Allium genus are prone to infection by Fusarium fungi. Chitinases of the GH19 family are pathogenesis-related proteins inhibiting fungal growth through the hydrolysis of cell wall chitin; however, the information on garlic (Allium sativum L.) chitinases is limited. In the present study, we identified seven class I chitinase genes, AsCHI1–7, in the A. sativum cv. Ershuizao genome, which may have a conserved function in the garlic defense against Fusarium attack. The AsCHI1–7 promoters contained jasmonic acid-, salicylic acid-, gibberellins-, abscisic acid-, auxin-, ethylene-, and stress-responsive elements associated with defense against pathogens. The expression of AsCHI2, AsCHI3, and AsCHI7 genes was constitutive in Fusarium-resistant and -susceptible garlic cultivars and was mostly induced at the early stage of F. proliferatum infection. In roots, AsCHI2 and AsCHI3 mRNA levels were increased in the susceptible and decreased in the resistant cultivar, whereas in cloves, AsCHI7 and AsCHI5 expression was decreased in the susceptible but increased in the resistant plants, suggesting that these genes are involved in the garlic response to Fusarium proliferatum attack. Our results provide insights into the role of chitinases in garlic and may be useful for breeding programs to increase the resistance of Allium crops to Fusarium infections.Vegetables of the  Allium genus are widely cultivated for their culinary and medicinal properties -C) and the GH19 catalytic domain (PF00182) with two active site signatures: CHITINASE_19_1 -F-Y-[ST]-x(3)-[FY]-[LIVMF]-x-A-x(3)-[YF]-x(2)-F-[GSA]) and CHITINASE_19_2 -[LIVMFY]-W-[FY]-W-[LIVM]) . In addiThe size of CBD1 (38 aa) was the same in all AsCHI proteins and those of the GH19 domains were 232 aa  and 233 aa . N-terminal signal peptides (19–21 aa) with the predicted cleavage site SxA-(Q/E)Q were detected in all AsCHI proteins .Multiple Em for Motif Elicitation (MEME)-based analysis revealed 10 conserved motifs in putative AsCHI enzymes, including motifs 5 (CBD1) and 8 . Motif 10 was characteristic for the C-terminus of clade 1 proteins AsCHI4, AsCHI5, и AsCHI6 .The total ratio of basic  to acidic (Asp and Glu) residues in putative AsCHI proteins was 27/17 (AsCHI1), 24/18 (AsCHI2), 19/19 (AsCHI3), 34/30 , 34/31 (AsCHI6), and 26/23 (AsCHI7). Considering the pI value determined for each studied chitinase , it can Functional annotation in gene ontology (GO) terms predicted that the AsCHI proteins had chitinase activity and chitin-binding ability, and were involved in the catabolism of chitin, cell wall macromolecules, polysaccharides and carbohydrates, and in plant response to fungal infection .Cocos nucifera  and Chimonanthus praecox , AsCHI2–with chitinases of Ananas comosus  and C. nucifera , AsCHI3—with chitinases of Elaeis guineensis  and C. nucifera , AsCHI4-6—with chitinases of E. guineensis , Phoenix dactylifera , and Acacia mangium , and AsCHI7—with chitinases of C. nucifera , E. guineensis , and Cinnamomum micranthum .In the NCBI database of non-redundant protein sequences, AsCHI1 had the highest degree of identity with chitinases of A. sativum cv. Ershuizao based on transcriptomics data  of SE145455 ). The anne stage .AsCHI2/AsCHI3 and AsCHI4–6 had a similar expression pattern within each clade, whereas those of AsCHI1 and AsCHI7 were rather different. AsCHI4–6 showed maximum expression in the pseudostem and shoots, and AsCHI2/AsCHI3—in the pseudostem. The AsCHI1 gene was strongly expressed in the roots and AsCHI7—in the roots, pseudostem, and flowers.AsCHI2, AsCHI3, AsCHI5, and AsCHI6 was quite weak, whereas that of AsCHI4 showed a bell shape, increasing up to stage 4 and then decreasing. The expression of AsCHI7, which was almost absent at stages 1 and 2, gradually increased, reaching a maximum at stage 7, whereas that of AsCHI1 peaked at stage 3 , which predictably included the promoter and 5′UTR, were amplified and analyzed for hormone- and stress-responsive elements. The search for sites important for gene transcription showed that the AsCHI1–7 regulatory regions contained 13 hormone-responsive elements , and 12 stress-responsive elements  (The nd WRE3) . Among tAsCHI1 contained six abscisic acid (ABA)-responsive elements (ABRE), whereas the other genes contained one or two; ABRE3a and ABRE4 as well as dehydration-responsive DRE1 were found only in the AsCHI1 gene -responsive elements were found in AsCHI2, AsCHI3, and AsCHI7  was present only in d AsCHI7 .AsCHI6, whereas gibberellin (GA)-responsive elements were absent in AsCHI1 and AsCHI7, and dehydration-responsive elements—in AsCHI3, AsCHI4, and AsCHI5. Low temperature-responsive elements were absent in AsCHI2, AsCHI5, and AsCHI7, drought- and salt/heavy metals-responsive elements—in AsCHI3-7, and W-box —in AsCHI4 and AsCHI7 , slightly increased (AsCHI2), or was the same (AsCHI3) compared to the control , 141T > C, 486T > A, 696G > A, 712A > G, 777C > T, 891C > A, and 915C > A (AsCHI3), 757G > A and 765C > G , and 198A > G, 330A > T, 414C > T, 699A > G, 834T > C, and 901G > A (AsCHI7).CDSs of d AsCHI7  were ampd AsCHI7 . The seqrshuizao , except Ershuizao chitinases but contained the following residue substitutions: neutral S174P (AsCHI2), T238A (AsCHI3), and V253I , and radical G301S (AsCHI7).AsCHI2, AsCHI3, AsCHI5, and AsCHI7 proteins did not differ in size from cv. AsCHI2, AsCHI3, and AsCHI7 genes, which were differently regulated in cv. Sarmat and Strelets in response to F. proliferatum infection. The AsCHI2 promoter differed from the corresponding cv. Ershuizao sequence by four nucleotide substitutions, and between Sarmat and Strelets sequences by 1 bp indel at position −451 relative to cv. ErshuizaoAsCHI2 promoter.We next amplified and analyzed the promoters (~1.0 kb before the start codon) of the AsCHI3 promoter differed from the corresponding cv. Ershuizao sequence by nine nucleotide substitutions and three indels . The 20 bp indel (TTGCTGACGTGAGAAAGGCA) was present at position −365 in cv. Sarmat and Strelets but at position −874 in cv. Ershuizao, which can be explained by inaccuracies in the assembly of the A. sativum genome. The AsCHI7 promoter was identical in both cv. Sarmat and Strelets, and differed from the corresponding cv. Ershuizao sequence by 12 SNPs and two indels .The AsCHI2, AsCHI3, and AsCHI7 transcription in cv. Sarmat and Strelets revealed the same cis-regulatory elements as in the AsCHI2 promoter of the corresponding genes in cv. Ershuizao  comprises 971 species, including economically important cash crops such as diploid garlic A. sativum, which has one of the largest genomes  among vegetables [Fusarium genus [Fusarium rot, which can make these enzymes a promising target in crop breeding programs.The genus getables ,41,42. Sum genus . The antA. sativum cv. Ershuizao genome [Sarmat and Strelets resistant and susceptible to Fusarium rot, respectively. All the genes were orthologues of the only known A. thaliana class I chitinase gene , suggesting redundancy or, conversely, functional divergence among AsCHI1–7, such as involvement in differential responses to various stresses and pathogens. Analysis of chromosomal localization showed that the garlic genome contained two clusters of tandemly arranged chitinase genes, AsCHI4–7 and AsCHI/AsCHI2, which may be a result of tandem duplication. All AsCHI1–7 genes had only two introns ; among  introns , which infection . This finfection ,46.A. thaliana chitinases [All putative AsCHI1–7 proteins contained chitin-binding and catalytic domains, suggesting functional conservation of garlic class I enzymes in regard to chitin hydrolysis. The presence of N-terminal signal peptides indicates the secretion of mature proteins, which is supported by GO analysis predicting the involvement of AsCHI1–7 in the secretory pathway  and is iitinases . The stritinases .Cucumis sativus chitinase genes [B. rapa class I chitinase genes [C. sativus, but unlike B. rapa, the AsCHI1–7 promoters contained elements associated with the response to ABA as well as those related to immune defense and response to elicitors and various stresses such as anaerobic conditions, dehydration, low temperature, salinization, heavy metals, and wounding  and pathogen attack ,50,51. Tse genes . Cis-actse genes . Similarwounding . Among tFusarium fungi should trigger JA signaling, including the activation of class I chitinases belonging to PR3, PR4, and PR11 families, because the AsCH1–7 promoters contain MeJA-responsive elements, indicating the participation of AsCHI1–7 enzymes in local acquired resistance. However, SA-responsive elements found in the promoters of AsCHI2, AsCHI3, and AsCHI7 genes may also suggest their role in systemic acquired resistance.In plants, pathogen attacks induce systemic or local acquired resistance associated with antagonistic SA and JA defense signaling, respectively ,55. The AsCHI1–7 promoters  significantly increased the resistance of transgenic lines to Sheath blight compared to wild-type plants [Coniothyrium minitans chitinase gene CmCH1 led to the upregulation of defense-related genes and enzymes in soybean plants, and thus enhanced their resistance to Sclerotinia sclerotiorum infection [The results obtained can be useful for breeding programs with the aim of increasing the resistance of onion crops to e plants . Heterolnfection .A. sativum cv. Ershuizao transcriptome sequence (NCBI accession number: PRJNA607255) and whole-genome shotgun contigs (NCBI accession number: PRJNA606385) [A. sativum class I chitinases (M94105.1 and M94106.1) were used as references. We selected sequences that contained start and stop codons and full-length CBD1 and GH19 domains characteristic of class 1 chitinases but did not have premature stop codons in CDSs.The search for class I chitinase genes was performed in the https://web.expasy.org/protparam/; accessed on 10 November 2020), conserved domains, sites, and motifs  [http://meme-suite.org/tools/meme, accessed on 1 December 2020), biological processes and molecular functions , subcellular localization , the functional importance of residue substitutions . The phylogenetic dendrogram was constructed based on protein sequences using the MEGA 7.0.26 (maximum likelihood (ML) method); confidence for tree topologies was estimated by bootstrap values of 1000 replicates.Multiple sequence alignment and structural and phylogenetic analyses of chitinase genes and encoded proteins were conducted with MEGA 7.0.26 . To predE 5.1.1) , http://PROVEAN; ), and siA. sativum cv. Ershuizao RNA-Seq data  [The expression of class I chitinase genes in garlic tissues was determined using A607255)  and visuA607255) .A. sativum cv. Ershuizao transcriptomic data (NCBI accession number: PRJNA607255) . Genomic DNA was isolated from young leaves of a single plant of each cultivar accession as previously described [® II Gel Extraction kit , cloned in the pGEM®-T Easy , and sequenced (3–5 clones for each accession) on ABI Prism 3730 DNA Sequencer  using the designed primers.To amplify full-length chitinase genes from garlic cultivars, gene-specific primers were designed based on A607255) ; manual escribed  and usedFusarium rot, respectively, were kindly provided by the Federal Scientific Vegetable Center  and F. proliferatum was kindly provided by the Group of Experimental Mycology, Winogradsky Institute of Microbiology .Accessions of garlic cv. Sarmat and Strelets resistant and susceptible to −2. Data on the resistance of cultivars to F. oxysporum infection were obtained from their originators .The choice of cultivars for comparative analysis was based not only on the difference in resistance to fungal diseases, but also on the similarity of other varietal morphological characteristics for the possible elimination of their influence on the experimental results. Both cultivars are winter garlic of Russian breeding. These are mid-season, shooting varieties: the leaf is green with a waxy bloom of medium intensity, leaf length/width is 50–51/2.0–2.3 cm; the bulb is round–flat, 65 g; the number of cloves is 5–7 (Strelets) or 7–11 (Sarmat); the structure of the cloves is simple; the color of dry scales is lilac–purple (Strelets) or lilac–white with anthocyanin streaks (Sarmat), leathery scales are brown (Strelets) or light pink (Sarmat), the flesh is white; productivity is 2.0 (Strelets) and 1.9 (Sarmat) kg mF. proliferatum strain was previously isolated from garlic bulbs (cv. Strelets); the pathogenicity test showed that the first signs of the decease appear on the surface of the treated cloves after five days of infection . Since this strain was identified in cv. Strelets, and, in addition, it was identified for the first time in Russia, it was decided to use this particular strain in the present study.The Fusarium infection experiment. In total, six bulbs of each cultivar were used; six cloves from each bulb were processed and halved for control and experiment.The number of cloves in a bulb (5–7 for cv. Strelets and 7–11 for cv. Sarmat) determined the number of cloves used per biological replicate in the F. proliferatum conidial suspension (~106 conidia mL−1) for 5 min . Then, rding to ,68.® Fluorometer, Thermo Fisher Scientific, Waltham, MA, USA) and qRT-PCR was performed in a CFX96 Real-Time PCR Detection System  with 3.0 ng of cDNA, SYBR Green RT-PCR mixture , and specific primers  using the RNeasy Plant Mini Kit , purified from genomic DNA , qualified by gel electrophoresis, and used for first-strand cDNA synthesis  with an oligo-dT primer. RNA and cDNA concentrations were quantified by fluorimetry . The data were expressed as the mean ± standard deviation (SE) based on three technical replicates of three biological replicates for each combination of cDNA and primer pairs. The unequal variance (Welch’s) t-test was applied to assess differences in gene expression; p < 0.05 was considered to indicate statistical significance.s, GAPDH  and UBQ PDH [UBQ , and qRTA. sativum cv. Ershuizao transcriptome sequence .The regulatory regions of chitinase genes were amplified by PCR using specific primers designed based on sequence , purifieA. sativum cv. Ershuizao and cloned AsCHI2, AsCHI3, AsCHI5, and AsCHI7 homologues from two garlic cultivars resistant and susceptible to Fusarium rot. The chitinase gene promoters contained hormone- and stress-responsive elements, including those associated with responses to fungal pathogens and their elicitors, suggesting that the putative garlic chitinases participate in local acquired resistance, whereas AsCHI2, AsCHI3, and AsCHI7 may be involved in systemic acquired resistance. Upon Fusarium attack, all AsCHI genes can be induced by ET, whereas AsCHI1 and AsCHI7 may be activated by ABA and auxin, respectively. AsCHI1–7 transcriptional profiling in resistant and susceptible garlic cultivars infected with F. proliferatum suggests that the expression of AsCHI2, AsCHI3, AsCHI5, and AsCHI7 genes could define the difference in chitinase-mediated response to Fusarium infection between resistant and susceptible plants. Our results provide useful insights into the functions of class I chitinases in A. sativum and Allium plants in general, and may be used in breeding programs to increase the resistance of Allium crops to Fusarium infections.We identified and characterized seven genes encoding class I chitinases in


Antibodies directed at so-called immune checkpoint molecules represent a substantial improvement in cancer therapy. These biological reagents highlight the exquisite interplay between cancer and our own immune system. Cancer progression is enabled by establishing a compartment of suppressive immune cells within the tumor. Therefore, elimination of these suppressor cells is an attractive strategy to augment beneficial anti-tumor immunity and to further improve the newly established immune checkpoint therapy. This review focuses on CCR8, a chemokine receptor highly expressed on suppressive immune cells, and its potential value as a novel target in cancer therapy.Immune checkpoint blockade inhibitors (CBIs) targeting cytotoxic T lymphocyte associated protein-4 (CTLA-4) and program death receptor-1 (PD-1) or its ligand-1 (PD-L1) have transformed the outlook of many patients with cancer. This remarkable progress has highlighted, from the translational point of view, the importance of immune cells in the control of tumor progression. There is still room for improvement, since current CBI therapies benefit a minority of patients. Moreover, interference with immune checkpoint receptors frequently causes immune related adverse events (irAEs) with life-threatening consequences in some of the patients. Immunosuppressive cells in the tumor microenvironment (TME), including intratumoral regulatory T (Treg) cells, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), contribute to tumor progression and correlate with a negative disease outlook. Recent reports revealed the selective expression of the chemokine receptor CCR8 on tumor Treg cells, making CCR8 a promising target in translational research. In this review, I summarize our current knowledge about the cellular distribution and function of CCR8 in physiological and pathophysiological processes. The discussion includes an assessment of how the removal of CCR8-expressing cells might affect both anti-tumor immunity as well as immune homeostasis at remote sites. Based on these considerations, CCR8 appears to be a promising novel target to be considered in future translational research. Numerous chemokines are involved in the development of cancer, reflecting the microcosmos of immune cells and tumor-associated tissue cells required for the establishment of tumors, their growth and, eventually, their dissemination to distant organs. The principal function of chemokines is the induction of cell migration via binding to chemokine receptors expressed on target cells ,2,3. Eig+ Th cells and cytotoxic CD8+ T cells (CTLs) that are capable of recognizing and killing tumor cells. Of note, T cell subsets are distinguished by the combination of cell surface chemokine receptors, indicating that their mobilization is not governed solely by a single chemokine receptor axis [It is unclear if chemokines already play a role in the initiation phase of cancer, well before the development of macroscopic tumors. However, it is well documented that a plethora of chemokines affect cancer at later stages of diagnosis and treatment. I will briefly summarize recent progress in our understanding of how chemokines affect cancer development and refer the reader to a selection of excellent recent reviews that cover this topic ,13,14,15tor axis ,6,7. Thi+ T cells, are the most effective anti-tumor immune cells, induction of the three CXCR3 ligands appears to be a valuable goal, possibly in combination with checkpoint blockage inhibitors (CBIs) that release the molecular brakes on tumor infiltrating lymphocytes (TILs). Of note, a “side-effect” of chronic IFN-γ exposure is the induction of PD-L1 in tumors, suggesting that IFN-γ may counteract anti-tumor T cell responses during long-term exposure to this cytokine [+ T cells. Additional immunosuppressive cells in the TME are tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) and neutrophils. Notably, hematopoietic (monocyte-derived) macrophages, as opposed to embryonic (tissue-resident) macrophages, as well as tumor-induced MDSCs express CCR2 [The functional spectrum of chemokines present in tumors is in part context-dependent and combines pro- and anti-tumor features. It is difficult to single out distinct chemokine-chemokine receptor axes, as they frequently synergize with other chemokine systems as well as immunomodulatory cytokines, growth and differentiation factors. Consequently, chemokines are “moving” targets and need to be discussed together with the ever-changing tumor landscape. The following brief discussion exemplifies the most striking findings in cancer-related chemokine research; for in-depth information, the reader is referred to additional articles that accompany this Special Issue in Cancers entitled “Emerging Roles of Chemokines in Cancer Immunotherapy” as well as excellent recent reviews ,13,14,15cytokine ,17. Tregess CCR2 ,15. Inhiess CCR2 ,19. Numeess CCR2 ,13,15,19+ T cells initiated during antigen-priming in secondary lymphoid tissues (iTregs). tTreg cells recognize self-antigens and primarily reside in peripheral tissues, where they inhibit Tconv cells recognizing local self-antigens, whereas iTreg cells probably provide a negative feedback loop for effector Tconv cells to prevent excessive inflammatory responses in target tissues. In healthy individuals, Treg cells constitute a minor subset of 1–4% among CD4+ T cells in blood and peripheral tissues, but their numbers are known to increase in chronic diseases. In cancer, they vary in numbers but generally represent the major immunosuppressive force. A recent CyTOF protein expression study, involving 32 patients with pancreatic cancer, revealed a frequency of 8 to >30% Treg cells present among CD4+ T cells extracted from tumor tissue, of which the majority were activated and exhibited strong suppressive activity [Treg cells are the major cellular compartment tasked with the suppression of excessive (or inappropriate) T and B cell responses that may cause tissue damage if left unchecked ,22,23,24activity .+ T conv cells [The identification of Treg cells in the context of cancer dates back many years. In fact, the concept of T cell-mediated suppression of anti-tumor immunity was first described 40 years ago by Berendt and colleagues . The fienv cells , and Fornv cells ,29,30. Lnv cells . FoxP3 inv cells ,22.Thanks to new technologies, including single cell RNA sequencing (scRNAseq) and mass cytometry (CyTOF), we now have a better understanding of the phenotype and tissue distribution of Treg cells in both mice and humans. In human tumors, canonical cell surface markers of Treg cells include immune checkpoints CTLA-4, PD-1, TIM3 and LAG3, and the co-stimulatory receptors GITR, ICOS and OX40 ,31,32, aTumor Treg cells suppress anti-tumor immune responses by a myriad of mechanisms . CTLA-4 + T cells [+ T cells and may include PD-1-expressing myeloid cells, as recently demonstrated in mouse tumor models [For the reasons discussed above, tumor Treg cells have become a prime target in translational research, culminating in several CTLA-4-inhibitory antibodies  currently approved for use in immunotherapy of patients with solid cancers ,17,46,47r models . A firm Recently, two reports described the selective expression of the chemokine receptor CCR8 on tumor-associated Treg cells recovered from patients with breast cancer (BC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and melanoma (ME) ,50. Subsccr8-targeted mouse models. Despite early conflicting reports [Future cancer-targeted therapies may benefit from a more detailed understanding of the involvement of CCR8 and its ligands in immunological processes. Human CCR8 was identified in 1997, either by means of low-stringency PCR screening  or by me reports ,68,69, r reports ,70 and a reports . It is n reports ,73,74,75 reports ,77,78,79+ cells among PBMC, confirming their relative scarcity in blood. The vast majority of CCR8+ cells are found among lymphocytes, especially among CD4+ αβ T cells, including FoxP3+ Treg cells and memory Tconv cells. CCR8 was absent on any other type of immune cell, except for a few γδ T cells and NK cells (see below). Interestingly, human blood monocytes were originally reported to respond to CCL1/I-309 [+ cells [+ cells among skin immune cells mirrored the situation in peripheral blood  expressing this chemokine receptor. CCR8 designates “skin-homing” T cells since circulating and skin-resident CCR8+ T cells co-express the cutaneous T cell antigen (CLA), an adhesion receptor known to guide immune cells into skin tissue. The small fraction of CCR8+ γδ T cells are Vδ1+ γδ T cells. The γδ T cell subset featuring the Vδ1-chain in their TCR is rare in blood but predominates in human skin. Similarly, CCR8 is also found on CD16-expressing NK cells that reside primarily in human skin but not in blood [+ Tconv cells [+ T cells are largely absent in other human tissues, as evidenced by recent RNA expression studies [Advanced staining reagents greatly accelerated human CCR8 research and refined early reports describing CCR8 expression in human blood Th2 and Treg cells ; Table 1L1/I-309  and to eL1/I-309 ; howeverL1/I-309 ,84. In f[+ cells ,77,78. Tal blood . Howeverin blood ,77. Howenv cells . In supp studies ,50.+ T cells bear many hallmarks of resident memory T (Trm) cells [Human skin CCR8m) cells ,89,90, im) cells . In contm) cells . Based om) cells , suggest+ and CD8+ αβ T cells with 56 ± 18% and 27 ± 18% being positive for CCR8, respectively  express CCR8 as opposed to conventional skin CD4ectively  [77,78,7iewed in ). Skin t T cells . The two T cells ). Both t T cells ,103,104  T cells ). CCR8 s T cells . In anal T cells ,110,111.+ tumor Treg cells could not only reduce suppression of anti-tumor immunity but also impair tumor repair mechanisms, which is another potentially beneficial effect of CCR8-targeted immunotherapy.Co-expression of CCR8 may also suggest overlapping functionalities in skin and tumor Treg cells that may go well beyond local immunosuppression. Obvious similarities between healthy skin and tumors are their regenerative or wound-healing capabilities. In fact, the concept that tumors are “wounds that do not heal” was proposed many decades ago (reviewed in ), and re+ Treg cells are heterogenous in terms of phenotype and function [+ Treg cells  are phenotypically similar to tumor CCR8+ Treg cells and share many TCR clonotypes with tumor CCR8+ Treg cells [Tumor-infiltrating FoxP3function . Of notefunction ,121,122.eg cells ,51,52. Ieg cells . These feg cells ,57,58,59eg cells ,58. Comb+ Treg cells within tumors. Unfortunately, mechanistic studies are hampered by the fact that CCL18 is known to interact with 3 different cell surface receptors, PITPNM3, GPR30 and CCR8. Similarly, absence of a mouse orthologue makes in vivo studies of CCL18 more challenging.The physiological significance of CCR8 on tumor Treg cells is not clear at present but, based on tumor models in mice, appears unlikely to be associated with the recruitment of tumor Tregs cells. Still, it is worth mentioning that CCL18, the second ligand for human CCR8, is strongly expressed in patients with solid tumors and reported to promote tumor progression by different means, including Treg cell and type-2 macrophage differentiation as well as mobilization of tumor cells , it is interesting to note that severe irAEs in T cell lymphoma patients undergoing mogamulizumab treatment were rarely observed  [Mogamulizumab, a non-fucosylated, humanized antibody specific for human CCR4, is another recent example of an approved antibody reagent causing irAEs in patients with cancer ,129. The severe) . + cells (other than lymphocytes), as summarized in In comparison to CCR4, T cells expressing CCR8 are much less common, being primarily restricted to skin T cells ; Table 1+ cells, which would require special management during CCR8-targeted therapies. Ultimately, this hypothesis will need to be tested in clinical trials involving patients with solid tumors.As suggested by the recent data from several mouse cancer models ,57,58,59


The RNAi pathway is also directly connected with the post-transcriptional gene silencing (PTGS) mechanism, and the pathway controls eukaryotic gene regulation during growth, development, and stress response. Nevertheless, genome-wide identification of RNAi pathway gene families such as DCL, AGO, and RDR and their regulatory network analyses related to transcription factors have not been studied in many fruit crop species, including banana (Musa acuminata). In this study, we studied in silico genome-wide identification and characterization of DCL, AGO, and RDR genes in bananas thoroughly via integrated bioinformatics approaches. A genome-wide analysis identified 3 MaDCL, 13 MaAGO, and 5 MaRDR candidate genes based on multiple sequence alignment and phylogenetic tree related to the RNAi pathway in banana genomes. These genes correspond to the Arabidopsis thaliana RNAi silencing genes. The analysis of the conserved domain, motif, and gene structure (exon-intron numbers) for MaDCL, MaAGO, and MaRDR genes showed higher homogeneity within the same gene family. The Gene Ontology (GO) enrichment analysis exhibited that the identified RNAi genes could be involved in RNA silencing and associated metabolic pathways. A number of important transcription factors (TFs), e.g., ERF, Dof, C2H2, TCP, GATA and MIKC_MADS families, were identified by network and sub-network analyses between TFs and candidate RNAi gene families. Furthermore, the cis-acting regulatory elements related to light-responsive (LR), stress-responsive (SR), hormone-responsive (HR), and other activities (OT) functions were identified in candidate MaDCL, MaAGO, and MaRDR genes. These genome-wide analyses of these RNAi gene families provide valuable information related to RNA silencing, which would shed light on further characterization of RNAi genes, their regulatory elements, and functional roles, which might be helpful for banana improvement in the breeding program.RNA silencing is mediated through RNA interference (RNAi) pathway gene families, i.e., Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) and their  Small RNAs (sRNAs) are considered novel riboregulators. These small regulatory RNAs are mainly of two types; microRNAs (miRNAs) and short interfering RNAs (siRNAs), both of which consist of 21–24 nucleotide and are generated from the synthesis processes of double-stranded RNAs (dsRNA) . Vast nuThe DCL proteins are the major constituents of the sRNAs biogenesis process and have significant roles in post-translational regulation in the RNAi pathway –16. TherC. elegens-specific group, manifest in multicellular and unicellular organisms  [AGO proteins play a crucial and ubiquitous role in the repression of a gene, which are the vital elements of RNAi pathway . AGO pro, fungi) –26. AGO , fungi) , epigene, fungi) , plant g, fungi) , and mer, fungi)  in diffeThe third major RNAi-related protein group is RDR which has a single conserved domain called RdRP ,32. The in silico characterized in many economically important plant species such as 28 genes in maize (Zea mays) [Solanum lycopersicum) [Oryza sativa) [Vitis vinifera) [Capsicum annuum L.) [Cucumis sativus L.) [Brassica sp.) [Hordeum vulgare) [Saccharum spontaneum) [Citrus sinensis) [Setaria italica) [A. thaliana), 20 RNAi pathways associated genes such as 4 AtDCL, 10 AtAGO, and 6 AtRDR genes have been identified and characterized [Through the advancement of high throughput integrated bioinformatics approaches, several RNAi related DCL, AGO, and RDR gene families have been identified and ea mays)  and tomaersicum) , 32 gene sativa) , 22 geneinifera) , and pepnuum L.) , 20 geneivus L.) , 51 geneica sp.) , 23 genevulgare) , 36 geneinensis)  and 38 gitalica) . In the cterized –57.Musa acuminata) is a perennial, monocotyledonous major fruit crop grown all over the tropical and sub-tropical country, especially in the African, Asia-Pacific, and Latin American and Caribbean regions [Banana , chromosomal localization, GO, sub-cellular localization, regulatory network and sub-network analysis between TFs and candidate genes, prediction of cis-acting regulatory elements of TFs in the banana genome. The complete banana genome sequence [Therefore, in the present study, we performed a comprehensive sequence  provideshttps://phytozome.jgi.doe.gov/pz/portal.html) by using the completed genome assembly (v1) of M. acuminata to identify candidate DCL, AGO, and RDR genes [A. thaliana  from The Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org) and collected sequences were used to search the protein sequences of M. acuminata. Furthermore, The Basic Local Alignment Search Tool (BLASTP) program was performed against M. acuminata in the Phytozome database  based on BLOSUM62 matrix, identity percentage (≥30%), coverage percentage (≥70%) and the significant E-values (≤10E-10). Only the primary transcript of the sequences was considered to avoid the redundancy of protein sequences in this study. Genomic information, including the primary transcript, genomic length, chromosomal position of a gene, and length of the open reading frame (ORF), protein length, was downloaded from the M. acuminata genome database deposited in Phytozome. The molecular weight of the identified protein sequences was determined and predicted by using the ExPASyComputepI/Mwtool (https://web.expasy.org/). Identified DCL, AGO, and RDR genes in M. acuminata genome were named according to the nomenclature based on the phylogenetic of the similar gene family members of the previously named A. thaliana genes.The retrieved candidate protein sequences from M. acuminata and A. thaliana were conducted by using the Clustal-W method [The multiple sequence alignments of DCL, AGO, and RDR protein sequences of both W method  through W method . FinallyW method  implemenW method .M. acuminata, protein sequences were retrieved and analyzed using the protein family database   that are similar to the A. thaliana AtDCL, AtAGO, and AtRDR proteins were selected.To investigate the conserved domains of DCL, AGO, and RDR gene families in am.org/) . The maxhttp://meme.sdsc.edu/meme4_3_0/cgi-bin/meme.cgi) [We used the Multiple Expectation Maximization for Motif Elicitation (MEME) webserver to investigate the conserved motifs in all of the predicted DCL, AGO, and RDR proteins (eme.cgi) . This anhttps://gsds.cbi.pku.edu.cn) [M. acuminata, the selected gene structures were compared with the A. thaliana gene structure. Chromosomal location of predicted MaDCL, MaAGO, and MaRDR genes was mapped using the online tool MapGene2Chromosome V2 (http://mg2c.iask.in/mg2c_v2.0/) and considered the physical position of the genes in different scaffold locations.The gene structure  of predicted genes were analyzed using the online program Gene Structure Display Server (GSDS2.0: .edu.cn) . Furtherhttp://planttfdb.cbi.pku.edu.cn//) [p-values by the Fishers test and Benjamini-Hochberg’s correction. The p-value <0.05 was considered to be a statistically significant level for GO enrichment results of the respective predicted genes. We predicted sub-cellular location of identified MaDCL, MaAGO, and MaRDR proteins into the various organelles of the cell using their protein sequences by a powerful and high accuracy web server Plant Sub-cellular Localization Integrative Predictor (PSI) [To determine the relationship of predicted RNAi-related genes with the different clusters of molecular pathways, the GO analysis was performed using the online tool of Plant Transcription Factor Database  . We deteor (PSI) .M. acuminata, we extensively studied the PlantTFDB (http://planttfdb.cbi.pku.edu.cn//) [M. acuminata. Then, constructed a regulatory network and visualized the network using regulatory network visualization tool Cytoscape 3.7.1 [To study the regulatory relationship and network analysis between TFs and RNAi-related genes in du.cn//) . Initialpe 3.7.1 .cis-acting regulatory elements of MaDCL, MaAGO, and MaRDR gene families, upstream region (1.5 kb genomic sequences) of the start codon (ATG) of each RNAi gene were retrieved. Then, we analyzed the stress-response associated promoter cis-acting regulatory elements through online prediction analysis using Signal Scan search program from the Plant CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [cis-acting regulatory elements into five groups; LR, SR, HR, OT, and unknown function. The known function and reported promoter cis-acting regulatory elements of MaDCL, MaAGO, and MaRDR are shown separately.To investigate the promoter e/html/) . We clasTo identify banana RNA silencing genes, AtDCL, AtAGO, and AtRDR retrieved protein sequences were used as query sequences to construct a Hidden Markov Model (HMM) . We idenOn the other hand, identified all 13 MaAGO genes exhibited an N-terminal PAZ and a C-terminal PIWI conserved domain in their polypeptide sequences which are the major components of the plant AGO protein family. The identified MaAGOs ORF ranged from 2841 to 15822 bp, which corresponded to MaAGO7 (GSMUA_Achr7T26400_001) and MaAGO5 (GSMUA_Achr6T04510_001) with the potential encoded 637 and 1237 aa . The genOur HMM analysis predicted the RdRP conserved domain in MaRDR gene family. The genomic length of the five identified MaRDRs varied from 3754 bp to 13305 bp, which are corresponding with MaRDR1b (GSMUA_AchrUn_randomT26010_001) encoded protein length 964 aa and 460 aa, respectively. The pI values of the MaRDRs proteins demonstrate that the proteins are more likely to be basic, where only the MaRDR6 has the highest pI value of 8.47, which showed the basic properties.The pI values are widely distributed (1.99–13.96) in various plant species . Also, pWe obtained the multiple sequence alignment by aligning the predicted MaDCL, MaAGO, and MaRDR protein sequences to the reference sequence of AtDCL, AtAGO and AtRDR Figs –4. The ath position were replaced to proline (P) and asparagines (N), which are replaced by the fourth serine (S) and histidine (H) residue at 798th position (th position was replaced by the aspartate (D) residue at 895th position   and four genes from Arabidopsis , but MaAGO4 is AGO4 subfamily on the basis of higher sequence similarity with the A. thaliana AGO protein AtAGO4. Moreover, the rest of the two banana genes (MaAGO6a and MaAGO6b) are included AGO6 subfamily on the basis of higher sequence similarity with the AtAGO6. Among Group V genes, MaAGO7 genes cluster with AtAGO2, AtAGO3, and AtAGO7 genes, but MaAGO7 exists in AGO7 subfamily on the basis of higher sequence similarity with the AtAGO7. Arabidopsis genome encodes 10 AGO proteins which have been involved in the RNA silencing mechanism [According to the phylogenetic tree analysis, AGO genes of flowering plant species are mainly divided into three clusters; Cluster 1 (AGO1/5/10), Cluster 2 (AGO2/3/7), and Cluster 3 (AGO4/6/8/9), which is similar to our study . We idenoup I-V) . In grouechanism ,91. Accoechanism ,92. Accoechanism ,93. Baseechanism  and deveechanism ,94.Arabidopsis (AtRDR1 and AtRDR2), but MaRDR1a and MaRDR1b is RDR1 subfamily on the basis of higher sequence similarity with the A. thaliana AGO protein AtRDR1. In addition, the MaRDR2 protein exists in the RDR2 subfamily based on the sequence similarity with the A. thaliana AGO protein AtRDR2. Similarly, the MaRDR6 protein is grouped (Group II) with the AtRDR6 protein and contained RDR6 subfamily according to the sequence similarity with AtRDR6. In Group III, the MaRDR5 protein is clustered with AtRDR3, AtRDR4, and AtRDR5 genes but closely clustered with AtRDR5. The MaRDR5 is included in the RDR5 subfamily on the basis of higher sequence similarity with a single Arabidopsis protein AtRDR5. RDRs proteins can regenerate dsRNAs from ssRNA to initiate a signal for RNA silencing mechanism [The phylogenetic tree analyses also revealed four groups of RDR genes (Group I-III) . The RDRechanism ,96. Baseechanism ,57,97,98echanism ,99. Accoechanism .In our analysis, MaDCL2, MaAGO2, MaAGO3, MaAGO8, MaAGO9, MaRDR3 and MaRDR4 were found to be absent in the whole banana genome. These results indicate their functional diversity which would be helpful for further banana improvement.Arabidopsis  as templates. A single conserved domain RdRP is present in RDR proteins which possesses a catalytic β’ subunit of RdRP motif ,115–117.Prediction of motifs insight into a protein sequence has the critical clues to further characterize their functional regulatory roles for gene expression . We predWe observed a maximum of 20 motifs in both proteins of MaDCL and AtDCL. Possibly, MaDCL1 will appear highly functional as like AtDCL1. However, MaDCL1 comprises of 20 motifs that are similar to the paralogs AtDCL1. The MaDCL3 and MaDCL4 contained 17 and 15 motifs, whereas 16 and 19 motifs were found in AtDCL3 and AtDCL4 proteins, respectively. We were not found motif 13 of DEAD domain in MaDCL3. Moreover, motif 6, 8, 13, and 17 of DEAD domain in MaDCL4 were absence as compared with the AtDCL4 protein. The absence of DEAD motifs in both MaDCL3 and MaDCL4 will possibly show a functional diversity between Arabidopsis and banana.Arabidopsis and banana. The motif 20 of ArgoL1 domain was not detected in AtAGO4 but present in MaAGO4. The motif 12 of PAZ domain also was not found in AtAGO5, whereas present in the MaAGO5. However, motif 20 of ArgoL1 domain of in MaAGO5 was absent in this analysis. The motif 17 of ArgoN domain was not detected in MaAGO6a, and MaAGO6b. Another motif 20 of ArgoL1 domain was absent in AtAGO6, whereas this motif was present in the MaAGO6a only. Importantly, motif 18 of PAZ domain was not found in AtAGO6 but were present in MaAGO6a and MaAGO6b in our analysis. The presence of conserved catalytic PAZ domain could lead to enhance the function of target RNA processing by endonucleolytic cleavage in these AGO proteins [On the other hand, we also predicted maximum of 20 motifs in MaAGOs. We found all 20 motifs in MaAGO1 , and MaAGO10 , which exhibited higher conservation with their paralogs AtAGO1 and AtAGO10s. These results reflect that the MaAGO1 and MaAGO10 proteins are highly homologous and will show functional similarities like AtAGO1 and AtAGO10s. However, some motif variability was observed in AGO4, AGO5, AGO6a, AGO6b, an AGO7 between the proteins . Out of Arabidopsis genes according to the gene structure analysis , chromosome 8 (MaDCL1), and chromosome 9 (MaDCL4). MaAGO genes are positioned in the chromosome 1 , chromosome 3 , chromosome 4 (MaAGO10b), chromosome 6 , chromosome 7 , chromosome 9  and unknown chromosome (MaAGO1c). Besides this, MaRDR genes appear in chromosome 2 (MaRDR6), chromosome 10 , and the unknown chromosome (MaRDR5). Gene pairs  and  were closely located to each other in the genomic location of chromosomes 6 and 10, respectively. These results suggest that these pairs of genes could be expressed in a diverse pattern due to their close genomic location, and further study can be performed under various stress conditions.p-value: 3.20E-20). Previous studies suggested that RNAi affects gene silencing through the PTGS mechanism in plants [p-value: 1.70E-06) . We also identified 5 genes  associated with RNA binding activity . Moreover, we identified 16 genes  which were linked with protein binding activities . These results indicate that RNAi proteins are involved in the conducted RISC and RNAi mechanism. Our predicted MaAGO proteins exhibited PAZ and PIWI domains, which are greatly responsible for their endonucleases activity [p-value: 6.00E-07), 9 genes  are related to negative regulation of gene expression . However, 8 genes  are associated with fragmentation of dsRNA , and 5 genes  are predicted to be produced of ta-siRNAs involved in RNAi . Among the predicted candidate RNAi associated banana genes, 9 genes  are responsible for responding to the virus . We drew the Ven Diagram to observe the shared GO terms for three clusters of the RNAi-associated gene families considering biological process, cellular components, and molecular functions   in the cactivity ,85,86. Gunctions . The Venunctions . We alsounctions . These rC. sinensis and predicted them in cell organelles, such as nucleus, cytoplasm, plasma membrane, mitochondria, and plastid [The biological processes of a eukaryotic cell are linked with the sub-cellular localization of specific proteins. The cellular location of protein helps us to understand their functional roles at the cellular level ,121. Sub plastid . The PTG plastid ,123. The plastid . The occ plastid ,123. Mor plastid . Our comTFs play a key role in various biological processes in living organisms, in particular, in plants. The plant TFs are involved in regulating diverse functions, e.g., responses to biotic and abiotic stresses, growth, development, metabolism, and defense against microbial infection –129. In We identified a total of 180 TFs which can regulate the candidate RNAi genes in the banana genome . Based oPratylenchus coffeae of banana [MusaNAC042 demonstrated a positive correlation related to salinity and drought resistance in Agrobacterium-mediated transgenic banana [OsRDR1 gene could enhance the response to the rice stripe virus (RSV) in rice (O. sativa) [M. acuminata represents a diagrammatic evolutionary model that could be explored in detail through the characterization of these predicted genes. Further gene function analysis studies are required to investigate the biosynthesis pathway of calmodulins and calmodulin-related proteins, which could be involved in RNA-related pathways in M. acuminata.We also analyzed the sub-network relationship between TFs and predicted MaDCL, MaAGO, and MaRDR genes . The subf banana . Howeverf banana . Prakashf banana . These Tf banana ,150–152.f banana ,154. An c banana . Another sativa) . Two ban sativa) . The regcis-regulatory elements (CAREs) are typically non-coding DNA composed of the short motif (5–20 bp), which is located in the promoter region of target genes [The et genes ,159. By et genes . Therefoin silico analysis of DCL, AGO, and RDR gene families in C. sinensis also identified almost similar LR motifs, which were predicted to be involved in the leaves’ photosynthesis process [Analyzed results showed that LR, SR, HR, and OT-associated motifs were present in the upstream regulatory regions of RNAi genes ; Fig 14. process . Therefo2 site, P-box, TGA elements, Aux RR core, GC motif, TATC box, TCA elements, TGA box, which are shared by most of the identified RNAi genes in banana. The prediction of HR-related motifs suggests their important biological functions in bananas.We detected AT-rich sequence, boxII-like sequence, HD Zip1, HD Zip3, MBS1, motif1, MSA-like, Non-box, RY elements associated with various plant biological functions are highly shared by many RNAi genes predicted in banana . Plant hMoreover, DRE core, LTR, and TC-rich repeats were shared with several predicted RNAi genes. Several research groups evaluated that TC-rich repeats, LTR elements, MBS, and DRE act as SR motif in different plant species ,175–178.in silico identification of RNAi pathway genes in the banana genome. In total, 3 DCL, 13 AGO, and 5 RDR RNAi pathway genes were identified in the banana genome. The phylogenetic analysis demonstrated that all subfamilies of RNAi genes maintain their maximum higher evolutionary relationship corresponding to banana and Arabidopsis RNAi genes. Conserved domain, motif, and gene structure revealed their functional similarity to those of banana and Arabidopsis RNAi genes. GO analysis revealed that most of the identified MaDCL, MaAGO, and MaRDR genes are connected with important biological functions; RNA silencing, defense against pathogens, and metabolic activity. Sub-cellular localization prediction exhibited that most of the MaDCL, MaAGO, and MaRDR proteins were abundant in the cytoplasm, and nucleus, where the PTGS process mainly occurred. Regulatory network and sub-network analysis were identified important TFs; ERF, Dof, C2H2, TCP, GATA and MIKC_MADS families, which are associated with MaDCL, MaAGO, and MaRDR genes. Also, analyzed CAREs associated with LR, SR, and HR were predicted to bind the TFs of putative MaDCL, MaAGO, and MaRDR genes. Therefore, our findings will provide valuable information on DCL, AGO, and RDR genes in the banana genome, which might be helpful for the cloning and characterization of banana RNAi genes in wet lab conditions and further improvement of these genes for the breeding programs of this economically important crop species. Moreover, these findings could upgrade the knowledge to in silico analysis of genes related to the various important biological pathways from others crop species.In this study, we performed a set of integrative bioinformatics approach to S1 File(TXT)Click here for additional data file.S2 File(TXT)Click here for additional data file.S3 File(TXT)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.


Reticuloendotheliosis virus (REV) is a type C avian retrovirus that causes immunosuppression, dwarf syndrome, and lymphoma in infected hosts. In this study, we used tandem mass tag (TMT) labeling and liquid chromatography–tandem mass spectrometry (LC-MS/MS) to characterize protein alterations in chicken bursa of Fabricius, before and after REV infection at 7, 14, 21, and 28 days. Our data showed that 1,127, 999, 910, and 1,138 differentially expressed proteins were significantly altered at 7, 14, 21, and 28 days after REV infection, respectively. Morphological analysis showed that REV infection reduced in cortical lymphocytes, bursal follicle atrophy, and nuclear damage. Bioinformatics analysis indicated these proteins were mainly involved with immune responses, energy metabolism, cellular processes, biological regulation, metabolic processes, response to stimuli, and multicellular organismal process. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway cluster analysis showed that post-infection, proteins were enriched in the cell cycle, Wnt signaling, antigen processing and presentation, cytokine receptor interaction, adenosine 3′,5′-cyclic monophosphate signaling pathway, and NF-κB signaling. In addition, we observed that peroxiredoxin 4 (PRDX4), peroxiredoxin 6 (PRDX6), glutathione peroxidase 3 (GPX3), catalase (CAT), and peroxidasin (PXDN) were involved in oxidative stress. Some heat shock protein (HSP) family members such as HSPH1, DNAJA4, HSPA8, and HSPA4L also changed significantly after REV infection. These findings help clarify interactions between REV and the host and provides mechanistic insights on REV-induced host immunosuppression. In poultry, reticuloendotheliosis virus (REV) infection leads to immunosuppression, acute reticuloma, short stature syndrome, and lymphoid and other chronic tumorous proliferative diseases . REV prein vitro tandem mass tag (TMT) peptide labeling, combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS) quantitative proteomics, has improved protein analysis and identification . Key proteins were analyzed by bioinformatics. When these data were combined with histopathological and electron microscopic observations, we gained valuable molecular pathological insights into REV infection in this organ, to generate a scientific theoretical basis for REV prevention and treatment in chickens.4.62/0.1 ml as determined by cell breeding. All animal procedures were approved by the Animal Experiment Ethics Committee of Northeast Agricultural University.Fourteen 1-day-old SPF chickens were purchased from Harbin Veterinary Research Institute of China Academy of Agricultural Sciences. REV-T (CVC No. CACCAV107) strains were purchased from the Center for Veterinary Culture Collection (CVCC). REV proliferation in chicken embryo fibroblasts, TCID50, was 10ad libitum access to food and water. At study end, control and infected chickens were euthanized by CO2. The bursa of Fabricius was rapidly excised, washed three times in chilled phosphate-buffered saline (PBS), frozen in liquid nitrogen, and stored at −80°C until required.Fifteen 1-day-old SPF chickens were randomly divided into five groups. Non-infected chickens were used as controls (Group C), and chickens at 7, 14, 21, and 28 days after REV infection were assigned in group I7, group I14, group I21, and group I28, respectively. Chickens in the infection groups were intraperitoneally injected with 100 μl of virus suspension and reared separately in isolation rooms. Chickens had Tissue was fixed in 10% formalin. Based on a previous method , tissue Tissue at −80°C was ground to powder in liquid nitrogen, after which 10% trichloroacetic acid/acetone was added. The sample was then precipitated at −20°C for 2 h. After this, the sample was centrifuged at 4,500 g for 5 min, the supernatant discarded, and the pellet washed in precooled acetone precipitation. After being dried, samples were ultrasonically lysed 1% sodium dodecyl sulfate (SDS) and 1% protease inhibitors and then centrifuged at 12,000 g for 10 min 4°C, and the supernatant was transferred to a new tube. Protein concentration was estimated using the bicinchoninic acid protein assay kit .Protein digestion was initiated by vortexing samples in precooled acetone and precipitation at −20°C for 2 h. Samples were centrifuged at 4,500 g for 5 min, the supernatant discarded, and the pellet washed twice in chilled acetone. Tetraethylammonium bromide (TEAB) at a final concentration of 200 mM was added after drying precipitation and ultrasonic dispersion precipitation, trypsin was added, and enzymatic hydrolysis was done overnight. Dithiothreitol (DTT) was added to a final concentration of 5 mM, with samples reduced at 56°C for 30 min. Then, iodine acetamide was added to a final concentration of 11 mM, and samples were incubated in the dark at room temperature for 15 min. The trypsin-hydrolyzed peptides were desalted on a Strata XC18 (Phenomenex) column and vacuum freeze-dried. For analysis, peptides were dissolved in 0.5 M of TEAB and labeled according to TMT kit  instructions. Briefly, labeling reagent was dissolved in acetonitrile, mixed with sample peptides, and incubated at room temperature for 2 h. Then, labeled peptides were mixed, desalted, and vacuum freeze-dried.via the PRIDE partner repository with the dataset identifier PXD024278.Peptides were dissolved in liquid mobile phase A and separated on an EASY-nLC 1,000 ultra-high performance liquid system. Mobile phase A contained 0.1% formic acid and 2% acetonitrile. Mobile phase B contained 0.1% formic acid and 90% acetonitrile. Peptides were separated by ultra-performance liquid chromatography (UPLC), injected into nanospray ionization (NSI) ion ionization source, and analyzed by QE plus mass spectrometry. The ion source voltage was 2.2 kV, and peptide parent ions and secondary fragments were detected and analyzed by high-resolution Orbitrap. The scanning primary range was 400–1,500 m/z, and the scanning resolution was 70,000. The secondary scanning range was fixed at a starting point of 100 m/z, and the scanning resolution was 17,500. The data acquisition mode used the data-dependent scanning (DDA) program. The signal threshold was 6.3E4 ions/s, the maximum injection time was 50 ms, and the dynamic exclusion time was 30 s. Three biological repetitions were performed. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium http://www.ebi.ac.uk/GOA/). GO enrichment analyses classified proteins according to cellular components, molecular functions, or physiological processes. The Fisher exact-test was used to test differentially expressed proteins in the context of identified proteins. A GO enrichment p-value < 0.05 was considered statistically significant. Pathway analyses were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) online service tool and the KEGG mapper to match proteins to corresponding pathways. We also performed cluster analysis of protein functional enrichment. Firstly, functional classification information and corresponding enrichment p-values were collected, and then the functional classification with significant enrichment  in at least one protein group was screened. The filtered p-value data matrix was first transformed by logarithm of –log10, and then the transformed data matrix was used to classify each function by Z transform. Finally, data sets were obtained after Z transform is used to do unilateral clustering analysis by hierarchical clustering method. Cluster relationships were visualized using Heatmap.2, a function in R language package gplots.Secondary mass spectrometry data were retrieved using Maxquant 1.5.2.8 search parameters. The database was Gallus_gallus_9031_PR_2020072 . Gene Ontology (GO) annotations were derived from the UniProt GOA database (Radioimmunoprecipitation assay (RIPA) lysis buffer  was added to tissue samples, homogenized using tissue homogenizer  at 120 Hz for 12 s, and centrifuged at 4,000 g for 15 min at 4°C; and supernatants were collected in centrifuge tubes. The bicinchoninic acid protein assay kit  was used for protein quantification. Extracted proteins were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System , and incubated overnight at 4°C with rabbit anti-HSP70 antibody (Biosynthesis Biotechnology Inc.), rabbit anti-P38 MAPK/MAPK14 (Biosynthesis Biotechnology Inc.), rabbit anti-CD40 (Biosynthesis Biotechnology Inc.), rabbit anti-AKT1 (Biosynthesis Biotechnology Inc.), and rabbit anti-eIF2B2 (Biosynthesis Biotechnology Inc.). Membranes were then washed three times in TBS + Tween20 (TBST) and incubated for 2 h at room temperature with goat anti-rabbit IgG/horseradish peroxidase (HRP) antibody (Biosynthesis Biotechnology Inc.). Finally, membranes were washed three times in TBST, and an enhanced chemiluminescence (ECL) kit  was used to visualize protein bands. β-Actin was used as an endogenous reference protein. Protein bands were imaged using a multifunctional imaging analysis system . Band intensity was quantified using ImageJ software .Histomorphological and ultrastructural changes in the bursa of Fabricius 21 days after REV infection are shown in p-value < 0.05, the variation threshold of significant up-accumulation was set as the variation of differential expression > 1.3, and the variation threshold of significant down-accumulation was set as the variation threshold < 1/1.3.Based on TMT labeling and LC-MS/MS approaches, we analyzed bursal protein changes in REV-infected chickens. From MS analysis, 666,226 spectra were obtained, of which 246,403 effective spectra were available. In total, 148,153 peptide segments were identified from spectral analysis, of which specific peptide segments numbered 138,043. In total, 8,193 proteins were identified, of which 7,842 were quantified . At a p-From In To further elucidate functional correlations of differentially expressed proteins in our samples, we enriched proteins using GO classifications and KEGG pathway analyses and clustered them. From For cell component classification, at 7 dpi, proteins were mainly distributed in the respiratory chain and nicotinamide adenine dinucleotide dehydrogenase complexes. At 14 dpi, proteins were mainly distributed in the oxidoreductase complex and organelle envelope. At 21 dpi, proteins were mainly distributed in the vacuolar lumen, the external side of the plasma membrane, membrane protein complexes, and the cell surface. At 28 dpi, proteins were mainly distributed in super-fraction complexes, cell–cell junctions, and the actin cytoskeleton.For molecular functions, at 7 dpi, proteins were mainly concentrated in phosphatase–triphosphatase activities, nucleotide binding, and myosin binding. At 14 dpi, proteins were mainly concentrated in antioxidant activities, peroxidase activities, and glutathione transferase activities. At 21 dpi, proteins were mainly concentrated in antigen binding, G protein-coupled serotonin receptor binding, and proteasome-activating ATPase activities. At 28 dpi, proteins were mainly concentrated in actin binding, enzyme activity, and extracellular matrix structural inhibitor.via the cytochrome P450 signaling pathway. At 21 dpi, proteins were mainly related to the immune response signaling pathway caused by exogenous pathogenic microorganisms such as Epstein–Barr virus infection. At 28 dpi, proteins were mainly associated with Fc γR-mediated phagocytosis and primary immunodeficiency signaling pathways.KEGG pathway analysis showed that proteins at 7 dpi were mainly related to antigen processing and presentation, actin cytoskeleton regulation, and the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) signaling pathway. At 14 dpi, proteins were related to xenobiotic metabolisms We identified 8,193 proteins using proteomics quantitative analyses . To screOur Mfuzz analyses indicated that proteins in cluster 1, including biological processes and molecular functions of movement of cell or subcellular components, actin cytoskeleton organization, supramolecular complex, cytoskeleton, structural molecule activity, and the immunoglobulin I-set domain, were decreased at day 7 after REV infection, and stable trends were observed at 7–28 days. Proteins in cluster 2, including intestinal immune networks for IgA production, calcium signaling pathways, NF-κB signaling pathways, adaptive immune responses, and lymphocyte-mediated immunity, exhibited increased expression after infection. Proteins in cluster 3, including intracellular protein transport, mitochondrial membrane organization, G protein-coupled receptor binding, and GTPase activity, were decreased within 7 days of REV infection but showed a steady increase from 7 to 21 days. In cluster 4, related biological processes and protein domains appeared in protein digestion and absorption, endoplasmic reticulum, globin, and Tudor domains. Protein expression decreased and then increased slowly at 21 dpi. Proteins in cluster 5, including antigen processing and presentation, intestinal immune networks for IgA production pathways and adaptive immune responses, lymphocyte-mediated immune biological processes, immunoglobulin receptor activity, and transmembrane signaling receptor activity, were increased within 21 dpi, with a slight decrease at 21–28 days. Proteins in cluster 6, including purine metabolism, platelet activation, ligase activity, intermediate filament cytoskeleton pathways, or molecular functions, showed a continuous decrease in expression within 28 dpi.To verify MS data accuracy, we randomly selected five proteins to investigate expression by western blotting . The resThe innate immune system recognizes pathogen-associated molecular patterns through the pattern recognition receptor (PRR), which is the first line of defense against pathogens. PRRs include toll-like receptors (TLRs), retinoic acid-inducible gene 1 (RIG-1)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-Leucin Rich Repeats (LRR)-containing receptors (NLRs), and C-type lectin receptors (CLRs). After the corresponding viral RNA or DNA was identified, signal transduction is initiated, and interferon (IFN) is expressed to induce antiviral effects . Both TLNF-κB plays key roles in mediating cell inflammatory and immune responses. Several NF-κB pathway activation factors are known, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-12, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), chemokines, adhesion molecules, and colony-stimulating factors . In thisIL-18 was also up-accumulated at the four time points after REV infection. IL-18 is as a pro-inflammatory cytokine and a member of the IL-1 cytokine family, which induces IFN-γ expression. It also exhibits multidirectional effects during inflammation . Previouvia interactions between viral protein Meq and PI3K p85 subunit, to delay cell apoptosis and promote virus replication  are produced; and when antioxidant clearance is exceeded, abnormal gene expression, receptor activity, aberrant cell proliferation, or cell death ensues . After iHeat shock proteins (HSPs) are highly conserved functionally related proteins, widely expressed in all cells. They are divided into different families based on molecular weight and include HSP100, HSP90, HSP70, HSP60, HSP40, and small HSP (15–40 kDa) –48. WhenIn summary, we used TMT-labeled quantitative proteomics to study dynamic changes in chicken bursal proteins at 7, 14, 21, and 28 dpi (compared with protein abundance before infection). GO, cluster, enrichment KEGG pathway, and pattern expression cluster analyses identified differentially expressed proteins and corresponding biological processes related to REV infection in chickens. This is the first time quantitative proteomics have been used to study protein changes in chicken bursa of Fabricius infected with REV. In future studies, we will focus on antioxidant treatments after REV infection and investigate relationships between REV infection, the HSP family, and endoplasmic reticulum stress.http://www.proteomexchange.org/, PXD024278.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by Animal Experiment Ethics Committee of Northeast Agricultural University.SZhe designed the experiments and revised manuscript. DY analyzed the data and drafted manuscript. XL performed the experiments of bioinformatics analysis. SZha performed the experiments of morphological analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes  Human betaherpesvirus 5) in cell culture results in mutations ranging from small substitutions, insertions, or deletions to large-scale deletions, duplications, and rearrangements. Some mutations appear to be stochastic, whereas others consistently disrupt specific genes and confer growth advantages in certain cell types or growth conditions [UL128, UL130, and UL131A [Passage of human cytomegalovirus  and entering retinal pigmented epithelial cells  with poor efficiency  was passhttps://www.pacb.com/support/software-downloads/) with default settings. Two-modal polymerase reads for TB40/EF  or TB40/EE  were indexed using pbindex, XML files of the subread counts were produced using dataset, and 16,920 HiFi reads were generated for TB40/EF and 6,985 HiFi reads for TB40/EE using CCS. The approximately three-fold difference in TB40/EF compared to TB40/EE reads is consistent with a three-fold difference in DNA concentration, which in turn reflects reduced levels of cell-free virus released from TB40/EE-infected ARPE-19 cells [In the current study, we used long-read PacBio sequencing to examine in detail the genetic changes potentially associated with adaptation to epithelial cells. DNA was isolated from TB40/EF and TB40/EE cell-free virions as described previously . HiFi SM19 cells . Final H19 cells , and the19 cells . The conL) and unique short (US) regions, each of which is flanked by inverted repeats in the arrangement ab-UL-bʹaʹcʹ-US-ca . Comparison of the TB40/EF and TB40/EE consensus genome sequences identified 12 single nucleotide substitutions and one single nucleotide deletion within noncoding regions in the a, b, or c inverted repeats , these loci represent only six unique differences. Although these mutations are in noncoding regions, the significant levels of enrichment suggest that they may provide a selective advantage in ARPE-19 cells. However, the inverted repeats have been reported to be particularly prone to mutation during passage of HCMV, with changes being generally replicated in all copies presumably as the result of recombination [The HCMV genome (236 kbp) consists of unique long  were anticipated. Indeed, targeted sequencing of TB40/EF had previously identified a suppressor substitution converting the UL128 stop codon (TGA) to TTA (encoding leucine), thereby extending UL128 by 19 residues [UL128 and resulting in truncation of UL128 (44% of genomes), and one containing a single nucleotide substitution in UL130 resulting in a C207S substitution in UL130 (9% of genomes)  with 26 novel residues, resulting in rapid degradation of the mutant protein and loss of endothelial cell tropism [Although it has not been demonstrated that the UL128 suppressor substitution disrupts the function of the PC, indirect evidence indicates that the UL130 C207S substitution is likely to have a negative effect. The crystal structure of the PC indicates the presence of a disulfide bond between C207 and C172 , and theUL26 (resulting in E98K in UL26), UL69 (H492Q in UL69), UL122 (D390H in IE2), and US28 (C320W in US28), and a two nucleotide insertion in gene UL141 introducing a frameshift truncating UL141. Examination of read frequencies revealed that most of these changes were enriched to a marginal or modest level: UL26 (from 53 to 100%), UL69 (from 44 to 52%), UL141 (from 44 to 69%) and US28 (from 88 to 100%). Thus, although these changes may be associated with improved replication in ARPE-19 cells, they may have been the consequence of stochastic effects. Moreover, both variants of UL69 and UL141 have been reported previously in consensus sequences of strain TB40/E, namely a partial TB40/E sequence (GenBank accession number AY446866.1), clone Lisa (KF297339.1), and the BAC clones TB40-BAC4 and TB40-KL7-SE  [UL69, clone Lisa and TB40-BAC4 encode Q492, whereas TB40-KL7-SE encodes H492. In UL141, the frameshift is absent from clone Lisa but present in the partial TB40/E sequence, TB40-BAC4, and TB40-KL7-SE. Thus, it appears that parental TB40/E stocks contained two variants of both genes, with capture of one or the other allele in the genomes of clone Lisa, TB40-BAC4, and TB40-KL7-SE resulting from cloning. In contrast, the prevalence of the D390H substitution in IE2 increased markedly from 5 to 87%  –13. In UUL128 and UL130 is consistent with the detected improvement in efficiency of epithelial cell entry [In summary, whole genome sequencing identified variants impacting IE2 and PC subunits UL128 and UL130 as being potentially selected during adaptation of HCMV strain TB40/E for growth in epithelial cells. Enrichment of viral genomes lacking disruptive mutations in ll entry , and, asll entry –25, may ll entry . It is n


There is evidence that sugammadex can facilitate extubation post-surgery and attenuate postoperative pulmonary complications resulting from postoperative residual neuromuscular blockade. However, it may induce adverse effects, including bronchospasm, laryngospasm, bradycardia, hypotension, and cardiac arrest. Here, we present a case of sugammadex-induced bradycardia and hypotension.An 82-year-old female received video-assisted thoracic surgery decortication and wedge resection of the lung for empyema. Post-surgery, she developed bradycardia, hypotension, hypoxia, and weakness.The patient was suspected to have sugammadex-induced bradycardia, hypotension, hypoxia and weakness.The patient received immediate treatment with atropine (0.5 mg) for bradycardia. Glycopyrrolate (0.1 mg) and neostigmine (1 mg) were administered to improve the train-of-four (TOF) ratio.Following initial management, we observed improvement in the hemodynamics of the patient. She was discharged without any sequelae.Sugammadex-induced bradycardia or cardiac arrest are rare; however, anesthesiologists must consider the possibility of the occurrence of such events and initiate appropriate management measures. Immediate treatment with atropine and inotropic or vasopressors is warranted if the patient presents with bradycardia. However, postoperative pulmonary complications, such as pulmonary hemorrhage, difficulty breathing, reintubation, and prolongation of the patient's length of stay could be attributed to postoperative residual neuromuscular blockade. In contrast to acetylcholinesterase inhibitors, sugammadex has been demonstrated to shorten extubation time, resulting in improved operating room turnover in clinical anesthesia settings and attenuation of postoperative pulmonary complications.,5 However, sugammadex has been associated with several adverse effects, including bronchospasm, pulmonary edema, desaturation, hypotension, laryngospasm, bradycardia, and cardiac arrest.–16 Here, we present a case of sugammadex-induced bradycardia, hypotension, and hypoxia in a patient following video-assisted thoracic surgery decortication and wedge resection of the lung. In addition, we analyzed adult cases of bradycardia or hypotension following sugammadex administration between January 2014 and January 2021 with a detailed literature review as well.Reversal agents are used to speed up recovery time from neuromuscular blockade and prevent postoperative residual neuromuscular blockade.2The patient was an 82-year-old woman  diagnosed with hypertensive heart disease with no known history of allergy. Upon being diagnosed with empyema, she was scheduled for video-assisted thoracic surgery decortication and wedge resection of the lung. Pre-procedural electrocardiography revealed a normal sinus rhythm. Cardiac echocardiography showed a left ventricular ejection fraction of 71% and impaired left ventricular relaxation. Abnormal laboratory data demonstrated a white blood cell count of 15500/uL and C-reactive protein level of 220.7 mg/L. Vital signs were unremarkable except for oxygen saturation being around 90% to 95% on room air.2, 99%; NMTM count, 4; and train-of-four (TOF) ratio, 64. The patient regained consciousness and spontaneous breathing. A dose of 200 mg sugammadex was next administered. One minute following sugammadex administration, the patient developed sinus bradycardia with 34 bpm without diffuse ST depression, hypotension (67/34 mmHg), hypoxia (SpO2: 65%), with an NMTM count of 0, and TOF ratio of 0. The patient was immediately administered atropine (0.5 mg) intravenously, following which her hemodynamics improved, with a corresponding heart rate of 65 bpm, SBP of 89/50 mmHg, and SpO2 of 93%. Subsequent administration of glycopyrrolate (0.1 mg) and neostigmine (1 mg) improved the TOF ratio to 69. Arterial blood gas values were as follows: pH, 7.291; PaO2, 75 mmHg; PaCO2, 51.4 mmHg; HCO3−, 22.8 mmol/L; lactate, 1.1 mmol/L; hemoglobin, 12.6 g/dL; and hematocrit, 36.7%. The anesthesiologist replaced the double-lumen tube with a single-lumen 4endotracheal tube, following which the patient was transferred to the intensive care unit for further care. The patient was extubated and discharged 4 and 10 days post-surgery, respectively, without any sequelae.Routine monitoring included electrocardiography (lead II), noninvasive blood pressure, pulse oximetry, end-tidal carbon dioxide measurement, entropy, and the use of neuromuscular transmission monitors (NMTM). General anesthesia was induced with thiamylal (225 mg), rocuronium (50 mg), lidocaine (40 mg), fentanyl (75 mg), and glycopyrrolate (0.1 mg). The patient was intubated with a 32 Fr left double-lumen tube, and inspected using a fiberoptic bronchoscope. Following tracheal intubation, a right radial arterial line and central venous catheter were inserted. Anesthesia was maintained with sevoflurane (0.5–0.9), minimum alveolar concentration and propofol (20 mL/h) ti4trated to effect under entropy. In addition, the patient received a continuous dose of rocuronium (15–10 mg/h) under NMTM. Ventilation was adjusted to maintain an end-tidal carbon dioxide of 35 to 45 mmHg. The surgery was completed uneventfully in 2 hours and 40 minutes. At the completion of the procedure, arterial blood pressure of the patient was estimated to be 124/85 mmHg; heart rate, 65 beats per minute (bpm); SpO3PubMed and Cochrane Database were searched for the terms “sugammadex AND (bradycardia operating room cardiac arrest)” in January 2021. Articles between January 2014 and January 2021 were included regardless of the type of publication or journal. The full text of the articles was retrieved. The authors assessed the articles using the following set of criteria:1.the article is written in English;2.the study is a case report or case series;3.the study includes a description of the dose of sugammadex used, its adverse effects, and the treatment strategy;4.the subjects are adult patients.Information such as the type of publication (case report), year of publication, description of adverse effects, treatment administered for the reaction, and patient outcome were extracted from the articles. The results have been summarized in Table –17 These cases were further categorized into cardiac events associated with bradycardia (9 out of 11),–11,14–17 cardiac arrest/asystole (5 out of 11),,10–13 hypotension (7 out of 11),,12,14–17 and desaturation (3 out of 11).,15,17 Amongst bradycardia patients, a total of 8 out of 9 cases returned to spontaneous circulation following initial treatment.–10,14–17 Desaturation was observed in 3 out of 11 cases; mask ventilation, cardiopulmonary resuscitation, defibrillation, and reintubation were performed in some of these cases.,15 In summary, cardiopulmonary collapse was reported in 8 out of 11 cases, wherein cardiopulmonary resuscitation was performed;,10–15 however, 2 cases resulted in death.,11 Case 1 reported by Bhavani et al returned to spontaneous circulation following resuscitation; however, the patient died on the 15th day after being transferred to the ICU. Mirza et al reported the death of another patient because of failure of resuscitation. In our case, the patient presented with bradycardia, hypotension, hypoxia, and weakness.We came across 11 cases of bradycardia and/or cardiac arrest.,10,14,16 or glycopyrrolate (1 out of 11) were administered. Although the mechanism of anticholinergic agents remains unknown, and their effects are limited, the use of anticholinergic agents to treat s44ugammadex-induced bradycardia is still being considered in some case reports. However, some patients did not respond to anticholinergic agents such as atropine.Different management practices were adopted according to clinical symptoms and signs. A total of nine cases were reported to have bradycardia. Therefore, atropine (4 out of 11),10–17 Additionally, other drugs, including lidocaine, calcium, and nicorandil, were administered to prevent further progression of cardiac ischemia and arrhythmia. Vasopressors have been preferred over anticholinergic agents for the treatment of sugammadex-induced bradycardia owing to the absence of known muscarinic effects of sugammadex and inadequate response to atropine. In our case, hypotension and bradycardia were effectively treated via administration of ephedrine and atropine.In cases where hypotension was reported, vasopressors with epinephrine, norepinephrine, or ephedrine were administered.,15 Another case was clinically diagnosed with hypersensitivity to sugammadex with tryptase and IgE levels within the normal range. These patients immediately experienced erythema, hypotension, or desaturation following the administration of sugammadex. Therefore, anaphylactic medications, such as methylprednisolone, and antihistamine drugs with hydroxyzine pamoate were administered.,17The etiology of cardiac and pulmonary adverse effects in all the other cases is unknown. Two cases were suspected to be associated with hypersensitivity to sugammadex, as indicated by serum tryptase level and/or skin prick test. reported that hypotension was observed in a study involving large doses of sugammadex. However, at three different doses of sugammadex  in pooled phase I-III patients, the incidence of marked bradycardia was found to be 1%, 1%, and 5%, respectively. Upon reviewing these 11 cases, we found that the dose of sugammadex ranged from 2.08 to 4.17 mg/kg.–17 In our case, the dosage of sugammadex used was 4.55 mg/kg. Therefore, the correlation between the dose of sugammadex and severity of bradycardia needs to be further investigated.Pühringer et alAdverse Event Reporting System database. These events include bradycardia (n = 160), cardiac arrest (n = 110), cardiorespiratory arrest (n = 16), hypotension (n = 83), and decreased oxygen saturation (n = 55). However, in this study, we have investigated only 11 case reports from previous literature.–17 Therefore, the incidence of adverse effects associated with sugammadex could have been underestimated.From 2014 to December 31, 2020, a total of 282 cases of major cardiac events were reported following sugammadex/sugammadex sodium/bridion administration as per the Food and Drug Administration 4Although sugammadex-induced bradycardia or cardiac arrest are rare, anesthesiologists should consider the possibility of the occurrence of such events. Immediate treatment with atropine and inotropic or vasopressors is recommended in such cases. Furthermore, advanced cardiac life support is required if initial management fails to manage the adverse effects.Investigation: I-Chia Teng, Yao-Tsung Lin, Zhi-Fu Wu.Supervision: Chin-Chen Chu, Jen-Yin Chen.Writing – original draft: I-Chia Teng, Zhi-Fu Wu.Writing – review & editing: Ying-Jen Chang, Zhi-Fu Wu.


Aurea helianthus is high, A. helianthus extract was more useful in modulating tumor activity compared to the two aforementioned substances. Notably, we established that the extract induced bioactive exosomes in EC cells, and profiling of miRNAs in the extract-inducing exosomes (EIE) indicated their potency to be developed as a biological drug. The extract and EIE contributed to the following five biological process categories for EC cells: (1) cell migration and invasion suppression, (2) cellular senescence activation by attenuating mitochondrial membrane potential and enhancing autophagy, (3) reproductive cancer activity attenuation, (4) drug susceptibility activation, and (5) EIE containing miRNAs associated with decreasing inflammation.Endometrial cancer (EC) cells metastasize to various regions, including the ovaries, fallopian tubes, cervix, blood, liver, bone, and brain. Various carcinogens are known to cause EC. Exosomes are released from several types of cells and contain various cellular components. In this study, flow cytometry and quantitative PCR were used to evaluate marker levels, cell migration, cell invasion, and mitochondrial membrane potential, and cellular senescence tests were used to estimate cancer activity. The microRNAs were profiled using next-generation sequencing. Although tocopherol-α and rutin content in  Endometrial cancer (EC) is classified into two types: type I, the most common type, and type II . Cancer HOX) gene, PI3K/AKT/mTOR, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor (VEGF), and vimentin [The typical characteristics of activated cancer cells include cell death evasion, healthy metabolism, unlimited replication, tissue invasion, and metastasis . Severalvimentin ,15,16,17Exosomes are extracellular vesicles released from several types of cells . In partAurea helianthus, belonging to the family Malvaceae, has various functions, including inflammation, fever, and tumor suppression; oxidation and melanogenesis; detoxification; lipid metabolism modulation; and immune regulation [A. helianthus extract (AH) protects human vaginal cells from oxidative and fungal stress and strongly suppresses CK8, Muc-16, and vimentin expression. Furthermore, it modulates monocyte differentiation and activates HPV peptide phagocytosis [gulation . Notablygulation . Tocophegulation . Additiogulation . Accordigulation , A. heliocytosis .Although no prior study has focused on the functions of phytoextract-inducing exosomes to date, the present study documented the potency of specific miRNAs in AH-induced exosomes (EIE), in addition to the functions of the extracts, including EC marker expression downregulation, and EC cell invasion and migration suppression. The functional miRNAs in this study play a key role in the development of a biological drug to protect against or heal gynecological cancers through in vivo experiments. In particular, the results of this study suggest that EIE robustly protects against and attenuates EC, and miRNAs in EIE may be utilized to develop liposomes for use as a biological drug.This study targeted to categorize the roles of AH and EIE in modulating tumor activity into five categories . FurtherThe extracts effectively downregulated SCC and UGF genes in EC cells a. In con+ cells in the presence of R25 and AH was two and four times lower than that of marker-positive control cells, respectively , mTORC2, and ABCB1 . Unlike tocopherol-α and rutin, AH, particularly AH1000, significantly downregulated SCC and UGF . SCC binAdditionally, SCC inhibits cellular apoptosis by irradiation, natural killer cells, anticancer drugs, and irradiation, and promotes migration and invasion by decreasing E-cadherin ,27,28. UThe extract and EIE modulate tumor activity around EC cells through their effects on five biological categories. In EC bearing and genetic models ,34,35, pPTGS2) and VEGFA, respectively. The PTGS2 protein level is modulated by beta-2 adrenergic receptor (ADRB2) receptor signaling, and PTGS2 silencing suppresses migration and invasion of ovarian cancer cells [First, tocopherol-α and the extracts significantly inhibited the malignant behavior of EC cells a,b. In per cells . VEGF prer cells . In the Second, the three substances activated senescence in EC cells, and senescence was intensely triggered under AH1000 d. Based ATP5A1 and ATP5I genes activate ATP synthesis on the electron transport system in mitochondria. Although cancerous activity is based on ATP synthesis activation, AH suppresses ATP synthesis by inhibiting mitochondrial membrane activity.The MMP significantly decreased in the presence of AH500 and AH1000, and was 2.92 times lower than that in the control. The exosomal results showed that anti-ATP5A1 and ATP5I hsa-miR-877-3p were 6.7 times upregulated . PreviouHSD17B12 hsa-miR-874-3p was significantly downregulated, whereas anti-STRN3 hsa-miR-125b-5p, anti-PHB2 hsa-miR-10a-5p, and anti-ISL1 hsa-let-7d-5p were significantly downregulated in EIE , synthesizes prostaglandins from arachidonic acid. The synthesized prostaglandin contributes to tumorigenesis, including proliferation, metastasis, and angiogenesis [Third, miRNAs in EIE affect endocrine modulation, such as estrogen and prostaglandin synthesis and signaling. Anti-d in EIE . Additiod in EIE b. Unlikeogenesis . The sigABCB1 expression was decreased by miR-451a in the AH-induced exosomes. When exposed to an anticancer drug, cancer cells alter their phenotypes to resist the drug by increasing the ATP binding cassette subfamily B member 1 (ABCB1) protein [NF-κB , mTORC2, and ABCB1  and mTORC2 induce multidrug resistance [Fourth, AH and EIE were involved in attenuating drug resistance and autophagy in EC cells. In the presence of AH,  protein . Further protein , NF-κB [ protein , and YB- protein . In contnd ABCB1 . Activat [NF-κB P, P52, mTsistance . These rFifth, the downregulated hsa-let-7e-5p contributed to suppressing the innate immune response by upregulating the POLR3D protein, a subunit of RNA polymerase . POLR3D A. helianthus flowers were exposed to infrared rays for 2 h and ground into a powder (400 mesh). The powder was extracted and concentrated under 0.08 MPa, 70 °C, and 2 h, and filtered with a 0.2 μm syringe filter . After treatment with dosages, the extracts  supported by Life Together , 5 μg tocopherol , and 25 µg rutin (Sigma), the endometrial cancer cells were cultured for 72 h at 37 °C in an atmosphere containing 5% CO2. The ovarian cancer cells were cultured with 50 μL/mL EIE for 72 h at 37 °C in an atmosphere containing 5% CO2. To estimate the exosomal functions, EC and ovarian cancer cells were cultured with 20 µL EIE. The total volume of medium per well was 1 mL.Human EC  and ovarian cancer  cells were purchased and cultured in Dulbecco’s modified Eagle’s medium  containing high glucose. After ripening at 10 °C for 48 h, Total RNA was isolated from cells exposed to the three substances using RiboEx reagent . cDNA was synthesized using Maxime RT PreMix , and quantitative PCR was performed with the primers  using thTo analyze the expression levels of markers, including CK8, Muc-16, and vimentin, the cells were fixed with 2% paraformaldehyde for 24 h, treated with 0.02 Tween 20 for 5 min, and incubated with three fluorescence-labeled three immunoglobulins, FITC anti-cytokeratin 8 , PE-anti-CA125 , and APC-anti-vimentin  for 48 h at room temperature with agitation at 85 rpm. The washed samples were analyzed using a flow cytometer, BD FACScalibur  and FlowJo 10.6.1 .TM 24-well cell migration assay kit . After excluding the gel spots, the seeded cells were cultured in all areas in the wells. After detaching the gels, the migration was estimated at specific time intervals  using an ECLIPSE Ts2 instrument  and the Nikon NIS Elements V5.11  imaging software.To estimate the migration inhibitory efficacy of the three substances, EC cells were cultured using the RadiusTM 24-well cell invasion assay kit . Invasiveness was estimated using a flow cytometer  and FlowJo 10.6.1 .To estimate the invasion inhibitory efficacy of the three substances, EC cells were cultured in a CytoSelectAfter exposure to the three substances for three days, EC cells were stained using the JC-1 Mitochondrial Membrane Potential Assay Kit  and the mitochondrial activity was estimated using a flow cytometer  and FlowJo 10.6.1 .After exposure to the three substances for three days, EC cells were stained using the CellEvent™ Senescence Green Flow Cytometry Assay Kit (Invitrogen). The stained cells were measured using a flow cytometer  and FlowJo 10.6.1 .n = 3) were summated for miRNA profiling in the presence of each substance. Small microRNAs were sequenced by ebiogen Inc.  to analyze exosomal functions. An Agilent 2100 bio-analyzer and the RNA 6000 PicoChip  were used to assess RNA quality. RNA was quantified using a NanoDrop 2000 spectrophotometer . The Agilent 2100 Bio-analyzer instrument for the high-sensitivity DNA assay  and NextSeq500 system single-end 75 sequencing  were used to prepare and sequence small RNA libraries. To obtain a bam file , the sequences were mapped using bowtie2 software , and the read counts were extracted from the alignment file using bedtools (v2.25.0)  and R language (version 3.2.2)  to determine the miRNA expression level. miRWalk 2.0  was used for miRNA target signal study, and ExDEGA v.2.0  was used to deduce various results, including van diagram, heatmap, scattering plots, and bar graphs.Semi-confluent EC cells were cultured for three days in the absence of FBS. The supernatants were filtered with 0.8 µm syringe filters , and the induced exosomes in the supernatants were isolated and their purity was estimated using the exoEasy Maxi kit  and FITC anti-CD63 immunoglobulin , respectively. The FITC anti-CD63-positive exosomes were estimated using a flow cytometer, Carlibur . The induced exosomes from the samples  with the post hoc test (Scheffe’s method) and independent t test using the SPSS software v26  and Prism 7 software .A. helianthus were high, AH modulated the environment more effectively than the two substances. The modulations by AH were summarized in five biological categories: migration and invasion suppression, increased drug susceptibility, decreased inflammation, reproductive cancer activity attenuation, and cellular senescence activation. Furthermore, the effects of EIE suggest a potency to protect against and heal gynecological cancers. These functional miRNA candidates should be further studied via in vitro and in vivo experiments with liposomes containing functional miRNAs. These candidates will help in performing in vivo and in vitro experiments to estimate the potency of a drug for gynecological cancers.In summary, AH significantly modulated the tumorigenic microenvironment in EC cells. Although α-tocopherol and rutin contents in