Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are becoming increasingly prevalent worldwide. Despite the different etiologies, their spectra and histological feature are similar, from simple steatosis to more advanced stages such as steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Studies including peroxiredoxin knockout models revealed that oxidative stress is crucial in these diseases, which present as consequences of redox imbalance. Protein tyrosine phosphatases (PTPs) are a superfamily of enzymes that are major targets of reactive oxygen species (ROS) because of an oxidation-susceptible nucleophilic cysteine in their active site. Herein, we review the oxidative inactivation of two tumor suppressor PTPs, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and T-cell protein tyrosine phosphatase (TCPTP), and their contribution to the pathogenicity of ALD and NAFLD, respectively. This review might provide a better understanding of the pathogenic mechanisms of these diseases and help develop new therapeutic strategies to treat fatty liver disease. The liver is the second-largest organ in the body and has functions in metabolism, excretion, and immunology . Fatty lProtein tyrosine phosphatases (PTPs) are a superfamily of protein phosphatases that play a key role in controlling signaling transductions . In cont2O2 [2H) or sulfonic (-SO3H) acid [Accumulating evidence on ALD and NAFLD pathology reveals that oxidative stress is one of the key factors in disease onset and progression ,16,17. R2O2 . All PTP2O2 ,21. Low 2•−) and hydroxyl radicals (HO•), whereas the most well-known nonradical is H2O2 [ROS are produced under physiological conditions and serve as signaling molecules that participate in cellular pathways, such as cell survival, proliferation, and differentiation ,23. ROS  is H2O2 ,23. Howe is H2O2 ,23. ROS  is H2O2 ,25,26.ROS generators comprise different organelles, including the endoplasmic reticulum (ER), mitochondria, and peroxisomes; oxidative enzymes, including NOX, xanthine oxidase, aldehyde oxidase, and cyclo-oxygenase; cytochrome P450 2E1 (CYP2E1); and environmental factors, including smoke, UV light, radiation, and certain drugs, such as antibiotics (nitrofurantoin) and methotrexate ,27. CellOxidative stress is one of the key drivers of the progression of ALD. ROS are excessively produced upon alcohol consumption, leading to the onset and progression of ALD. Research on the metabolism of ethanol in the liver is very critical for understanding the generation of ROS. The gastrointestinal tract is responsible for the absorption of ethanol, which is primarily metabolized in the liver; however, 3–5% is secreted through other means, such as breath, urine, and sweat ,30. In t2 to NADP+ and H2O, which generates ROS. ROS are excessively produced and cause cellular damage during oxidization of ethanol by CYP2E1 [+ ratio, which subsequently decreases the ETC and facilitates the formation of O2•− and HO• [ROS are overproduced upon ethanol consumption via the MEOS system and mitochondrial dysfunction. It has been reported that the activation of MEOS increases during chronic alcohol consumption due to the increased stabilization of CYP2E1 , which py CYP2E1 . Additio and HO• . Mitocho and HO• .NAFLD pathogenesis is characterized by two hypotheses: the “two-hit” hypothesis and the “multiple-hit” hypothesis ,38,39. TDisequilibrium in lipid metabolism induces oxidative stress via the overproduction of ROS, which affects ROS generators, including the mitochondria, ER, and NOX. In NAFLD, mitochondria have been well-studied for their role in lipid metabolism and are one of the most important sources of ROS. In addition, the participation of the ER and NOX has been described, but the degree of involvement remains unclear. In hepatic cells, the mitochondria adapt to the increase in FFA intake using fatty acid oxidation (FAO), electron transport chain (ETC) activity, and oxidative phosphorylation efficiency . Most st2O2, alkyl hydroperoxides, and peroxynitrite into water and alcohol, thus maintaining cellular redox homeostasis [2O2 and confers a defensive function against toxic peroxides. Most Prxs have an additional resolving cysteine (CR) in the COOH terminus; Prxs are divided into three classes based on this cysteine: the 2-Cys, atypical 2-Cys, and 1-Cys subfamilies [2O2, whereas Prxs V and VI reduce alkyl hydroperoxides and peroxynitrite. Prxs have been shown to regulate the ROS-scavenging functions and redox regulation of PTEN [The antioxidant enzymes, Prxs, are thiol-dependent peroxidases that function as intracellular antioxidants to reduce Heostasis ,52,53,54eostasis . Prxs cofamilies . In addifamilies . Prxs I families . Accumul of PTEN .Research on Prx knockout has emphasized the importance of oxidative stress in ALD and NAFLD, with mitochondrial ROS being a significant contributor. Pyrazole is known as a substrate of CYP2E1 and is used to study CYP2E1-induced oxidative stress . A previThe role of Prx V in regulating adipogenesis and NAFLD has been widely reported ,62. Prx PTEN is a well-known tumor suppressor that is frequently deleted in various cancers ,64. This124 is most susceptible to oxidation by intracellular oxidants. Unlike TCPTP, when ROS inactivate PTEN at Cys124, a disulfide bond is formed with the neighboring cysteine, preventing irreversible inactivation by other oxidized species [124 and Cys71 residues  and antimycin A , PTEN oxidation levels were increased compared with those in untreated cells . These fResearch on the PI3K/PTEN/Akt signaling pathway has also shown its involvement in alcohol-induced liver injury ,90. InhiPTPN2, which encodes the TCPTP protein, is located on chromosome 18p11.2–p11.3, which was found in 1992 [PTPN2 deletion is characteristic of acute lymphoblastic leukemia and breast cancer, suggesting that TCPTP functions as a tumor suppressor [ in 1992 . Studiesppressor ,14. In appressor . The strppressor ,94. HoweTCPTP, a member of the PTP family, regulates various signaling pathways via its tyrosine-specific phosphatase function. TCPTP dephosphorylates various substrates, including receptor tyrosine kinases ,96, Janu2H or SO3H [215 and the nitrogen atom of an adjacent residue. This intermediate causes structural alterations resulting in the inhibition of substrate binding and the exposure of the oxidized cysteine to the cellular environment [216 of TCPTP and the Cys215 of PTP1B [It has been shown that PTPs are regulated by ROS, which inhibit their activity. For classical PTPs, such as PTP1B, there is a mechanism to prevent the irreversible oxidation of the nucleophilic cysteine by SO or SO3H . During ironment ,110. In ironment ,111. Resof PTP1B . Further2O2. The interaction was determined by the formation of intermolecular disulfide bonds between Trx and PTP1B when the second cysteine of Trx was mutated to serine (Trx1-C35S) and stabilized this mixed disulfide intermediate [The Trx system has been shown to reduce sulfenamide species. Among PTPs that form cyclic sulfenamide during oxidation, PTP1B is effectively reduced by Trx . PTP1B irmediate . Furtherrmediate . These fTCPTP was initially studied as a negative regulator of the insulin signaling ,114. TCP3H. These oxidized PTPs included PTP1B and TCPTP, which suggests the consequential redox regulation of TCPTP in obesity-induced oxidative stress [Obesity is a known risk factor for NAFLD, and it increases the levels of ROS and oxidative stress. A previous study demonstrated PTP oxidation in mice fed an HFD (23% fat) . PTPs wee stress . The oxie stress . These r3H in the HFD mice livers that developed hepatic steatosis. However, the redox regulation of PTPs in the development of NASH and HCC remains unexplored. Different mouse models, including a standard chow diet model, an HFD model , and a choline-deficient HFD model , were used to examine whether PTP oxidation was associated with NASH [As observed in a previous study, PTPs, including TCPTP, were oxidized to -SOith NASH . In partith NASH . This fiAlb-Cre;fl/flPtpn2) were fed an HFD to further investigate the role of TCPTP in NASH and HCC. These mice developed NASH, which involved hepatocyte ballooning; lymphocytic infiltrates, including CD4+ and CD8+ T cells and immunoglobulin A; and ectopic lymphoid-like structures [Alb-Cre;fl/flPtpn2 mice with HFD showed progressive tumors with many characteristics of aggressive HCC. Since both STAT-1 signaling and STAT-3 signaling were enhanced upon TCPTP deletion, the function of these signaling pathways in NASH and HCC was analyzed using mice with TCPTP-deficient hepatocytes fed an HFD. STAT-1 heterozygosity (fl/+Stat-1) repressed hepatic inflammation, which was indicated by the reduction in STAT-1 target genes such as Cxcl9 and Lcn2. Immune cell recruitment was reduced, as indicated by the decrease in CD4+ and CD8+ T cells. fl/+Stat-1 also repressed fibrosis in HFD Alb-Cre;fl/flPtpn2 mice [fl/+Stat-3) did not reverse the increase in Cxcl9 or Lcn2 expression, immune cell recruitment, and fibrosis in mice with TCPTP-deficient hepatocytes. fl/+Stat-3 in HFD-fed Alb-Cre;Ptpn2fl/fl mice inhibited the development of HCC, unlike the control mice [Mice with TCPTP-deficient hepatocytes  when exposed to ethanol in vivo and in vitro. In addition, the deficiency of Prx III, a mitochondrial ROS scavenger, increased the oxidation of PTEN and lipid accumulation, which emphasized the importance of increased mitochondrial ROS generation in the progression of PTEN-mediated hepatic alcoholic steatosis. Further research, in which redox regulation of PTEN is well examined, is needed to investigate more advanced stages of ALD, such as ASH or HCC. Additionally, studies have shown that TCPTP was inactivated and oxidized to the irreversible -SOSeveral studies showed that inhibiting Akt, downstream of PTEN, or STAT, downstream of TCPTP, could be potential treatments for fatty liver disease. For instance, mice were treated with an Akt inhibitor, MK-2206, that prevented NAFLD and liver cancer progression . MK-2206


Sjögren’s syndrome (SjS), characterized by keratoconjunctivitis sicca and dry mouth, is a common autoimmune disease, especially in middle-aged women. The immunopathogenesis of SjS is caused by the sequential infiltration of T and B cells into exocrine glands, including salivary and lacrimal glands. Effector cytokines produced by these immunocytes, such as interferons (IFNs), IL-17, IL-22, IL-21, IL-4, TNF-α, BAFF and APRIL, play critical roles in promoting autoimmune responses and inducing tissue damages. Epigenetic regulations, including DNA methylation, histone modification and non-coding RNAs, have recently been comprehensively studied during the activation of various immunocytes. The deficiency of key epigenetic enzymes usually leads to aberrant immune activation. Epigenetic modifications in T and B cells are usually found to be altered during the immunopathogenesis of SjS, and they are closely correlated with autoimmune responses. In particular, the important role of methylation in activating IFN pathways during SjS progression has been revealed. Thus, according to the involvement of epigenetic regulations in SjS, target therapies to reverse the altered epigenetic modifications in auto-responsive T and B cells are worthy of being considered as a potential therapeutic strategy for SjS. Sjögren’s syndrome (SjS) is the second most common chronic autoimmune rheumatic disease, characterized by dry eyes (sicca) and dry mouth. It is caused by abnormal infiltration and activation of immunocytes in the lacrimal glands (LGs) and salivary glands (SGs) due to multiple genetic and environmental factors. These infiltrated immunocytes lead to substantial progressive damage of the acinar structures responsible for saliva production and secretion. In addition, as indicated by some studies, the autoimmune responses may already occur before the immunocyte infiltration . For exaSjS generally develops in middle-aged women, but it can occur at any age throughout the population. Its prevalence is estimated to be around 3% in subjects aged 50 years and older, with a female to male ratio of 9:1 . X-chromSjS is a multi-factorial disease caused by genetic, environmental and epigenetic abnormalities. The molecular etiology of autoimmune responses in SjS is still quite elusive. Genetic factors associated with SjS include particular HLA-DR allele subtypes and specific polymorphisms of genes, such as STAT4, IL-12A, TNIP1, IRF5 (type I interferon related), BAFF and BLK (related to B-cell activation) ,11. EnviEpigenetic modifications are mitotically and/or meiotically heritable and reversible alterations that induce upregulated or downregulated gene expression, without any changes in underlying DNA sequences. In addition, they also play a vital role in silencing or promoting the expression of non-coding sequences . EpigeneMethylation changes on DNA include both methylation and demethylation, which are, respectively, regulated by DNA methyltransferases, DNMT1, 3A and 3B, and Ten Eleven Translocation (TET)-1, 2 and 3 . During + T cells [Tbx21 and Ifng loci display demethylation and increased 5hmC level [Dnmt1 deficiency in T cells, or treating T cells with demethylation agents, results in enhanced Th1 effector cytokine expression [Ifng locus stably silenced in Th17. The deletion of Dnmt3a results in demethylation at the Ifng locus of Th17 cells. Thus, under IL-12 stimulation, Dnmt3a-deficient Th17 become pathogenic and express higher levels of IFN-γ [Dnmt1-deficient Treg lose their suppressive function both in vitro and in vivo [Dnmt1 in Treg die after 3 to 4 weeks due to severe systemic autoimmunity [DNA methylation and demethylation play critical roles in regulating the activation and differentiation of CD4 T cells . The mosmC level . Dnmt1 dpression . DNMT3A of IFN-γ . For Tre in vivo . Mice coimmunity .Ifng locus to keep it demethylated, while Tet2 deficiency results in reduced IFN-γ expression [Tet2 deficient leads to reduce 5hmc and RORγt binding at the Il17a locus, thus resulting in reduced IL-17 expression [Foxp3 locus, where they ensure the expression of Foxp3. Both TET1 and TET2 are required for the conversion of 5mC to 5hmC at the Foxp3 locus, while their joint depletion results in impaired Treg differentiation and function [Tet2 or Tet3 does not significantly impact Foxp3 expression, depleting them both leads to severely impaired Treg differentiation and stability [Foxp3 locus [On the other hand, TET enzymes are also involved in T-cell differentiation. In Th1, T-bet recruits TET2 to the pression . In Th17pression . In Tregfunction . TET enztability . TET expp3 locus ,24.Dnmt3a and Dnmt3b in B cells does not affect their development and maturation; however, it results in the abnormal accumulation of plasma cells in the spleen and bone marrow. Compared to normal plasma cells, the Dnmt3-deficient plasma cells display reduced DNA methylation at over a thousand gene loci, and thus are unable to limit the activation and differentiation of B cells [Tet2 and Tet3 deficiency in activated B cells results in substantially reduced 5hmC levels and defective IgG1 switching [During Tfh differentiation, the involvement of TET and DNMT family members is still quite elusive. Nevertheless, in comparison with other Th cells, Tfh displays significantly reduced 5hmC at Bcl6 binding sites . DNMT fa B cells . In addiwitching .Histones play important regulatory roles in DNA replication and gene expression, highly relayed on their unique amino acid sequences. Histones are enriched with basic lysine and arginine residues, especially in the N-terminal tails, which are feasible for several post-translational modifications (PTMs), including methylation, acetylation, phosphorylation and ubiquitination . HistoneHdac1-deficient Th1 display increased STAT1 phosphorylation and IFN-γ expression [Hdac11-deficient CD4+ T cells express higher Eomes and T-bet, and produce more IFN-γ [Sirt1 in T cells protects mice from EAE, due to the reduced pathogenic differentiation of Th17 [Hdac6 or Hdac9 deficiency leads to the increased suppressive function of Treg. As major microbial metabolites in the gut, short-chain fatty acids (SCFAs) can passively diffuse across the cell membrane of T cells and inhibit the activity of HDAC [Foxp3 locus of Treg by inhibiting HDAC, and thus promotes the transcription factor stability and activity [+ T cells [Multiple enzymes are involved in histone modifications, such as histone deacetylases (HDACs), histone acetyltransferases (HATs) and histone methyltransferase (HMTs). Several HDAC members have been reported to be involved in T- and B-cell responses . For exapression . Additiore IFN-γ . Sirtuin of Th17 . In addi of HDAC ,33,34. Sactivity . In addi T cells . In addi T cells .AICDA/Aicda (encoding AID) mRNA, and upregulated miR-23b, miR-30a and miR-125b, which silenced PRDM1/Prdm1 (encoding Blimp-1) mRNA [Faslpr/lpr mice, ameliorating the disease and extending mice survival. Thus, they also provide a therapeutic rationale and potential for other autoimmune diseases [HDACs are also involved in regulating B-cell responses. The master regulator for plasma cell differentiation is Blimp-1. In undifferentiated B cells, Bcl-6 suppresses Blimp-1 expression by interacting with HDAC4, -5 or -7 and forming stable complexes . When th-1) mRNA . Throughdiseases .Non-coding RNAs (ncRNAs) are single-strand RNA molecules transcribed from the genome. They do not encode proteins, but are implicated in cell development, proliferation and metabolism, playing important regulatory roles. Small non-coding RNAs (miRNAs) are 19–22 nucleotides in length, affecting gene expression at post-transcriptional levels, whereas long non-coding RNAs (lncRNAs) are defined as being above 200 nucleotides in length . The miRThere have been many studies showing the crucial role of miRNA in the development and differentiation of T and B cells ,46. One There have been numerous studies indicating that certain miRNAs, such as miR-21, miR-27, miR-31, miR-138, miR-155, miR-146a, miR-181a and the miR-17~92 cluster, regulate T-cell development, differentiation and effector function . AbnormaParticular and abundant miRNAs are present at different stages of B-cell development. At an early stage, the miR-17∼92 cluster can promote the degradation of PTEN mRNA to increase PI3K activity, which further promotes B-cell survival . In addiMiRNAs also regulate the development and activation of innate immunocytes, such as ILCs and macrophages . MiR-142+ T cells constitute the predominant infiltration in SGs, while at a later stage, B cells are gradually accumulated [+ T and B cells are closely correlated with damages in the glandular tissues of SjS patients. In addition, CD8+ T cells are also crucial players in the immunopathogenesis of SjS, contributing to acinar injury. Overall, the infiltration of T and B cells are usually closely associated with SjS progression [Though the precise mechanism for the pathogenesis of SjS remains largely unclear, the infiltration of immunocytes in exocrine glands is usually considered associated with disease progression. Immunocytes infiltrated in SGs of SjS patients mainly include T and B cells. At an early stage, CD4umulated ,86. Thisgression . In addiInfiltrated T cells exist throughout the progression of SjS and act as key drivers of the disease. The aberrant activation of T cells is then critical for the progression of the disease especially at an early stage, through recruiting other immunocytes, secreting pro-inflammatory cytokines and promoting B-cell activation and autoantibody secretion ,89,90,91Type 1 and type 17 T-helper cells (Th1 and Th17), as major sources of IFN-γ and IL-17 in many autoimmune diseases ,96,97,98B cells are gradually accumulated at a late stage of SjS . T-folliAlthough Treg infiltration in the exocrine glands of SjS patients has been substantially studied, it is still unclear whether Treg play anti-inflammatory roles in the disease ,109. Sev+Foxp3+CXCR5highPD-1highBlimp-1+. They exert a suppressive role for Tfh proliferation and GC B-cell activation. The ratio of Tfr/Tfh in the blood of SjS patient is a biomarker for disease severity [A particular subpopulation of Treg, follicular regulatory T cell (Tfr), is substantially increased in the SGs and blood of SjS patients ,113. Theseverity ,113. Howseverity . In gene+ T cells exert pathogenic roles by secreting cytotoxic molecules, such as granzyme B (GzmB) and perforin, and inflammatory cytokines, including interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) [+ T cells are enriched around the apoptotic acinar epithelial cells of SjS patients. IFN-γ in SGs is involved in the recruitment of more CD8+ T cells. Ifng deficiency was reported to abrogate CD8+ T-cell infiltration and decrease gland destruction in SjS mice. CD8+ T cells infiltrated in SGs displayed a tissue-resident phenotype CD69+CD103+/− and exhibited dramatically increased IFN-γ production. In a p40−/−Il2ra−/− murine model of SjS, CD8+ T-cell depletion, via either genetic Cd8a deficiency or antibody-mediated depletion, fully protected the mice from pathologic manifestation [+ T-cell depletion successfully restored the secretory function of SGs, suggesting it as a potential therapeutic strategy. Thus, the correlation between CD8+ T cells and gland destruction in SjS patients should receive more attention in the future.CD8 (TNF-α) . Recent estation . MoreoveB cells are central to the pathophysiology of SjS at a late stage . In SjS B cells promote the progression of autoimmune diseases mainly through their roles as cytokine producers, antigen-presenting cells or autoantibody secretors. B cells possess the capacity to produce a range of cytokines, including either the pro-inflammatory cytokines IL-6 and TNF-α or the anti-inflammatory cytokines TGF-β and IL-10 . The difA key feature of SjS is that target organs, including salivary and lachrymal glands, are involved in B-cell activation, especially the formation of GC-like structures within the epithelium and plasma cell niches . Thus, i+ Th subsets Th1, Th2 and Th17 [Innate lymphoid cells (ILCs) are tissue-resident innate lymphocytes that play crucial roles in regulating tissue homeostasis . Unlike and Th17 . The disand Th17 . For exaand Th17 . ILC3 isand Th17 .In addition to B cells, other antigen-presenting cells, such as DCs and macrophages, are also increased in the SGs of SjS patients ,128. BotAs previously mentioned, the immunopathogenesis of SjS is initiated by Th1 and Th17, and progressed by Th2 and Tfh, which then ignite B-cell responses, resulting in more severe tissue lesions . In lineAfter processing certain autoantigens, antigen-presenting cells are activated to produce IL-7, IL-12, IL-18 and type I IFNs, which effectively promote Th1 responses . IFN-γ aHigh levels of IFNs and the elevated transcription of IFN-stimulated genes (ISGs) are found in the SGs and serum of SjS patients, as well as SjS animal models, indicating an important role of IFNs in the pathogenesis of SjS ,48,49,50Ifnar1−/−) display significantly reduced susceptibility to SjS-like disorders [In the inflamed SGs, IFN-α are mainly produced by plasmacytoid dendritic cells (pDCs), after they recognize autoantigens through the Toll-like receptors (TLRs) TLR-7 and TLR-9 ,138. On isorders ,148.+ T cells towards Th1 [In addition to type I IFNs, type II IFN IFN-γ also play a crucial role in inducing tissue lesions in SjS patients. Thus, the lesion tissue is usually characterized by both type I and type II IFN signatures ,150. In ards Th1 ,153. In ards Th1 ,153, wheards Th1 ,155,156.ards Th1 ,158. MorType III IFN, IFN-λ, shows a certain functional similarity to type I IFNs . HoweverThe inflammatory milieu in SjS patients is characterized by a dysregulated cytokine network, especially pro-inflammatory interleukin overexpression. The major pro-inflammatory interleukins include IL-17, IL-22, IL-21 and IL-4, and exhibit aberrant upregulation in the SGs or serum of SjS patients ,160.Il17a-deficient mice fail to develop SjS [Pro-inflammatory cytokine IL-17A is mainly expressed by Th17 cells in the inflamed SGs of SjS patients, and considerable evidence suggests IL-17A as a pathogenic factor in SjS . The levelop SjS , while melop SjS .Il22 reduces the infiltration of B cells in the SGs of SjS mice [IL-22 is another pro-inflammatory cytokine released by Th17 ,169. IL-SjS mice .+ Th cells [+ T cells, which induces damages of SGECs [Accumulating evidence suggests that IL-21 plays a critical pathogenetic role in SjS. High levels of IL-21 and IL-21R are detected in the SGs and serum of SjS patients . AdditioTh cells ,175. IL-Th cells . In addiof SGECs . Consistof SGECs .Il4 or Stat6 impedes antibody switches to IgG1 isotype against muscarinic acetylcholine type-3 receptor (anti-M3R) [IL-4 level is found to increase in SGs of SjS patients, especially those exhibiting significant B-cell infiltration . IL-4/STnti-M3R) . In addinti-M3R) .The TNF superfamily is composed of more than 20 structurally related protein ligands . They exTNF-α is involved in numerous autoimmune diseases, while anti-TNF-α treatment has also showed great promises as a therapeutic strategy . In SjS BAFF and APRIL share similar biological roles in promoting B-cell differentiation, survival and autoantibody secretion ,195. TheAccording to the role of epigenetic regulations in immunocyte activation, epigenetic changes can also be involved in the immunopathogenesis of SjS. Indeed, DNA methylation, histone modifications, as well as ncRNAs play important roles in the progression of SjS. They are involved in regulating the aberrant infiltration and activation of T and B cells and promote the production of pro-inflammatory cytokines during SjS immunopathogenesis .Numerous studies have confirmed the important role of the IFN pathway in the immunopathogenesis of SjS. In pSS patients, disease progression can be divided into three key stages: activation of the innate immune system, especially via the IFN pathway; the activation of T and NK cells due to HLA-related predisposition and through IL-12–IFN-γ pathways; and the activation of B cells, presumably through CXCR5-mediated recruitment to lymphoid follicles and B-cell receptor activation. Epigenetic modifications are critical in activating the IFN pathway .+ T cells of 11 pSS patients and 11 healthy cohorts in an epigenome-wide study [STAT1, IFI44L, USP18 and IFITM1. In another comprehensive study of DNA methylation of whole blood, CD19+ B cells as well as minor SG biopsies from pSS patients, prominent hypomethylation on IFN-regulated genes was also confirmed, which correlated with their increased expression. Furthermore, the top 12 differentially methylated CpG sites in pSS patients, such as MX1, IFI44L, PARP9, PLSCR1, IFIT1, IFITM1 and HLA-A, were found to be mainly influenced by the presence of anti-Sjögren’s Syndrome antigen A (SSA) and/or anti-Sjögren’s Syndrome antigen B (SSB) antibodies [+ T cells and CD19+ B cells of pSS patients and age-matched health cohorts using 450K BeadChip suggested that the major methylation alterations were present in B cells and genetic at-risk loci [IFITM1, IFITM3, IFI44L and IRF5, were validated. Intriguingly, the methylation status of B cells was closely correlated with disease progression. Differentially methylated genes in patients positive for at least one pSS-specific autoantibody  showed a significantly enriched IFN signature [The correlation between DNA methylation and disease progression in pSS patients has been substantially studied. DNA methylation was compared between the naïve CD4de study . The hyptibodies ,200. Theisk loci . The DNAignature .+ T cells and CD19+ B cells from pSS patients and healthy cohorts [Histone modifications in SjS patients are still quite elusive, while expression changes on miRNAs are reported in some studies. The differential expression of miRNAs, including hsa-miR-378a-3p, hsa-miR-222-3p, hsa-miR-26a-5p, hsa-miR-30b-5p and hsa-miR-19b-3p, were discovered in a comparative study of CD4 cohorts ,201. Of  cohorts . Additio cohorts .As mentioned above, in SjS patients, methylation changes in B cells are more obvious than in T cells, suggesting an important role of B cells in SjS progression. The hypomethylation of IFN-induced genes, including STAT1, IFI44L and IFITM1, was found in peripheral blood B cells, especially from SSA and/or SSB antibody-positive patients . The hypSjS patients often exhibit increased SSA and SSB autoantigens, correlated with the aberrant expression of the miRNAs targeting them. In minor SGs and peripheral blood of SjS patients, a large number of miRNAs display differential expression patterns. The miRNA difference also exists in T and B cells from the peripheral blood of SjS patients, especially those positive for anti-SSA autoantibodies. The expression of 372 miRNAs in purified blood cells from SjS patients and health cohorts were analyzed in a recent study. A total of 21 miRNAs showed significant expression differences in T cells, while 24 differentially expressed miRNAs were identified in B cells . Among tTreg exerting immune suppressive functions are quantitatively and qualitatively defective in SjS patients. Foxp3 (forkhead box p3) is the master transcription factor of Treg, which dictates their development and function . Its expGWAS has been used to assess the association of genetic variation and unique traits in SjS patients, and an increasing number of susceptible SNPs have been identified ,207,208.SjS manifests in various ways, typically characterized by keratoconjunctivitis sicca and dry mouth. Fatigue infections also usually threaten the patients. Thus, the major therapeutic aim for most SjS patients is to improve the quality of their lives by ameliorating the sicca and fatigue symptoms. Anti-inflammatory treatments, such as topical corticosteroids, topical cyclosporine and oral secretagogues, are commonly implied to prevent the pathogenesis of the disease . In addi+ T cells is also found in RA, associated with their autoimmune responses. Methotrexate (MTX), a first-line drug for RA treatment through antagonizing folate metabolism, affects one-carbon metabolism to disrupt the methyl transfer process of CpG methylation [The cellular and molecular etiology of SjS is quite complicated, considering the immunocytes and effector cytokines involved in the autoimmune responses. In addition, they are also dynamically changing along with the immunopathogenesis of the disease. Thus, new therapies targeting pro-inflammatory cytokines or autoreactive immunocytes in SjS patients are still worth exploring. Based on our description, epigenetics is intensively involved in the pathogenesis of SjS, and it shows broad impacts on the abnormal activation of T and B cells, as well as their aberrant production of pro-inflammatory cytokines. Accordingly, we particularly considered the possibility of taking epigenetic targets as a strategy for SjS therapy. Actually, epigenetic drugs have already been used in other autoimmune diseases, especially RA and SLE. Increased DNA methylation in CD4hylation . In addihylation . Additiohylation .Given the involvement of epigenetic regulators, such as DNMT1, DNMT3A, DNMT3B, TET2, TET3, HDAC1, HDAC11, SIRT1 and miR-146a, in SjS immunopathogenesis, they can all be possibly considered as therapeutic targets. In particular, as previously mentioned, the IFN pathway plays critical roles in promoting SjS progression, while it is well studied that methylation changes in T and B cells are closely associated with the activation of IFN-related genes. Thus, perhaps targeting therapies towards the abnormal methylation changes in the auto-responsive immunocytes of SjS patients should be particularly considered in future.SjS is a common chronic autoimmune rheumatic disease, caused by complicated cellular and molecular etiologies. Disease progression is companied by the abnormal activation of T and B cells sequentially, including Th1, Th17, Tfh and autoreactive B cells. Key effector cytokines produced by these immunocytes are closely correlated with the autoimmune responses and the damages induced in glandular tissues. The involvement of epigenetic regulations during immune cell activation has attracted considerable attention and has been intensively studied in the past. In particular, epigenetic changes in auto-responsive immunocytes from SjS patients have also been discovered. Thus, unraveling the involvement of epigenetic changes in SjS progression has provided many new insights into its immunopathogenesis and therapy. Especially, these studies reveal that IFN-related genes are hypomethylation in multiple immunocytes, such as T and B cells, as well as SGECs, which play critical roles in the immunopathogenesis of SjS. These discoveries suggest that epigenetic targeting could be considered as a potential therapeutic strategy of SjS. The application of epigenetic treatment in other autoimmune diseases, such as RA and SLE, further demonstrates this possibility. Nevertheless, more studies about the epigenetic modification changes and their impacts on pro-inflammatory cytokine production and SjS progression are still required to precisely design and adjust the epigenetic therapy.


Protobothrops is a genus of pit vipers, which are venomous and inhabit the Nansei (Southwest) islands of Japan, Taiwan China, Vietnam, Thailand, Myanmar, Nepal, Bhutan, and India. Our previous study decoded the genome of Protobothrops flavoviridis, a species endemic to the Nansei Islands, Japan, and revealed unique evolutionary processes of some venom genes. In this study, we analyzed genes that are highly expressed in venom glands to survey genes for candidate enzymes or chaperone proteins involved in toxin folding and modification. We found that, in addition to genes that encode venom proteins and ribosomal proteins, genes that encode protein disulfide isomerase (PDI) family members (orthologs of human P4HB and PDIA3), Selenoprotein M (SELENOM), and Calreticulin (CALR) are highly expressed in venom glands. Since these enzymes or chaperones are involved in protein modification and potentially possess protein folding functions, we propose that P4HB, SELENOM, CALR, and PDIA3 encode candidate enzymes or chaperones to confer toxic functions upon the venom transcriptome.Genes encoding snake venom toxins have been studied extensively. However, genes involved in the modification and functioning of venom proteins are little known.  Protobothrops flavoviridis , Ovophis okinavensis , P. elegans (Ishigaki and Iriomote islands of the Sakishima Islands), P. mucrosquamatus (Taiwan), and Trimeresurus stejnegeri (Taiwan) (The Reptile Database: http://www.reptile-database.org/ (accessed on 31 March 2019)). From the point of view of snake envenomation in Japan, the habu, P. flavoviridis, is the greatest threat to people, with more than 1000 people bitten per year, and a fatality rate of 1% [Several crotalid snakes inhabit the Nansei (southwestern) Islands of Japan  and Taiwan . They inTo clarify snake venom repertoires, as well as the evolution and physiological function of these venoms, many proteomic and transcriptomic studies have been performed ,4,5. TheP. flavoviridis genome in 2018 [2 (PLA2), three-finger toxins (3FTX), and cysteine-rich secretory proteins (CRISP)) show accelerated evolution [Since genomic information is essential for further studies on snake venom, we decoded the  in 2018 . In that in 2018 . The habvolution .2s, which are major habu venom components, contain seven disulfide bonds. Type III svMPs, such as flavorase, HR1A, HR1B, and HVI1, possess 17 disulfide bonds, and a metalloprotease domain with disintegrin-like and cysteine (Cys)-rich domains at the C-terminus. In addition, CTLP, 3FTXs, and CRISP family proteins also contain four or more disulfide bonds. Furthermore, a certain type of CTLP forms an interchain disulfide bond with type III MPs, resulting in type IV MPs [P. flavoviridis snake venom proteins are Cys-rich proteins. It is thought that venom proteins pass through several modification processes to assume fully functional forms.However, there are many questions to be answered in order to understand snake venom evolution, physiology, and pharmacology. One important question concerns how venom proteins are processed post-translationally to acquire their toxic functions. For example, one of the salient characteristics of venom proteins and peptides from venomous snakes, scorpions, spiders, and sea snails is that they are rich in disulfide bonds that stabilize their 3D structures ,12. PLA2e IV MPs . Thus, mCrotalus adamanteus) and found that genes for protein disulfide isomerases (PDIs) are highly expressed in the venom glands. In addition, Luna et al. (2013) [Bothrops jararaca and found that proteins from the endoplasmic reticulum (ER), such as PDI and GPR78, and cytoplasmic proteins, such as 40S ribosomal protein, are enriched in venom glands. We expect that more genes involved in protein-modification functions are also highly expressed in venom glands of habus. Thus, to identify genes for candidate enzymes or chaperones of venom protein modification in P. flavoviridis, we carried out comprehensive transcriptomic analyses of venom glands and other organs. Here, we propose P4HB, PDIA3, SELENOM, and CALR as candidate enzyme genes for regulating/modifying venom proteins, since these genes are suggested to be strongly associated with the oxidative folding of venom proteins with multiple disulfide bonds.As mentioned above, although studies of venom transcriptomes have been conducted for many venomous snakes ,4,5, the. (2013)  carried Protobothrops flavoviridis whole-genome decoding project, we carried out thorough transcriptomic analyses of 20 organs and tissues of an adult snake, which included the venom gland (sample name: venom gland-specimen#1), venom fang-forming tissue, lung, liver, kidney, small intestine, colon, stomach, pancreas, heart, masseter muscle, brain (brain-specimen#1), eye, nose, pit organ, spleen, and ovary . In adda et al. . We expeWe first identified the 50 genes that are most highly expressed in habu venom glands . Based oCNP01), svPLA2-02 and -03, svMP06, svPLA2-01, -07, and -08, svMP07 and -08, svCTLP06, and -07, svMP04, svCLTP08, svMP05, svSP04 and LAAO01 , also known as the beta-subunit of prolyl 4-hydroxylase (P4HB), was the ninth most highly expressed gene in one adult venom gland  were ranked as the 19th, 23rd, and 31st most highly expressed genes, respectively  genome and in transcriptomes of a related snake (P. mucrosquamatus) are summarized in As already mentioned, major venom proteins, such as PLA members  in the hT, PDIA2 , PDIA5 PDI familyPDI family genes were estimated with ORTHOSCOPE using human PDIA3  Other PDI family-related membersERP44 analyzed by ORTHOSCOPE using other PDI family member human ERP44  that contained one habu ortholog each, habu1_s32648_g23040.t2 for the ERP44 clade, habu1_s10239_g19902.t1 for the PDIA4 clade, and habu1_s1390_g04983 for the PDIA3 clade.The molecular phylogenetic tree of an ERP44  as queryAGR2 and AGR3 using human genes as queries are shown in AGR2 and AGR3 trees both consisted of three main clades , and each clade contained one habu ortholog, habu1_s49803_g23446.t1 for the AGR3 clade, habu1_s49803_g23445.t1 for the AGR2 clade, and habu1_s2744_g09787.t1 for the TXNDC12 clade.Molecular phylogenies of TMX1, TMX2, TMX3, and TMX4 , each of which includes a single habu ortholog: habu1s_10239_19902.t1 for the PDIA4 clade, habu1_s604_g02858.t1 for the PDIA6 clade, and habu1_s3682_g12384.t1 for the TXNDC5 clade. The tree for TXNDC12 contains the three main clades   , and eacCASQ1 and CASQ2 each using the human gene as a query showed that only a single habu ortholog exists in each: habu1_s2370_g08604.t2 for the CASQ1 clade and habu1_s8721_g18867.t1 for the CASQ2 clade (Molecular phylogenetic trees for Q2 clade .(c) Calreticulin membersCALR members in the habu genome, a molecular phylogenetic tree of CALR was constructed using ORTHOSCOPE, with human CALR as a query sequence  Redox partner proteins of PDI family proteins, Ero1α and Ero1βPDI gene family  and habu1_s4277_g13858.t1 (Ero1β). Although the habu ortholog of Ero1β was a single copy, the gene was divided into two scaffolds tentatively annotated as habu1_s114618_g23966  and habu1_s4277_g13858 .The molecular phylogeny of Ero1  indicate(e) TXN and PRX membersTXN) members using ORTHOSCOPE, with human TXN as query sequences is shown in TXN2, TXN, TXNL, and TXNL1, and clades TXN2, TXN, and TXNL1 each contained one habu ortholog: habu1_s6210_g16942.t1 for TXN2 clade, habu1_s3891_g12904.t1 for TXN clade, and habu1_s4406_g14149 for TXNL1 clade. However, we failed to find an ortholog for TXNL.The molecular phylogeny of thioredoxin (PRX) isoforms genes were analyzed, with human PRX1  is a unique, rare amino acid encoded by the codon, UGA. It is highly conserved in selenoproteins. We therefore performed an alignment with habu Selenoprotein M and confirmed that the position of selenocystein (U) is highly conserved in all gene sequences, including habu Selenoprotein M  than in other organs. However, except for SELENOM, they all showed high and ubiquitous expression in all organs  = 8572.4/13,303.9) than in other organs, in the range of (TPM =) 213–4084 (40 times higher than brain). CALR and PDIA3 also showed high expression levels in venom glands  and in other organs. SELENOM showed high expression levels in venom glands (1514.7/1928.2), and in other organs, except for the liver, ovary, kidney, stomach, and small intestine. In addition to these genes of the four protein-modification enzyme candidates, PDIA4, PDIA6 (P5), TXNDC12, and CASQ1, also showed high expression levels in venom glands , while the expression levels of other protein-modification enzymes genes, such as PDIA2, PDIA5, ERp46, ERp27, ERp29, TMX1, TMX2, TMX3, TMX4, AGR2, AGR3, DNAJC10, and CASQ2, in the venom gland were lower than those in other organs.For example, PDIA6 tends to have ubiquitously high expression, and especially in venom glands and fetal fibroblasts, specifically . Additionally, in habus, all organs, including venom glands, showed high expression of PRX4 (TPM = 229.4).Among the redox partner proteins of PDIs, PRX4 is an ER-localized PRX isoform that reduces hydrogen peroxide produced during Ero1-mediated oxidation of P4HB ,24,25. PEro1 in various habu organs showed that two kinds of paralogs of Ero1, Ero1α, and Ero1β, exhibit different expression profiles. Ero1α was ubiquitously expressed in all organs, but at low levels (TPM = 0.3–29.3). The expression level of Ero1α was especially low (TPM = 3.3) in venom glands, whereas Ero1β was comparatively highly expressed in venom glands (TPM = 75.1 to 247.6) as well as in the ovary, spleen, pancreas, and nose (TPM = 182.8–498.9). Thus, the expression levels of Ero1α and Ero1β differ, depending on organs and tissues, as noted in other vertebrates [On the other hand, the expression level of tebrates .svMPs exhibit accelerated evolution when examined by A/KSK analysis. In contrast, as shown here, genes involved in the modification of venom proteins are present as single copies in the habu genome. However, since these genes are also highly expressed in the venom gland, we examined whether accelerated evolution occurred in these genes by calculating A/KSK values using the Nei–Gojobori method.Our previous study of the habu genome revealed that most venom protein genes have been extensively duplicated and that major venom genes including P4HB and PDIA3. However, A/KSK analysis against P4HB and PDIA3 genes showed no accelerated evolution of the P4HB and PDIA3 genes , Selenoprotein M (SELENOM), and Calreticulin (CALR), are highly expressed in habu venom glands. Since these enzymes or chaperones are involved in protein modification and folding in the cellular secretory pathway, the present results suggest the most likely involvement of PDI, PDIA3, Selenoprotein M, and Calreticulin in protein folding and modification during snake venom production as candidate modification enzymes or chaperones. Therefore, it is likely that PDI, PDIA3, Selenoprotein M, and Calreticulin are critical to confer toxic functions upon the disulfide bond-rich venom transcriptome.In this study, in addition to genes that encode venom and ribosomal proteins, genes that encode PDI family proteins , Rokyta et al. (2012) [Bothrops jararaca venom glands [P4HB, PDIA3, SELENOM, and CALR, which are non-toxin genes, are among the 50 most highly expressed venom gland genes. Consistently, the 20 most highly expressed non-toxin transcripts listed by Rokyta et al. (2012) [Although studies of venom transcriptomes or proteomes have been conducted for many venomous snakes ,4,5, the. (2012)  demonstrm glands  found th. (2012)  includedNaja naja), venom of which is rich in post-synaptic neurotoxins, Suryamohan et al. (2020) [PDI (log (CPM) = 13.27~15.65 (n = 6)), as well as the moderately high expression of PDIA3 [P4HB and PDIA3, these genes were more highly expressed in venom glands than in other organs. This seems reasonable since PDIs are expressed ubiquitously due to their essential functions as protein modification enzymes or chaperones. DUG analysis is undoubtedly useful to find genes of venom proteins since venom proteins are exclusively expressed in venom glands. However, this type of analysis may not always be useful to find genes that are ubiquitously and highly expressed, but more so in specific organs. When focusing on essential genes encoding non-toxic proteins, we need to consider the most appropriate analytical method. Nevertheless, all data shown herein for various snake species allow us to speculate that PDIs are essential for the modification and folding of venom proteins with abundant cysteines and multiple disulfide bonds.In this context, with transcriptomic data for the Indian cobra (. (2020)  showed tof PDIA3 . While tPDI accompanies high expression of habu Ero1. Indeed, our transcriptomic data from habu venom glands indicate high co-expression of Ero1β and PDI, whereas Ero1α is expressed to a much lesser extent. This was an unexpected result. However, previous studies demonstrated that Ero1β is constitutively and abundantly expressed in professional secretory tissues and can be strikingly induced in the course of the unfolded protein response [PDI together with Ero1β supports our notion that habu PDI could be the primary venom protein modification enzyme.Given that PDI works together with Ero1 as the primary catalyst for disulfide bond formation , we surmresponse ,29. Moreresponse . Thus, iresponse . The prePDI family orthologs and paralogs and their redox partner genes, SELENOM and CALR. This program is a species tree-based ortholog or paralog relationship identification tool, and all orthologs and paralogs for a specific gene are estimated automatically using this software [A/KSK analysis indicates that these genes never undergo accelerated evolution. In contrast to habus, proteomic analysis of cone snails, which have conotoxin, by Safavi-Hemami et al. (2011) demonstrated multiple PDI isoforms in cone snail venom glands, suggesting that cone snail PDI does undergo accelerated evolution [In this study, ORTHOSCOPE, the leading tool for phylogenetic analysis, enabled us to estimate phylogeny for all software . Our prevolution . TherefoBothrops jararaca, this cycle continues for about 30–50 days [Variables relative to venom gland sampling conditions can also affect results. Luna et al. (2013)  indicate–50 days . Moreove–50 days . Thus, iP. flavoviridis, namely P4HB, PDIA3, SELENOM, and CALR, and in general all of which encode protein folding enzymes or molecular chaperones in the ER. These are single-copy genes and are highly conserved among Protobothrops species, in contrast to the multiple copies and accelerated evolution of venom protein genes. Gene for a redox partner protein of PDI, Ero1β is also highly expressed in venom glands. The highly active expression of P4HB, PDIA3, SELENOM, and CALR in habu venom glands, which is also supported by previous reports, together with the disulfide bond-rich nature of venom proteins and the high co-expression of PDIs’ redox partner genes, suggests that PDI, PDIA3, Selenoprotein M, and Calreticulin are strongly associated with venom protein modification. It is most likely that folding enzymes or molecular chaperones such as habu PDIs are critical to endow them with toxic functions.In this study, we identified four non-toxin genes quite highly expressed in venom glands of ® Ultra™ Directional RNA Library Prep Kit for Illumina . RNA quality was checked with an Agilent Technologies 2100 Bioanalyzer using an Agilent RNA 6000 Nano Kit. Sequencing was performed using an Illumina Hiseq2500. De novo assembly of whole RNA sequence reads was performed using a de Bruijn graph-based program, Trinity [For transcriptomic analyses, a total of 22 samples were used as follows: 20 samples for 18 tissues and organs from two adult animals, and 2 samples for 2 tissues and organs from one neonate, including two venom glands specimens from two adult ; two brains specimens from two adult ; one specimen of eye, nose, infrared sensing pit organ, and venom fang-forming tissue, from one female adult; one specimen of lungs, liver, kidneys, small intestine, colon, stomach, pancreas, heart, masseter muscle, spleen, ovary, and accessory glands from the other female adult; one neonate venom gland tissue, as well as fibroblast cells, from one neonate. We extracted total RNA using a standard TRIzol protocol , and prepared cDNA libraries using an NEBNext Trinity ,33. AsseRaw Illumina reads were quality filtered and trimmed with Trimmomatic (v0.36) . After aThe 50 most highly expressed genes in venom glands were identified, and high-expression profiles of genes in the venom gland are listed in order .We performed molecular phylogenetic analysis using ORTHOSCOPE, which is a novel pipeline for molecular phylogenetic analysis . This unP4HB, PDIA3, SELENOM, and CALR, but also the other PDI family genes and PDI related redox partner genes, phylogenetic analysis was conducted with protein-coding DNA sequences of human PDIA3, PDIA4, P4HB, PDILT, PDIA2, CALR, both of Ero1α and Ero1β, TXN, PRX1, PRX2, PRX3, PRX4, SELENOM, PDIA5, PDIA6, ERP27, ERP29, ERP44, AGR2, AGR3, TMX1, TMX2, TMX3, TMX4, TXNDC5, TXNDC12, CASQ1, CASQ2, CALR3, CANX, and Danio rerio CALRL/CALR3b as queries, respectively, using ORTHOSCOPE, a species tree-based ortholog identification tool [−3; number of hits to report per genome, 3; aligned site rate threshold within unambiguously aligned sites, 0; data set, DNA (Exclude 3rd); rearrangement BS (bootstrap) value threshold, 60%. In ORTHOSCOPE analysis, to construct neighbor joining (NJ) trees of all the above genes, all of the following analysis were carried out: amino acid sequences of each query gene were aligned with MAFFT (v7.221) [To identify orthologs or paralogs of not only ion tool  with the(v7.221)  using th(v7.221)  using th(v7.221) . For phy(v7.221)  implemen(v7.221)  for DNA (v7.221)  for amin(v7.221) , with a (v7.221) . AnalyseA/KSK analysis, DNA sequences were aligned by using the ClustalW multiple alignment program ) [A/KSK analysis, all alignments were manually curated. For pairwise comparisons of nucleotide sequences of each the organism genes, the number of nucleotide substitutions per synonymous site (KS) and per non-synonymous site (KA) for protein-coding regions was computed according to the Nei–Gojobori method [http://www.gen-info.osaka-u.ac.jp/~uhmin/study/sqdifPlot/index.html (accessed on 31 August 2021)).We conducted the alignment of amino acid sequences of Selenoprotein M in a representative vertebrate using MUSCLE (v.3.8.31) alignment algorithms . For KA/t 2021)) . Due to i method  using th


Inonotus hispidus mushroom is a traditional medicinal fungus with anti-cancer, antioxidation, and immunomodulatory activities, and it is used in folk medicine as a treatment for indigestion, cancer, diabetes, and gastric illnesses. Although I. hispidus is recognized as a rare edible medicinal macrofungi, its genomic sequence and biosynthesis potential of secondary metabolites have not been investigated. In this study, using Illumina NovaSeq combined with the PacBio platform, we sequenced and de novo assembled the whole genome of NPCB_001, a wild I. hispidus isolate from the Aksu area of Xinjiang Province, China. Comparative genomic and phylogenomic analyses reveal interspecific differences and evolutionary traits in the genus Inonotus. Bioinformatics analysis identified candidate genes associated with mating type, polysaccharide synthesis, carbohydrate-active enzymes, and secondary metabolite biosynthesis. Additionally, molecular networks of metabolites exhibit differences in chemical composition and content between fruiting bodies and mycelium, as well as association clusters of related compounds. The deciphering of the genome of I. hispidus will deepen the understanding of the biosynthesis of bioactive components, open the path for future biosynthesis research, and promote the application of Inonotus in the fields of drug research and functional food manufacturing. Inonotus hispidus (Bull.) P Karst. (Hymenochaetaceae) is a well-known edible and medicinal mushroom with a long history as a health food and ancient folk medicine in Europe -6,7-vinyl]-3,5-dioxafluoren-5-one (5), inoscavin C (6), hypholomine A (7), inoscavin E (8), inonoblin A (9), inonophenol A (10), inonophenol B (11), hispolon (12), hispinine (13), methyl 5--3-hydroxypenta-2,4-dienoate (14), MBP (15), interfungin C (16), interfungin A (17), inonophenol C (18), inonotusin A (19), inotolactone B (20), eburicoic acid (21), hispindic acid B (22), 3β-hydroxy-lanosta-8,24-dien-21-al (23), inonotusol F (24), inonotusol G (25), inonotusane F (26), cerevisterol (27), 4,6,8(14),22(23)-tetraen-3-one-ergostane (28), 7(8),22(23)-dien-3-one-ergostane (29), inonotsutriol E (30), inonotsutriol A (31), inonotusane E (32), inotolactone A (33), and inonotusol E (34) (1–19) and the lanosterol-type triterpenoids (20–34), which correspond to the distinct clusters formed in the network  , Table 3 network , Table 3 species .Sanghuang is a well-known macrofungal medical herb in China, Japan, Korea, and other Asian countries. It has a long history of medicinal usage and health benefits [Compendium of Materia Medica. Indeed, sanghuang is recorded in various medical documents as a generic term for a group of medicinal macrofungi with specific biological activity and similar morphology, which is difficult to reconcile with modern species classification systems, seriously limiting modern medicinal research on sanghuang. Although the establishment of Sanghuangporus genus containing 15 species [Sanghuangporus species, several medicinal fungi from the genera Inonotus and Phellinus, such as I. hispidus [P. gilvus [sanghuang in traditional medicine. Such fungi are more aptly known as sanghuang-like fungi. These valuable medicinal macrofungi should not be excluded from the Chinese medicinal fungus sanghuang because of the development of Sanghuangporus genus. The ability of genome sequencing to reliably correlate morphological traits to the genome enables the differentiation of sanghuang-like fungi from Sanghuangporus species, which is difficult to separate morphologically.benefits ,67,68, w species ,69,70 hahispidus  and P. g. gilvus , are stiI. hispidus have traditionally been used as medicines for the treatment of indigestion, cancer, diabetes, and gastric diseases by the residents of Xinjiang province and Northeast China [1) [2) [15) [19) [20) [I. hispidus are essentially polyphenols [I. hispidus. Indeed, polyphenolic compounds with a styrylpyrone backbone are abundant in the genera Phellinus and Inonotus [Sanghuangporus [Sanghuangporus species. Furthermore, the molecular network constructed based on GNPS clearly displays the differences in the chemical composition of the fruiting bodies and mycelium of I. hispidus. This may serve as a precise guide for the targeted isolation of specific compounds  , phellib [1) [2) , MBP (15[2) [15) , inonotu19) [20)  (Figure yphenols , which aInonotus , as wellangporus , reflectompounds . The molompounds .G. lucidum identified multiple biosynthetic genes necessary to produce ganoderic acids, which provided vital information to uncover the biosynthetic pathways of these essential medicinal components [A. cinnamomea yielded critical candidate gene information to unravel antrocamphins biosynthesis in phase II clinical trials. Although polyphenols with a styrylpyrone moiety are a characteristic component of sanghuang-like fungi, the biosynthesis for these chemicals has received little attention [I. hispidus indicated that BGC12, with g1457 as a core gene, and BGC13, with g1510 as a core gene (Genome sequencing of mponents . The genttention ,71,75. Core gene , Table 2I. hispidus is thought to be related to the multiple P450s spread throughout the chromosomes, notably the over 50 P450 members of unidentified gene families (Secondary metabolite biosynthesis genes, especially post-modification genes, found in mushrooms tend to be scattered across chromosomes, similar to plants, rather than clustered like bacteria. The presence of high oxidation of polyphenolic compounds and triterpenoids in families .I. obliquus, a homology of I. hispidus, has more than 20 sesquiterpene synthases [I. hispidus only has 15 sesquiterpene synthases and one sesquiterpenoid xylaritriol [I. hispidus are inactive.Basidiomycetes-derived sesquiterpenes are a class of important natural products with diverse structures and various activities, and sesquiterpene synthases are a class of critical genes for natural product biosynthesis . Accordiynthases  and eighynthases , but I. aritriol , suggestInonotus hispidus is a well-known medicinal mushroom that exhibits anti-cancer and immunomodulatory activities, as well as a long history of usage as a medicinal fungal material with various health benefits. Here, for the first time, we provide the de novo assembled complete genome of I. hispidus. Chromosome-level assembly and functional annotation described in this study provide useful clues for subsequent gene functional research. According to comparative genomic analysis, the genus Inonotus has different gene compositions. Phylogenomic and evolutionary analysis of the genus Inonotus reveals evolutionary traits. The investigation of mate locus and CAZyme facilitates artificial cultivation. A thorough examination of secondary metabolite biosynthesis genes showed a wide range of biosynthetic potential. Further molecular network-based metabolite analysis revealed differences in chemical composition and concentration in fruiting bodies and mycelia. This work not only covers a vacuum in I. hispidus genetic information but also gives crucial insights into the biological aspects of the medicinal-edible fungus I. hispidus, such as growth characteristics and biosynthesis routes of bioactive components. A thorough grasp of I. hispidus’ genome will pave the way for its future application in pharmacological research and functional food development. In short, the genome sequencing of I. hispidus sheds light on the biosynthesis and medical applications of its metabolite.


Nearly all anti-PD-1 antibodies are of the IgG4 isotype, and thus possess residual FcR effector functions. Such anti-PD-1 antibodies are also associated with immune tolerance and escape due to instability of the CH3 domain and Fc-Fc interaction. In this trial, we examined the efficacy and safety of penpulimab, a novel IgG1 anti-PD-1 antibody that does not bind to the Fc receptor, in patients with refractory or relapsed classical Hodgkin lymphoma (R/R cHL).Adult patients (≥18 years of age) with R/R cHL received 200 mg penpulimab once biweekly until disease progression or unacceptable toxicities for a maximum of 24 months. The primary endpoint was objective response rate (ORR) based on the Independent Radiology Review Committee per Lugano 2014 criteria. Secondary endpoints included progression-free survival (PFS), overall survival (OS), treatment-related adverse events (TRAEs) and immune-related adverse events (irAEs).A total of 94 patients were enrolled. The median follow-up was 15.8 months. The ORR was 89.4%  in the full analysis set (85 patients). Forty (47.1%) patients achieved complete remission, 36 (42.4%) patients achieved partial remission. The 12-month PFS rate was 72.1%  and the 18-month OS rate was 100%. Totally 97.9% (92/94) of patients experienced at least one TRAE. The rate of grade 3 and above TRAEs was 26.6% (25/94). In addition, 51 (54.3%) patients experienced an irAE, and 4 (4.3%) patients developed grade 3 or above irAEs. No irAE-related death occurred.Penpulimab was effective and safe in patients with R/R cHL. Despite+ macrophages, induce antibody-dependent cell-mediated phagocytosis (ADCP), damage PD-1+ T cells and stimulate the release of IL-6, TNF-α, and other inflammatory cytokines, which may play a role in the development of immune-related adverse events (irAEs)  –18.Penpulimab, also known as AK105, is a human IgG1 anti-PD-1 antibody. A unique feature of penpulimab is a Fc mutation that eliminates Fc receptor and complement-mediated effector function, thus avoiding antibody-dependent cell-mediated cytotoxicity (ADCC), ADCP, and complement-dependent cytotoxicity (CDC), which are induced by binding of Fc to Fc receptor FcγRIIIa, FcγRIa, and C1q, respectively . In addist line chemotherapy and at least one line of multi-agent systemic chemotherapy in subsequent lines of chemotherapy. We also included patients with refractory cHL, defined as failure to achieve CR after at least 4 cycles of treatment, or PR after at least 2 cycles of treatment, or progressive disease (PD) as the best objective response (BOR) or the cause of discontinued medication on any number of cycles of treatment.Adult patients (≥18 years of age) with pathologically proven R/R cHL were eligible. We included patients who had received combination chemotherapy followed by high-dose chemotherapy and stem cell transplant and relapsed while on or after their last line of therapy, or patients who had received multi-agent systemic chemotherapy as the 118FDG-PET) according to the 2014 Lugano criteria; 4) adequate organ function. The main exclusion criteria were 1) nodular lymphocyte-predominant HL or gray zone lymphoma; 2) active immune disease and inflammatory bowel disease; 3) central nervous system (CNS) involvement; 4) receipt of allogeneic organ transplant and allogeneic hematopoietic stem cell transplant, or receipt of ASCT within 90 days of first penpulimab dose; 5) receipt of prednisone  or immune modulators for at least 2 weeks prior to the start of penpulimab therapy; 6) receipt of concurrent chemotherapy, immunotherapy, biological therapy or hormone therapy; 7) receipt of any anti-programmed death 1 (PD-1), anti-PD-1 ligands 1 (PD-L1) or anti-cytotoxic T-lymphocyte antigen 4 (CTLA4) antibodies, or any other antibody or drug targeting T-cell costimulation or checkpoint pathways; 8) known allergy to penpulimab or its excipient, or antibodies of murine or human origin. We also excluded pregnant or lactating women. Detailed eligibility requirements are described in Supplementary Methods.Other inclusion criteria were 1) an Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1; 2) life expectancy ≥ 3 months; 3) at least one measurable lesion .The study had an 8-week safety run-in period (Part A). Three patients were infused with 200 mg penpulimab once every 2 weeks and assessed for dose-limiting toxicities (DLTs) 28 days after receipt of the first penpulimab infusion. DLTs were graded and recorded according to NCI CTCAE version 4.03 and defined as any grade 3 or above treatment-emergent toxicities. If DLTs occurred in no more than one patient during the first 28-day treatment cycle, 3 additional patients were infused with 200 mg penpulimab once every 2 weeks and assessed for DLTs. If DLTs occurred in no more than one patient among the 6 evaluable patients during the safety run-in evaluation, patients started to receive 200 mg penpulimab (infusion over 60 min) once every 2 weeks for a maximum of 24 months (Part B). Penpulimab was administered until disease progression, unacceptable toxicities, withdrawal of consent, or investigator decision per protocol, for a maximum of 24 months. At the initial PD, penpulimab was continued until confirmation of PD per Lugano 2014 criteria, absence of continuous clinical benefit judged by the investigator. Other types of anti-tumor therapy were not allowed until PD was confirmed. Supportive care was provided at the discretion of the investigators. Patients with PD on initial evaluation were allowed to continue treatment if cHL did not rapidly progress, and the patients were judged to continue to benefit from and could tolerate penpulimab therapy and had stable ECOG performance status scores, and penpulimab therapy did not interfere with treatment of severe complications such as CNS metastasis.Patient demographics including age, gender, and ethnicity, previous history including history of cancer and cancer treatment were recorded. Responses were evaluated by an independent radiology review committee (IRRC) per the 2014 Lugano classification based on contrast-enhanced computed tomography (CT) or fluorodeoxyglucose positron emission tomography (PET)-CT scans. Contrast-enhanced CT scans were conducted at baseline and weeks 8, 16, and 24 for the first 24 weeks and every 12 weeks thereafter and every 16 weeks after week 48 until initiation of new antitumor therapy, PD, death, or withdrawal of consent. PET-CT was conducted at baseline and weeks 8, 16, and 24, and when CR or PD was determined by CT.The patients were followed up every three months upon completion of the final penpulimab dose. Survival was evaluated every three months. The primary efficacy outcome was ORR per the 2014 Lugano classification, which was the proportion of patients whose BOR was CR or PR. Secondary efficacy outcomes included duration of response (DoR), disease control rate (DCR), PFS, and time to tumor response (TTR).Physical examinations including vital signs and ECOG performance status, routine blood chemistries and hematological tests, thyroid function tests for thyroid-stimulating hormone (TSH), free and total T3 and T4, and urinary tests were done on days 1 and 15 of each cycle. Urinary hCG test was done at baseline and when clinically indicated in women of reproductive age. In addition, ECG was obtained at baseline and every 2 cycles, and ultrasonography was done when clinically indicated. AEs were graded and recorded according to NCI CTCAE version 4.03 and coded using MedDRA 22.0. AEs included treatment-emergent AEs (TEAEs), TRAEs and irAEs.Patients). ORR and DCR and their 95% confidence interval (CI) were estimated using the Clopper Pearson method. Exploratory analyses of the heterogeneity of treatment effects among subgroups were performed. ORR and its 95% CI were presented graphically using forest plots.Assuming an ORR of 35% using the null hypothesis and that the ORR would reach 55% after penpulimab treatment , a sample size of 70 was required to detect a difference of 20% with a power of 92%. Considering dropout and exclusions as a result of the central pathology review, a sample size of 80 patients was used for the study. Part A enrolled 6 patients and Part B enrolled 74 patients. Efficacy outcomes were examined in the full analysis set (FAS) that included patients (1) who had received at least one dose of penpulimab, (2) who had a diagnosis of cHL confirmed by the central pathology review panel, and (3) who met the definition of R/R cHL .Between August 13, 2018, and November 8, 2019, the trial enrolled 94 patients from 23 study sites (Appendix I). The FAS included 85 patients (The median duration of follow-up was 15.8 months (range 12.1-26.9). Based on the review by the IRRC, 40 (47.1%) patients achieved CR, 36 (42.4%) patients achieved PR, and 6 (7.1%) patients had SD. The ORR was 89.4% , and the DCR was 96.5% . At the last follow-up, 73.7% patients remained in remission. The median TTR was 1.8 months . DoR lasted for 12 months in 74.9%  patients and the median DoR was not yet reached , sex, ECOG performance status score (0 or 1), B symptoms, previous lines of therapy (<3 or ≥3), time from initial diagnosis to the first dose (<1 or ≥1 years), previous radiotherapy and prior ASCT  and 96.8%, 91.5%, and 76.6% patients had total exposure time ≥3, 6, and 12 months, respectively. The median number of penpulimab infusions was 28.0  (TEAEs and TRAEs (including unlikely related) with an incidence ≥10% and CTCAE grade 3 and above TEAEs and TRAEs are shown in Fifty-one (54.3%) patients experienced an irAE, but only 4 (4.3%) patients developed grade 3 and above irAEs . The dosing regimen was mainly based on the results of an earlier phase Ia study (AK105-101) showing that penpulimab at a dose of 3 mg/kg body weight could saturate peripheral PD-1 rapidly (within 24 h) and maintain an occupancy rate above 90% for PD-1 at day 15 (before the second dose). Based on the safety and pharmacokinetics (PK) data of the phase I study, a dose of 200 mg penpulimab  was recommended for the phase II trial.versus other anti-PD-1 monoclonal bodies for Chinese R/R cHL patients   of the patients remained in remission after a median follow-up of 15.8 months. The 12-month PFS rate was 72.1% and the 18-month OS rate was 100%. The treatment benefit was seen across all patient subgroups including previous lines of therapy (<3 or ≥3) and ASCT (yes or no). When compared with historical controls, this study showed that penpulimab as a single agent is more effective than traditional chemotherapies in the third line and above setting , 23. MorThe mechanisms underlying the development of irAEs are not fully clarified and one possible reason is the production of cytokines such as IL-6 and TNF-α induced through Fc-binding –18. Fc mvia Fc-Fc interactions . These findings need to be validated in randomized controlled studies with long term follow-up. Second, no subjects had received prior therapy with brentuximab vedotin as the drug had not been approved for CD30-positive lymphoma in China at the time of this trial. Another limitation of this study is that only 16.5% patients had received ASCT, which is in line with the low rate of ASCT in cHL patients across China and comparable to the rate of ASCT for tislelizumab (18.6%), sintilimab (19%), and camrelizumab 12%) , 12.% , 12.12Currently, monotherapy with brentuximab vedotin or anti-PD-1 monoclonal antibodies remain the predominant treatment for R/R cHL. Brentuximab vedotin was not yet approved in China at the time of this trial. There was also no data on pembrolizumab and nivolumab or any other anti-PD-1 monoclonal antibodies for Chinese cHL patients and clinical access to these agents remained extremely limited. The current findings are of clinical relevance in China and resource-constrained countries. Earlier studies have shown that anti-PD-1 monoclonal antibody monotherapy has noticeable efficacy in R/R cHL patients who have failed multiple lines of chemotherapy, which prompted us to pursue a single arm design for the current trial, with ORR being the primary efficacy measure. One feature of immunotherapy is the remarkable durability of remission once it has been achieved. We will report the longer term results of penpulimab treatment for R/R cHL when they become available, which will shed further light on the efficacy and safety of penpulimab in this patient population. Meanwhile, we are conducting confirmatory phase III trials to validate the findings of the current study. In conclusion, the novel anti-PD-1 antibody penpulimab achieved a high ORR, long survival with an acceptable safety profile in this trial, providing an important efficacious and safe treatment option for Chinese R/R cHL patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Peking University Cancer Hospital & Institute; The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital; Cancer Hospital Affiliated to Guangzhou Medical University Tianjin Medical University Cancer Institute and Hospital; National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, the Sino-US Center for Lymphoma and Leukemia Research; Qilu Hospital, Shandong University; The First Affiliated Hospital with Nanjing Medical University, Jiangsu Province Hospital, Collaborative Innovation Center for Cancer Personalized Medicine; The First Affiliated Hospital of Nanchang University; The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China Tumor Hospital of Xiangya School of Medicine of Central South University; Liaoning Cancer Hospital and Institute; Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University; Second Affiliated Hospital of Dalian Medical University; Shanghai First People’s Hospital, Shanghai Jiaotong University; West China Hospital, Sichuan University; Heilongjiang Provincial Hospital; The Fourth Hospital of Hebei Medical University; Weifang People’s Hospital; Union Hospital, Tongji Medical College, Huazhong University of Science and Technology; Shaanxi Provincial Cancer Hospital; Shanghai Tongren Hospital; The Affiliated Hospital of Qingdao University; The First Affiliated Hospital of Guangxi Medical University; The Third Affiliated Hospital of Suzhou University, The First People’s Hospital of Changzhou. The patients/participants provided their written informed consent to participate in this study.Conception and design: JZ, YS, YX, BL; Development of methodology: JZ, YS, XJ, MZ; Acquisition of data: YS, KZ, CJ, ZQ, MH, LF, FL, KD, HZ, XL, BC, XHS, XMS, MJ, QZ, LHL, GY, YH, ZZ, LGL, HX, JL, BH, JZ; Analysis and interpretation of data: YS, KZ, CJ, ZQ, MH, LF, FL, KD, HZ, XL, BC XHS, XMS, MJ, QZ, LHL, GY, YH, ZZ, LGL, HX, JL, BH, XJ, MZ, JZ; Writing, review, and/or revision of the manuscript: YS, KZ, CJ, ZQ, MH, LF, FL, KD, HZ, XL, BC, XHS, XMS, MJ, QZ, LHL, GY, YH, ZZ, LGL, HX, JL, BH, XJ, MZ, JZ; All authors reviewed and approved the final version of the manuscript.Funding for this study was provided by Akeso Biopharma Co., Ltd.XJ, MZ, BL and YX are employees of Akeso Biopharma Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The authors declare that this study received funding from Akeso Biopharma Co., Ltd. The funder had the following involvement with the study: sponsored this trial, and participated in the study design, data collection, statistical analysis, and provided medical writing support.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Purpose: This study assessed the safety, feasibility, and tolerability of mesenchymal stem cells for patients diagnosed with COVID (Coronavirus disease 2019-induced ARDS (acute respiratory distress syndrome)). Materials and Methods: Critically ill adult COVID-19 patients who were admitted to Wonju Severance Christian Hospital were enrolled in this study. One patient received human bone marrow-derived mesenchymal stem cell (hBMSC) transplantation and received a total dose of 9 × 107 allogeneic hBMSCs via intravenous infusion. The main outcome of this study was to assess the safety, adverse events, and efficacy following transplantation of hBMSCs in COVID-19- induced ARDS patients. Efficacy was assessed radiologically based on pneumonia improvement, changes in PaO2/FiO2, and O2 saturation. Results: A 73-year-old man visited Wonju Severance Christian Hospital presenting with fever and fatigue. A throat swab was performed for real-time polymerase chain reaction to confirm COVID-19, and the result was positive. The patient developed ARDS on Day 5. MSC transplantation was performed on that day and administered on Day 29. Early adverse events, including allergic reactions, were not observed following MSC transplantation. Subsequently, clinical symptoms, signs, and laboratory findings, including PaO2/FiO2 and O2 saturation, improved. Conclusion: The results of this case report suggest that intravenous injection of MSC derived from the bone marrow is safe and acceptable and can lead to favorable outcomes for critically ill COVID-19 patients. Acute respiratory distress syndrome (ARDS) is a life-threatening illness and is the primary cause of death and disability among critically ill patients; the mortality rate ranges from 26–50% ,2,3. ARDThe causes of ARDS include bacterial or viral pneumonia and severe traumatic lung injury. ARDS has been managed conservatively, including mechanical ventilation and treatment of the underlying disease. Despite these treatments, ARDS is usually associated with a high mortality rate. Previous experimental investigations indicate that intra-tracheal or intravenous administration of bone marrow-derived stem cells can reduce lung inflammation, accelerate lung repair, restore alveolar fluid clearance, and improve arterial oxygenation ,6,7.Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initially reported in Wuhan, Hubei, China, in December 2019 . COVID-1Recently, some studies suggest that cell-based therapy provides therapeutic effects for critically ill patients with pneumonia due to COVID-19 ,13. The We hypothesized that stem cell therapy can be effective for patients with ARDS caused by COVID-19. Therefore, the purpose of this study was to evaluate the safety, feasibility, and tolerance of mesenchymal stem cells in COVID-19-induced ARDS patients.Adult COVID-19 patients in critical condition admitted to Wonju Severance Christian Hospital were enrolled in this study. COVID-19 infection was confirmed using reverse transcription polymerase chain reaction (RT-PCR). The inclusion criteria were as follows: (1) 50–90 years old; (2) patients with pneumonia confirmed by chest radiography or computed tomography (CT); (3) patients with a respiratory rate of 30 breaths or more, severe dyspnea, or <90% oxygen saturation; (4) patients diagnosed with ARDS per WHO guidelines for definition and classification of ARDS ; and (5)5 cells/cm2 in T-75 flasks. The cultures were maintained at 37 °C in a humidified environment containing 5% carbon dioxide. Non-adherent cells were eliminated by changing the media after 5 days, and adherent cells were cultivated for an additional 3 days (passage 1). Upon achieving 70–80 percent confluence, adherent cells were separated using TripLE Express (Gibco) and replated at a density of 4–5 × 103 cells/cm2 in T-175 flasks. To prepare a Master Cell Bank, cultured cells (passage 2) were harvested, frozen using a cryostabilizer , and kept in a vapor phase liquid nitrogen tank . Cells were thawed from the MCB, cultivated for 3–5 days (passage 3–6), harvested from passage 6, and then frozen to prepare the Working Cell Bank (WCB). WCB cells were thawed and cultured until passage 8. Eighth passage cells were cultured in a hypoxia-conditioned incubator  for 72 h. Following hypoxic preconditioning, hBMSCs were collected and washed twice with PBS and once with Plasma Solution A inj. . hBMSCs were resuspended in Plasma Solution A inj. to a final concentration of 9 × 107 cells. The release of hBMSCs for clinical use was contingent upon the absence of microbial contamination , 80% viability in a trypan blue exclusion assay, 0.5 EU/mL endotoxin level, and immune phenotyping via flow cytometric analysis, demonstrating the expression of CD73 and CD105 surface markers and the absence of CD14, CD34, CD45, and HLA-DR.The Institutional Review Board of the Public Institutional Bioethics Committee  approved the study protocol which was performed according to the Declaration of Helsinki. Samples of bone marrow were taken from healthy volunteers. All production and testing processes for human bone marrow-derived mesenchymal stem cells (hBMSC) were conducted per appropriate manufacturing practices . Bone marrow mononuclear cells were extracted using density gradient centrifugation  and phosphate-buffered saline washing . The cells were resuspended in Dulbecco’s modified Eagle’s medium-low glucose  with 10% PLUSTM human platelet lysate (hPL)  and 20 g/mL gentamicin (Gibco). The cells were plated at a density of 1.0–1.5 × 107 allogeneic hBMSCs via an intravenous route. The infusion time was approximately 50 min. The patient received standard medications according to the patient’s condition.One patient received hBMSC transplantation. The patient was administered 9 × 10Vital signs measurement, chest radiography, and laboratory tests, including arterial blood gas analysis (ABGA), chemistry, complete blood counts (CBC), and urine analysis, were performed on days 0, 1, 3, 5, 7, 14, 21, and 28 after administration. Chest CT was performed on days 3, 14, and 28 after administration, if the patients’ condition was tolerable.2/FiO2, and O2 saturation.The primary outcomes of this study were the safety, side effects, and effectiveness of hBMSC transplantation in COVID-19-induced ARDS patients. Common Terminology Criteria for Adverse Events (CTCAE) (version 5.0) are used to describe the severity of side effects . Early s9/L), lymphocyte (1.34 × 109/L), elevated procalcitonin level (0.07 ng/mL), high C-reactive protein (CRP) (7.16 mg/dL), and creatinine (1.41 mg/dL) levels. A throat swab was performed for RT-PCR to confirm COVID-19, and the result was positive. Treatment with chloroquine and lopinavir/ritonavir was initiated.On 3 March 2020, A 73-year-old man with a history of diabetes mellitus, hypertension, cerebral infarction, and hyperlipidemia visited our emergency department with fever and fatigue. His initial blood pressure and pulse rate were within normal ranges and his temperature was 37.7 °C. Physical examination revealed no specific signs. Lung auscultation revealed normal breathing sounds, and chest radiography showed ground-glass opacities in both lower lung fields. Laboratory tests showed normal leukocyte (5.14 × 102: 30%), and the CRP level increased to 28.1 mg/dL.On Day 4 of hospitalization, the patient developed a fever of 38.6 °C shortness of breath requiring oxygen supplementation via nasal high flow system . Chest radiography revealed increased haziness in both lower lung fields , and septic shock was considered. After initiation of the inotropic agent, blood pressure returned to normal, and immunoglobulin administration via the venous route was initiated. The inotropic agent was discontinued on Day 7.On Day 9, fever persisted at 38.3 °C, and cefepime and teicoplanin were added considering bacterial superinfection.2: 54.1) was aggravated although high O2 was applied (FiO2: 90%). Chest radiography showed haziness in both lower lung fields, which was dominant in the left lower lung field. High-pressure mechanical ventilation was continued . Intravenous steroids were administered on the same day.On Day 14, hypoxemia  (27 U/L), alanine aminotransferase (ALT) (32 U/L), creatinine (0.9 mg/dL), elevated total bilirubin (1.42 mg/dL), CRP (3.46 mg/dL), and leukocytosis (16.72 × 109/L). MSC transplantation was performed on that day and administered the next day.Ventilator weaning was attempted since Day 19 but was unsuccessful. On Day 28, the PaOEarly adverse events, including allergic reactions, were not observed following MSC transplantation. Three days after MSC transplantation, a chest CT was performed, which showed ground-glass opacity (GGO) in both lungs, consolidation, and a cavity in the right upper lobe .Five days after MSC transplantation, seizures with eyeball deviation were observed and anterior cerebral artery territory infarction was suspected on CT. Aspirin was then added to prevent further infarction. This was confirmed via magnetic resonance image.2O and pressure-controlled mode (pressure setting:8 cmH2O) with a value of FiO2:0.5. PaO2/FiO2 was 129.1 mmHg, and O2 saturation was 94.4. Chest radiography showed slight regression of the GGO in both lower lung fields  with values of FiO2:0.4. PaO2/FiO2 was 249.5 mmHg, and O2 saturation was 97.7. Chest radiography showed slight regression in the GGO in both lower lung fields.Fourteen days after MSC transplantation, the patient was mechanically ventilated at PEEP: 6 cmH2: 0.4, and a flow rate of 30 L/min. PaO2/FiO2 was 279.4 mmHg, and O2 saturation was 98.2%.The ventilator was weaned on Day 25 after the MSC transplantation. On Day 28 after the administration, high-flow nasal oxygen therapy was applied to the patient with values of FiODetails of the method for respiratory support, chest radiography findings, arterial blood gas analysis results, and observed adverse events on the day of follow-up after MSC transplantation are described in Isolation was discontinued on Day 55 after transplantation because the RT-PCR results were negative. Chest CT performed on Day 106 showed decreased GGO in both lungs, decreased consolidation, and a cavity in the right upper lobe relative to the previous CT scan . The patNo specific COVID-19 treatment is currently available. Although mild cases may require supportive therapy, severe cases have been managed using approved or experimental therapies. These include chloroquine, which is used for prophylaxis and treatment of malaria; lopinavir/ritonavir, which is a combination therapy for HIV; remdesivir and favipiravir; and tocilizumab (IL-6 antagonist). In accordance with the severity or stage of the infection, various combinations of these drugs have been introduced and used in the treatment guidelines of various countries ,18,19.Multiple aspects of the pathophysiology causing ARDS can be targeted by cell-based therapy, a potentially novel therapy. In recent years, cell-based therapies have been introduced into preclinical studies of ARDS. Several cell types, including embryonic stem cells, induced pluripotent stem cells, MSC, pulmonary epithelial progenitor cells, and endothelial progenitor cells, have been investigated as potential therapeutic candidates . MSCs arRecently, stem cell therapy has been used for critically ill patients with COVID-19. Several clinical trials using MSC, such as phase 1 clinical trials (NCT 04252118), phase 1/2 clinical trials (NCT 04288102), and phase 2 clinical trials (NCT 04269525), are being conducted. Leng et al.  reportedSeveral mechanisms have been suggested to explain the efficacy of MSCs. MSCs stimulate tissue recovery and regeneration by secreting several paracrine substances with anti-inflammatory, immunomodulatory, angiogenesis-promoting, antifibrotic, antibacterial, and structural repair capabilities ,23. NotaMSCs were administered via the intravenous route in this study. Wu et al. demonstrated that the colonization and differentiation of BMSCs in the organ are mainly induced by the microenvironment in the injured organ . MSCs ha2/FiO2 and O2 saturation, improved. A recent systematic review reported that cell-based therapy reduces mortality rates and improved recovery [2/FiO2 ratio after stem cell infusion, 7 of the 14 enrolled studies reported an increase in oxygen saturation, and 15 of the 17 studies showed an improvement in lung images such as chest CT or chest radiography. Additionally, 11 of the 14 studies demonstrated discontinuation of oxygen support (intubation or ECMO) after cell infusion. Twenty-seven of the enrolled studies in the systematic review did not report any adverse events [Here, we report the case of a patient with ARDS due to COVID-19 who was successfully treated with MSC transplantation. Although COVID-19 typically reaches its peak within 7–14 days and then improves, our patient experienced rapid disease progression and no recovery despite all supportive and pharmacological interventions, including chloroquine and lopinavir/ritonavir. After the administration of MSC, neither immediate nor delayed adverse events were detected. In addition, clinical symptoms, signs, and laboratory findings, including PaOrecovery . In parte events .This case report has several limitations. As this is a report of a single case, it is impossible to draw conclusions about the treatment outcomes. Second, although 28 days post-treatment follow-up revealed no adverse effects, the study’s timetable did not allow us to investigate potential long-term side effects. Third, since several treatments were attempted after MSC transplantation, we cannot conclude that MSC transplantation has an absolute effect on improving patient outcomes.Despite these limitations, this study is meaningful because the patient, showing no signs of recovery despite several treatment strategies, achieved favorable clinical outcomes after MSC transplantation. To our knowledge, this is the first report in South Korea of the effectiveness of MSC for patients with ARDS due to COVID-19.The results of this case report suggest that intravenous injection of MSC derived from the bone marrow is safe and acceptable and can lead to favorable outcomes for critically ill COVID-19 patients. However, we cannot conclude that MSC transplantation for COVID-19-induced ARDS is effective and completely safe. Large randomized controlled trials are required to prove the therapeutic potential of MSC for the treatment of this disease.