerocytes . Althougerocytes . Despiteescribed , only setal vein ,52. Notaperiment , demonstnstrated . It is instrated . Here, tDectin-1 by LPS, possibly responsible for the higher responses against BG when simultaneously presented with LPS. The LPS–BG responses of hepatocytes were severe enough for the more profound upregulation of HIF-1α and fibronectin, the important liver profibrotic genes [Dectin-1, but the inflammatory responses against LPS–BG were potent enough for a more prominent collagen type III upregulation compared with LPS activation alone [The synergy or additive effect of BG presentation upon LPS responses is intensively demonstrated through the activation of several innate immune cells  ,59,60,61ic genes ,73, whenon alone . Hence, Our data support the crosstalk among several organs in CKD through leaky gut, which might be a common situation in several medical conditions . TherefoThe approved protocol of the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (CU-ACUP No. 018/2562), according to the National Institutes of Health (NIH) criteria , using 8Candida albicans (5/6 Nx + Candida) to explore the impact of gut fungi using C. albicans from the American Type Culture Collection (ATCC 90028) , which was cultured overnight on Sabouraud dextrose broth (SDB)  at 35 °C for 48 h before enumeration using a hemocytometer. The C. albicans at 1 × 106 CFU in 0.5 mL PBS or PBS alone was orally administered on alternate days starting from 4 weeks after the right nephrectomy until 20 weeks. Another group of mice underwent the sham operation to identify renal vessels before abdominal closure (Sham group). The Candida-administered sham mice have not been demonstrated here because of the lack of difference from the sham control reported in a previous publication [First, 5/6 nephrectomy (5/6 Nx) surgery was performed via flank approach under isoflurane anesthesia by removing the upper and lower poles of the left kidney before the right nephrectomy 1 week later, as previously described ,42. Only® silica; 00G-4274-E0, Phenomenex®, Torrance, CA, USA) and high-performance liquid chromatography , as previously described [© software, Bethesda, MD, USA) in a 200× magnification field with 10 fields per sample. Because of the interest in the pro-inflammatory effect of LPS and BG in each organ, the gene expression of TLR-4 and Dectin-1 in each organ was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) relative to β-actin (a house-keeping gene) with the 2−ΔΔCT method, as previously described [All mice were sacrificed at 20 weeks post right nephrectomy, and the 24 h urine samples were collected at 48 h before sacrifice using a metabolic cage . The mouse systolic blood pressure and hematocrit were measured by tail-cuff plethysmography   and the escribed . Serum cescribed ,80. The escribed ,81,82,83®, St. Louis, MO, USA) was orally administered before measuring FITC-dextran in serum 3 h later by fluorospectrometer . Meanwhile, serum LPS and BG was evaluated by HEK-Blue LPS Detection Kit 2  and Fungitell® assay , respectively, and values less than 0.01 EU/mL (for LPS) and 7.8 pg/mL (for BG) were recorded as 0, due to the lower limit of the standard curve of the test.Because the detection of fluorescein isothiocyanate (FITC)-dextran  in serum after an oral administration, or the spontaneous elevation in serum of LPS or BG without systemic infection, indicate gut permeability defects (gut leakage), these parameters were used as previously described ,58,84,85Candida, respectively, on kidneys and liver, HK2 renal proximal tubular cells (ATCC 237 CRL-2190) and HepG2 hepatocytes  were used as the representatives. Briefly, HK2 cells or HepG2 at 1 × 106 cells/well maintained in Dulbecco’s modified Eagle Medium (DMEM) were incubated LPS (Escherichia coli O26:B6) (Sigma-Aldrich) at 100 µg/mL with or without BG (CM-Pachyman)  at 100 µg/mL for 24 h (HK2 cells) or 4 and 72 h (HepG2 cells) under 5% CO2 at 37 °C before the determination of supernatant cytokines (TNF-α and IL-6) by ELISA . Additionally, the expression of several genes related to inflammatory responses and fibrosis was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) relative to β-actin (a house-keeping gene) with the 2−ΔΔCT method using cDNA (SuperScript™ Vilo™ cDNA synthesis assay) (Invitrogen™) prepared from 50 ng of TRIzol-extracted total RNA (invitrogen™) by a qPCR machine (LightCycler® 2.0) (Roche Diagnostics) with the primers , determined using GraphPad Prism version 9.0 software . Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s analysis. The time-point experiments were analyzed by repeated measures ANOVA. All statistical analyses were performed with Stata 16.0 software  and Graph Pad Prism version 7.0 software . A Our results suggest the crosstalk among several organs in CKD through a leaky gut. Uremia-induced leaky gut in advanced CKD causes the translocation of LPS and BG from the gut into the systemic circulation. As such, LPS and BG additively induce the pro-inflammatory and pro-fibrotic pathways through the activation of TLR-4 and Dectin-1, causing internal organ fibrosis. These enhanced inflammatory responses during leaky gut could worsen uremic complications in patients with advanced CKD. Gut abundance of Gram-negative bacteria and fungi and/or the levels of LPS and BG in the blood might be interesting factors in predicting the severity of systemic inflammation during CKD-induced leaky gut.