Protein ubiquitination, which is catalyzed by ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin ligases, is a crucial post-translational modification to regulate numerous cellular functions in a spatio–temporal-specific manner. The human genome encodes ~100 deubiquitinating enzymes (DUBs), which antagonistically regulate the ubiquitin system. OTUD1, an ovarian tumor protease (OTU) family DUB, has an N-terminal-disordered alanine-, proline-, glycine-rich region (APGR), a catalytic OTU domain, and a ubiquitin-interacting motif (UIM). OTUD1 preferentially hydrolyzes lysine-63-linked ubiquitin chains in vitro; however, recent studies indicate that OTUD1 cleaves various ubiquitin linkages, and is involved in the regulation of multiple cellular functions. Thus, OTUD1 predominantly functions as a tumor suppressor by targeting p53, SMAD7, PTEN, AKT, IREB2, YAP, MCL1, and AIF. Furthermore, OTUD1 regulates antiviral signaling, innate and acquired immune responses, and cell death pathways. Similar to Nrf2, OTUD1 contains a KEAP1-binding ETGE motif in its APGR and regulates the reactive oxygen species (ROS)-mediated oxidative stress response and cell death. Importantly, in addition to its association with various cancers, including multiple myeloma, OTUD1 is involved in acute graft-versus-host disease and autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and ulcerative colitis. Thus, OTUD1 is an important DUB as a therapeutic target for a variety of diseases. O. OSkp2, e stress . The sloin NSCLC . OTUD1 sMCL1 is a fast-turnover Bcl-2 family protein that plays important roles in cell survival, proliferation, differentiation, and tumorigenesis . The effOtud1-deficient mouse embryonic fibroblasts (MEFs) and mice, as compared to the responses in the wild-type (WT) counterparts. These results suggest that OTUD1 is involved in the host defense against RNA viruses. In this process, OTUD1 negatively regulates the protein levels of mitochondrial antiviral-signaling protein (MAVS) and the TNF receptor-associated factor 3 (TRAF3) and TRAF6 E3s. Furthermore, OTUD1 regulates the ubiquitination and protein level of a HECT-type E3, Smurf1, and the OTUD1–Smurf1 axis destabilizes MAVS/TRAF3/TRAF6 levels and decreases IFNβ induction during RNA virus infection , a regulator of endoplasmic reticulum (ER) stress-induced apoptosis and autophagy, on the antiviral immune response during an infection with bovine viral diarrhea virus (BVDV), a single-stranded RNA virus . A. A41]. ACandida albicans fungal infection, C-type lectin receptors (CLRs) expressed on macrophages and dendritic cells recognize the fungal cell wall glucans and then activate Src family Tyr kinase and protein kinase Cδ (PKCδ). Subsequently, the PKCδ-mediated phosphorylation of CARD9 induces the CARD9–BCL10–MALT1 (CBM) complex, which activates the NF-κB and MAPK signaling pathways [Otud1 deficiency impairs cytokine production and antifungal immune responses in vivo. Furthermore, Wu et al. report that Otud1-deficient mice show increased susceptibility to dextran sulfate sodium (DSS)-induced colitis, with upregulation of inflammatory cytokines due to the increased NF-κB signaling [Otud1 is 1 of 13 key genes that are crucial for LPS-induced cardiac dysfunction and sevoflurane-induced cardioprotection in mice [Upon pathways . TRIM62,pathways . OTUD1 ipathways   is the only E3 that can generate the N-terminal Met1(M1)-linked linear ubiquitin chain . LUBAC amulation B. Thus, e stress   is a malignancy of immunoglobulin (Ig)-producing plasma cells and characteristically secretes an abnormal serum monoclonal Ig named M protein. Proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib are reportedly effective in the treatment of MM. Vdovin et al. found that the expression levels of intracellular Ig and OTUD1 are associated with survival after the onset of MM, since OTUD1 regulates Ig production, myeloma cell proliferation, and tumor growth . FurtherOtud1, as well as Otud4 and Otud5, were significantly upregulated in allogeneic T cells, but not syngeneic T cells. Moreover, the authors determine that OTUD1 is abnormally upregulated in CD4+ T cells, but not CD8+ T cells, of aGVHD patients. OTUD1 levels are positively correlated with the incidence and severity of aGVHD, and, therefore, Otud1 deficiency suppresses T cell activation and proinflammatory cytokine production, resulting in the alleviation of aGVHD pathogenicity. As the molecular basis, they show that OTUD1 selectively deubiquitinates K1770 on the intermediate Notch-intracellular domain (Notch-ICD) of Notch2, but not Notch1, Notch3, or Notch4, protects it from degradation, and regulates the expression of Notch2 target genes  is a serious complication that affects patients who have had a stem cell transplant using cells from a donor, due to the allogeneic response mediated by donor T cells, which induces uncontrolled T cell activation, proliferation, and upregulation of inflammatory cytokines. Recently, Cheng et al. identified OTUD1 as an essential regulator of aGVHD . These aet genes B. The NoThe ubiquitin system plays an important role in ribosome-associated quality control (RQC), and the ZNF598 E3 (Hel2 in yeast)-mediated ubiquitination stalls ribosomes on a polyA stretch through the K63-ubiquitination of uS10 ,54. OTUDOTUD1 mRNA is upregulated in the aortic endothelium of Ang II-treated mice, and vascular remodeling and collagen deposition are attenuated in Ang II-treated Otud1-deficient mice [Angiotensin II (Ang II), a peptide hormone involved in vasocontraction and increasing blood pressure, also affects hypertension and hypertensive vascular remodeling. Huang et al. recently showed that among OTU family DUBs, only the ent mice . FurtherOTUD1 is encoded by a gene that is characteristically induced by the rearing nutritional status and stress of animals such as pigs, chickens, and fish. Thus, OTUD1 was identified as one of the upregulated genes in muscle tissues from 20% rapeseed meal (RSM)-treated Norwegian Landrace growing–finishing pigs, as compared with the soybean-meal-fed pigs . This coIn the muscle tissue of high-yielding broiler line chickens, several muscle myopathies such as woody breast myopathy were identified, and a high level of OTUD1 expression was detected in the musculoskeletal system at day 21 . Althougotud1, as well as trib3 and dusp5, was identified as a hub gene of cold-induced damage [When zebrafish larvae were exposed to lethal cold stress (10 °C) and rewarmed to normal temperature (28 °C), d damage . The genC10ORF67–OTUD1 intergenic region of chromosome 10p12, that are strongly associated with sarcoidosis in African-American women [A genome-wide association study (GWAS) by Cozier et al. identified two SNPs, rs1398024 (G>T) and rs11013452 , on the an women . SarcoidIn this review, we comprehensively summarized the functions of OTUD1, a DUB that has recently received keen attention. OTUD1 exhibits strong activity against the K63 ubiquitin chain in vitro, but also reportedly cleaves K6, K27, K33, K48, and K63 ubiquitin chains in cells. These activities reveal that OTUD1 is involved in the regulation of numerous cellular functions, such as oncogenic protein regulation, antiviral signaling, inflammation, innate and acquired immunity, cell death, and ribosomal function. In addition, there are OTUD1-related diseases, such as various cancers, autoimmune diseases, sepsis, inflammatory bowel disease, aGVHD, arteriosclerosis, and so on. It is highly likely that ROS production and oxidative damage are involved in the regulation of these cell functions and diseases related to OTUD1, and the KEAP1–OTUD1 axis plays an important role. The development of OTUD1-targeted inhibitors, activators, and KEAP1-binding inhibitors is expected to provide new therapeutic drugs. Dapagliflozin, an SGLT-2 inhibitor for type 2 diabetes treatment, reportedly has the potential to inhibit OTUD1, and its binding model has also been proposed . Detaile


V, KV, CaV, and TRP families, are the major targets of peptide neurotoxins. Insight into the mechanism of neurotoxins acting on ion channels contributes to our understanding of the function of both channels and centipede venoms. Meanwhile, the novel structure and selective activities give them the enormous potential to be modified and exploited as research tools and biological drugs. Here, we review the centipede venom peptides that act on ion channels.Centipedes are one of the most ancient and successful living venomous animals. They have evolved spooky venoms to deter predators or hunt prey, and are widely distributed throughout the world besides Antarctica. Neurotoxins are the most important virulence factor affecting the function of the nervous system. Ion channels and receptors expressed in the nervous system, including Na Craterostigmomorpha, Geophilomorpha, Lithobiomorpha, Scutigeromorpha, and Scolopendromorpha, making them one of the oldest and the richest lineages of living venomous terrestrial predators [Throughout human history, venomous creatures such as snakes, scorpions, centipedes, caterpillars, and poisonous frogs have existed in the human living environment and civilization. In most ancient cultures, venomous creatures were either deified and associated with the divine, or vilified as inflictors of pain and distress due to their mysterious and destructive power. As one of the most ancient and successful venomous predators, centipede venom is an excellent model for insight into venoms’ functional and molecular evolution. Centipedes originated around 440 million years ago with almost worldwide distribution except for Antarctica, and comprise approximately 3300~3500 extant species that are divided into five categories: redators .For centuries, centipedes have been frequently involved in human accidents ,2. Most V) channels, voltage-gated potassium (Kv) channels, voltage-gated calcium (CaV) channels, and transient receptor potential cation channels (TRP).From the view of survival strategy and clinical features, neurotoxins affecting the nervous system might play an important role in centipede envenomation ,13,14,15For different strategies and purposes, venomous animals have evolved complex venom systems that have independently evolved more than one hundred times from non-toxic ancestral peptides . AccordiScolopendra subspinipes mutilans L. Koch by peptidomics and cDNA library, further strengthening the diversity of centipede venoms [+, K+ and Ca2+ channels [Most proteins in centipede venoms are homologous with that of other venomous animals, while some centipede peptides surprisingly bear no or few resemblances to any other characterized venomous arthropod peptide family . In 2014e venoms . Of theschannels ,9. TheseScolopendra viridis [S. viridis contain peptides acting on Ca2+ and K+ channels, allergens, metalloproteases, and β pore-forming toxins [S. viridis (FL) has been compared with previously published S. viridis (MO) in transcriptomes and proteomes, which unexpectedly exhibit extreme levels of variation of S. viridis [Furthermore, similar results were also found by Rokyta et al. on  viridis . Accordi viridis ,18. The g toxins . Fourteeg toxins ,10,18. SHemiscolopendra marginata) and confirmed it as the first sample of centipede venom variation based on sex [H. marginata [H. marginata than in male venom, which has been reported to enhance platelet aggregation and hemolysis [In addition, the work of Gunnar S. Nystrom et al. filled a gap in sexually dimorphic traits in centipede venom that were reported in invertebrate venoms, such as scorpions and spiders. They identified 47 toxins and 717 nontoxic transcripts by RP-HPLC, venom proteomes, and venom-gland transcriptomes on both female and male eastern bark centipedes  is composed of α1 subunit-containing pore-forming and voltage-sensing domains, and several α2δ, β, and γ regulatory subunit isoforms [V3.1–3.3) channels are denoted as low-threshold channels, while superfamilies 1 and 2 are high-threshold channels, including L-type (CaV1.1/4), N-type (CaV2.2), P/Q-type (CaV2.1), and R-type (CaV2.3) channels [V2.2  channels are known to be the most varied channels. According to the structure of ɑ-subunits, potassium channels are further divided into four categories: voltage-gated potassium (Kv) channels, calcium-activated potassium (KCa) channels, inwardly rectifying potassium (Kir) channels, and two-pore potassium (K2P) channels [V channels selectively mediate the potassium transmembrane transportation induced by voltage or other physiological mediators. They are found in almost all cell types and involved in various vital activities, including the release of neurotransmitters and hormones and controlling membrane properties [+ (KATP) channels are expressed in pancreatic β cells that secrete insulin and is regarded as the regulator between the level of blood glucose and insulin secretion [V1.3 channel is highly expressed in immune cells and is considered an alternative target in treating AID (autoimmune disease) [Scolopendra mutilans, which exhibited lethal toxicity in hematological and respiratory systems by potently inhibiting KCNQ (voltage-gated potassium channel family 7) channels  channel [50 from 0.1 to 0.3 M [Potassium  ,35. In 2channels A,B [6,36 channel . The Shato 0.3 M . With thto 0.3 M . Based oATP were identified from the venom of Scolopendra subspinipes dehaani. Functionally, four of these proteins (SsdTx1-3 and SsTx) inhibit hKATP channels by blocking hKir6.2 with a Kd of <300 nM [50 of 42.5 nM at 140 mV [50 of 89 nM and 360 nM at 140 mV, respectively. This work confirmed that SsTx-4 is the first known peptide toxin antagonistic to the Kir4.1 channel [50 of Kir4.1 [Further, a similar family of peptides that inhibit hK <300 nM . In addi <300 nM . SsTx-4 t 140 mV . Meanwhi channel . To conf channel . They fo channel . On the  channel . At the  channel . The binf Kir4.1 . These wEuphorbia resinifera and Einhorbia poissonii, researchers have achieved the first cloning and characterization of TRPV1, which brought insight into the function of TRPV1 in itch, cancer, and weight loss [TRPV1 is a well-studied channel known as an autologous and environmental noxious stimuli receptor, for instance, high temperature above 40 °C, pH below 6.0, peptide toxins, and the most classic, capsaicin, the hot chemical in chili . As reveght loss . Accordight loss . We identified RhTx C, a pept50 of 38.35 ± 4.61 μM, which is nearly 100 times that of RhTx [Furthermore, in 2020, we once more identified a peptide that is identical to RhTx except for four additional residues (NSKY) at the N terminus, hence named RhTx2 . Analogo of RhTx . Further of RhTx . With si of RhTx . With RhDespite the P2X receptor having been verified to be involved in a broad range of physical performance disturbances, including hypertension, bladder incontinence, chronic cough, inflammatory and immune disorders, megrim, pain, IBS (irritable bowel syndrome), epilepsy, atherosclerosis, depressive disorder, diabetes mellitus, and cancer, purinergic receptors have been seldom considered and involved in the development of exploiting novel molecules, or new tools from animal toxins that modulate ion channels ,53,54,55V channel, NaV channel, CaV channel, and other channels [Scolopendra subspinipes mutilans L. Koch exhibits no similarity to any known peptide toxin families from any venomous animal [V1.7, TTX-S NaV channel, and CaV channels respectively [V1.3 channels, which are considered to mutually intensify the inhibitory effect [Humans have strugglingly and progressively coexisted with venomous creatures for centuries. With technology development, people have intensively studied some well-known venomous animals, such as snakes, spiders, and frogs . Howeverchannels ,8,15,38.s animal . Three oy effect . Further


Hyper-IgE Syndrome (HIES) is a heterogeneous group of primary immune-deficiency disorders characterized by elevated levels of IgE, eczema, and recurrent skin and lung infections. HIES that is autosomally dominant in the signal transducer and activator of transcription 3 (STAT3), and autosomal recessive mutations in phosphoglucomutase 3 (PGM3) have been reported in humans. An early diagnosis, based on clinical suspicion and immunological assessments, is challenging. Patients’ metabolomics, proteomics, and cytokine profiles were compared to DOCK 8-deficient and atopic dermatitis patients. The PGM3 metabolomics profile identified significant dysregulation in hypotaurine, hypoxanthine, uridine, and ribothymidine. The eight proteins involved include bifunctional arginine demethylase and lysyl hydroxylase (JMJD1B), type 1 protein phosphatase inhibitor 4 (PPI 4), and platelet factor 4 which aligned with an increased level of the cytokine GCSF. Patients with STAT3 deficiency, on the other hand, showed significant dysregulation in eight metabolites, including an increase in protocatechuic acid, seven proteins including ceruloplasmin, and a plasma protease C1 inhibitor, in addition to cytokine VEGF being dysregulated. Using multi-omics profiling, we identified the dysregulation of endothelial growth factor (EGFR) and tumor necrosis factor (TNF) signaling pathways in PGM3 and STAT3 patients, respectively. Our findings may serve as a stepping stone for larger prospective HIES clinical cohorts to validate their future use as biomarkers. Hyper-IgE syndrome (HIES) is a group of heterogeneous primary immune-deficiency disorders characterized by elevated levels of IgE, eczema, and recurrent infections as described by Davis et al. in 1966 ,3. RecenAlthough the IgE levels are commonly elevated in these conditions, they have different clinical presentations. STAT3 deficiency is a multisystem disease involving the immune system, dentition, skeleton, and connective tissues . PatientIncreased serum IgE levels are characteristic but not specific to allergic diseases. HIES exhibits an unusual constellation of clinical features. Diagnosis can be confusing and difficult, especially during early childhood, and it is potentially life-threatening, although curable with bone marrow transplantation. Thus, the diagnosis of HIES is critical and should be sought at an early definitive therapy. Severe atopic dermatitis (AD) and HIES share some clinical symptoms, including eczema, eosinophilia, and increased serum IgE levels. AD is a chronic inflammatory disease with remission that commonly occurs in early infancy. Clinical manifestations in patients with AD include food allergy, asthma, and allergic rhinitis, probably in response to impaired innate and adaptive immunity and environmental stimuli . EczematHigh throughput “omics” technologies are indispensable for understanding HIES disease’s underlying molecular mechanisms and in-depth pathogenesis as a complex disorder. Multi-omics technologies have been used in several disease’ biomarker discoveries . Signifi® xMAP® protocols. The PGM3 and STAT3 distinctive profiles were compared to AD and DOCK8 profiles from previous studies for specificity [Herein, we describe 4 patients with HIES, two of the four with documented mutations in STAT3 and the other two in PGM3. In the current investigation, we combined metabolomics, proteomics, and serum cytokine levels data utilizing a ‘systems biology’ approach to characterize alterations at the biochemical and molecular level in HIES patients using established mass spectrometry and Luminexcificity ,19,20. W12/L) and 4.2 ± 1.4 (109/L), whereas, in STAT3 patients, they were 4.7 ± 0.02 (1012/L) and 5.5 ± 1.9 (109/L), respectively. While eosinophilia was present in all patients, the counts were significantly higher in the PGM3 cohort than in STAT3. On the other hand, no significant difference in neutrophil counts was noted between the two cohorts. Comparatively, the CD4/CD8 ratio in the PGM3 cohort was 1.6 ± 0.7; for one of the STAT3 patients, the ratio was 1.1 (we could not get the ratio for patient P1).The genetic and laboratory characteristics of the study cohorts are represented in p-value < 0.05) and fold change (FC > 2) analyses showed the significant molecules for each immunodeficiency. Partial least square-discriminant analysis (PLS-DA) demonstrates considerable and distinct separation between the five study group  were collected from these platforms and analyzed, as shown in dy group E profiledy group A–C and Sdy group D–F were dy group A The prody group B. The cySimilarly, STAT3 patients revealed significant alterations in eight metabolites, including increased 2-protocatechuic acid, alanine, and aminobutyric acid and decreased methionine sulfoxide and ferulic acid, among others D. The prWe carried out an integrated approach using ingenuity pathway analysis (IPA) software to analyze the ‘omics’ data by incorporating the significantly differential proteins and metabolites in PGM3  and STAT13C-dansyl chloride was available from the University of Alberta) ). For proteomics analysis, the analytical solvents Dithiothreitol (DTT), and Iodoacetamide (IAA) were purchased from Sigma-Aldrich  and RapiGest SF from .LC-MS grade reagents were purchased from Fisher Scientific , and n = 10), AD (n = 9), STAT3 (n = 2), PGM3 (n = 2) and healthy controls (n = 33). All recruited subjects attended the allergy/immunology clinics at King Faisal Specialist Hospital and Research Centre (KFSHRC) [Serum samples were collected from infants, children, and adults with the clinical and laboratory-confirmed diagnosis of the hereditary DOCK8-deficiency ((KFSHRC) ,18. Patin = 10), AD (n = 9), STAT3 (n = 2), PGM3 (n = 2), and control samples (n = 33) underwent testing using the CIL LC-MS metabolomics platform. The samples were labeled with 12C-dansyl chloride (DnsCl), while a pooled sample was generated by mixing all individual samples and then labeled with 13C-DnsCl. Liquid chromatography with ultraviolet detection (LC-UV) quantitation was performed to determine the total concentration of dansyl-labeled metabolites. Each 12C-labeled sample was mixed with the same molar amount of the 13C-labeled pooled sample and injected into LC-MS. Serum samples were analyzed using a Thermo Fisher Scientific Dionex Ultimate 3000 UHPLC System  linked to a Bruker Maxis II quadrupole time-of-flight (Q-TOF) mass spectrometer , as previously described [Serum samples from DOCK8-deficient (escribed .n = 10), AD (n = 9), STAT3 (n = 2), PGM3 (n = 2), and healthy controls (n = 33) underwent proteome analysis; 100 µg total protein was subjected to in-solution tryptic digestion, as previously described [Similarly, serum samples from DOCK8-deficiency , AD (n = 9), STAT3 (n = 2), PGM3 (n = 2), and control samples (n = 15) were quantitatively profiled using the MILLIPLEX MAP kit (Millipore) according to the manufacturer’s instructions, where phycoerythrin-conjugated detection was used to form sandwich complexes. The fluorescence data were acquired on the FLEXMAP 3D [Patient samples from DOCK8-deficient  metabolism, as noted in the differential levels of uridine, hypoxanthine, and xanthine. Purines and pyrimidine are building blocks for synthesizing DNA, RNA, metabolic signals, energy transducers, and essentcoenzymes. Studies in animal models have noted that PGM3-mediated sugar nucleotide synthesis is essential for hematopoiesis and overall development . A deficTwo other patients with the clinical phenotype of HIES were identified to have autosomal dominant STAT3 mutations. STAT3 belongs to the STAT family of transcriptional regulators, which are known to play key roles in cell development and differentiation, in addition to cell death. It is also a key regulator of many immunologic pathways transducing signals downstream of many cytokines, including but not limited to IL-6, IL-10, IL-21, GCSF, and the interferon (IFN) family  ,31. ThesDespite having a small cohort, our case report is the first to carry out an integrated multi-omics systems biology approach toward profiling patients with HIES. Our findings showed distinct metabolites, proteins, and cytokines acting in concert to regulate distinct metabolic and signaling pathways that can be targeted for effective treatment and prevention strategies. Future improvements in the application of multi-omics, the generation of larger data sets in more diverse populations, and customized analysis of data types will promote discoveries and shorten the time taken from data generation to publication and translation to clinics. Identifying these mutations will improve the timing and precision of diagnosis and may permit earlier definitive identification.An integrated metabolomics analysis identified a distinct molecular pattern in PGM3 and STAT3 patients. The generated integrative data provides a holistic view of a network of interactions and regulatory events underlying disease processes. Our study highlights the importance of using metabolomics, proteomics, and cytokine profiles for the characterization of overlapping but distinct HIES disorders and identifying potential distinguishing biomarkers for early diagnosis. Our findings may form the basis of larger, prospective, externally validated studies in clinical cohorts for their future use as biomarkers.


Cells were subjected to differentiation toward iTregs using a transforming growth factor β (TGF-β)-based protocol, with or without SCFAs . Thereafter, they were subjected to flow cytometric phenotyping or a suppression assay. During differentiation, cells were collected for chromatin-immunoprecipitation (ChIP)-based analysis of histone acetylation. The enrichment of the TGF-β-based protocol with butyrate or propionate potentiated the in vitro differentiation of human naïve CD4+ non-Tregs towards iTregs and augmented the suppressive capacity of the latter. These seemed to be at least partly underlain by the effects of SCFAs on the histone acetylation levels in differentiating cells. GITR, ICOS, CD39, PD-1, and PD-L1 were proven to be potential markers of human iTregs. Our results might boost the further development of Treg-based therapies against autoimmune, allergic and other chronic inflammatory disorders.Regulatory T cells (Tregs) control immune system activity and inhibit inflammation. While, in mice, short-chain fatty acids (SCFAs) are known to be essential regulators of naturally occurring and in vitro induced Tregs (iTregs), data on their contribution to the development of human iTregs are sparse, with no reports of the successful SCFAs-augmented in vitro generation of fully functional human iTregs. Likewise, markers undoubtedly defining human iTregs are missing. Here, we aimed to generate fully functional human iTregs in vitro using protocols involving SCFAs and to characterize the underlying mechanism. Our target was to identify the potential phenotypic markers best characterizing human iTregs. Naïve non-Treg CD4 Regulatory T cells (Tregs) control immune system activity and inhibit the inflammatory process. In subjects suffering from autoimmune disorders, Tregs are compromised ,2,3. TheShort-chain fatty acids (SCFAs), the major end products of bacterial fermentation in the human colon, play an important role in protection against several chronic inflammatory or autoimmune diseases, such as asthma, allergies, inflammatory bowel disease, and type 1 diabetes, mostly through their putatively significant role in Treg induction ,21,22,23+Foxp3+ subpopulation in human peripheral blood (PB) possessing no suppressive capacity [Forkhead box P3 (Foxp3) is currently considered to be the most specific marker for naturally occurring Tregs, especially those generated in the thymus. However, growing evidence from humans suggests that this issue is more complex. For instance, there is also a CD4capacity . Some hucapacity ,32. In acapacity ,34,35, Ccapacity ,37,38, icapacity ,40, progcapacity ,42, intecapacity , and humcapacity , have beEstablishing further human iTreg generation strategies and a better understanding of their biology will boost the development of Treg-based therapeutic approaches. Hence, we aimed to successfully generate fully functional human iTregs in vitro using protocols involving SCFAs and to characterize the mechanism underlying this differentiation. In parallel, we wanted to identify the potential phenotypic markers best characterizing human iTregs.+CD45RO−CD25−CD127hi) were stimulated with anti-CD3/anti-CD28 in the presence of interleukin-2 (IL-2) and transforming growth factor β (TGF-β), with or without SCFAs, for 5 days (To compare the iTreg cell induction patterns between cord blood (CB) mononuclear cells (CBMCs) and adult peripheral mononuclear cells (PBMCs), FACS-sorted naïve non-Treg cells  Foxp3 is mainly an activation marker for human iTregs; (b) GITR, ICOS, CD39, PD-1, PD-L1, and CTLA-4 are potential markers for human iTregs; and (c) butyrate and propionate, but not acetate, potentiate the expression of phenotypic markers of human TGF-β-induced Treg cells in vitro.+CD25− responder cells (freshly bead-isolated) under various ratios in vitro. Then, on day 3, they were analyzed for the differences in the proliferation of responder cells.To test the suppressive capacity of iTreg cells treated with different SCFAs, these iTreg cells were co-cultured with allogeneic CFSE-labeled CD4+ cells. To isolate activated (purified CD25high) cells for the in vitro suppression assay, the whole population of cultured cells incubated under various conditions  was subjected to CD25-based positive selection. Butyrate or propionate-treated iTreg cells reduced the in vitro proliferation of allogeneic CD4+CD25−responder cells compared to the control iTreg cells , significant differences were masked in the higher suppressor/responder cell ratios among different conditions. In contrast to lower proportions of iTregs, the differences became more obvious and statistically significant.These results demonstrate that the addition of butyrate and propionate, but not acetate, augments the suppressive capacity of in vitro induced human Treg cells.+ non-Treg cells derived from CBMCs or PBMCs, cultured toward iTregs in the presence or absence of SCFAs and harvested on days 1 and 3 or day 3, respectively  might explain the controversies regarding the suppressive effectiveness of human iTregs [In the present study, we tested a human iTreg differentiation protocol applying the SCFAs acetate, butyrate, or propionate, in addition to the classically used IL-2 and TGF-β ,48,49,50n iTregs ,52,53, an iTregs .+ cells were unable to fully differentiate into Tregs, and there was a significant non-activated subpopulation with a smaller cell size. Among all of the culture conditions, butyrate-treated iTregs had the most non-activated cells, around 40% of the whole cultured cells. We speculated that this population was nonresponsive and did not share iTreg characteristics. Indeed, these cells had the minimal expression of all the phenotypic markers. We then compared the suppressive capacity between activated iTreg cells and their pair-matched whole cultured cells generated from adult naive CD4+ cells. As expected, the activated iTregs demonstrated a higher percentage of suppression compared to their non-activated counterparts. In butyrate-treated iTregs, these differences were much greater when the large proportion of non-activated cells was taken into account. Interestingly, this differential induction pattern was not present in the iTregs generated from CB naïve CD4+ cells. All naïve non-Tregs sorted from CB naïve CD4+ cells showed full differentiation into iTregs. These findings are further supported by the fact that the histone acetylation changes most probably at least in part resulting from SCFAs treatment were observed in CB cells earlier than in adult cells. This reasoning is limited by the lack of early time point epigenetic data for the latter. Nevertheless, when analyzing iTregs generated from CB naïve CD4+ cells and the activated, rather than the whole, iTregs generated from adult PB naïve CD4+ cells, we were able to reproduce the results of the relevant animal studies.In our study, naïve non-Treg cells isolated from adult CD4− T cells are truly “naïve” in adults, especially compared to CB. Human naïve T cells express CD45RA, but lack the expression of CD45RO, and the expression of at least one additional naïve T cell marker, such as CCR7, CD62L, or CD27, is necessary to distinguish naïve from terminally differentiated T cells [+ T cell pool tends to be maintained by both thymic output and peripheral homeostatic proliferation throughout adulthood [Considering the lack of methodological differences between our experiments conducted using cells deriving from CB and PB, the question arises as to whether CD45RO T cells ,55. The  T cells . Althougdulthood ,57, therdulthood . In addidulthood ,57,58,59dulthood . In addidulthood ,62. HoweENTPD1) encoding this molecule [Our epigenetic investigations demonstrated the possible mechanism underlying the SCFA-mediated augmentation of the differentiation towards human iTreg cells. Butyrate, propionate, and, to a lesser extent, acetate most probably at least partly enhanced histone acetylation at the important regulatory regions of several Treg-associated loci, thus increasing the accessibility of these genes to the transcriptional machinery ,64,65. Omolecule ,69,70. TAlong with the previous reports ,31,32, tIn conclusion, the successful SCFA-augmented in vitro generation of human iTreg cells and its mechanistic characterization reported here adds to the current knowledge on iTreg biology, creates a potentially useful tool for further research, and might boost the further development of Treg- and, considering the origin of natural SCFAs in human organisms, personalized nutrition-based therapeutic strategies against autoimmune, allergic, and other chronic inflammatory disorders.This research was reviewed and approved by the Human Research Ethics Committee (HREC) of Nepean Blue Mountains Local Health District according to the Declaration of Helsinki. All participants were recruited on a volunteer basis and signed a written informed consent prior to donating blood.To determine the effect of SCFAs on Tregs differentiation from naïve non-Treg cells, CBMCs were also used in the study in addition to adult PBMCs. Shielded through the placenta, CB is more genuinely “naïve” as it has limited environmental antigen exposure.In total, 13 healthy adult volunteers (average age of 29.15 ± 1.92 years old with an average of 48.09% naïve CD4 T cells in PBMCs) and 12 healthy term pregnant women were recruited. Individuals with any history of autoimmune diseases or allergies were excluded. Adult blood samples were drawn from the antecubital vein. CB samples were collected via the sterile aspiration of the umbilical vein from the delivered placenta immediately after an elective caesarean section (CS). All 12 CS were due to repeated CS, malpresentation or other non-medical issues. Women with gestational diabetes, chorioamnionitis, placenta previa, chronic villitis, or twin pregnancies were excluded. The overall design of the study is presented in All blood samples were processed within 2 h of collection. Human MCs were isolated from blood using Ficoll-Paque PLUS density gradient medium . Blood collected in heparinized vacuum tubes was diluted in a 1:1 ratio with sterile phosphate-buffered saline . This mixture was carefully layered over Ficoll-Paque PLUS medium at a 2:1 ratio before centrifugation at 1700 rpm for 30 min, without brake. The layer containing MCs was carefully aspirated and washed twice in PBS with 0.5% fetal calf serum  at 1300 rpm for 8 min. MCs were then re-suspended in PBS containing 0.5% FCS. After trypan blue-based counting, MCs were subjected to cell sorting.TM (Becton Dickinson) cell sorter. For Treg induction, the pre-enriched CD4+ T cells (PBMCs were labeled with a Miltenyi CD4+ T cell Isolation Kit #130-091-155 and separated on a Cell Separation Magnet) were further sorted for CD4+CD45RO−CD25−CD127hi with a purity of ≥99%.Freshly isolated cells were surface stained with the appropriate fluorochrome-conjugated antibody, including anti-CD127 , anti-CD45RO , anti-CD4 , and anti-CD25 . The cells were sorted on a BD FACSAria5/48 wells at 37 °C for 5 days under standard culture conditions . Recombinant human IL-2 , TGF-β , and, in selected cultures, sodium acetate , sodium butyrate , or sodium propionate  were added on day 0 and day 3. On day 5, the cells were harvested for phenotyping or further functional studies. The concentrations of SCFAs were chosen based on the immune cell viability (FACS-sorted naïve non-Treg cells of both adult and CB origins were cultured in cell culture medium (RPMI-1640 (Thermo Fisher Scientific) containing L-Glutamine , penicillin , streptomycin  plus 1% HEPES (Thermo Fisher Scientific), and 10% FCS in 48-well plates, with immobilized anti-CD3  and mobilized anti-CD28  for 3.5 h at 37 °C.+CD25+ Regulatory T Cell Isolation Kit . Carboxyfluorescein succinimidyl ester (CFSE) was used to trace cell proliferation. Responder cells were washed  three times in RPMI-1640 in the absence of FCS at room temperature. T cells were then incubated (labeled) with 0.5 µM CFSE at 37 °C for 7 min in the dark. The addition of 200 μL of FCS could terminate the reaction. Afterwards, the cells were washed 3 times with RPMI-1640 with FCS. Non-CFSE labeled iTregs  positive selection) and CFSE-labeled responder cells were co-cultured at a ratio between 1:1 and 1:16 and stimulated with Treg Suppression Inspector  in 96 U-bottom plates. The cells were cultured in medium for 3 days under standard culture conditions and subsequently harvested for flow cytometry. The percentage of suppression was determined as the difference in the proliferation between the negative control and the test sample, expressed as a percentage of the former.Allogenic responder cells for the suppression assay were enriched using a CD4Flow cytometric analyses using BD FACSVerse  were conducted for all of the cultured cells, and a LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit  was used to determine the viability of all of the cultured cells prior to the fixation and permeabilization required for intracellular antibody staining. Surface and intracellular staining was performed according to the manufacturer’s instructions. For the intracellular staining of Foxp3 and CTLA-4, a True-Nuclear™ Transcription Factor Buffer Set  was used. The antibodies used included CD4 , CD39 , Foxp3 , GITR , ICOS , PD-1 , PD-L1 , and CTLA-4 . The gating strategy is shown in .The acquired sample (fcs) files were analyzed in FlowJo Version10 . Consistent gating strategies were used for all samples in the same set of experiments. An isotype control was included to help with the gating. The mean fluorescence intensity (MFI) was calculated using the geometric mean.RPL32), separately for the mock (IgG) and H4 antibody. Then, the locus-specific percent enrichment to the input control obtained for IgG was subtracted from the corresponding values for the H4 antibody. Finally, the calculated IgG-corrected percent enrichment was divided for each gene into that of RPL32, resulting in a relative enrichment value [Chromatin fixation, isolation, shearing, and immunoprecipitation, as well as DNA de-cross linking and purification, were conducted using the established and thoroughly validated in-house method described elsewhere ,72,73. Fnt value ,72,74. Tnt value . All sampost hoc) were used. For all analyses, a p value of less than 0.05 was deemed statistically significant.Statistical analyses were performed in GraphPad Prism 9 . All of the data were not normally distributed due to the small sample size. Therefore, for comparisons among two or more groups, the Mann–Whitney test or Friedman test with Dunn’s multiple comparison test (


We report the case of a 34-year-old male patient, a bodybuilding trainer and user of anabolic androgenic steroids (AASs) for 16 years. He was found in cardio-respiratory arrest in his home. By performing a medico-legal autopsy, a severe form of COVID-19, aortic atherosclerotic plaques, and an old myocardial infarction was found. The SARS-CoV-2 RT-PCR test on necroptic lung fragments was positive, with a B.1.258 genetic line. The histopathological examinations showed microthrombi with endothelitis in the cerebral tissue, massive pulmonary edema, diffuse alveolar damage grade 1, pulmonary thromboembolism, hepatic peliosis, and severe nesidioblastosis. The immunohistochemical examinations showed SARS-CoV-2 positive in the myocardium, lung, kidneys, and pancreas. ACE-2 receptor was positive in the same organs, but also in the spleen and liver. HLA alleles A*03, A*25, B*18, B*35, C*04, C*12, DRB1*04, DRB1*15, DQB1*03, DQB1*06 were also identified. In conclusion, death was due to a genetic predisposition, a long-term abuse of AASs that favored the development of a pluriorganic pathological tissue terrain, and recent consumption of AASs, which influenced the immune system at the time of infection. Anabolic androgenic steroids (AASs) are widely used illicitly to improve body appearance and increase strength , but theThe aim of the study was to analyze the autopsy results of a young bodybuilder patient, who consumed AASs regularly and was affected by a severe form of COVID-19.Histopathology. For the histopathological examination of the organ fragments collected at autopsy, the samples were fixed in formaldehyde 7% for 5 days, after which the samples were oriented and placed in cassettes. Tissue processing was performed using a vacuum infiltration processor, Tissue-Tek VIP 5 Jr . Paraffin embedding and sectioning were performed using the Tissue-Tek TEC 6 system  and Accu-Cut SRM 200 Rotary Microtome . Slide staining was performed using the automated slide stainer Tissue-Tek Prisma Plus  according to the internal staining protocol, using Mayer Modified Hematoxylin  and Eosin solution (10 g Eosin B in 1000 mL distilled water).®, Granada, Spain) using ethylenediaminetetraacetic acid (EDTA), at pH  =  9, for antigen retrieval. For the immunohistochemical assessment, we used Anti-SARS Coronavirus NP Mouse anti-Virus antibody  at a 1:100 dilution, and anti-ACE antibody  at 1:100 dilution.Immunohistochemistry was performed automatically on 3-μm-thick sections of formalin-fixed and paraffin-embedded tissues with MD Stainer (Vitro Master DiagnosticaMicroscopic examination was performed by an experienced pathologist (D.G.) using an Olympus BX46 clinical microscope  with dedicated image acquisition camera and software. All sections were examined at 400× magnification.Molecular biology. During autopsy, 3 lung fragments were collected to perform the SARS-CoV-2 RT-PCR test. Total nucleic acids isolation was performed with MasterPure™ Complete DNA and RNA Purification Kit , according to the manufacturer’s instructions, using the protocol for total nucleic acids purification from tissue samples. A Pearl Nanophotometer  was engaged in determining the DNA and RNA concentration and purity. The RNA sample was amplified on a QuantStudio™ 5 RT-PCR System  after reverse transcription, using aSARS-CoV-2 Real-TM kit . This multiplex RT-PCR assay uses four simultaneous amplification reactions: those of the E gene region common for all SARS-like coronaviruses (FAM channel), the specific SARS-CoV-2 E gene (ROX channel) and the specific SARS-CoV-2 N gene (Cy5 channel), as well as the amplification of the nucleic acid sequence of the Internal Control-RNA (HEX channel). The assay also included a positive control of amplification (cDNA C+). The following program was used: reverse transcription for 20 min at 35 °C, initial denaturation for 5 min at 94 °C, 5 amplification cycles (10 s at 94 °C and 25 s at 64 °C) followed by 45 amplification cycles (10 s at 94 °C and 25 s at 64 °C with fluorescence detection).Using the initial solution of isolated total nucleic acids, the identification of SARS-CoV-2 variant was performed. RNA preparation and amplification were carried out in accordance with protocols published by the ARTIC network, using the V3 version of the ARTIC primer set from Integrated DNA Technologies  to create tiled amplicons across the SARS-CoV-2 genome. Libraries were prepared using the Nextera DNA Flex library preparation kit and MiSeq reagent cartridge V2 .Using the same initial solution, the identification of HLA-A, B, C, DRB1 and DQB1 was performed. HLA-FluoGene ABC kit and HLA-FluoGene DRDQ kit  were used according to the manufacturer’s instructions, based on the Sequence Specific Priming Polymerase Chain Reaction (SSP-PCR). DNA amplification was carried out on a G-Storm thermal cycler  and the mixture containing the extracted DNA sample was submitted to 40 amplification cycles (15 s at 96 °C and 60 s at 60 °C) after an initial denaturation step for 2 min at 95 °C. Detection of the PCR products was performed by measuring fluorescence signals on a FluoVista Analyzer , the endpoint fluorescence of the various fluorochromes before and after PCR was automatically calculated using the FluoGene analysis software.A 34-year-old patient, a fitness and bodybuilding trainer, was found by the ambulance in cardio-respiratory arrest in his home. The patient did not respond to resuscitation maneuvers. A medico-legal autopsy was requested to establish the causes of death.postmortem heteroanamnesis, the following information was recorded:-4 days before death, the patient presented infectious symptomatology with sudden onset ; about 15 min before death, the patient presented anxiety, obnubilation, severe dyspnea; death occurred suddenly;-3 days before the onset of the mentioned symptomatology, the patient encountered COVID-19-positive subjects in the gym room;-during the 4 days of illness, the patient self-medicated with paracetamol (3–4 tablets/day), ibuprofen 400 mg (1–2 tablets/day), aspirin in antiplatelet doses (2 tablets/day); the patient refused to be medically evaluated during the illness;-cardiovascular pathology was noted in the patient’s family history—essential arterial hypertension in both parents and polyglobulia (father);-the patient’s personal pathological history was essential hypertension grade II , controlled under monotherapy with bisoprolol 5 mg/day, frequent palpitations with the subjective perception of an irregular heart rhythm, tachycardia, visible apex shock in the left V intercostal space on the medio-clavicular line in orthostatism;-the patient had been constantly practicing strength sports (powerlifting and bodybuilding) for the past 16 years. To improve his performance, the patient had used AASs in a continuous cycle since the age of 18. In the last 6 months before his death, the patient had used Sustanon 250 mg/mL , Nandrolone decanoate 100 mg/mL , Trenbolone acetate 100 mg/mL alternatively with Methenolon enanthate (2 doses/week). The patient also used, more or less regularly, fast-acting insulin, growth hormones and derivatives , multivitamins, omega 3-6-9 fatty acids (3–4 capsules daily), linseed oil, high molecular weight carbohydrate powder, isolated protein powder 2 g/body kg/day, creatine, caffeine-based energy drinks. In the last 2–3 years, the patient was doing 5 bodybuilding workouts/week, without cardio type workouts, and his diet consisted of 3 main meals with a total nutritional value of less than 3000 kcal.From the Macroscopic autopsy findings. Necropsy was performed 24 h postmortem. On external examination—male corpse, 165 cm tall, 85 kg, BMI 31.22, marked muscle hypertrophy , violet color; on the pleuro-pulmonary surface, bilaterally, multiple reddish hematic suffusions, with a diameter of 0.1–0.2 cm, could be detected C. When pThe aorta showed numerous yellowish atherosclerotic plaques, raised on the surface of the intima E. The heThe results of the histopathological examination showed microthrombi with endothelitis (arrows) in cerebral tissue, on a cerebral edema background A, in pulResults of the immunohistochemical examinations are presented in the Molecular biology. The RNA test was positive for SARS-CoV-2, and the amplification of all three targets were observed: SARS-like coronaviruses gene , E gene  and N gene . The test for identification of SARS-CoV-2 variant indicated the genetic line B.1.258.The following HLA allele were identified: A*03, A*25, B*18, B*35, C*04, C*12, DRB1*04, DRB1*15, DQB1*03, DQB1*06.Genetic data. Data literature show that the HLA allele pairs identified by us in the studied patient correlate with severe forms of the disease (HLA-DRB1*04) [DRB1*04) , and proDRB1*04) . In a grDRB1*04) . One stuDRB1*04)  and anotDRB1*04) . InspectDRB1*04) ,11.The genetic line identified in our case, B.1.258, had been circulated in Central Europe since August 2020, long before the import of B.1.1.7 .Testosterone and COVID-19. Synthetic analogues of testosterone are the most widely used AASs [sed AASs .Regarding the correlation between testosterone levels and severe forms of COVID-19, a prospective study of 358 COVID-19 patients described a correlation between low serum testosterone and a poor prognosis, finding that low testosterone was linked to more severe forms of COVID -19, a need for intensive care and death . HoweverHowever, the link between androgen signaling and SARS-CoV-2 infectivity has been established and is believed to explain, at least in part, why men tend to have more severe forms of COVID-19 compared to women . AlthougAuthors believe that the thromboembolic phenomena of this case A,D are dTestosterone and immunity. Sexual dimorphism in immunity has been widely studied, and the consensus is that females have more active immune systems compared to males, meaning that they are better equipped to combat various infections, while at the same time being more susceptible to autoimmune diseases. This is a result of both hormonal and genetic factors, and their individual contributions could not be fully separated [eparated ,27. A reeparated .The effect of AASs use on immunity has not been widely studied. A small study on 13 healthy bodybuilders using AASs showed marginal reductions in serum IgG, IgA and IgM levels compared to non-AASs users . Some caWhile the influence of testosterone or AASs on the immune response requires further study, they both have immunosuppressive effects that may reasonably lead to poorer outcomes when fighting infection. Thus, the case studied had a low immune status at the time of infection.Histopathological changes. Among the cardiovascular disorders caused by AASs abuse, myocardial infarction is widely reported, and is most often triggered by atherosclerosis (clerosis F, increaclerosis G has beeReports of nesidioblastosis H in adulThe pulmonary histopathological changes observed B–D are aThe lack of laboratory examinations and antemortem radio-imaging investigations, as well as the lack of serum determination of the postmortem AASs concentration, represent limitations to our study.In the case analyzed, the severe form of COVID-19 was due to several factors: genetic predisposition, long-term abuse of AASs, which favored the development of a pluriorganic pathological tissue terrain, and recent consumption of AASs, which influenced the immune system at the time of infection. The main reason of death was COVID-19. Severe lung damage was followed by multiple organ failure and death.


Escherichia coli (ETEC) is one of the main causes of diarrhea in weaned piglets, and ornithine α-ketoglutarate (OKG) as a food supplement has been shown to improve intestinal immune status in animals and humans. However, it remains unknown whether OKG alleviates inflammation through the regulation of gut microbiota and its metabolites on ETEC-infected piglets. This study was conducted to explore the impact of OKG on growth performance, immunity, and ileal mucosa microbiota and its metabolites in piglets infected with ETEC. On a total of 40 pigs, a 2 × 2 factor design was performed; the major factors were diet  and challenge (E. coli or LB Broth). The results showed that ETEC-infection inhibited growth performance, and OKG supplementation alleviated growth performance. Interestingly, ETEC-infection increased the serum TNF-α and IL-6, decreased the serum IL-10, downregulated the mRNA expression of IL-1β, IL-6, MyD88, and improved the mRNA expression of IL-8, IL-18, and TLR4. OKG inhibited serum IL-6, suppressed the phosphorylation of downstream signals of NF-κB/JNK in the ileum, and enhanced serum IL-10 and ileum SIgA in ETEC-challenged piglets. OKG supplementation enhanced the mRNA expression of IL-1β and IL-10 and reduced NF-κB and MyD88 in the ileum. Importantly, OKG reversed intestinal microbiota dysfunction, including the diversity of ileal microbiota, the relative abundances of Actinobacillus, Turicibacter, and [Acetivibrio]_ethanolgignens_group, which significantly affected arachidonic acid metabolism and primary bile acid biosynthesis. Collectively, our results suggest that OKG improves growth performance, regulates immunity, and ileal mucosa microbiota and its metabolites in ETEC-infected piglets.Enterotoxigenic  Escherichia coli (ETEC), to invade or colonize the gut. ETEC is a universal intestinal inhabitant for diarrhea in young animals, resulting in significant growth retardation and economic losses in pig production due to severe diarrhea, morbidity, mortality, and impaired growth during weaning transition , increases the serum of glutamate, proline, aspartate, threonine, and branch chain amino acid levels, and alleviates growth-suppression induced by D-galactose chronic oxidative stress (Ornithine α-ketoglutarate (OKG) is a nutritional compound that consists of one molecule of α-ketoglutarate and two molecules of ornithine, a precursor of glutamine, arginine, and proline, which benefit the immune response by altering inflammatory processes during times of challenge –12. Moref chicks . Adminisf chicks . Interese stress . Dietarye stress . Also, Oe stress . The benn = 20/diet, NRC2012, n = 10/treatment group). Pigs in the ETEC and OKG ETEC received orally 10 ml (2 × 109 CFU/ml) of the enterotoxic strain of E. coli K88, respectively, whereas pigs in the CON and OKG groups were given an equal volume of LB Broth on day 15. The piglets were housed individually and had free access to diets and water. Three days after the challenge, all animals were euthanized. Samples of serum, ileum, ileal mucosa, and feces were immediately frozen at −80°C for analysis. All animal procedures were approved by the Animal Care Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences.Forty healthy piglets  weaned at 28 days were used. After 3 days of adaption, all pigs were fed a standard diet. Then, the pigs were randomly assigned to either a basal (CON) or 1% OKG , tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interleukin-10 (IL-10), interleukin-6 (IL-6), as well as the ileum level of secretory immunoglobulin A (IgA) were determined by a commercially available ELISA kit  according to the manufacturer’s instructions .Target–Ctβ –actin) treatment–(CtTarget–Ctβ –actin) control (mRNA was isolated from frozen liquid nitrogen and ground ileum tissues with TRIzol reagent. The expression of β-actin (house-keeping gene) and intestinal immune-associated genes were determined by RT-PCR according to our previous study –19. Prim control . RelativSequencing procedures and data analyses were performed by a commercial company . In brief, total genome DNA from ileal mucosa samples was extracted for amplification using the specific primer with the barcode (V3-V4 regions) , 21. Theg at 4°C for 20 min. The supernatants were further transferred to a new Eppendorf tube, centrifuged at 15,000 g at 4°C for 20 min, and injected into the LC-MS/MS system analysis   . Data we, China) .The detailed process of Western blot was described according to our previous study . The prit-test or factorial ANOVA and p < 0.05 was taken to indicate statistical significance (IBM SPSS statistics 20 software). The statistical model contained the effects of infect (LB or ETEC), diet , and their interaction. Data were expressed as mean ± standard error of the mean (SEM). The Pearson correlation analysis was used to measure the correlation between ileal mucosa microbiota and metabolites by using GraphPad Prism 7.Data in pigs were evaluated using independent samples p < 0.05). Similarly, ETEC-challenge significantly increased the feces score in pigs fed with the basal or OKG diet. Compared with the CON group, in the OKG group, the feces score was decreased after ETEC-infection (p < 0.05). Meanwhile, ETEC-challenge markedly increased the serum TNF-α and IL-6 and decreased the serum IL-10, while OKG treatment markedly increased the serum IL-10 and ileum SIgA and decreased serum IL-6 (p < 0.05). We further measured the ileal gene expression of ILs, TNF-α, nuclear factor kappa B (NF-κB), myeloid differentiation primary response gene 88 (MyD88), and transmembrane Toll-like receptors (TLR2 and TLR4) by RT-PCR. ETEC-challenge markedly downregulated the gene expression of IL-1β, IL-6, and MyD88 and improved the mRNA expression of IL-8, IL-18, and TLR4, whereas OKG treatment markedly improved the gene expression of IL-1β and IL-10 and downregulated the mRNA expression of NF-κB and MyD88 (p < 0.05).The growth performance, diarrhea, serum, and small intestinal immunity are shown in p < 0.05). Dietary supplementation OKG treatment or not presented a clear separation clustering of microbial community composition in beta-diversity . OKG ETEC groups had a higher proportion of Proteobacteria, but a lower proportion of Firmicutes (p < 0.05). At the order levels, Lactobacillales were decreased in relative abundance by ETEC-challenged pigs (p < 0.05). OKG treatment markedly increased the proportion of Enterobacterales but decreased the proportion of Peptostreptococcales-Tissierellales. Similar alterations were observed for Actinobacillus, [Acetivibrio]_cthanolgignens_group, and Turicibacter at the genus level (p < 0.05).The overall microbial composition at the phylum, order, and genus levels is presented in p < 0.05, p < 0.05).Phylogenetic Investigation of Communities by Reconstruction of Unobserved States analysis showed that ETEC treatment markedly increased ribosome and mitochondrial biogenesis  in the four pairwise comparisons . Compared with the OKG ETEC group, the main metabolic pathways in the OKG group that were enriched are beta-alanine metabolism, bile secretion, glutathione metabolism, protein digestion and absorption, and arginine and proline metabolism, and the main metabolic pathways in the ETEC group that were enriched are taurine and hypotaurine metabolism (p < 0.05).Metabolome analyzed a total of 156 positive ions and 39 negative ions with functional annotations. Among them, 21 (20 up and 1 down), 50 (13 up and 37 down), 53 (30 up and 234 down), and 71 (47 up and 24 down) metabolites with functional annotations were significantly different (p < 0.05). Compared with the OKG ETEC group, sulfoacetic acid and hypotaurine were clearly increased in the ETEC pigs, and spermidine, tyramine, cholesterol, L-histidine, cadaverine, serotonin were significantly increased, while spermine and D-proline were markedly decreased in the OKG group (p < 0.05).Based on the comparison of the data with KEGG, 14 identified endogenous metabolites were characterized. These metabolites were ergocalciferol, leukotriene C4, 16(R)-HETE, thromboxane B2, chenodeoxycholic acid, sulfoacetic acid, hypotaurine, spermidine, tyramine, spermine, cholesterol, L-histidine, cadaverine, serotonin, and D-proline. Among these metabolites, ergocalciferol was increased significantly in the ETEC pigs, and leukotriene C4, 16(R)-HETE, thromboxane B2, chenodeoxycholic acid were increased clearly in the OKG pigs compared to the controls (r) between metabolites and microbiota are shown in p < 0.05): cadaverine vs. Actinobacillus . The results indicated that OKG treatment suppressed the phosphorylation of JNK and p38 -HETE, thromboxane B2, and chenodeoxycholic acid. Leukotriene C4, 16(R)-HETE, and thromboxane B2, a product of arachidonic acid, can improve anti-inflammation, enhance intestinal health, and cure disease –55. For Actinobacillus, [Acetivibrio]_ethanolgignens_group, and Turicibacter. Further studies with the use of OKG in healthy animals and clinical application are anticipated.In conclusion, dietary supplementation with 1% OKG regulates serum levels of TNF-α, IL-6, and IL-10, alters the genes expression related to ileal immunity, and alleviates the growth-suppression of piglets infected by ETEC, which may be associated with positive alterations in the composition of gut microbiota and modulation in the gut metabolites, especially for PRJNA808781.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: NCBI, The animal study was reviewed and approved by Committee on Animal Care of the Institute of Subtropical Agriculture, Chinese Academy of Sciences . Written informed consent was obtained from the owners for the participation of their animals in this study.YL and KY conceived the study. YL wrote the original draft of the manuscript. All authors have contributed to the development of the methodology, design of the study, reviewed, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Th and Ddc expression. MiR-551b-5p regulated the expression of a variety of genes including Slc18a2 and Igfbp3. The upregulation of Pcsk9 expression in HFD intervention on AD mice might be closely related to the increase of cholesterol in brain tissues, while Huanglian Jiedu Decoction significantly downregulated the expression of Pcsk9. Our data showed the close connection between the alterations of transcriptome and translatome under the effect of HFD, which emphasized the roles of translational and transcriptional regulation were relatively independent. The profiled molecular responses in current study might be valuable resources for advanced understanding of the mechanisms underlying the effect of HFD on AD.Different studies on the effects of high-fat diet (HFD) on Alzheimer’s disease (AD) pathology have reported conflicting findings. Our previous studies showed HFD could moderate neuroinflammation and had no significant effect on amyloid-β levels or contextual memory on AD mice. To gain more insights into the involvement of HFD, we performed the whole-transcriptome sequencing and ribosome footprints profiling. Combined with competitive endogenous RNA analysis, the transcriptional regulation mechanism of HFD on AD mice was systematically revealed from RNA level. Mmu-miR-450b-3p and mmu-miR-6540-3p might be involved in regulating the expression of   Alzheimer’s disease (AD) was a progressive neurodegenerative disease related to aging, characterized by the pathological hallmarks of extracellular accumulation of amyloid-β (Aβ) plaques and intracellular accumulation of neurofibrillary tangles. AD was caused by the complex interaction of multiple mechanisms, and the etiology was still unclear. AD was generally considered to be related to genetic and environmental factors . Diet anHuanglian Jiedu Decoction (HLJDD) was composed of Rhizoma coptidis, Radix scutellariae, Cortex phellodendri, and Fructus gardenia at a ratio of 3:2:2:3. HLJDD was a classic prescription for clearing away heat and toxic materials in past dynasties. Alkaloids, flavonoids and iridoid glycosides were the mainly active ingredients in the prescription . Modern ABCA7, BIN1, CASS4, CD33, CD2AP, CELF1, CLU, CR1, DSG2, EPHA1, FERMT2, HLA-DRB5-DBR1, INPP5D, MS4A, MEF2C, NME8, PICALM, PTK2B, SLC24H4 RIN3, SORL1, ZCWPW1, PLD3, and TREM2 (Genome-wide association studies (GWAS) identified the following genes associated with AD risk: nd TREM2 . The locnd TREM2 . APP, PSnd TREM2 , while And TREM2 , in addind TREM2 . RNA seqMRAK088596, MRAK081790, and MAPK10 and downregulation of BC092582, MRAK050857, and S100A8 in AD rats (Competing endogenous RNAs (ceRNAs) were RNAs in the complex network of transcriptional regulation in organisms, including protein-coding mRNA, long non-coding RNA (lncRNA), pseudogene, and circular RNA (circRNA). The regions of these RNAs could be bound by systematically functionalizing microRNA (miRNA) response element (MRE)-harboring non-coding RNAs. Competing to bind common miRNAs through common MREs, the RNAs interacted and regulated the expression of target gene transcripts. Thus, through the miRNA, these RNAs could interact with each other to form complex miRNA-mediated ceRNA networks. The interaction relationship showed the possible functions of the lncRNAs and circRNAs. Significant changes in lncRNA were also observed in AD models, with studies reporting the upregulation of  AD rats . CircRNA AD rats  and play AD rats . MiRNAs  AD rats .in vivo translation and coupled, translational events (SwDI/NOS2–/–) and Tg2576 (APPSw), by tandem RNA-seq and Ribo-seq. AD model mice had similar levels of transcriptome regulation, but differences in translatome regulation , in which next-generation sequencing used to identify ribosome-protected mRNA fragments, thereby revealing the positions of the full set of ribosomes engaged in translation, has emerged as a transformative technique for enabling global analyses of l events . Ribo-sel events –25. The gulation .Previously, we detected that long-term HFD intervention altered the levels of cholesterol and polyunsaturated fatty acids in the brain tissue of APP/PS1 mice and influenced the secretion of peripheral bile acids . Translaad libitum and a 12 h light-dark cycle and kept under a consistent temperature of 21°C. Wild type mice were assigned to the normal group  and the Nor_HFD group (fed a HFD diet). The HFD diet contained 63.6% basic feed, 15% lard, 20% sucrose, 1.2% cholesterol, and 0.2% cholate . APP/PS1 mice were randomly allocated into 3 groups: one was fed a normal chow diet (the AD group), and one was fed a HFD diet (the AD_HFD group), and another was fed HFD diet and the powder of HLJDD (the H_H group). The HLJDD powder was prepared in our laboratory as previously described  transgenic mice and C57BL/6J wild type mice  were used in this study. All experiments and animal care in this study were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals  and the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation. The study was approved by the Institutional Animal Care and Use Committee of the Beijing animal science Co., Ltd., and the animal ethics approval number was IACUC-2018100605. Animals were housed in a single cage with chow and water The Morris water maze (MWM) test was performed to detect spatial memory as previously described with a slight modification . Mice paAfter the MWM test, all mice rested for 4 days under normal conditions. After anesthesia with 10% chloral hydrate, serum was collected from the heart, followed by removal of brain tissue on a sterile table, rinsing with pre-cooled RNAse-free saline at 4°C, blotting up. Then put the sample into 1.5 mL labeled RNase-free EP tubes, which were rapidly frozen in liquid nitrogen for 30 min and stored at −80°C in the refrigerator until use. Whole brain had been ground in liquid nitrogen.Western blot (WB) analysis for brain tissues were lysed in precooled RIPA buffer with the protease inhibitor PMSF (Amresco), and protein concentrations were determined using a BCA protein assay kit. Protein samples were separated on 12% sodium dodecyl sulfate polyacrylamide gels electrophoresis (SDS-PAGE) and transferred onto NC membranes. Then, membranes were blocked in 5% non-fat milk for 30 min at room temperature and incubated with primary antibodies overnight at 4°C. Membranes were then washed and incubated with HRP conjugated goat anti-rabbit and HRP-conjugated goat anti mouse  secondary antibodies for 40 min at room temperature followed by development using ECL detection. The obtained bands were then scanned and analyzed using ImageJ software, and band density was assessed using Total Lab Quant V11.5 .Total RNA was extracted from brain tissue using TRIzol reagent  according to the manufacturer’s instructions. RNA concentrations were equalized and converted to cDNA using the EntiLink™ 1st Strand cDNA Synthesis Kit . Gene expression was measured using a StepOne™ Real-Time PCR system. The sequences of primers used in these experiments were listed in the The experimental procedure and data analysis were listed in the http://bigd.big.ac.cn/gsa/, CRA007307.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 here: t-test for two groups and one-way ANOVA for more than two groups, followed by the LSD and Games-Howell post-test. Statistical calculations were performed using SPSS 20 software. Differences with statistical significance were denoted by P-value less than 0.05.The results were expressed as the mean ± standard error of the mean (SEM). The significance of difference among the groups was assessed by Student’s P < 0.05). The platform crossing number, percent distance and time spent in the target quadrant in the AD_HFD group were higher than those in the AD group, indicating that it had improvement tendency toward the cognitive impairment with the HFD intervention. There is no significant difference between the H_H group and AD group . The expression of TNF-α and IL-1β was decreased in the AD_HFD and H_H groups compared to the Nor group, and IL-1β levels was significantly reduced (P < 0.001)  . Taken tR-values of Nor, AD, AD_HFD, and H_H groups were 0.62, 0.63, 0.65, and 0.7, indicating a moderate correlation between mRNA abundance and Ribo sequence abundance in four groups. Principal component analysis (PCA) of mRNA-seq and Ribo-seq were shown in At the mRNA sequence profiling, 18490 detected genes were identified. At the Ribo sequence profiling, 17433 detected genes were identified. The gene expression levels for both the transcriptome and the translatome were similar with normal distribution. The distribution of expression abundance among the samples was shown in P < 0.05 and | log2FC| ≥ 0.585 as significant differentially expressed genes (DEGs). 87 genes were up-regulated and 125 genes were down-regulated in the AD group compared to the Nor group. Compared to the AD group, 116 and 120 genes were up- and down-regulated in the AD_HFD group, respectively. 30 genes were significantly differentially expressed in both AD and AD_HFD groups compared to the Nor group. 13 of them were reduced and 16 were significantly increased in AD mice. In addition, Gpr151 was reduced in the AD group but increased in the AD_HFD group. The volcano plot and Wayne plot of differentially expressed genes between the groups were shown in Based on the HISAT2 comparison results, we reconstructed the transcripts using StringTie and calculated the expression of all genes in each sample. Using the reads count data of gene expression levels of each sample, we analyzed the difference between groups using DESeq2 software with KEGG analysis (Nor vs. AD group) identified the significant enrichment pathways including apoptosis, MAPK signaling pathway, neuroactive ligand-receptor interaction, purine metabolism, dopaminergic synapse, serotonergic synapses, etc. The AD and AD_HFD groups were significantly enriched in the KEGG at dopaminergic synapse, synaptic vesicle cycle, cholinergic synapse, estrogen signaling pathway, neuroactive ligand-receptor interactions, MAPK signaling pathway, TNF signaling pathway, galactose metabolism, serotonergic synapse, starch and sucrose metabolism . DEGs amThe results of GO enrichment analysis results were shown in Dusp1, Gpr151, Th, Ddc, and Npas4 were upregulated, while Ccl21b and Slc1a1 were downregulated in the AD_HFD group compared with the AD group. Dual specific phosphatase (DUSP) played an important immunomodulatory function through the DUSP-MAPK phosphatase pathway  was a catecholamine rate-limiting enzyme that catalyzed the conversion of tyrosine to dihydroxyphenylalanine and regulated the production of dopamine, noradrenaline and epinephrine neurotransmitters. Aromatic L amino acid decarboxylase (DDC) catalyzed the conversion of dopa to dopamine. Both were more highly expressed under HFD, indicating that HFD intervention would strengthen the dopamine neurotransmitter synthesis in the brain tissue of AD mice. Neuronal PAS domain binding protein 4 (NPAS4) mRNA was upregulated in the AD_HFD group, suggesting that HFD could enhance the regulation of glutamatergic and GABAergic synapses. In addition, GPR151 was associated with pineal synaptic function and nicotinic uptake. pathway . DUSP1 p pathway . IncreasP < 0.05 were shown in In order to further understand the effect of HFD in AD, GSEA analysis was performed. GSEA analysis of KEGG revealed that 94 of the 324 gene sets were upregulated in the AD group compared to Nor group, including glycosphingolipid biosynthesis-ganglio series; 9 gene sets were downregulated in the AD group, including the specific pathways of folate biosynthesis, antigen processing and presentation, phenylalanine metabolism, tryptophan metabolism, alanine metabolism, ascorbate and aldarate metabolism, DNA replication, GPI-anchor biosynthesis, and carbon metabolism. 170 gene sets were upregulated in the AD_HFD group compared to AD group, while 154 gene sets were downregulated in the AD_HFD group. The results of the GSEA analysis with Differential genes in AD patients were mainly enriched in immune and metabolic pathways . Meta-anSlc6a3, Chrnb4, Fos, Hspa1b, Igfbp3, Gm45837, Dusp1, Slc18a2, Hspa1a, Itk, Gucy2c, Wnt9b, and Chrna6.A total of 8 patterns of gene trends among the Nor, AD and AD_HFD groups were plotted , with prSlc6a3 encoded the dopamine transporter and its variant carriers reduced cognitive performance and were at greater risk of developing dementia  inflammation  gene family, bile acid biosynthesis, transport, secretion and metabolism were screened . Low expPcsk9, a cholesterol transport-related gene, was significantly decreased in the H_H group. PCSK9 promoted low density lipoprotein (LDL) degradation. The upregulation of Pcsk9 expression in the AD_HFD group might be closely related to the increase in cholesterol in brain tissues, while HLJDD significantly downregulated Pcsk9 expression. The expression of Slc10a4, a bile acid transport-related gene, was significantly reduced in the AD group and significantly increased in the AD_HFD group and significantly reduced in the H_H group. SLC10A4 was a family of bile acid sodium cotransport proteins that were activated by proteases to transport bile acids  was non-synonymous SNV, while the main variants location of SNP was in intronic. The SNP mutation types were transition (80.22%) and transversion (19.78%). A->G in transition accounted for the largest proportion. G->T in transversion accounted for the largest proportion. Among the analysis of alternative splicing, skipped exon accounted for the most in four groups .Differential translation genes (DTGs) between groups were performed using edgeR software. Compared to the Nor group, 336 DTGs were significantly up-regulated and 881 DTGs were down-regulated in the AD group; 603 and 194 DTGs were significantly up- and down-regulated in the AD_HFD group compared to the AD group, respectively; while H_H resulted in 851 and 292 DTGs being significantly up- and down-regulated, respectively.Zbtb16 and Tmem121b, all genes were significantly modulated by HLJDD.A total of 382 differentially translated genes were co-varied among the Nor, AD and AD_HFD groups, of which 59 were up-regulated and 323 down-regulated in the AD group, while the HFD intervention significantly back-regulated changes in translated gene expression in the AD group. A total of 201 differentially translated genes were co-varied among AD, AD_HFD and H_H groups, 90 translated genes were down-regulated and 111 translated genes were up-regulated in the AD_HFD group, respectively. Except for Sgk1, Myo1f, Oip5, and Cst7 were both up-regulation; Iqschfp, Gm45837, Itga2b, Alb, Npas4, Fos, Ccn1, and Dusp1 were both down-regulation; Npy, Ptchd4, Clcc1, Thbs4, and Cdh12 were up-regulation in transcriptome and down-regulation in translatome; Grid2ip, Gucy2c, Th, Eva1a, Ngb, Slc10a4, Hs3st3b1, and Hspb1 were down-regulation in transcriptome and up-regulation in translatome. Homodirectional genes were enriched in learning, memory, cognition, regulation of cell death, response to lipid, response to cAMP, negative regulation of p38MAPK cascade, negative regulation of microglial cell activation, nervous system development and regulation of neuroinflammatory response. The pathways of homodirectional genes were significantly enriched in fluid shear stress and atherosclerosis and MAPK signaling pathway. Gene ontology-biological process (GO-BP) of opposite genes were enriched in neuron development, negative regulation of response to oxidative stress, neuron differentiation and regulation of cellular response to oxidative stress. The pathways of opposite genes were enriched in tyrosine metabolism, VEGF signaling pathway, regulation of lipolysis in adipocyte, adipocytokine signaling pathway and dopaminergic synapse.There were 212 DEGs and 1217 DTGs between the Nor and AD groups, and 25 genes that changed at both levels. The combination of transcriptional and translational analysis revealed that Ecm1, Reep4, and Cmtm3 were both up-regulation; Gbp5, H1f3, and H1f4 were both down-regulated; Sspo, Hoxb5, and Ccm2 were up-regulation in transcriptome and down-regulated in translatome; Slc1a1, Glt8d2, Serinc2, Cd34, C1ra, Thbs4, Lct, Gm45208, Ltf, and Cnpy1 were down-regulated in transcriptome and up-regulated in translatome. The GO-BP of homodirectional genes had function at cellular process and positive regulation of biological process. H1f3, H1f4 were closely associated with histone modification. The pathways of homodirectional genes were significantly enriched in nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway. The GO-BP of opposite genes had function at metabolic process, cellular process, biological regulation and developmental process. Ccm2, Cd34, Thbs4, and Slc1a1 were closely associated with blood vessel development. Opposite genes were enriched in phagosome, galactose metabolism, carbohydrate digestion and absorption, synaptic vesicle cycle.There were 236 DEGs and 797 DTGs between the AD and AD_HFD groups, and 19 genes that changed at both levels. The combination of differences based on transcriptional and translational analysis revealed that Alms1, Lcmt2, Ryr3, and Ppp1r10 were both up-regulated; Zfp968, Ccn1, Npas4, Fos, Dusp1, and Mpeg1 were both down-regulated; C1ra, Mpp4, and Thbs4 were up-regulated in transcriptome and down-regulation in translatome; Otof, Abl2, Glra1, Hoxb5, and Nrap were down-regulated in transcriptome and up-regulated in translatome. The GO-BP of homodirectional genes were enriched in response to endogenous stimulus, learning, positive regulation of ceramide biosynthetic process, regulation of ceramide biosynthetic process, regulation of metabolic process, response to lipid and cognition. The pathways of homodirectional genes were significantly enriched in MAPK signaling pathway, Th1 and Th2 cell differentiation, IL-17 signaling pathway, TNF signaling pathway and dopaminergic synapse. The GO-BP of opposite genes were enriched in endothelial cell-cell adhesion, negative regulation of transmission of nerve impulse and behavior. The pathways of homodirectional genes were significantly enriched in ErbB signaling pathway and ECM-receptor interaction  of each gene was calculated. It exhibited a very weak correlation between TE and transcription abundance in four groups. 63 genes between the Nor and AD groups were significantly different at TE and transcription and had the opposite trends (opposite). The opposite genes were mainly enriched in the aspects of aging, neurotransmitter loading into synaptic vesicle, response to endogenous stimulus and dopamine metabolic process terms. The pathways were significantly enriched in cocaine addiction, amphetamine addiction, alcoholism, tyrosine metabolism, dopaminergic synapse and caffeine metabolism.In total of 62 genes between the AD and AD_HFD groups were opposite. The genes were mainly enriched in neurotransmitter loading into synaptic vesicle, aminergic neurotransmitter loading into synaptic vesicle, response to nicotine, neurotransmitter transport, regulation of neurotransmitter levels terms in biological process ontology. The pathways were significantly enriched in neuroactive ligand-receptor interaction, dopaminergic synapse, synaptic vesicle cycle, alcoholism, and tyrosine metabolism.Zfp968 and Ccn1 between the AD_HFD and H_H groups were significantly different at both levels and had the same tendency. 60 genes between AD_HFD and H_H groups were opposite. The genes were mainly enriched in skeletal system morphogenesis, embryonic skeletal system morphogenesis and neuropeptide signaling pathway terms. The pathways were significantly enriched in neuroactive ligand-receptor interaction, galactose metabolism, carbohydrate digestion and absorption.P < 0.05 and | log2FC| ≥ 0.585 were screened as significant DEGs. According to the screening criteria, 114 or 137 were up- or down-regulated differentially expressed lncRNAs (dif-lncRNAs), 154 or 159 were up- or down-regulated differentially expressed circRNAs (dif-circRNAs), 11 or 5 were up- or down-regulated differentially expressed miRNAs (dif-miRNAs) in the AD group compared to the Nor group. There were 126 and 138 dif-lncRNAs, 193 and 202 dif-circRNAs, 17 and 4 dif-miRNAs up and down regulated in the AD_HFD group compared to the AD group. 150 and 129 dif-lncRNAs, 174 and 207 dif-circRNAs and 7 and 29 dif-miRNAs up and down regulated in the H_H group compared to the AD_HFD group  to obtain potential ceRNA pairs, and then screened for ceRNA pairs with Igfbp3 (ENSMUSG00000020427), Slc18a2 (ENSMUSG000000 25094), Tmem265 (ENSMUSG00000106715), Gbx2 (ENSMU SG00000034486), Lhx9 (ENSMUSG00000019230), ENSM UST00000219444, and MSTRG.3992.1. Mmu-miR-211-5p was competitively integrated by Lhx9 (ENSMUSG00000019230), Tmem26 (ENSMUSG00000060044), Tmem265 (ENSMUSG 00000106715), Gbx2 (ENSMUSG00000034486), ENSMUST000 00219444, and MSTRG.3992.1. Mmu-miR-6540-3p was competitively integrated by Slc18a2 (ENSMUSG00000025094), Lhx9 (ENSMUSG00000019230), Ddc (ENSMUSG00000020 182), novel_circ_014145, novel_circ_000302, ENSMUST0000 0129510, ENSMUST00000227816. Mmu-miR-221-3p was competitively integrated by Chrna6 (ENSMUSG00000031491), Shox2 (ENSMUSG00000027833). Pou4f1 (ENSMUSG0 0000048349) was regulated by mmu-miR-200a-3p, mmu-miR-182-3p. Mmu-miR-450b-3p was competitively integrated by Th (ENSMUSG00000000214), Lhx9 (ENSMUSG00000019230), Abhd17b (ENSMUSG00000047368), novel_circ_002080, ENSMUST00000129204, MSTRG.3992.1  was a family of proteins with high affinity for insulin-like growth factor (IGF). IGF-1 and IGFBP-3 were associated with oxidative stress and longevity , MSTRG.3992.1 in the ceRNA network were competed with Igfbp3 to bind mmu-miR-551b-5p. In addition, Pou4f1 in the competition network inhibited neuronal apoptosis, Slc18a2 negatively regulated neurotransmitter transport, Shox2 and Irx5 were associated with neurodevelopment, and the transmembrane protein TMEM also played an important role in human immune-related diseases as well as tumor development  can be found here: The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the Beijing Animal Science Co., Ltd., and the animal ethics approval number was IACUC-2018100605.WG, JZ, XF, LW, and XG performed the experiments. WG analyzed the data and wrote the original manuscript. YZ and HZ revised the manuscript. NS and HW contributed to the work. BB and HZ conceived and designed the experiments. All authors reviewed the manuscript and approved the submitted version.


As one of marine tetrahydroisoquinoline alkaloids, renieramycin T plays a significant role in inhibiting tumor metastasis and invasion. However, the effect of renieramycin T on inflammation-related tumor metastasis and invasion is still unknown, and its mechanisms remain unclear. Here we established an inflammation-related tumor model by using the supernatant of RAW264.7 cells to simulate B16F10 mouse melanoma cells. The results indicate that renieramycin T suppressed RAW264.7 cell supernatant-reduced B16F10 cell adhesion to a fibronectin-coated substrate, migration, and invasion through the matrigel in a concentration-dependent manner. Moreover, Western blot results reveal that renieramycin T attenuated the phosphorylation of STAT3 and down-regulated the expression of Nrf2. Together, the above findings suggest a model of renieramycin T in suppressing B16F10 cancer cell migration and invasion. It may serve as a promising drug for the treatment of cancer metastasis. A malignant tumor has a higher metastatic capacity; the way of metastasizing includes tissue, blood and lymph, etc. . MelanomThe tumor microenvironment comprises a variety of nonmalignant stromal cells that play a pivotal role in tumor progression and metastasis . Among tAs a transcription factor, NF-E2-relatedfactor 2 (Nrf2) mediate tumor progression via accelerated tumor proliferation ,13,14. IIt is reported that aberrant STAT3 activation in tumor cells is associated with cell proliferation, cell survival, invasion, angiogenesis, and metastasis ,21,22,23Xestospongia sp., for this study, was self-synthesized by Dr. Xiaochuan Chen from Sichuan University  were analyzed using the GraphPad Prism software . Details of each statistical analysis used are provided in the figure legends.In conclusion, we established an inflammation-related tumor model by using the supernatant of RAW264.7 cells to simulate B16F10 mouse melanoma cells. We showed that RT suppressed RAW264.7 cell supernatant-reduced B16F10 cell adhesion to fibronectin-coated substrate, migration, and invasion through the matrigel in a concentration-dependent manner. We explored the underlying mechanism and demonstrated that RT attenuated the phosphorylation of STAT3 and down-regulated the expression of Nrf2. The above findings suggest a model of RT for suppressing B16F10 cancer cell migration and invasion. It may serve as a promising drug for the treatment of cancer metastasis.