Drosophila, canonical dosage compensation is implemented by the male-specific lethal (MSL) complex functioning in all male somatic cells. This complex contains acetyl transferase males absent on the first (MOF), which performs H4K16 hyperacetylation specifically in the male X chromosome, thus facilitating transcription of the X-linked genes. However, accumulating evidence points to an existence of additional, non-canonical dosage compensation mechanisms operating in somatic and germline cells. In this review, we discuss current advances in the understanding of both canonical and non-canonical mechanisms of dosage compensation in Drosophila.Dosage compensation equalizes gene expression in a single male X chromosome with that in the pairs of autosomes and female X chromosomes. In the fruit fly  Drosophila melanogaster with abnormal chromosome ploidy have shown that for several X-linked genes analyzed, amount (per cell) of the protein product in 3X;2A metafemales was almost the same as in 2X;2A  females and in XY;2A  males [Since heterogametic males of mammals and of most insects have an XY karyotype while females have an XX karyotype, there is a problem of imbalance of X-chromosomal gene expression between the two sexes. To eliminate this imbalance, special mechanisms of dosage compensation (DC) have emerged to ensure equal expression of X-linked genes in males and females. Early studies in l) males ,2. Howevl) males ,3,4,5,6.l) males ,8,9,10. l) males . The imbDrosophila, this twofold activation is partially mediated by the male-specific lethal (MSL) complex, which specifically binds to the male X chromosome and performs its H4K16 hyperacetylation, thus promoting transcription of the X-linked genes [Drosophila cells [Drosophila have been published recently [Drosophila, focusing on the hypotheses explaining canonical and non-canonical mechanisms.In different organisms, DC is realized via somewhat similar principles . In mammed genes ,21,22,23ed genes ,25. Moreed genes , but pared genes ,28,29,30ed genes . Finallyed genes ,32. Basela cells ,34,35. Arecently ,37,38, tDrosophila MSL complex were initially identified in the genetic screening for male-specific lethality [roX1 or roX2 long non-coding RNAs [roX RNAs as well as JIL-1, CLAMP, and TopoII are localized very close to each other on the male X chromosome [The components of ethality ,39. Lateing RNAs ,48,49,50ing RNAs ,54,55,56romosome . Thereforomosome ,59.roX1 and roX2 RNAs are functionally nearly redundant since double mutations of roX1 and roX2 genes cause male lethality and loss of coverage by the MSL complex of the male X chromosome, whereas mutations of either roX1 or roX2 genes are phenotypically neutral [roX1 or roX2 RNAs affects expression of different gene sets, thus showing their incomplete interchangeability [roX RNAs into the MSL complex [Brief characteristics of the core MSL complex components are provided below. The  neutral ,48,49,60eability . MSL1 ineability ,63,64. Meability ,66,67. Deability ,69. MLE eability  necessar complex ,71. MOF  complex ,19,21. A complex ,46,72,73 complex .msl3, mle, and mof null and roX1 and roX2 double-null mutant males retain occupancy of these sites by the MSL1-MSL2 subcomplex [The current model explaining the delivery of the MSL complex to virtually all expressed genes on the male X chromosome includes at least two stages . At the bcomplex ,77,82. Ebcomplex ,77, yet bcomplex . This rabcomplex ,73. Recebcomplex . Moreovebcomplex . Therefobcomplex ,86. Howebcomplex ,86. Yet,bcomplex . Hence, bcomplex ,88. Morebcomplex . Therefobcomplex ,88. In abcomplex ,89.roX1 and roX2 gene positions on the X chromosome coincide with two HAS/CES, roX RNAs were supposed to be incorporated in the incomplete MSL complex at the sites of their nascent transcription. Apart from this, roX RNAs may be incorporated in the MSL complex in the nucleoplasm (before it is bound with chromatin), or already on chromatin of other HAS/CES [roX RNAs, making them accessible for interactions with the MSL2 subunit [roX RNAs in the MSL complex triggers its spreading from HAS/CES to the nearby regions [roX transgenes create new HAS/CES at the sites of insertions from which the MSL complex spreads bi-directionally at hundreds of kilobases into autosomal chromatin [roX RNAs were involved in this process. Recent work has shed light on this puzzle. It was found that MSL2 contains intrinsically disordered regions (IDRs) at the C-terminus, which are able to form phase-separated droplets with either roX2 or, less efficiently, with roX1 RNAs [roX RNAs, MSL2, and other MSL subunits within these droplets, which envelope HAS/CES and nearby chromatin, may facilitate binding of the MSL complex to the low-affinity sites and its further spreading to H3K36me3-modified chromatin of active genes. Additionally, the acetyltransferase activity of MOF is necessary for the MSL spreading, since the mof mutation specifically eliminating this activity also blocks the spreading [Since  HAS/CES ,75,90. F subunit ,93,94,95 regions ,71,75. FoX1 RNAs . The incroX RNAs in the initial binding of the MSL1–MSL2 subcomplex to HAS/CES [roX RNA involvement in the MSL targeting HAS/CES is that the replacement of the C-terminal domain of Drosophila MSL2, carrying IDRs, with its counterpart from mouse MSL2, lacking IDRs, results in the loss of binding of the hybrid MSL complex to HAS/CES [Drosophila MSL2 also contained the CLAMP-binding domain, which participates in the cooperative recognition of HAS/CES by MSL2 and CLAMP [roX2 expression level and MSL2 occupancy at HAS/CES [roX2 overexpression partially restored the MSL complex recruiting to the X chromosome upon simultaneous mutations in the CXC and CLAMP-binding domains of MSL2 [roX RNAs may enhance MSL2 binding to HAS/CES has gained support in the studies of MSL targeting the male X chromosome in D. virilis [D. melanogaster, while roX RNAs are more instructive in specifying MSL2-binding sites. The authors suppose that interactions between roX RNA and MSL2 may allosterically modulate MSL2 conformation, thus enhancing MSL2 binding capacity towards HAS/CES.There are somewhat contradictory results from different groups regarding the involvement of  HAS/CES ,96. One nd CLAMP ,88; this of MSL2 . The ide virilis . In thisroX2 transgene insertion by ~2.6 Mb, with no detectable binding within a ~1.7-Mb region in between these sites [Recent works analyzing genome architecture have uncovered additional layers of regulation of the MSL complex recruiting and spreading within the male X chromosome ,101,102.se sites . Furtherse sites . These dA more detailed analysis of genome architecture in sex-sorted embryos revealed some differences in the X chromosome organization in males and females ,101. TheDrosophila males differs from autosomes and from X chromosomes of females by a drastic increase in H4K16 acetylation [mof gene demonstrate the male-specific lethality [msl mutants, thus pointing to the causative role of MOF and H4K16 acetylation in the canonical DC. Finally, upon lack of maternally supplied H4K16ac, caused by MOF depletion in oocytes, DC was compromised in late embryos [How does the MSL complex perform transcriptional activation of the male X-linked genes? Almost 30 years ago, it was found that polytene X chromosome of tylation ,106. Lattylation ,107,108.tylation , and H4Ktylation ,111. Bastylation ,21,112. tylation ,25. Moreethality ,113,114, embryos .Whether the increased H4K16 acetylation facilitates initiation or elongation of transcription, or both, has been a matter of debate, since when using ChIP-seq, the 1.2-fold increase in the Pol II occupancy was revealed at both the promoters and gene bodies of the X-linked genes compared to autosomal genes ,117, wheDrosophila males [If the increased H4K16 acetylation enhances transcription of the X-linked genes, then what is the mechanism that restricts this enhancement to only a 2-fold value? Recent work has shown that nucleoporin Mtor is involved in this process . Mtor wala males . Howeverla males . Importala males . Therefola males , thus shla males .roX RNAs, the formation of phase-separated droplets induced by binding between roX RNAs and MSL2, the creation of “hard-wired” 3D organization of the X chromosome shaped by CLAMP, the spreading of the MSL complex in 3D from HAS/CES to the active X-linked genes due to MSL3 association with their H3K36me3 mark, the stimulation of transcription of the X-linked genes by MOF , and, finally, the restriction of transcriptional activation to a twofold value by Mtor.Altogether, these studies draw the complicated picture of how the MSL complex realizes its mission in the canonical DC . This piDrosophila embryos, the MSL complex is not yet bound with the X chromosome, whereas partial DC is revealed [roX1 and roX2 RNAs) are absent or only barely expressed, and H4K16 acetylation is equally represented on the X chromosome and on autosomes [The canonical DC mechanism which relies on MSL complex activity does not ensure twofold upregulation of the X-linked genes. For example, depletion of MSL complex subunits in the male somatic S2 or SL-2 cells did not lead to complete loss of DC, but only ~1.4-fold downregulated X-linked gene expression ,25. Furtrevealed . Additioutosomes ,29. Neveutosomes ,28,119. utosomes . Collectutosomes ,25,30.What might be the nature of non-canonical mechanisms? It was supposed that non-canonical DC may be mediated by an “inverse dosage” effect  or by “bDrosophila spermatogenesis is a useful model for the analysis of non-canonical DC mechanisms, since male germline cells have the same karyotype as male somatic cells, but the MSL complex does not operate in these cells [se cells . Recent se cells  providesse cells . A similse cells ,100,101.se cells , or by dse cells . What isse cells , may detse cells  and is cse cells , is actise cells . Therefose cells ,101, andse cells  (Figure Another idea is that the enhanced interactions within the active compartment of the male X chromosome may be achieved due to increased plasticity of the single X chromosome stemming from the lack of its paired homologue. Indeed, computer modeling has shown that the absence of a paired homologue makes the X chromosome less rigid and more prone to interactions within the active compartment, which may facilitate transcription . In suppOne more hypothesis is that binding of peripheral chromatin with the nuclear lamina may enhance chromatin interactions between active regions located in the nuclear interior. It was shown that in early germline cells of testes, X chromosome is more strongly bound with the nuclear lamina than autosomes . This efDrosophila cells needs further studies.It should be mentioned that the hypotheses regarding non-canonical mechanisms of DC are not mutually exclusive. However, the question of which of them are actually realized in Drosophila is one of the first organisms where these mechanisms have been studied and where many important results clarifying DC have been obtained. Nevertheless, while the canonical DC has become more clearly interpreted over time, the non-canonical mechanisms are far from being understood. There are many hypotheses, but they were not tested directly. In light of the similarity of DC mechanisms in Drosophila and humans, studies on the Drosophila model become more relevant. The advent of modern technologies opens avenues for future research in this field.Despite several decades of research on DC in various organisms, many questions still remain unresolved.


Of importance, we and others demonstrated significant cross-talk between TRPM7, RTKs, and TK signaling in different cell types including vascular smooth muscle cells (VSMCs), which might be a link between TKIs and their cardiovascular effects. In this review, we summarize the implications of RTK inhibitors (RTKIs) and TKIs in cardiovascular toxicities during anti-cancer treatment, with a focus on the potential role of TRPM7/Mg2+ as a mediator of RTKI/TKI-induced cardiovascular toxicity. We also describe the important role of TRPM7 in cancer development and cardiovascular diseases, and the interaction between TRPM7 and RTKs, providing insights for possible mechanisms underlying cardiovascular disease in cancer patients treated with RTKI/TKIs.Receptor tyrosine kinases (RTKs) are a class of membrane spanning cell-surface receptors that transmit extracellular signals through the membrane to trigger diverse intracellular signaling through tyrosine kinases (TKs), and play important role in cancer development. Therapeutic approaches targeting RTKs such as vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR), and TKs, such as c-Src, ABL, JAK, are widely used to treat human cancers. Despite favorable benefits in cancer treatment that prolong survival, these tyrosine kinase inhibitors (TKIs) and monoclonal antibodies targeting RTKs are also accompanied by adverse effects, including cardiovascular toxicity. Mechanisms underlying TKI-induced cardiovascular toxicity remain unclear. The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed chanzyme consisting of a membrane-based ion channel and intracellular α-kinase. TRPM7 is a cation channel that regulates transmembrane Mg The activation of RTKs leads to phosphorylation and activation of numerous tyrosine kinases such as Abl, c-Src, Ras, PI-3K, JAK, and ALK, which regulatecellular processes, such as cell migration, differentiation, apoptosis, contraction, metabolism and survival (Receptor tyrosine kinases (RTKs) are a class of membrane-spanning cell-surface receptors that transmit extracellular signals through the membrane to trigger diverse intracellular signaling . In humasurvival . ActivatAbnormal expression and overactivation of RTKs, including vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR) are associated with tumor invasion, metastasis, and tumor angiogenesis . Inhibit2+, Mg2+ and Ca2+, and the α-kinase domain phosphorylates downstream substrates including annexin-1, eukaryotic elongation factor 2 (eEF2)'s cognate kinase (eEF2K), phospholipase Cγ2 (PLCγ2), myosin IIA, SMAD2, tropomodulin 1, myelin basic protein (MBP), cAMP response element binding protein (CREB), and RhoA  possesses both ion channel and enzymatic functions. TRPM7 channel is permeable to divalent cations such as Znand RhoA . TRPM7 pand RhoA –19, withand RhoA –28.In this review, we summarize the implications of RTK and TK inhibition in human cancers, with a focus on their cardiovascular toxicities. We also describe the important role of TRPM7 in cancer development and cardiovascular diseases, and the interaction between TRPM7 and RTKs, providing insights for possible mechanisms whereby anti-cancer drugs targeting RTKs induce cardiovascular toxicity.Under physiologic conditions, RTK activity is tightly controlled. However, dysregulated signaling through RTKs promotes an imbalance between cell proliferation and cell death, which is implicated in cancer development. Mechanisms underlying the aberrant activation of RTKs are associated with overexpression , mutatioTKIs are a class of pharmacologic agents that block multiple signal transduction pathways . In 2001VEGF signaling plays a critical role in angiogenesis, cell proliferation and survival. Following advances in knowledge about the role of angiogenesis in promoting tumor growth , 95, mulde novo or worsening of previously controlled hypertension , with cardiac ischemia/infarction (67.4%) as the most common events for ATE , particularly cardiac ischemia, and venous thromboembolism (VTE). The risk of ATE associated with anti-VEGF TKIs is greater than that of VTE, with an incidence generally <3% , 103. A  for ATE . However for ATE . Several for ATE , 107. He for ATE .Meta-analysis of trials of VEGFR-TKIs  including 10,647 patients demonstrated a pooled incidence of asymptomatic HF of 2.4% and symptomatic HF of 1.2%. Notably, there was no apparent difference in the risk of cardiovascular toxicity between the relatively specific VEGFR-TKIs  and those targeting against a broader range of tyrosine kinases  . MechaniThe incidence of QT prolongation associated with VEGFR-TKIs varies widely among individual drugs. Vandetanib has the highest incidence and the most significant prolongation, with up to 8% of patients exhibiting a corrected QT (QTc) interval duration of >500 ms . Meta-anEGFR is a cell surface protein that binds to epidermal growth factor, thus inducing receptor dimerization and tyrosine autophosphorylation leading to cell proliferation. Mutations in this gene have been associated with a number of cancers, including adenocarcinoma of the lung , glioblain vivo rat cardiomyocytes, Korashy et al. showed that Gefitinib, a selective inhibitor of EGFR, induces cardiovascular toxicity through modulating the cardiac PTEN/AKT/FoxO3a pathway and the formation of CYP1A1-mediated reactive metabolites , indicated that the incidence of asymptomatic decline of left ventricular ejection fraction  and symptomatic HF was 7.5 and 1.9%, respectively . A case-abolites .In a retrospective study based on the world health organization (WHO) pharmacovigilance database VigiBase, among 98,765 adverse reactions reported with NSCLC-targeted therapies including EGFR-TKIs, 1,783 (1.8%) were cardiac arrhythmias . The mosPDGFR signaling pathway plays a critical role in promoting cardiomyocyte proliferation and heart regeneration . VariousTRPM7 human gene is located on the long arm of chromosome 15, consisting of 19 exons and encoding a 1,863 amino acid protein with a molecular weight of 210 kDa . The bas2+ homeostasis and cell growth  influx and exerting effects on cell migration and proliferation  and cardiomyocytes , 139. Inl growth . We receferation . TRPM7 wferation , 141. Inferation . The phygh TRPM7 , 143. Adgh TRPM7 . TRPM7 apression , 146.via a mineralocorticoid receptor (MR)-dependent genomic signaling cascade involving serum- and glucocorticoid-regulated kinase 1 (SGK1) and a functional TRPM7 α-kinase domain (via molecular mechanisms involving phospholipase C (PLC), protein kinase C (PKC) and c-Src (2+ entry in chondrocytes and stimulate bone growth via activating natriuretic peptide receptor 2 (NPR2)  , 147–149e domain . BradykiTRPM7 deletion before embryonic day 9 led to congestive heart failure and death by embryonic day 11.5 (Growing evidence indicates an impoartnt role for TRPM7 in cardiac development . TRPM7 pday 11.5 . These f2+ dependent effects (2O2)  , 153. Inroblasts , 153. Giroblasts . Moreove channel .via its Mg2+ permeability. Despite inconsistent findings to support the correlation between serum Mg2+ and hypertension, Mg2+ supplementation at a dose of 368 mg/d for 3 months has been shown to lower blood pressure in adults , which is most commonly sustained arrhythmia and a major cause of morbidity and mortality , 162–164human AF . These fhuman AF . TRPM7 mhuman AF . Importahuman AF . Consist+ influx .2+, which is regulated by TRPM7 cation channel activation, is an essential cation and second messenger involved in tyrosine kinase signaling and regulation of RTKs -dependent manner , EGF increases TRPM6 activity and surface expression, while in cancer patients receiving anti-EGFR treatment, serum level of Mgecreased –171. In ecreased , 172. PDecreased . Consistecreased . In a huecreased . The regeoblasts . Moreovet manner . Taken t2+ influx through the TRPM7 channel and consequently regulates VSMCs function and vascular morphology. Of importance, the effects of EGF on TRPM7 and VSMCs were attenuated by gefitinib, a TKI that exclusively targets EGFR. In addition, we also found that in VSMCs from TRPM7 kinase-deficient mice (TRPM7kinase+/Δ) and aortic tissues from TRPM7-kinase dead (TRPM7R/R) mice, EGFR expression and EGFR phosphorylation (Y845) were reduced, respectively, supporting a significant cross-talk  stresses, increased ROS production and dysregulation of cellular metabolism , inhibiting Ca2+ influx resulting in defective insulin secretion (2+ interferes with intracellular signaling pathways mediated by IRS1 and IRS2 activation (Mgecretion . Furthertivation , 189. Oftivation .It should be highlighted that while it is clear that there is important cross-talk between RTK/TK and TRPM7 at the functional level, direct interacting p-tyr sites between RTKs/TKs and TRPM7 still awaits confirmation. However, there is growing evidence that both TRPM6 and TRPM7 possesses phosphorylation sites, which potentially may link to the RTK/TK pathway , 191. Mo2+, it also influences intarcellular Ca2+ homeostasis and downstream kinases and substartes that may also play a role in TRPM7-RTK/TK crosstalk. Unraveling these TRPM7-dependent processes might provide greater insights into the molecular mechanisms that underlie cardiovascular disease in patients treated with RTKI/TKIs.RTKs such as VEGFR, EGFR and PDGFR are important therapeutic targets for human cancers, however, inhibitors of RTKs and TKs are also accompanied by a profile of adverse cardiovascular effects through unclear mechanisms. Since the cross-talk between RTKs and TRPM7 exists in multiple cell types, and TRPM7 is critically involved in both cancers and cardiovascular diseases, we suggest that TRPM7 might play a role in the cardiovascular toxicity associated with RTKI/TKIs . TRPM7 iQL and SL wrote the manuscript. YQ and JZ provided support for the tables and figures. FR, ZZ, and RT provided guidance and corrected the manuscript. All authors contributed to the article and approved the submitted version.


Glioma is the most common primary malignant brain tumor in adults and accounts for approximately 80% of brain and central nervous system tumors. In 2021, the World Health Organization (WHO) published a new taxonomy for glioma based on its histological features and molecular alterations. Isocitrate dehydrogenase (IDH) catalyzes the decarboxylation of isocitrate, a critical metabolic reaction in energy generation in cells. Mutations in the IDH genes interrupt cell differentiation and serve as molecular biomarkers that can be used to classify gliomas. For example, the mutant IDH is widely detected in low-grade gliomas, whereas the wild type is in high-grade ones, including glioblastomas. Long non-coding RNAs (lncRNAs) are epigenetically involved in gene expression and contribute to glioma development. To investigate the potential use of lncRNAs as biomarkers, we examined lncRNA dysregulation dependent on the IDH mutation status. We found that several lncRNAs, namely, AL606760.2, H19, MALAT1, PVT1 and SBF2-AS1 may function as glioma risk factors, whereas AC068643.1, AC079228.1, DGCR5, FAM13A-AS1, HAR1A and WDFY3-AS2 may have protective effects. Notably, H19, MALAT1, PVT1, and SBF2-AS1 have been associated with temozolomide resistance in glioma patients. This review study suggests that targeting glioma-associated lncRNAs might aid the treatment of glioma. Gliomas are primary brain tumors derived from neuroglial stem cells or neuroglial progenitor cells , which do not encode proteins and are more than 200 nucleotides in length, regulate cellular biological processes in cancer such as cell proliferation, metastasis, invasion, motility, and drug resistance, and their intracellular locations reflect their functions  methylation, 1p/19q co-deletion, IDH, and epidermal growth factor receptor have been widely used as glioma biomarkers H In cases of IDH1 mutation, the homodimer formed by two IDH1-mutant monomers does not have enzymatic activity, but the heterodimer composed of the IDH-wildtype and mutant monomer has enzymatic activity sufficient to convert α-KG to 2-HG  and myostatin (MSTN), and it has been experimentally demonstrated that both BMP and MSTN directly stimulate AC068643.1 expression  and AC079228.1, were suggested as predictors of TMZ efficacy in IDH-mutant type LGGs  modification, is also associated with poor prognosis. It was also demonstrated that MALAT1 overexpression promotes the malignant glioma phenotype by inducing epithelial–mesenchymal transition (EMT) and tumor necrosis factor (TNF) signaling through nuclear factor kappa B (NF-κB) activation  is an overexpressed lncRNA in glioblastoma and promotes cell growth and tumorigenesis. For example, it has been demonstrated that lncRNA MALAT1 promotes the expression of SRY-box transcription factor 2 (SOX2) by suppressing miR-129 to promote glioma stem cell viability, proliferative abilities, and tumorigenesis , which plays an oncogenic role in various tumors, may function as a biomarker in diffuse LGG. Kaplan-Meier analysis showed that the prognosis of LGG patients with high SBF2-AS1 expression was poorer than those with low expression. Cox regression analysis showed that SBF2-AS1 is an independent prognostic factor of poorer overall survival in LGG  is expressed at low levels in IDH-wildtype gliomas. Kaplan-Meier analysis showed that WDFY3-AS2 overexpression was associated with longer overall survival than low WDFY3-AS2 expression, and Cox regression analysis showed its expression was independently correlated with overall survival. In addition, gene ontology (GO) and gene set enrichment analysis (GSEA) revealed that WDFY3-AS2 is involved in the regulation of synaptic transduction, glutamate receptors, and TNF signaling pathways  plays cancer-dependent roles. For example, although DGCR5 levels were unrelated to WHO malignancy grade in glioma, they were more downregulated in IDH-wildtype glioma than in IDH-mutant glioma. Furthermore, DGCR5 increased the levels of Smad7 and phosphatase and tensin homolog (PTEN) by sponging miR-21 and miR-23a, respectively, in glioma cells. It is also reported that Smad7 inhibits the migratory and invasive activities of glioma, and PTEN inhibits cell proliferation and enhances apoptosis. Patients with low DGCR5 expression levels show poorer overall survival (He et al. Glioma classification based on histological features and IDH mutation status helps with diagnosis and prognosis. Although advances in techniques, technologies, surgical resection, chemotherapy, and radiotherapy have significantly improved treatment efficacy outcomes fall far short of satisfactory. Furthermore, accumulating evidence indicates lncRNA dysregulations are involved in glioma chemo- and radioresistance. This review compiles several differentially expressed lncRNAs in IDH-mutant and IDH-wildtype gliomas, and it has been shown that lncRNAs SBF2-AS1, PVT1, MALAT1, and H19 significantly contribute to the induction of TMZ resistance.TMZ is a lipophilic anticancer drug, which is widely used to treat brain tumors. TMZ can be readily delivered to brain tissues as it crosses the blood–brain barrier and induces cell death by causing single- or double-strand breaks by incorporating mismatched base pairs. However, more than 50% of glioma patients do not respond to TMZ, and lncRNAs appear to be involved in TMZ resistance by regulating chemoresistance-associated gene expression, enhancing cell survival, and inhibiting apoptosis Lee . FurtherIn glioma, increased TMZ resistance, conferred by the upregulations of risky lncRNAs such as H19, MALAT1, PVT1, and SBF2-AS1 , is likeThe dysregulation of lncRNA contributes to the development of several types of cancer, and lncRNA promotes glioma development and induces chemo- and radioresistance. IDH mutation status is a widely-accepted biomarker for glioma classification, but additional markers are desired to determine effective treatment strategies. This review focuses on risky and protective lncRNAs according to IDH mutation status and the mechanisms involved in TMZ resistance. We suggest five lncRNAs acting as risk factors, namely, AL606760.2, H19, MALAT1, PVT1, and SBF2-AS1, and another six protective lncRNAs, that are AC068643.1, AC079228.1, DGCR5, FAM13A-AS1, HAR1A, and WDFY3-AS2. The risky H19, MALAT1, PVT1, and SBF2-AS1 increase TMZ resistance . Identif


B. pseudolongum (Bp7 and Bp8) on alleviating colitis and to explore its possible potential mechanisms. Our results displayed that Bp7 and Bp8 intervention effectively reduced dextran sodium sulfate (DSS)-caused body weight loss and the release of several pro-inflammatory factors (interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α)) and increased the activities of antioxidant enzymes  and the concentrations of tight junction proteins . Moreover, Bp7 and Bp8 intervention drastically down-regulated the expression of colonic MyD88, NF-κB, iNOS and COX2 and drastically elevated the expression of colonic STAT3, Nrf2, and PPARγ. Gas chromatography-mass spectrometry results revealed that the cecal levels of isobutyric, butyric, and isovaleric acids were drastically increased in colitis mice intervened with Bp7 and Bp8. Moreover, 16S rRNA sequencing revealed that Bp7 and Bp8 intervention modulated the intestinal microbiota structure, particularly by enhancing the proportion of Eubacterium coprostanoligenes group, Marvinbryantia, Enterorhabdus, Faecalibaculum, Coriobacteriaceae UCG 002, Alistipes, and Bifidobacterium, which are relevant to the levels of cecal isobutyric acid, butyric acid, isovaleric acid, and inflammatory cytokines. Collectively, these findings suggest that Bp7 and Bp8 intervention alleviates the intestinal barrier function, possibly by blocking the secretion of proinflammatory cytokines, maintaining the intestinal physical barrier integrity, activating the PPARγ/STAT3 pathway, and modulating intestinal microbiota composition. Our study also suggested that B. pseudolongum is a promising probiotic for colitis treatment.Colitis has become a major health concern worldwide. The objective of the present study was to determine the probiotic influence of different strains of  Inflammatory bowel diseases (IBDs), comprising Crohn’s disease (CD) and ulcerative colitis (UC), are chronic refractory inflammatory diseases of the gastrointestinal tract. According to a previous investigation, IBD affects millions of individuals, and the global prevalence and occurrence of IBD are rapidly rising . MultiplEscherichia, Streptococcus, and Veillonella) is commonly increased and the relative abundance of beneficial bacteria  is reduced in patients with IBD ).Thirty-two 7-week-old male SPF C57BL/6J mice (19–21 g) were procured from Jiangsu Laboratory Animal Center  and housed under a standardized environment . After 7 days of adaptation, all mice were equally divided into 4 groups. The control (NC) and model (DSS) groups were fed with 0.2 mL of skim milk  was measured every day according to a previous study . BrieflyColonic TNF-α, IL-1β, IL-6, IL-10, IL-22, and mucin2 (Muc2) levels were measured using commercial kits  following the manufacturer’s instructions. The activities of myeloperoxidease (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were analyzed using commercial kits .Histopathological examination of colon tissues was carried out as reported previously . BrieflyImmunohistochemical analysis was carried out following our previous study . BrieflyTotal RNA was obtained from the colon using a commercial kit  and reverse-transcribed into complementary DNA (cDNA) using a commercial kit . The mRNA transcription levels were estimated using an RT-qPCR system . The sequences of the primers used in this study are listed in Cecal SCFA concentrations were obtained following a method from a previous study . Gas chrGenomic DNA was obtained from frozen fecal samples using a DNA isolation kit  following the manufacturer’s instructions. The 16S rRNA genes (V3–V4 region) were amplified using forward (338F) and reverse (806R) primers. The PCR products were purified using a commercial kit , quantified using a NanoDrop ND-1000 spectrophotometer , and sequenced using an Illumina MiSeq sequencer at Jiangnan University .The raw data from high-throughput sequencing were filtered using the QIIME software package (v 1.8.0). The results were annotated to operational taxonomic units (OTUs) when the value of similarity was greater than or equal to 97%. Principal coordinates analysis (PCA) scores and hierarchical clustering analysis (HCA) were implemented using SIMCA (v 14.1.0). STAMP (v 2.1.3) software was applied to identify intestinal microbial phylotypes with obvious difference among the groups. The metabolic pathways of intestinal microbiota were performed using PICRUSt2. The predictive associations were explored by Pearson correlation coefficients and the network was visualized by R software and Cytoscape v 3.6.1, respectively.p < 0.05 was considered statistically significant.To calculate the significance of the differences between groups, the data were analyzed using one-way ANOVA with GraphPad Prism (Ver. 7.0) software, followed by Tuckey’s multiple comparison test. In addition, p < 0.05), whereas Bp7 or Bp8 intervention resulted in a sharp reduction in DAI score . The colon length notably shortened in the DSS group (7.05 ± 0.9 cm) relative to that in the NC group (7.88 ± 0.20 cm). Compared with the DSS group, colitis mice treated with Bp7 or Bp8 partially reversed the effect of DSS treatment .Relative to the NC group, the body weight was drastically reduced in colitis mice. However, the oral administration of Bp7 and Bp8 inhibited body weight loss . The DAIp < 0.05). As expected, colitis mice treated with Bp7 and Bp8 exhibited lower concentrations of TNF-α and IL-1β (p < 0.05) and higher concentrations of IL-10 and IL-22 relative to the DSS group (p < 0.05). Moreover, Bp7 intervention significantly reduced the colonic IL-6 levels in mice with DSS treatment (p < 0.05).As shown in p < 0.05) (p < 0.05). Moreover, the activities of T-AOC, SOD, and GSH were remarkably decreased in the DSS group relative to those in the NC group (p < 0.05), whereas Bp7 and Bp8 intervention significantly elevated the activities of T-AOC and GSH (p < 0.05). Nevertheless, there was no remarkable discrepancy in SOD activity among all mice with DSS treatment (p > 0.05).To further explore the ameliorative effects of Bp7 and Bp8 on intestinal barrier damage, the levels of colonic oxidative stress were analyzed. DSS treatment drastically increased the MPO activity and MDA levels in the mice of the DSS group relative to those in the NC group ( < 0.05) A. IntereB. pseudolongum (Bp7 and Bp8) attenuated DSS-induced histopathological changes in the colon.The results of the colon tissue staining are shown in p < 0.05) (p < 0.05), but Bp8 intervention did not affect the number of goblet cells (p > 0.05)  C. Furthe > 0.05) D.B. pseudolongum intervention as part of the latter’s effect on the integrity of the intestinal barrier. Relative to the NC group, the transcription levels of occludin, claudin-1, and ZO-1 were remarkably decreased in the DSS group (p < 0.05) (p < 0.05). These data were also confirmed by immunohistochemical analysis of the colon  was modulated by  < 0.05) A. Neverthe colon B,C, whicp < 0.05), but the expression of colonic STAT3, Nrf2, and PPARγ were remarkably inhibited (p < 0.05). However, Bp7 and Bp8 intervention drastically down-regulated the expression of Tlr4, NF-κB, iNOS, and COX2 and drastically up-regulated the expression of STAT3, Nrf2, and PPARγ in colitis mice (p < 0.05).To deeply analyze the influences of Bp7 and Bp8 intervention on mice with DSS treatment, the mRNA levels of genes related to colitis were measured in the NC, DSS, Bp7, and Bp8 groups . The traB. pseudolongum intervention could prevent DSS-induced disturbances in intestinal microbiota composition. The results of PCA showed that the intestinal microbiota composition was altered in the DSS group compared with that in the NC group  was applied to determine alterations in the intestinal microbiota following Bp7 and Bp8 intervention. The relative abundances of NC group . After BSS group A. Similaacterium B. Altogen-glycan biosynthesis, peroxisome, protein digestion and absorption, proteasome, phosphonate and phosphinate metabolism, valine, leucine and isoleucine degradation and biosynthesis, starch and sucrose metabolism, apoptosis, glycosaminoglycan degradation, streptomycin biosynthesis, pantothenate and CoA biosynthesis, glycine, serine and threonine metabolism, lipoic acid metabolism, histidine metabolism, TCA cycle, and galactose metabolism in the Bp7 group were enriched remarkably , but there were no obvious differences in cecal valeric acid levels between the NC and DSS groups (p > 0.05). Notably, Bp7 intervention significantly elevated the cecal isobutyric, butyric, and isovaleric acid levels (p < 0.05). Bp8 treatment significantly elevated the cecal propionic, isobutyric, butyric, valeric, and isovaleric acid levels compared with those in the DSS group (p < 0.05).Cecal contents were analyzed to explore the influence of Bp7 or Bp8 supplementation on the levels of SCFAs . The cecStreptococcus, GCA 900066575, Ruminiclostridium 9, Romboutsia, Adlercreutzia, Turicibacter, Escherichia Shigella, Parabacteroides, Pseudomonas, Oscillibacter, Ruminiclostridium, Mucispirillum, Ruminiclostridium 5, Acinetobacter, and uncultured Bacteroidates bacteria were positively related to the colonic IL-6, MDA, MPO, IL-1β, and TNF-α levels but were negatively related to body weight, colon length, SCFA levels , IL-10 levels, and the activities of antioxidant enzymes . The abundance of A2, Intestinimonas, Roseburia, Eubacterium ventriosum group, Eubacterium xylanophilum group, Lactobacillus, Marvinbryantia, Bifidobacterium, Coriobacteriaceae UCG 002, Enterorhabdus, Clostridium sensu stricto 1, Erysipelatoclostridium, Eubacterium coprostanoligenes group, Eubacterium nodatum group, Alistipes, and Faecalibaculum were negatively associated with colonic IL-6, MDA, MPO, IL-1β, and TNF-α levels. Moreover, the abundance of A2, Intestinimonas, Roseburia, Eubacterium ventriosum group, Eubacterium xylanophilum group, Lactobacillus, and Marvinbryantia were positively associated with acetic acid, colon length, and antioxidant enzyme activity .The potential relationship between the intestinal microbiota composition and inflammatory cytokine related to colitis was further analyzed using Spearman rank correlation A,B. The B. pseudolongum (Bp7 and Bp8) effectively mitigated intestinal barrier damage in colitis mice via regulating the PPARγ/STAT3 pathway and modulating the intestinal microbiota composition, recovering the antioxidant enzyme activities, and inhibiting pro-inflammatory cytokines.Colitis is regarded as a life-threatening disease in developed and developing countries. Conventional therapies result in tremendous economic costs and adverse side effects. Consequently, it is important to seek and develop effective and safe therapeutic agents. In this study, we showed that B. pseudolongum. It is widely accepted that the accumulation of inflammatory cytokines is generally accompanied by decreased activities of antioxidant enzymes. Some studies have revealed that MPO serves as a pro-oxidative and pro-inflammatory enzyme. Moreover, its activities are strongly associated with the degree of tissue damage and progression of inflammation, and its expression directly reflects the neutrophil infiltration in colitis mice [Milk is commonly used as a protective matrix for probiotics in animal experiments. Milk exhibits favorable characteristics regarding the survival of probiotics during storage. In addition, no data were found regarding the use of milk in the treatment of colitis and irritable bowel syndrome ,16. Thertis mice . Furthertis mice . Our fintis mice . Thus, iBifidobacterium promotes the secretion of IL-22 by inhibiting the expression of MyD88 [Oral administration of DSS can destroy the intestinal barrier function in mice. The intestinal barrier is a complex structure that plays an essential role in host response to gut pathogens and tolerance to the intestinal microbiota. Goblet cells are specialized cells of the intestinal epithelium, and they play a vital role in maintaining the mucosal barrier and the mucosal immune homeostasis . The funof MyD88 . In the The mRNA levels of colonic genes related to inflammation were analyzed in order to uncover the molecular mechanisms underlying the ameliorative effects of Bp7 or Bp8 on gut inflammation in colitis mice. Bp7 and Bp8 intervention elevated the expression of STAT3 and PPARγ in colitis mice. The up-regulation of STAT3 elevated the transcription of PPARγ, which preserves intestinal homeostasis through controlling the differentiation of colon epithelial cells and regulating the function of adaptive immune effector T cells ,25. A reLactobacillus, Eubacterium xylanophilum group, Eubacterium ventriosum group, Roseburia, Marvinbryantia, and A2 compared with that in mice without DSS treatment. Among those, Lactobacillus, Roseburia, and Marvinbryantia belonging to the Firmicutes phylum and are thought to be probiotic [Lactobacillus is a gram-positive bacterium that relieves the symptoms of inflammation by regulating the NF-κB pathway. Lactobacillus intervention maintains intestinal mucosal integrity, elevates the secretion of tight junction proteins, and inhibits the expression of immune factors [Roseburia and Marvinbryantia are butyrate-producing bacteria, and their abundance is strongly related to the concentration of butyrate in the gastrointestinal tract [Mucispirillum and Pseudomonas are considered pathobionts because they can cause several diseases under certain conditions, especially IBD [Acinetobacter are rare in the intestinal microbiota of healthy individuals, and they stimulate proinflammatory responses in monocolonized mice [Eubacterium coprostanoligenes group leads to increased excretion of 7α-dehydroxylase, which in turn elevates the conversion of intestinal cholesterol to bile acid [Enterorhabdus can hydrolyze primary bile acids, which facilitates the excretion of bile acid [Marvinbryantia and Coriobacteriaceae UCG 002 have recently attracted growing interest due to their anti-inflammatory role. High abundance of Marvinbryantia and Coriobacteriaceae UCG 002 results in the elevation of SCFA concentration, which suppresses the differentiation of naive CD4+ T cells and thus impacts the differentiation of helper T (Th) cells [Eubacterium coprostanoligenes group, Marvinbryantia, Enterorhabdus, Faecalibaculum, Coriobacteriaceae UCG 002, Alistipes, and Bifidobacterium in colitis mice significantly increased after B. pseudolongum intervention. Notably, the association between Alistipes and the host health is still disputed. A recent report suggested that Alistipes could produce succinic acid, which helps to maintain intestinal homeostasis by accelerating cell differentiation, promoting tight junction assembly, and increasing the expression of proglucagon in intestinal L cells [Faecalibaculum, a kind of lactic acid producer, is known to be strongly negatively related to inflammatory cytokines (IL-6 and TNF-α) [B. pseudolongum (Bp7 and Bp8) intervention regulates the intestinal microbiota structure, and its collective effects suppress intestinal inflammation.Alterations in intestinal microbiota composition are closely associated with the development of several diseases, for instance, colitis, obesity, and diabetes. Among those, colitis is regarded as a result of an inappropriate response of the mucosal immune system induced by the intestinal microbiota, characterized by the increase of potentially harmful bacteria and the decrease of potentially beneficial bacteria. A previous study showed that the changes in intestinal microbiota structure exacerbate low-grade mucosal inflammation . It is wrobiotic . Lactoba factors . Rosebural tract . Butyratal tract . Our resal tract . In contally IBD . Acinetozed mice . These sile acid . Enterorile acid . Marvinbh) cells . High co L cells . In addid TNF-α) . Taken tB. pseudolongum (Bp7 and Bp8) in the pathogenesis and development of colitis. However, additional metabolomic and metagenomic studies are needed to further explore the specific metabolites derived from B. pseudolongum that are involved in ameliorating the damage to the intestinal barrier function.Bp7 and Bp8 intervention ameliorated the intestinal barrier function by activating the PPARγ/STAT3 pathways and regulating the intestinal microbiota composition. Our data reveal the essential role of


Obvious DNA methylation and moderate alteration frequency of PDIA5 were observed in human cancers. The expression level of PDIA5 was significantly correlated with infiltrated immune cells, immune pathways, and other immune signatures. We found that cancer cells and macrophages exhibited high PDIA5 expression in human cancers using the single-cell RNA sequencing analysis. We also demonstrated the interaction between PDIA5 and immune cells in glioblastoma multiforme (GBM). Multiplex immunofluorescence staining showed the upregulated expression level of PDIA5 and the increased number of M2 macrophage markers-CD163 positive cells in pan-cancer samples. Notably, PDIA5 silencing resulted in upregulated expression of PD-L1 and SPP1 in U251 cells. Silencing of PDIA5 in hepG2 cells, U251 cells, and PC3 cells contributed to a decline in their ability of proliferation, clone formation, and invasion and inhibited the migration of cocultured M2 macrophages. Additionally, PDIA5 also displayed predictive value in the immunotherapy response of both murine and human cancer cohorts. Overall, our findings indicated that PDIA5 might be a potential target for immunotherapies in cancers.The aberrant protein disulfide isomerase A5 (PDIA5) expression was relevant to the poor prognosis of patients with human cancers. However, its relationship with the epigenetic and genetic alterations and its effect on tumor immunity is still lacking. In the present study, we comprehensively analyzed the immune infiltration role of PDIA5 in human cancers based on large-scale bioinformatics analyses and  Since the critical role of the immunological landscape of tumor microenvironment (TME) in tumorigenesis has been increasingly recognized, cancer immunotherapies such as immune checkpoint inhibitors are promising , 2. AlthProtein disulfide isomerase (PDI) is a cluster of endoplasmic reticulum (ER) enzymes with both thiol-disulfide oxidoreductase and chaperone activities and plays a pivotal role in the modulation of proteostasis in the ER. PDI proteins mediate the formation, breakage, and rearrangement of protein disulfide bonds . PDI is More significantly, the involvement of PDI proteins in several disease states, particularly cancer, has also been gradually identified. PDI proteins impact the survival and apoptosis of cancer cells through a complex unfolded protein response (UPR) pathway, and the overexpression of PDI is related to the tumorigenesis, invasion, and metastasis of various cancers –13. AmonProtein disulfide isomerase A5 (PDIA5), also named protein disulfide isomerase-related (PDIR) protein, belongs to the human PDI family and has isomerase and chaperone activities , 17. A pAlthough the correlations between PDIA5 overexpression and immune infiltration in gliomas have been revealed , the imphttps://www.ncbi.nlm.nih.gov/geo/) and BioProject dataset (https://www.ncbi.nlm.nih.gov/bioproject). The scRNA-seq datasets of bladder urothelial carcinoma (BLCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), prostate adenocarcinoma (PRAD), skin cutaneous melanoma (SKCM), and stomach adenocarcinoma (STAD) were acquired from GSE145137, GSE125449, GSE81861, GSE138794, GSE103322, GSE125449, GSE118828, GSE137829, GSE72056, and GSE183904 in the GEO database, respectively. The scRNA-seq dataset of breast invasive carcinoma (BRCA) was downloaded from GSE75688 and GSE118389. The scRNA-seq dataset of kidney renal clear cell carcinoma (KIRC) was obtained from GSE121636 and GSE171306. In addition, the scRNA-seq dataset of lung adenocarcinoma (LUAD) was downloaded from the BioProject (#PRJNA591860).We obtained pan-cancer samples of 33 cancer types from The Cancer Genome Atlas (TCGA) database. We downloaded the single-cell RNA sequencing (scRNA-seq) dataset of 13 cancer types from the Gene Expression Omnibus (GEO) database (http://bioinfo.life.hust.edu.cn/web/GSCALite/) was employed to analyze the methylation profiles, single nucleotide variation (SNV), and copy number variation (CNV) of PDI family genes   .http://timer.cistrome.org/) (http://cis.hku.hk/TISIDB/) examined the association between PDIA5 expression and immune subtypes of cancers   . The TIS cancers . The cors (GSVA) . Correlas (GSVA) .https://www.targetvalidation.org/) (https://string-db.org/cgi/input.pl) was used to predict the protein-protein interaction network of PDIA5. Besides, the immunotherapy response was predicted by the Tumor Immune Syngeneic MOuse database (http://tismo.cistrome.org), TIDE database (http://tide.dfci.harvard.edu), and ROC Plotter (http://www.rocplot.org/).The involvement of PDIA5 in human disease was identified by the OPENTARGET platform (on.org/) , and theThe integration of GSE75688 and GSE118389 for BRCA and GSE121636 and GSE171306 for KIRC were performed using the Anchors function of R package “Seurat” .The scRNA-seq analysis was conducted as previously described . After sSingle-cell pseudotime trajectories analyses were performed using the R package “Monocle” ; cells iWe obtained the tissue microarray from the Outdo Biotech company  and the ethics was approved. Briefly, the paraffin sections of pan-cancer samples were deparaffinized and then were blocked with 3% H2O2 and 2% BSA after antigen retrieval. Anti-PDIA5 antibody , anti-CD68 antibody , and anti-CD163 antibody  were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody  incubation and tyramide signal amplification (TSA)  incubation. And then, 4’,6-Diamidino2-phenylindole dihydrochloride (DAPI) was applied for the nuclei staining. The stained slides were visualized for multispectral images under the Pannoramic Scanner . DAPI glows blue by UV excitation wavelength 330-380nm and emission wavelength 420nm in the fluorescence spectra. CY5, FITC, and CY3 glow pink, green, and red. The excitation wavelength was 608-648nm, 465-495nm, and 510-560nm, respectively, with an emission wavelength of 672-712nm, 515-555nm, and 590nm.2.Human LIHC cell line hepG2, GBM cell line U251, and PRAD cell line PC3 were cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C in 5% CO2. THP-1 cells cultured in 6-well plates (2.5 × 105) were stimulated with 320nM phorbol 12-myristate 13-acetate  and incubated at 37°C for 6h to induce M0 macrophages. For M2 macrophage polarization, M0 macrophages were treated with M2-polarizing reagents (20 ng/ml IL-4 and 20 ng/ml IL-13) and hatched at 37°C for 72h.Human monocyte cell line THP-1 was cultured in 1640 complete medium containing 10% FBS at 37°C in 5% COFor siRNA knockdown, the siRNA target sequence of siRNA-PDIA5-1 and siRNA-PDIA5-2 was 5′- GCAAGAAGATGAAAGTTGA-3′and 5′- GACGGTTCTTGTTCCAGTA-3′, respectively. PDIA5 expression in HepG2, U251, and PC3 cells was silenced with PDIA5 siRNA using a siRNA transfection kit , according to the manufacturer’s instruction.HepG2, U251, and PC3 cells were processed for western blotting. Immunoblot analyses were performed using the following primary antibodies: anti-PDIA5 , anti-secreted phosphoprotein 1 , anti-programmed death-ligand 1  and anti-β-actin . HRP goat anti-mouse IgG  and HRP goat anti-rabbit IgG  were used as the secondary antibody. The proteins were visualized using an enhanced chemiluminescent (ECL) detection kit.4 cells per well. The CCK8 reagent  was subsequently added per the manufacturer’s descriptions. Later, the si-NC and a si-PDIA5 group of hepG2, U251, and PC3 cells were cultured for 24h, 48h, and 72h, respectively. The absorbance was measured at 450nm after being incubated at 37°C in 5% CO2.HepG2, U251, and PC3 cells were seeded in a 96-well plate at the density of 1×10The si-NC and si-PDIA5 groups of hepG2, U251, and PC3 cells were plated in 6-well plates (200 cells per well) and cultured at 37°C in 5% CO2 for 2 weeks. The colonies were fixed with 4% methanol (1 ml per well) for 15min and were then stained with 0.5% crystal violet for 30min at room temperature. The numbers of colonies larger than 2mm were counted.5) were seeded on the Matrigel in the upper chamber with a serum-free medium. The lower chamber was filled with a complete medium containing 10% FBS. After 24h, cells were fixed with 4% paraformaldehyde for 25min and stained with 0.1% crystal violet for 20min.Cell invasion assay was carried out with a 6-well Transwell . The matrigel was diluted with DMEM at the ratio of 1:8 on the ice. Each upper chamber was added with 50uL diluted Matrigel and then incubated at 37°C for solidification. The si-NC and si-PDIA5 groups of hepG2, U251, and PC3 cells (5 × 105) were added to the lower chamber, respectively, and 5 × 105 M2 macrophages were added to the upper chamber. After coculturing for 24h, M2 macrophages in the upper chamber were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet.Cell migration assay was performed with a 6-well Transwell . The si-NC and si-PDIA5 groups of hepG2, U251, and PC3 cells (5 × 10https://www.r-project.org/). We used correlation analyses to measure the degree of correlation between certain variables. For data that followed a normal distribution, Student’s t-test and one-way analysis of variance (ANOVA) were performed to compare two groups and among more than two groups, respectively. When data didn't follow a normal distribution, we used Mann–Whitney test and Kruskal–Wallis test to compare two groups and among more than two groups, respectively. A p < 0.05 indicated statistical significance.Statistical analyses were performed using the GSCALite platform and R software  than in other cancer types  in the majority of cancers. Conversely, there were negative correlations between PDIA5 and the abundance of CD8+ T cells in some cancer types, like BRCA, HNSC, LUSC, and testicular germ cell tumors (TGCT) , mesothelioma (MESO), sarcoma (SARC), and uterine carcinosarcoma (UCS)  efficacy –40. We uSince studies have underlined the critical role of immune checkpoint genes in tumor immunotherapy , we inveNeoantigen refers to the mutated peptides expressed only by tumor cells, and neoantigen may contribute to the spontaneous antitumor immune responses and form a biomarker in cancer immunotherapy , 43. Pos). The cells were identified as aneuploid cells, and diploid cells were defined as cancer cells using the R package “CopyKAT” and aneuploid cells. And the R package “InferCNV” identified the cells as immune cells, malignant cells, and stromal cells . Different clusters of infiltrated immune cell types  in each tumor sample were identified by immune cell-specific molecular markers .The correlations between PDIA5 expression and specific signaling pathways were further illustrated. Overall, neoplastic cells with high expression of PDIA5 exhibited interaction with T cells Using pan-cancer samples, we detected the PDIA5 expression in laryngeal squamous cell carcinoma (LSCC), THCA, BLCA, urinary tract urothelial carcinoma (UTUC), LGG, GBM, UCEC, CESC, ovarian serous papillary cystadenocarcinoma (OPV), OV, penis squamous cell carcinoma (PSCC), TGCT, and PRAD. As indicated in via a loss-of-function assay. Firstly, a plasmid containing different PDIA5 siRNA sequences was constructed and transfected into human GBM cell line U251 cell. The western blotting analysis indicated that siRNA-PDIA5-1 and siRNA-PDIA5-2 contributed to a notable decrease in the protein level of PDIA5 compared with siRNA-NC  of human immunotherapy cohorts was visualized in PDIA5 could significantly predict immunotherapy response in 5 murine immunotherapy cohorts . And high PDIA5 levels seemed to suppress the activity of CD8+ T cells and Treg cells in some tumor types. These observations indicated that PDIA5 might be closely related to immunosuppression and T cell dysfunction in human cancers. The subsequent correlation analyses about the immune subtypes and immune pathways further demonstrated the broad involvement of PDIA5 in tumor immunity regulation. As the specific mechanism regarding the effect of PDIA5 on immune cells in TME was largely unknown, more wet experiments were expected to be performed.Our study revealed the close relationships between PDIA5 and immune checkpoint genes in multiple human cancers, strengthening the hypothesis that PDIA5 might enhance immunotherapy responses by synergizing with known immune checkpoint inhibitors in cancers. We also found that PDIA5 impacted neoantigens, TMB, MSI, MMR, and DNA methyltransferase in the TME. Collectively, these findings supported the possible intimate association between PDIA5 and TME and antitumor immunity.In pan-cancer analysis, scRNA-seq has emerged as a potent tool to identify the cellular heterogeneity of TME within tumors , 53. WanPseudotime trajectories analysis is always applied to evaluate correlations between genes and specific cell clusters in human cancers as it can capture and dissect the transcriptional alteration in cells during tumor progression . And celTo validate the relationships between PDAI5 and immune pathways in cancers, we investigated the effect of PDIA5 on the programmed death-1 (PD-1)/PD-L1 signaling pathway and SPP1 signaling pathway. The PD-1/PD-L1 pathway is responsible for the tumor immune tolerance and immune escape in human cancers, serving as one of the targets of ICB therapy . SPP1 caTAMs account for up to 50% of the cell population of the TME in some solid human malignancies, like breast cancer , gastricWe have previously concluded that PDIA5 could predict patients’ response to immunotherapies in gliomas as high PDIA5 displayed high anti-tumor immune activity , and theOur study also has several limitations. First, validation in other cancer types or a large cohort is warranted in further research. Second, the underlying mechanisms by which PDIA5 takes part in immune regulation remain largely unknown; more loss-of-function assay is necessary to elucidate the specific role of PDIA5 in human cancers. Third, previous studies found that PDIA3 could mediate the induction of T cell tolerance, the differentiation of regulatory T cell, and the activation of macrophage/microglia in cancers. In the present study, our results demonstrated that PDIA5 was able to mediate the migration of M2 macrophages. Therefore, we think that both of PDIA5 and PDIA3 promote the progression of human cancers through affecting the infiltrated immune cells in tumor microenvironment, and the underlying mechanism remains further exploration.In conclusion, our study provided comprehensive insights into the DNA methylation, genetic alterations, and immune infiltration role of PDIA5 across human cancers. These observations suggested that PDIA5 might serve as a potential target for the immunotherapy of human cancers.The datasets generated for this study are available on request to the corresponding author/s.RC, YC, and JH designed the study, interpreted data, and revised the manuscript. QC provided foundation support and supervised the study. HZ supervised the study. LZ revised the manuscript. ZW, ZD, XL, and HZ acquired and analyzed data. WW, PL, JZ, YP, ZL, and NZ drafted the manuscript and revised for submission quality. All authors read and approved the final manuscript.This work was supported by the National Natural Science Foundation of China (NO.82073893), Hunan Provincial Natural Science Foundation of China (NO.2022JJ20095), Hunan Provincial Health Committee Foundation of China (202204044869).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.


Salvia miltiorrhiza Burge (Danshen), a member of the Lamiaceae family, has been used in traditional Chinese medicine for many centuries as a valuable medicinal herb with antioxidative, anti-inflammatory, and antifibrotic potential. Several evidence-based reports have suggested that Salvia miltiorrhiza and its components prevent vascular diseases, including myocardial infarction, myocardial ischemia/reperfusion injury, arrhythmia, cardiac hypertrophy, and cardiac fibrosis. Tanshinone IIA (TanIIA), a lipophilic component of Salvia miltiorrhiza, has gained attention because of its possible preventive and curative activity against cardiovascular disorders. TanIIA, which possesses antioxidative, anti-inflammatory, and antifibrotic properties, could be a key component in the therapeutic potential of Salvia miltiorrhiza. Vascular diseases are often initiated by endothelial dysfunction, which is accompanied by vascular inflammation and fibrosis. In this review, we summarize how TanIIA suppresses tissue inflammation and fibrosis through signaling pathways such as PI3K/Akt/mTOR/eNOS, TGF-β1/Smad2/3, NF-κB, JNK/SAPK (stress-activated protein kinase)/MAPK, and ERK/Nrf2 pathways. In brief, this review illustrates the therapeutic value of TanIIA in the alleviation of oxidative stress, inflammation, and fibrosis, which are critical components of cardiovascular disorders. Salvia miltiorrhiza Bunge, known as Danshen in Chinese, is a member of the Labiatae family. The dried root of the rhizome of Salvia miltiorrhiza Burge has been widely used in traditional medicine in China and other oriental regions, especially for treating cardiovascular diseases like coronary heart disease, myocardial infarction (MI), angina pectoris, and atherosclerosis [clerosis –3. The aclerosis , 5. Althclerosis .Endothelial dysfunction describes a series of pathogenics promoting the development of hypertension and atherosclerosis, including oxidative stress, vascular endothelium injury, inflammation, and loss of smooth muscle elasticity , 7. In hSalvia miltiorrhiza extract, recognized for their therapeutic activity in the treatment and control of various diseases, including cardiovascular diseases, hepatitis, chronic kidney injury, and even dysmenorrhea [Tanshinones are a group of nonpolar lipid-soluble components in enorrhea . TanIIA enorrhea , 13. Tanenorrhea . FurtherSalvia miltiorrhiza has a long history of application in traditional medicine, the search for possible pharmacological components began in the 1930s [Salvia miltiorrhiza were reported to have benefits in microcirculation and could increase blood flow, dilate coronary arteries, and prevent myocardial ischemia, atherosclerosis, calcification, and aortic aneurysm formation. Owing to their different chemical structures, their pharmacological activities, pharmacokinetics, and clinical applications are different. Among these active ingredients, tanshinones are one of the most well studied compounds [Although he 1930s . These dhe 1930s –16 and lhe 1930s , 18. Wathe 1930s . In conthe 1930s , 21. Theompounds . In thisSalvia miltiorrhiza roots by Nakao in 1930 [Salvia miltiorrhiza [Tanshinones are quinone diterpenes that were first isolated from  in 1930 , and mor in 1930 . Tanshin in 1930 , 25. Taniorrhiza . The coriorrhiza . TanIIA iorrhiza , 26. Anoiorrhiza –29. STS iorrhiza –30. Bothiorrhiza . Howeveriorrhiza –35.In detail, the metabolic reaction of TanIIA and STS is similar in rat bile except for the oxidation in side chain, which occurred in TanIIA but not STS . STS suptβ1/2)” of 0.91 ± 0.21 hour but “elimination half-life” (tγ1/2) for 13.45 hours when 2 mg/kg of STS were injected into male Sprague–Dawley rats [Notably, TanIIA and STS exhibit different pharmacokinetic patterns. For intravenous injection, TanIIA showed elimination half-life of 1.0 ± 0.7 hour at 20 mg/kg and 1.8 ± 0.6 hour at 60 mg/kg dosage in the serum of male Sprague–Dawley rats . STS sholey rats , while tley rats . The extFor the tissue distribution, both TanIIA and STS can both found in organs like the heart, liver, spleen, and lung. For intravenous injection, TanIIA tends to reside in the lung and liver and STS tend to distribute in the kidney and liver , 38. IntDespite there being differences in pharmacokinetics and some molecular activities, both TanIIA and STS suppress endothelial dysfunction, the focus in this review. In this review, we summarized the studies using STS and TanIIA in vitro and in vivo in order to further explore the functional properties of these compounds Tables  and 2.Reactive oxygen species (ROS) are essential components of metabolism, immune responses, and cell signaling. However, excessive ROS threatens the normal function of cells. For example, oxidative stress damages DNA and alters protein structure into misfolded forms, causing stress to the cells and leading to cell senescence, apoptosis, or even necrosis , 42. In − free radicals from NO oxidation, (2) increased ICAM/VCAM expression, (3) increased intracellular Ca2+ levels activating the MAPK and Akt pathways, and (4) increased collagen disposition [NOX expression can be triggered by physical stimuli, such as shear stress, growth factors, cytokines, and metabolic factors such as hyperglycemia. Downstream metabolites include (1) ONOOposition .2O2 and xanthine oxidase, reduced the RNA level of procollagens but boosted MMP-2, MMP-9, MMP-13, and fibronectin in cardiac fibroblasts [ROS promote vascular fibrosis through several mechanisms. In fibroblasts, NOX4 promotes survival but suppresses cell death in models such as hypoxia , 45. Furroblasts . In endoroblasts . Finallyroblasts , indicatroblasts . This maroblasts .Endothelial cells are critical regulators of vascular tension. Mechanistically, the endothelium releases vasoconstrictors such as endothelin-1 (ET-1) and vasodilators such as nitric oxide (NO) . EndotheκB activation in endothelial cells [κB in endothelial cells upregulates VCAM-1, which binds to very late antigen-4  to recruit leukocytes [The endothelium of blood vessels plays a critical role in inflammatory responses. For example, NO suppresses ET-1 expression and TNF-driven NF-al cells . Endotheal cells . Furtherukocytes . Mechaniukocytes . Second,ukocytes . A classukocytes . Targetiukocytes .Below, we summarize the mechanism of action of TanIIA in the regulation of endothelial dysfunction and its potential implications for treating tissue inflammation and fibrosis  and 2.Salvia miltiorrhiza is well documented in cardiovascular diseases and anticancer therapy [The antioxidative activity of  therapy , 60. Pre therapy , 62. Not therapy . TanIIA  therapy .Salvia miltiorrhiza aqueous extract could alleviate ROS-dependent cell apoptosis in adriamycin-induced cardiomyopathy [The antioxidative effect of TanIIA has therapeutic potential in diseases other than CVD. For example, TanIIA reduced oxidative stress in the serum of tumor-bearing mice by combining intermittent hypoxia. TanIIA promotes apoptosis of tumor cells, which may be related to the activation of Nrf2 . Similarmyopathy , which fβ-driven chondrocyte apoptosis, inflammation, and extracellular matrix degradation in cellular models and protects against bone erosion in an osteoarthritis model [α signaling pathways, which are downstream of TNF-α [Tanshinones possess anti-inflammatory effects and ameliorate many diseases. For example, tanshinone I possesses potency similar to that of celecoxib to suppress IL-1is model . TanIIA of TNF-α .κB pathway, a potent proinflammatory circuit, can be inhibited by TanIIA, leading to the reduction of inflammatory mediators such as MCP-1, TGF-β, and TNF-α. The suppression of these cytokines also reduced macrophage infiltration into the infarcted myocardium [κB blockade also prevents the adhesion of TNF-α-driven endothelial progenitor cells (EPCs) by lowering the expression of ICAM-1 and VCAM-1 on the cell surface [β and IL-18 in response to oxidative stress and danger signals upon myocardial infarction [β pathway and reduction of miR-155 [κB and NLRP3 inflammasomes but restores other signaling pathways.TanIIA also suppresses inflammatory responses in blood vessels, which are known to exacerbate endothelial dysfunction, atherosclerotic plaque formation, and vascular injury. The NF-ocardium , 73. NF- surface . In addifarction . TanIIA farction . Moreove miR-155 . TanIIA β binds to a heterodimeric receptor composed of TGF-βRI and TGF-βRII. Upon TGF-β activation, TGF-βRI is phosphorylated by the kinase activity of TGF-βRII, providing a docking site for Smad2/3. Smad2/3 is phosphorylated by TGF-βRI and then binds to the co-Smad-like Smad4, which forms a complex and then translocates into the nucleus [α-SMA  [βRI [TGF- nucleus . Profibr nucleus . The Sma nucleus . Snail,  nucleus . For exae actin) . In contin) [βRI .β pathway. In rat cardiac fibroblasts, TanIIA inhibits the phosphorylation of Smad2/3, leading to the reduction of nuclear translocation of Smads and downregulated expression of fibronectin genes [α-SMA, collagen type I and III, periostin, and TGF-β but elevated matrix metalloproteinase-1 (MMP-1) in AngII-treated cardiac fibroblasts [TanIIA controls fibrosis by interfering with the Smad-dependent TGF-in genes , 84. Simroblasts –87, suggroblasts .The suppressive effect of TanIIA on Smad phosphorylation may be Nrf2-dependent . TanIIA β also transmits noncanonical signals through the phosphorylation of MAPK family members, PI3K, Rho A, Rac, c-Abl, and PKC. Downstream genes include Snail1, Snail 2, and Twist1 [β2 drives EndMT in human cutaneous microvascular endothelial cells via collaborating with the Smad, MEK, PI3K, and p38 MAPK signaling pathways [In addition to Smad phosphorylation, TGF-d Twist1 . For exapathways . JAK2 alpathways .β1-activated human atrial fibroblast-to-myofibroblast differentiation by suppressing both phosphorylation of Smad3 and ERK1/2 [STS has been shown to inhibit AngII-induced cardiomyocyte hypertrophy and lower systolic blood pressure in cultured neonatal rat myocardial cells in vitro and in rats in vivo, mainly through the suppression of c-Fos, c-Jun, ERK, and MEK –98. The d ERK1/2 , suggestd ERK1/2 .κB, Akt, and p21 [The Notch pathway is essential for the development of vascular smooth muscle  and card and p21 , 102. In and p21 . For exa and p21 . By usin and p21 .μM, TanIIA limits the growth, migration, and invasion of astrocytoma cells. At the molecular level, TanIIA boosts the expression of Notch-1 and caspase-3/9 but reduces the phosphorylation of c-Myc, MMP-9, and Bcl2 [μM was found to promote the survival of spinal cord endothelial cells (SCMEC) in an oxygen-glucose deprivation model by boosting Notch signaling. In this case, Notch upregulation decreases inflammatory cytokines such as IL-6, TNF, and IL-1β in SCMECs. In the murine spinal cord injury model, STS can (1) boost the expression of Notch, (2) rescue the microvessel, (3) maintain the blood-spinal cord barrier, and (4) protect the structural and functional integrity of the nervous system [TanIIA is known to suppress tumor progression through activation of the Notch signal pathway . At an iand Bcl2 . In the and Bcl2 . Moreoves system . Howeverβ-catenin is constantly quenched by a destruction complex, comprising proteins such as disheveled and GSK-3β, and then subjected to proteasomal degradation. Upon Wnt signal activation, β-catenin accumulates and translocates into the nucleus [β-catenin binds to T cell factors (TCFs) to induce the transcription of genes such as Twist [β-catenin signaling is crucial for heart cushion formation through EndMT activity [β-catenin, TCFs, and SMA in vitro and in vivo, respectively [β-catenin, and snail expressions than those from normal subjects. The results fit the elevation of fibrosis markers such as SMA and FSP-1 in DCM patients [There are more than 10 Wnt genes in mammals, which share a conserved feature: palmitoylation on their polypeptide chain . The lip nucleus . In the as Twist . Wnt sigactivity . Neverthectively . In clinpatients .μM, TanIIA ameliorates ischemic skin flap mice by inducing the expression of β-catenin and stem cell markers such as SOX2, Nanog, and OCT4 in epidermal cells [μM, STS inhibited the expression levels of COX-2, β-catenin, and VEGF, resulting in growth inhibition of HC8693 colon cancer cells [The therapeutic potential of the TanIIA-Wnt axis can be found in skin transplantation and cancer studies. At a dosage of approximately 5 al cells . Neverther cells . The regμM) rescues the expression of β-catenin and the phosphorylation of GSK-3β in high-glucose-treated HUVECs. In contrast, STS treatment reduced apoptosis and the expression of CXCL1 in HUVEC [β-catenin, and WISP-1. In addition, TanIIA readily reduces hypertrophy and fibrosis of the heart, especially the left ventricle. At the molecular level, TanIIA reduced the mRNA levels of Col1a1 and Col3a1. Moreover, TanIIA reduced cardiac injury markers such as troponin, NOX4, and ADMA, but elevated cardioprotective NO and eNOS. Functionally, TanIIA controls systolic blood pressure in SHR animals, regardless of dosage [TanIIA exerts protective effects by boosting the Wnt pathway in endothelial cells. For example, STS (~10 in HUVEC . Furtherin HUVEC . Howeverin HUVEC . Intrapef dosage . AlthougEndothelin-1 (ET-1) is encoded by EDN1. Full-length preproendothelin-1 is processed by furin, chymase, and neprilysin cleavage, leaving a 21-amino-acid peptide. ET-1 is the most potent member of the three endothelins found in mammals, largely derived from vascular endothelial cells in all types of blood vessels, although other cells such as macrophages, enteric glial cells, and some neurons express ET-1 as well. In addition to a constant expression, ET-1 in endothelial cells can be further boosted by pathophysiological stimuli. After release from the endothelium, ET-1 largely binds to a GPCR named ETA, but less so to the other GPCRs, ETB , 122. ETTanIIA suppresses ET-1-driven endothelial dysfunction at both the cellular and organ levels. In endothelial cells, TanIIA inhibits the expression of ET-1 in HUVEC under cyclic strain . In smooκB p65 [β crosstalk can be found in vascular smooth muscle cells (VSMCs). CAV-1 is phosphorylated by c-Src upon TGF-β stimulation. Y14-phosphorylated CAV-1 activates Rho-GTP/ROCK signaling, which inhibits PTEN and phosphatase PPM1A. As a result, Smad2/3 phosphorylation is maintained and PAI-1 expression is increased in atherosclerotic plaques [Caveolin-1 (CAV-1) is essential for the formation of caveolae and secretion of substances in the apical direction of vesicles. CAV-1 oligomerizes and is coated onto the surface of invaginated membrane structures . In the κB p65 . CAV-1 a plaques .β pathway. Whether TanIIA directly controls the activity of CAV-1 in endothelial cells or VSMCs remains unknown. A previous study suggested the role of TanIIA in CAV-1-related endocytosis. In neuron progenitor cell lines, CAV-1 is essential for the endocytosis of TanIIA, promoting its biological activity in boosting MAPK42/44 and CREB activities. Furthermore, the entry of TanIIA elevates the expression levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in these cell lines and promotes neuronal differentiation [TanIIA is known to control the downstream TGF-ntiation .Salvia miltiorrhiza for the treatment of coronary heart disease was conducted after integrating the protein–protein interaction data. Tanshinones are proposed to regulate blood circulation through guanylate cyclase soluble subunit alpha-1 and guanylate cyclase soluble subunit beta-1 [As a broad-spectrum therapeutic compound, TanIIA also protects cardiovascular function through other mechanisms. In the hearts of the canine MI model, TanIIA restored PPAR-alpha expression but limited lipid accumulation . TanIIA t beta-1 , thus prRecently, the medicinal value of Tan IIA, especially water-soluble STS, has been intensively explored. Cardiovascular application of TanIIA in combination with classical treatments dominates recent clinical trials. TanIIA has been found to improve the prognosis of coronary artery diseases by reducing injury markers, such as cardiac troponin-I, and the incidence of major adverse cardiovascular events –143. As Salvia miltiorrhiza with substantial therapeutic potential for cardiovascular disorders (Tables Taken together, TanIIA, which possesses multifaceted roles involving antioxidation, anti-inflammation, and antifibrosis, could be a key component of s Tables  and 2. I


Background: New tools for the assessment and prediction of the severity of hospitalized COVID-19 patients can help direct limited resources to patients with the greatest need. Circulating levels of calprotectin (S100A8/S100A9) reflect inflammatory activity in multiple conditions, and have been described as being elevated in COVID-19 patients, but their measurement is not routinely utilized. The aim of our study was to assess the practical and predictive value of measuring circulating calprotectin levels in patients at admission and during their hospitalization. Methods: Circulating calprotectin levels were measured in 157 hospitalized patients with COVID-19 using an automated quantitative chemiluminescent assay. Results: Circulating calprotectin levels were strongly correlated with changing respiratory supplementation needs of patients. The overall trajectory of circulating calprotectin levels generally correlated with patient improvement or deterioration. Conclusions: Routine measurement of circulating calprotectin levels may offer a valuable tool to assess and monitor hospitalized patients with COVID-19, as well as other acute inflammatory conditions.  Although increasing immunity levels due to past infection and vaccination have resulted in declining COVID-19 mortality in most regions, high infection rates continue, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus continues to evolve, and individuals remain at risk of progressing to severe disease. This is of particular relevance in light of emerging variants of concern (VOCs) such as Delta B.1.617.2) or, most recently, the Omicron variant B.1.1.529) . Levels 29 . Leve17.2 or, Several studies have shown an association between cCP and intensive care unit (ICU) admission, mechanical ventilation, and mortality, but there has been little examination of its potential to monitor improving or deteriorating patient status ,8,9,10. ®, Inova Diagnostics, San Diego, CA, USA; Investigational Use Only in USA, CE (Conformitè Europëenne) marked for in vitro diagnostics) on the BIO-FLASH® instrument . This assay utilizes paramagnetic beads coated with calprotectin-specific antibodies to capture calprotectin. Briefly, after incubation with the patient specimen, followed by washing steps, bound calprotectin is detected by an anti-calprotectin monoclonal antibody conjugated to isoluminol. Upon exposure to a triggering reagent, bound isoluminol generates a luminescent signal, which is detected by the instrument as relative light units (RLUs). The RLU values are proportional to the amount of calprotectin captured on the paramagnetic beads. Using a predefined, lot-specific master curve and the results of three calibrators, the instrument software calculates μg/mL for each sample. The analytic measuring range of the assay is 0.18 to 22.76 µg/mL. Samples with cCP over 22.76 µg/mL can be automatically diluted 10-fold and re-run by the instrument to allow values up to 227.60 µg/mL to be measured. According to the manufacturer, the precision of the cCP assay evaluated according to CLSI EP05-A3 was as follows: repeatability (CV 2.1–3.3%), inter-run (CV 1.3–2.6%), inter-day (CV 2.1–4.5%), and intra-laboratory precision (CV 3.1–5.1%).Sera collected from 157 patients admitted to University of Michigan Hospital during spring 2020 with a diagnosis of COVID-19 confirmed by an FDA-approved RNA test were tested with a sensitive, quantitative, automated circulating calprotectin chemiluminescent immunoassay (QUANTA Flashn = 29), nasal cannula , high-flow oxygen , and mechanical ventilation . RA, NC, HFO, and MV would be equivalent by the 10-point WHO classification scheme to scores of 4, 5, 6, and 7–9, respectively, and by the alternative 8-point system to scores of 3, 4, 5, and 6–7, respectively [All patients were classified by ventilation status at the time of the first specimen collection, as follows: room air  and GraphPad Prism  were used for statistical analysis and graphical presentation. Wilcoxon, Mann–Whitney, and ANOVA tests were used to compare categorical variables; the Mann–Whitney test was used to analyze differences between groups. Receiver operating characteristic (ROC) analysis was conducted to assess the diagnostic performance of cCP ; The characteristics of the study population are summarized in p < 0.0001)) (Patients with COVID-19 had increased median levels of cCP (4.8 µg/mL) compared to the manufacturer’s cutoff of 2 µg/mL for healthy individuals. In the present study, the median cCP level measured in 39 healthy controls was 1.2 µg/mL. Higher cCP levels were significantly associated with increased need for oxygen supplementation ) a. ROC an0.0001)) b. At a cp = 0.0017). ROC analysis showed an AUC of 0.866   b. PatienExamining 20 patients with longitudinal measurements showed that increasing or declining cCP levels were generally correlated with respiratory deterioration or improvement, respectively . CirculaIn addition to cCP, results for D-dimer, fibrinogen, CRP, ferritin, and LDH were available for a small subset of the longitudinally followed patients. Examination of these patients revealed a number or different profiles. In Despite progress in understanding the pathogenesis of COVID-19, the development of vaccines, and increasing experience in managing patients, COVID-19 remains a critical healthcare problem worldwide. While intense efforts to understand and predict the differential impact of SARS-CoV-2 infection on individuals have resulted in an expanding trove of data on gene, protein, and cellular processes in patients with COVID-19, front-line care of patients still focuses on the use of practical, well-understood, and easily automated routine laboratory tests ,14,15,16Interest in calprotectin as a biomarker of inflammation has surged with its increasing availability on ELISA and automated platforms, and with better understanding of the important pre-analytical requirements for reproducible measurement ,18,19,20The trajectory of cCP levels generally appears to be correlated with patient outcome. Moreover, monitoring changes may provide actionable information to predict patient improvement or deterioration, and may help to guide clinical management. While a recent publication questioned the incremental benefit of cCP as a predictor of severe COVID-19 compared to CRP, the study used a different cCP assay, and focused on a review of 3280 ambulatory patients where only 6.8% had a final diagnosis of COVID-19 . In this