Ventx is a homeobox transcription factor and has a DNA-interacting domain that is evolutionarily conserved throughout vertebrates. It has been extensively studied in Xenopus, zebrafish, and humans. The Ventx family contains transcriptional repressors widely involved in embryonic development and tumorigenesis in vertebrates. Several studies have documented that the Ventx family inhibited dorsal mesodermal formation, neural induction, and head formation in Xenopus and zebrafish. Moreover, Ventx2.2 showed functional similarities to Nanog and Barx1, leading to pluripotency and neural-crest migration in vertebrates. Among them, Ventx protein is an orthologue of the Ventx family in humans. Studies have demonstrated that human Ventx was strongly associated with myeloid-cell differentiation and acute myeloid leukemia. The therapeutic potential of Ventx family inhibition in combating cancer progression in humans is discussed. Additionally, we briefly discuss genome evolution, gene duplication, pseudo-allotetraploidy, and the homeobox family in Xenopus.The Ventx family is one of the subfamilies of the ANTP ( Xenopus laevis is an established animal model for studying vertebrate development, neurobiology, immunology, cell biology, and toxicology ..Xenopus NTXP2 13q.1, VENTX(3p24.3) ,36,37,38(3p24.3) ,40,41,42ebrafish ,44,45. Iat St.11 ,45,46,47. Th. ThDrosot manner ,13,26. At manner ,9,27. Hot manner .Xenopus, the Ventx family has six members, including Ventx1.1, Ventx1.2, Ventx2.1 Ventx2.2, Ventx3.1, and Ventx3.2  during gastrulation. The detailed functional and molecular mechanisms of these members are discussed below.In Ventx3.2 A. The phs laevis B,B’. AllXenopus embryos. Moreover, high levels of ventx1.1 expression have been reported in the ventral marginal zone and ectoderm in gastrulation stage embryos. A study demonstrated that BMP4/Smad1 signaling enhanced the expression of ventx1.1 while blocking BMP4 signaling via a constitutively inactive, truncated form of BMP4 receptor I (DN-BR/tBR) that eliminated ventx1.1 expression in uncommitted ectodermal explants of Xenopus embryos . Th. ThXenopignaling . Human Vic cells . In addiferation . These sVentx led to macrophage differentiation, depending on the cytokines involved, such as CSFs and IL-3. The inhibition of Ventx in primary monocytes suppressed monocyte-to-macrophage differentiation, suggesting that Ventx plays a significant role in macrophage differentiation and the ensuing activation of cytokines . A recenVentx in grafted HSC/MPP cells also increased the engraftment potential of HSC/MPP in NOD/SCID/IL2Rγ2null murine models. This suggested that Ventx could be a potent target for the application of HSC/MPP-based therapy. Recently, a study demonstrated that the expression of Ventx robustly increased in normal myeloid cells and in AML patients [+ stem and progenitor cells. Moreover, the transplantation of retrovirally-engineered Ventx-expressed bone marrow progenitor cells increased the population of primitive erythroid cells to trigger acute erythroid leukemia in transplanted mice. Furthermore, the concomitant expression of AML1-ETO ) usually occurs in acute myeloid leukemia (AML) with granulocytic differentiation, FAB M2 subtype [ink4a-Rb and p53–p21 tumor-suppression pathways [Ventx enhanced cell senescence and ameliorated the camptothecin (CPT) resistance in lymphocytic leukemia cells. Ventx-associated chemotherapeutic resistance and its involvement in the cell-senescence process suggested that Ventx could be a novel, potential target for cancer prevention. A recent study confirmed the anticancer role of Ventx and showed that Ventx triggered apoptosis in a p53-independent manner in cancer cells (HCT116 p53KO) but not in healthy cells, in vitro [−/− knockout cells, leading to the suppression of tumor growth.An elevated level of Ventx was reported in immature human hematopoietic cells. The knockdown of the Ventx expression significantly expanded the population of human stem cells and multipotent progenitor cells (HSC/MPP) and promoted the survival of HSC, ex vivo . The suppatients . The ove subtype , and Ven subtype . Ventx i subtype . A genompathways . Overexpin vitro . In addi−/− knockout cells. Ventx’s involvement in hematopoiesis, leukemogenesis, cell senescence, and macrophage differentiation is illustrated in +/+ and p53−/− cells, and Ventx might be a novel therapeutic target for certain human cancers, working in a p53-independent manner. In summary, Ventx is deeply involved in hematopoiesis and leukemogenesis in humans, and it could be a future therapeutic target for combating tumorigenesis.Furthermore, the treatment of doxorubicin increased the level of Ventx in both p53 (WT) and p53Nanog (p53 is a direct inhibitor of Nanog). Nanog and p53 mutually inhibit each other expression in glioma tumors . However, p53 promoted Notch1 expression in early-stage oral squamous cell carcinoma. Notch1 is a positive regulator of Nanog [Nanog is a well-established stem-cell pluripotent marker and homeobox transcription factor which creates stem-cell-like properties in cancer cells . Earlierof Nanog . The Nanof Nanog ,120. MeaAlthough, Ventx synteny is absent in rodent, but a recent study has been demonstrated that Nanog acts like a Ventx ortholog in rodents . It showThese studies showed that Nanog and Ventx not only share functional similarities in maintaining cell pluripotency to delay cell-lineage commitments during embryonic development, but they are also similarly involved in cancer progression and leukemogenesis in humans.Ventx and Vega families in the ventral regions of Xenopus and zebrafish embryos, respectively. The Ventx family is known for its transcriptional repressor activity, which drives the proper axial formation during embryonic development. The Ventx family not only inhibited dorsally expressed genes but also activated the transcription of ventral specific genes, leading to axial patterning and cell-fate determination in zebrafish and Xenopus [Xenopus [Xenopus embryos.The Ventx and Vega families function downstream of BMP4/Smad1/5/8 signaling in vertebrates. BMP4/Smad1 upregulates the expression of  Xenopus ,83,121. [Xenopus ,49. Addiventx1.1 and ventx1.2, in a LEF1/TCF-dependent manner in Xenopus [gsk3β mRNA, leading to embryonic ventralization in Xenopus [Xenopus [Xenopus. In humans, Ventx boosted cell proliferation and hematopoietic cell-terminal differentiation, including myeloid cell differentiation [−/− knockout cell lines and activates cell senescence, which is followed by the activation of caspase-3, p53-p21, and p16ink4-Rb pathways. In p53−/− cells, doxorubicin augmented the Ventx level to trigger apoptosis in a caspase-3-dependent manner. Therefore, the Ventx family acts as a key player in dorsoventral axial patterning, pluripotency, cell-fate determination, hematopoiesis, apoptosis, and CNC migration. Additionally, the inhibition of Ventx family members suppresses cell proliferation and tumor growth in humans.In the Ventx family, the transactivation domain (TAD) of Ventx2.2 transactivated the Ventx genes, including  Xenopus ,22,24,87 Xenopus . However[Xenopus . Ventx2.ntiation ,32. Studntiation ,51. Ventntiation . An elevntiation ,115. Venventx1.2 and bmp4 in Xenopus [Xenopus embryos. The expression of GATA2 was also directly controlled by BMP4/Smad1 signaling and remained high in VBIs to induce differentiation of the hematopoietic mesoderm in Xenopus laevis [GATA2 were also reported in AML cell lines, patients’ samples, and tumors [WT1 —in KG1a cells and in clinical patients’ AML cells, triggering apoptosis. The heterozygous knockout of GATA2+/− in mice caused the inhibition of abnormal myeloid-progenitor-cell proliferation and differentiation and leukemia inhibition in Cbfb-MYH11 transgenic mice. Interestingly, transplanted leukemic cells drove leukemogenesis more aggressively in Cbfb-MYH11 and GATA2+/− mice, but not in normal mice. GATA2 inhibition minimized the mutation frequency of genes associated with leukemogenesis and delayed the onset of leukemogenesis in Cbfb-MYH11 knock-in mice. Collectively, the knockdown of GATA2 and Ventx attenuated cell proliferation and tumor growth, improved patient survival, and boosted the immunosuppression by drugs in tumor samples. Kumar et al. (2019) have reported that Ventx1.1 repressed the expression of ventx2.2 and ventx1.2 in a BMP4-independent manner in Xenopus embryos [ventx2.2, leading to ventx2.2 inhibition in Xenopus embryos (unpublished data). A previous study has reported that overexpressing Ventx1.1 suppressed GATA2 mRNA levels in the VMZ during hematopoiesis of Xenopus embryos [Xenopus embryos. However, the physiological outcome of this interaction remains unknown [Xenopus, it might be that the ectopic expression of Ventx1.1 inhibits the Ventx–GATA2 complex in humans and blocks GATA2- and Ventx2.1-induced leukemogenesis. Based on the collective evidence in Xenopus and humans, we concluded that Ventx1.1 may also inhibit the transcription of Ventx and GATA2, leading to the inhibition of human leukemogenesis. Xenopus Ventx1.1 could be a novel therapeutic pathway to target Ventx- and GATA2-associated cancers.A study has shown that Ventx2 interacted with GATA2 to promote the functions of  Xenopus . Moreoves laevis ,122,123.d tumors ,125,126.d tumors ,125. The embryos . This in embryos . A study unknown . Since V


Glioblastoma multiforme persists to be an enigmatic distress in neuro-oncology. Its untethering capacity to thrive in a confined microenvironment, metastasize intracranially, and remain resistant to the systemic treatments, renders this tumour incurable. The glial cell type specificity in GBM remains exploratory. In our study, we aimed to address this problem by studying the GBM at the cell type level in the brain. The cellular makeup of this tumour is composed of genetically altered glial cells which include astrocyte, microglia, oligodendrocyte precursor cell, newly formed oligodendrocyte and myelinating oligodendrocyte. We extracted cell type-specific solid tumour as well as recurrent solid tumour glioma genes, and studied their functional networks and contribution towards gliomagenesis. We identified the principal transcription factors that are found to be regulating vital tumorigenic processes. We also assessed the protein–protein interaction networks at their domain level to get a more microscopic view of the structural and functional operations that transpire in these cells. This yielded the eminent protein regulators exhibiting their regulation in signaling pathways. Overall, our study unveiled regulatory mechanisms in glioma cell types that can be targeted for a more efficient glioma therapy. Astrocytes are the most abundant (approximately 20–40%) irregular star-shaped cell type in the brain Although some cancers have well-defined series of events that lead to their genesis, the development of GBM is driven by a complex network of various genetic and molecular perturbations, resulting in critical changes in signaling pathways In this study, we conducted an analysis on differential gene expression profiles of GBM primary solid tumour (TP) and recurrent solid tumour (TR) in astrocytes, microglial cells, OPCs, NFOs, and MOs. Our investigation enabled us to dissect top transcription factor regulators and scrutinize protein-domain level network interactions in each of these GBM TP and TR specific cell types. This allowed us to make some noteworthy observations, and revealed common as well as unique mechanisms that occur in these glial cells. These findings can be further validated to inspect their relevance as targets for drug therapy in the hope of a sustainable treatment against GBM.22.1https://gdac.broadinstitute.org/) provided us with RNA-Seq by Expectation-Maximization (RSEM) normalized expression data for normal brain and TP, TR cancer from GBM patients. The DESeq2 package, which is part of the integrated Differential Expression and Pathway analysis (iDEP v.90) software p-adjusted-value ≤ 0.05 and log2 fold change 1 (absolute fold change: 2) https://biit.cs.ut.ee/gprofiler/orth). Ultimately, we went ahead with the genes that were available in human organism and performed our investigation.The Cancer Genome Atlas (TCGA) firehouse , an index of 1639 known and probable TFs https://hbiostat.org/R/Hmisc/). We then formatted the correlation matrix that consists of the cormat matrix of the correlation coefficients, and the pmat matrix of the correlation p-values, by using the function flattenCorrMatrix. The TF co-regulatory and TF-TF correlation networks were visualized in Cytoscape v3.8.0 We curated transcription factors (TFs) from the above-mentioned subsets of genes by using the human TF atlas v1.01 (2.4http://instruct.yulab.org), a 3-dimensional structurally resolved library of high-quality protein interactome networks containing 6585 interactions at protein domain level. INstruct consists of interactions that were derived from some of the most prominent interaction databases and screened to present only binary interactions that met its stringent quality standards http://signor.uniroma2.it), the SIGnaling Network Open Resource We extracted the human protein domain interactions (PDIs) data from the INstruct database (2.5We used METASCAPE 2.6https://www.r-project.org/) to conduct data visualization and statistical analysis. Hierarchical clustering analysis was done using the Euclidean similarity metric and visualized as dendrogram using factoextra R package. Volcano plots illustrating the top DEGs in and across glioma-specific cell types were generated using the ggplot2, ggrepel, EnhancedVolcano, gghighlight, and dplyr R packages. The differential expression analysis of GBM and the top protein regulators in PDINs were visualized as barplots using the ggpubr R package that implemented the ggpbarplot function. Unique and common glioma-specific genes/TFs in cell types were pictured as barplots using the UpSet R package. The Pheatmap R package was used to render the heatmaps.In this study, we used R statistical software v3.6.3 . The bulk RNA and single-cell sequencing gene datasets of the brain cell types- astrocyte, microglia, OPC, NFO and MO- were mined from Zhang Y et al GBM is widely studied at tissue level, however, here we scrutinized the events that occur in the cell types of this tumour at its primary as well as recurrent stages. The workflow we followed in this study is briefly illustrated as a pipeline in Fig. 13.2B-C). We detected 6639 DEGs in GBM TP, out of which 3465 were upregulated and 3174 were downregulated (D). In GBM TR, we identified 579 DEGs, out of which 310 were upregulated and 269 were downregulated (E).To elucidate the functional dynamics that occur due to the gene expression in GBM, we performed the transcriptomic examination. The hierarchical clustering analysis of GBM TP and TR expression profiles evidently showed clear partitioning of the normal brain and cancer patient samples B-C. We degulated D. In GBMegulated E.A-B.Our next step involved overlapping of the GBM TP, TR, astrocyte, microglia, OPC, NFO and MO gene datasets in order to derive cell type-specific glioma genes. This allowed us to cull down subsets of unique cell type-specific glioma genes and common glioma genes across the cell types as shown in Fig. 2In total, we obtained 29 subsets of TP glioma genes and 19 subsets of TR glioma genes. These include genes that are unique to the cell types and common across various combinations of these cell types. The genes in each subset were categorized based on their differential expression . The DEG3.3To enhance our knowledge on the involvement of these cell-specific DEGs in GBM, we performed pathway enrichment analysis of upregulated and downregulated genes separately in TP and TR. These analyses revealed that in TP astrocytes, genes participate in hormone synthesis and metabolism . In TP mCorrespondingly, in TR astrocyte, genes are involved in signaling cascades . In TR mThe pathway enrichment analysis was also conducted for genes that were common between the cell types and GBM. In sum, the common TP upregulated genes are involved in signaling, cell adhesion and metabolic pathways . SimilarFurthermore, the TR upregulated genes that are common across different combinations of cell types participate in signaling pathways, cell growth, apoptosis, and immune response (3.4Transcription factors (TFs) play a critical role in the regulation of gene transcription and expression processes. This corresponds as a vital element in an intricate network system that governs healthy cell development and function A). In TP microglia, out of 14 total TFs, 11 are complemented with ChIP-seq . In TP MO, out of a total of 15 TFs, 11 were supported by ChIP-seq data . In TP NFO, out of a total of 23 TFs, 13 are corroborated with the ChIP-seq data . In TP OPC and NFO, out of a total of 26 TFs, 16 were complemented with ChIP-Seq data . In TFs common across TP NFO and MO, a total of 13 were observed out of which 8 were enriched with ChIP-Seq data . In TP astrocyte, OPC and NFO, a total of 7 TFs were found, out of which 4 were supported with ChIP-Seq data . Lastly, in TP OPC, NFO and MO, a total of 12 TFs were identified, out of which 8 were corroborated with ChIP-Seq data (A-D). The common novel putative TFs were also observed in TP OPC and NFO (n = 10), NFO and MO (n = 5), astrocyte, OPC and NFO (n = 3) and OPC, NFO and MO (n = 4) (E-H). For these novel TFs we did not find evidences in ChIP-seq datasets from ENCODE. Overall, this analysis revealed enriched common and unique transcription factors in glioma-specific cell types.We initiated our examination with putative TF-target co-regulation analysis. This was done via CoRegNet that enriched the networks with the ENCODE ChIP-sequencing data as additional evidence Supplementary Fig. S1Seq data . FinallySeq data A-D. The  (n = 4) E-H. For 3.5A). Similarly, in TP microglia, correlation between 11 TFs was seen, with LTF being the prominent TF . In TP MO, correlation between 11 TFs was found, with DMRTA2 being the notable TF influencer, followed by TCF7L1and FOXS1 . In TP NFO, correlation between 13 TFs was noted, with CENPA being the influential TF followed by E2F8 and EN2 . In OPC and NFO, correlation between 16 TFs was detected with E2F7 being the eminent TF influencer followed by FOXJ1 . Similarly, in TFs common between NFO and MO, correlation between 8 TFs was witnessed, with ASCL1 being the principal TF, followed by SOX11 and CSRNP3 . In OPC, NFO and MO, correlation between 8 TFs was observed, with TEAD2 being the chief TF followed by DLX1 and ST18 . Lastly, in astrocyte, OPC and NFO, correlation between 4 TFs was seen, with BCL11A being the salient TF influencer . In sum, TF-TF network analysis revealed top TF regulators in each glioma-specific cell type.To gain a deeper understanding of the functionality and association between the TFs, we conducted a TF-TF correlation analysis by computing the correlation coefficient to generate networks displaying positive (activation) and negative (inhibition) interactions. We further refined this investigation by conducting TF transcriptional influence activity analysis using the CoRegNet package Fig. 63.6A). Consequently, in TP microglia, LTF, the significant TF regulator is found to regulate signaling pathways like TNF, TLR, chemokine, B-cell receptor and processes such as transcriptional misregulation, natural killer cell mediated cytotoxicity, phagocytosis and carbon metabolism in cancer . Moreover, the TF BATF is found to significantly regulate the highest number of processes such as negative regulation of immune system, positive regulation of adaptive immune response and NFκB TF activity, B cell activation, T cell migration etc. In TP MO, the top regulator, DMRTA2, is seen to be involved in CAMs . In TP NFO, the eminent regulator, CENPA, is observed to be involved in cell cycle whereas the third top regulator, EN2, is found to be involved in apoptosis, phosphatidylinositol signaling system, complement and coagulation cascades. However, NKX6.2 is seen to participate in the highest number of processes like pathways in cancer, beta-alanine metabolism, RIG-I-like signaling pathway etc. . In addition, ASH1L is seen to regulate the highest number of processes such as apoptosis, NFκB signaling pathway, carbon metabolism in cancer etc. Similarly, in TP OPC and NFO, the TF SP9 is witnessed to significantly regulate highest number of processes such as CAMs, and cAMP, MAPK signaling pathways .Additionally, to better acquire knowledge of the processes the TFs participate in each of the cell types, we performed a pathway enrichment analysis. This evaluation revealed that in TP astrocyte, TFs ZNF878 and CREB3L4 are involved in highest number of processes such as signal transduction downstream of smoothened protein which is involved in hedgehog signaling Fig. 7F). Additionally, the TF TEAD3 is found to regulate highest number of signaling pathways such as extrinsic apoptotic via death domain receptors, NFκB and insulin-like growth factor receptor. In TP astrocyte, OPC and NFO, the notable TF, BCL11A, is involved in microRNAs in cancer, whereas the TF SIX5 is seen to regulate highest number of processes such as regulation of GTPase activity, cell growth, complement activation, lectin pathway etc (G). Finally, In TP OPC, NFO and MO, the second most significant TF influencer DLX1, is noted to be involved in cGMP-PKG signaling pathway and the third top TF influencer, ST18, is noted to be involved in the highest number of processes that include cAMP, oxytocin and MAPK signaling pathways (H). Taken together, the TF network pathway analysis gave us a thorough view of the important cancer-related pathways they regulate in each glioma-specific cell type.Furthermore, in TFs common between TP NFO and MO, the prominent TF regulator, ASCL1, is found to be involved in endocytosis and cytokine-cytokine receptor interaction, whereas the second top TF influencer, SOX11, is found to regulate signaling pathways such as TGF-β, MAPK, calcium, phosphatidylinositol and apelin F. Additihway etc G. Finallpathways H. Taken 3.7Protein-protein interactions (PPIs) are the fundamental driving force behind the working of a complex network of processes. The interactions occur when the respective domains of each protein physically associate with each other, thereby corresponding to facilitate a particular functionA). In TP microglia, we observed signaling pathways that regulate pluripotency of stem cell, MAPK signaling pathway, complement and coagulation cascades as some of the important processes (B). We further winnowed down MAPK14 (domain- Pkinase), NFKBIA (domain- Ank), BCL3 (domain- Ank), SYK and TRADD (domain- Death) as the significant protein regulators that are regulating 4, 2, 2, 2 and 2 proteins, respectively. The top TF influencer LTF, is found to regulate BCL3. In TP OPC, the participation of the PDIs in pathways and processes needs to be studied further (C). In TP NFO, the PDIs were seen to take part in processes such as cell cycle, signaling pathways regulating pluripotency of stem cells, Rap1 and PI3K-Akt signaling pathways, complement and coagulation cascades (D). The two most prominent protein regulators were found to be CDK1 (domain- Pkinase) and PTPN12 (domain- Y_phosphatase) regulating 4 and 3 proteins, respectively. In TP MO, we witnessed PDIs engaging in signaling pathways such as T-cell receptor, MAPK, and sphingolipid (E). In this PDIN, PDPK1 (domain- Pkinase) was found to be the chief protein regulator that is regulating 2 proteins. PDPK1 is regulated by the most significant TF influencer DMRTA2 in this cell type.In TP astrocyte, some important processes like neurotrophin signaling pathway and endocytosis were seen A. In TP rocesses B. We fur further C. In TP cascades D. The twngolipid E. In thiF). In TP microglia and OPC, a PDI is observed to engage in cell cycle (G). In TP microglia and NFO, the vital processes witnessed were Rap1, NFκB signaling pathways and natural killer cell mediated cytotoxicity (H). PRKCA (domain- Pkinase) and SYK were noted to be the influential protein regulators regulating 6 and 2 proteins, respectively. In TP OPC and MO, a PDI takes part in axon guidance (I). In TP OPC and NFO, vital processes like cell cycle, transcriptional misregulation, fluid sheer stress and signaling pathways such as Jak-STAT, hippo, PI3K-Akt, AGE-RAGE, apelin, and cAMP were observed (J). The 3 notable protein regulators were winnowed down to SRC, PDGFRB and PLK1 (domain- Pkinase) that are regulating 12, 3 and 3 proteins, respectively. PLK1 was found to be governed by the top TF influencer i.e. E2F7. In TP NFO and MO, the PDIs participate in Ras, Rap1, TNF, phospholipase D signaling pathways and natural killer cell mediated cytotoxicity (K). In TP astrocyte, OPC and MO, we noted the PDIs but their involvement in processes needs further evaluation (L). In TP astrocyte, OPC and NFO, the PDIs were observed to take part in Notch and AMPK signaling pathways (M). In TP microglia, NFO and MO, T-cell receptor and Rap1 signaling pathways were seen with LCK (domain- Pkinase_tyr) being the principal protein regulator governing 2 proteins (N). Lastly, in TP OPC, NFO and MO, the PDIs are involved in cell cycle and p53 signaling pathway with CDK2 (domain- Pkinase) as the salient protein regulator governing 5 proteins (O).Similarly, this analysis was also conducted for TP proteins common across cell types. In TP astrocyte and MO, the PDI was found to participate in CAMs process F. In TP ll cycle G. In TP toxicity H. PRKCA guidance I. In TP observed J. The 3 toxicity K. In TP aluation L. In TP pathways M. In TP proteins N. Lastlyproteins O.A). Lastly, in TR NFO, the PDI is found to take part in T-cell receptor signaling pathway (B).This analysis was similarly conducted on TR cell types. However, the SIGNOR result was available for only TR NFO, and TR OPC and NFO proteins. In TR OPC and NFO, the participation of the PDI needs further examination A. Lastly pathway B.Fig. 9POverall, this analysis manifested a comprehensive view of the domain-level interactions of the proteins, their regulation, and the pathways they control.A).For some proteins, the SIGNOR visualization and functional funct data from enrichment analysis was unobtainable. However, we found that these How shared PDIs between TP astrocyte and OPC participate in cell proliferation, differentiation, migration, apoptosis, oncogenesis, act as modulator/transducer in various transmembrane signaling pathways and assists in autophagy. The PDIs common between TP astrocyte and NFO take part in processes such as invasive growth, cell migration and signal transduction inhibition. In TP microglia and MO, the PDI is involved in regulation of Akt/AKT1 activity, mitochondrial import, regulation of cell cycle and signal transduction proteins. The PDIs in TP astrocyte, NFO and MO are involved in cell interaction with ECM, neurite formation and arborization. In TP microglia, OPC and NFO, the PDIs take part in catalyzing the covalent attachment of ubiquitin and inhibition of cysteine proteinases. The PDI shared between microglia, OPC, NFO and MO, is engaged in the complement system. The PDI between TP astrocyte, microglia and OPC, takes part in cell division and inhibits adenylate cyclase activity. The common PDI between astrocyte, OPC and MO is involved in cell cycle, DNA replication and elongation. Lastly, the mutual PDI among astrocyte, microglia and NFO takes part in the cell motility process A.Fig. 10B).Similarly, the PDIs in TR microglia takes part in cell growth, survival, motility, immune response, apoptosis, T cell activation, homeostasis, proliferation and signaling pathways such as NFκB and TNF. The TR MO PDI participates in innate immunological memory and telomerase regulation. PDI shared between TR astrocyte and NFO is involved in phagocytosis, cell migration, gene expression, TNF and NFκB signaling pathways. The PDI in TR microglia and NFO engages in PI3K signaling pathway whereas mutual PDI between TR astrocyte, OPC and NFO, is involved in cell cycle arrest, cell migration, negative regulation of apoptosis, PDGFR and Wnt signaling pathways. Finally, the PDI common between TR OPC, NFO and MO engages in ubiquitination. B.Taken together, this analysis revealed the key protein domain interactions and functions regulated in GBM TP and TR by proteins across cell types.3.8Somatic aberrations induce the transformation of healthy glial cells into cancerous cells. Our findings give crucial insight into the various unique and common molecular mechanisms that undergo alterations in the glial cell types leading to the inception of GBM . The dif4Due to its heterogenic nature and ability to evade any form of therapy regimens, GBM remains to be the most invasive and detrimental brain tumour. Designing a compelling treatment strategy against this lethal glioma has been a challenging issue globally. The studies being conducted are maximally based and focused on the tumour tissue as a whole. This prompted us to renew the standard approach and perform an extensive in-silico analysis at the cell type level.Glial cells or neuroglia are the non-excitable group of support cells in the nervous system that predominate the neurons in abundance. The CNS consists of astrocyte, microglia, and oligodendrocytes B-E). We compared this dataset with the gene sets from bulk RNA and single-cell sequencing datasets of astrocyte, microglia, OPC, NFO and MO. This winnowed down subsets of TP and TR glioma genes that are unique as well as common to the cell types (A-B). Differential expression analysis allowed us to segregate upregulated and downregulated genes  that monitor the above-mentioned genes. We mined the TFs from the Human TF atlas The TFs found in TR astrocyte, SIX3 has been discovered to suppresses glioblastoma cell growth and invasion via the WNT pathway A-H). In TP, this sequestered out 9 (astrocyte), 11 (microglia), 11 (MO), 13 (NFO), 16 (OPC & NFO), 8 (NFO & MO), 8  and 4  experimentally validated TFs, thereby excluding out 6 (astrocyte), 2 (microglia), 4 (MO), 10 (NFO), 10 (OPC & NFO), 5 (NFO & MO), 3  and 4  novel TFs, respectively (A-H). Further investigation of the TFs based on their correlation and TF influence activity revealed that the downregulated GLIS3 is the significant TF regulator in TP astrocyte. The increased expression of this factor has been studied to influence the glioma cells’ invasion, migration and proliferation activity, whereas its insufficient expression has an inhibitory effect on NF-κB signaling pathway Fig. 6A-H).Given our findings in TP dataset, we conducted the TP TF co-regulatory network analysis A-H. In Tectively A-H. FurtA-H). In TP astrocyte, we discovered that TFs ZNF878 and CREB3L4 are involved in highest number of processes such as signal transduction downstream of smoothened protein, activation of phospholipase C activity and peroxisome, positive regulation of translation, respectively. Phospholipase C (PLC) has been determined to control a range of cell functions such as cell motility, transformation, differentiation, and proliferation, and PLC also regulates cancer cells in part by serving as signaling intermediates for cytokines like EGF and interleukins In addition, we conducted an in-depth study of the functional operations regulated by these TFs in their co-regulatory networks A-H. In TA). In TP microglia, the significant protein regulators such as MAPK14 (domain- Pkinase) activates MAPKAPK3 (domain- Pkinase), binds to PXN, inhibits MAX (domain- HLH) and EIF4EBP1 (domain- eIF_4EBP). The next protein regulator NFKBIA (domain- Ank) inhibits NFKB1 and activates RELA (domain- RHD), whereas regulator BCL3 (domain- Ank) activates NFKB1 and indirectly binds to NFKB2 (domain- Ank). BCL3 is regulated by LTF, the prominent TF influencer detected in TP microglia. In addition, protein regulator SYK activates SH3BP2 and VAV1, and regulator TRADD (domain- Death) activates FADD (domain- Death) and RIPK1. Some fundamental signaling pathways like MAPK, regulation of pluripotency of stem cells and processes such as cytokine-cytokine receptor interaction, complement and coagulation cascades etc. are seen to be transpiring in this cell type . The eminent protein regulators in TP NFO are CDK1 (domain- Pkinase) that activates CDC25A, inhibits WEE1 (domain- Pkinase), CASP9 (domain- Peptidase_C14), CASP8 (domain- Peptidase_C14), and regulator PTPN12 (domain- Y_phosphatase) that inhibits JAK2 (domain- Pkinase_Tyr), BCAR1 and PTK2B (domain- Pkinase_Tyr). These PDIs are observed to participate in essential processes like cell cycle, Rap1, PI3K-Akt signaling pathways, regulation of pluripotency of stem cells, complement and coagulation cascades . In TP MO, the notable protein regulator was detected to be PDPK1 (domain- Pkinase) that activates PRKCE (domain- Pkinase) and PRKCA. This protein regulator is found to be governed by the prime TF influencer in this cell type i.e. DMRTA2 . The PDI common between TP astrocyte and MO is involved in cell adhesion . The PDI common between TP microglia and OPC participates in cell cycle . The significant protein regulators in the PDIN common between TP microglia and NFO are PRKCA (domain- Pkinase) that activates NCF1, CYBA, inhibits LCK (domain- SH3_1), binds to ITGB2, SNAP23, DGKZ (domain- C1_1), and regulator SYK that activates PRKCA (domain- Pkinase) and inhibits LCK (domain- SH3_1). This PDIN is engaged in NFκB, Rap1 signaling pathways and natural killer cell mediated cytotoxicity . Whereas the PDI shared between TP OPC and MO participates in axon guidance . The three influential protein regulators in PDIN common between TP OPC and NFO are SRC that activates EPHA2 (domain- Ephrin_Ibd), IGF1R, LRP1, DLG4, JUP, CTNNB1 (domain- Arm), CDH5, RAC1 (Ras), TIAM1 (domain-PH), inhibits ITGAL and binds to GRB2, MMP14, PTPN12. Followed by PDGFRB that binds to GRB2, SRC and FYN, and lastly, PLK1 (domain- Pkinase) that activates CHEK2 (domain- Pkinase), binds to CTNNB1 (domain- Arm) and BRCA2 (domain- BRCA2). PLK1 regulator has been found to be regulated by the chief TF influencer detected i.e. E2F7. This PDIN is associated with vital signaling pathways such as JAK-STAT, PI3K-Akt, cAMP, apelin, AGE-RAGE, hippo and processes such as cell cycle, transcriptional misregulation in cancer, fluid sheer stress . The PDIs mutual between TP NFO and MO are engaged in TNF, Rap1, Ras, Phospholipase D signaling pathways and process such as natural killer cell mediated cytotoxicity . Whereas the PDIs common between TP astrocyte, OPC and NFO are involved in Notch, AMPK signaling pathways . We found notch, AMPK signaling pathways regulated in astrocyte, OPC and NFO in TP . In TP microglia, NFO and MO, the chief protein regulator was recognized as LCK (domain- Pkinase_Tyr) that activates PRKCD and PTPN6 (domain- SH2). This PDIN is witnessed to take part in glioma, Rap1 and T-cell receptor signaling pathways . Finally, in the PDIN shared between all the three cell types of oligodendrocytes, the principal protein regulator is inferred to be CDK2 (domain- Pkinase) that activates UBE2A, MCM2, MCM3, and inhibits CDKN1A (domain- CDI), TPX2 (domain- Aurora-A_bind). Cell cycle and p53 signaling pathway are the crucial processes affiliated to this PDIN . Correspondingly, the SIGNOR data was available for only two TR subsets of proteins. In TR NFO, the PDI is observed to be involved in T-cell receptor signaling pathway (B).Once we had conducted our analysis at the transcriptomic level, we inspected the proteome activities undertaken in the GBM brain cells types individually. Given the importance of the association of domains in protein–protein interactions for a functional outcome to eventuate, we retrieved protein-domain interaction (PDI) data by logging the cell type-specific gene datasets onto the INstruct database . We furtFig. 8 pathway B.A-B). The following functional processes are seen to be operated by the TP proteins in the cell types: invasive growth, cell migration, signal transduction inhibition, apoptosis, oncogenesis, cell cycle, autophagy, cell attachment/migration/differentiation, and complement system. Analogously, the processes driven by the TR proteins in cell types are: apoptosis, T cell activation/homeostasis/proliferation, cell growth/survival/adhesion/motility, phagocytosis, cell cycle arrest, ubiquitination, signaling pathways like TNF, NFκB, PI3k, PDGFR and Wnt. In sum, our analysis gives a comprehensive and wider perspective of the various phenomena that emerge in the GBM cell types.A similar analysis of TP and TR proteins was performed that lacked the SIGNOR visualization and regulation data A-B. The 5In this study, we examined the brain cell types in GBM. Astrocyte, microglia, OPC, NFO and MO specific TP and TR glioma genes were extracted from the openly accessible transcriptome dataset of GBM. We studied the GBM grade 4 gene expression in each of the glial cell types and performed comprehensive network analyses to understand cell type specific pathways. This underlined cell type specific prominent transcription factors regulating significant pathways and also identified prime protein regulators in protein-domain interaction networks. Furthermore, we observed that the chief TF influencers, LTF, DMRTA2 and E2F7, govern the eminent protein regulators BCL3, PDPK1 and PLK1, respectively. Some distinctive as well as mutual processes and functional pathways have been deduced to take place in these GBM borne glial cell types. Our study opens avenue for inspection and modulation of these mechanisms in the brain cell types in order to refine the therapeutic routes to treat this aggressive cancer.Samreen Fathima: Software, Data curation, Investigation, Resources, Formal analysis, Writing- original draft, Writing- review & editing. Swati Sinha: Formal analysis, Supervision, Project administration, Writing – review & editing. Sainitin Donakonda: Conceptualization, Software, Methodology, Project administration, Supervision, Formal analysis, Writing – original draft, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Musa spp.) plant produces elongated and edible fruit. The two main parthenocarpic species of banana are Musa accuminata Colla and Musa balbisiana Colla. There are several health-promoting and disease-preventing effects of Musa accuminata Colla, which are attributed to its important bioactive compounds, including phenolics, carotenoids, biogenic amines, phytosterols, and volatile oils, found in the stem, fruit, pseudostem, leaf, flower, sap, inner trunk, root, and inner core. Banana possesses numerous pharmacological activities, such as antioxidant, immunomodulatory, antimicrobial, antiulcerogenic, hypolipidemic, hypoglycemic, leishmanicidal, anthelmintic, and anticancer properties. Various individual studies have reported anticancer effects of different components of the banana plant. However, according to our understanding, an up-to-date, systematic, and critical analysis of existing scientific results has not yet been carried out.The banana (This review aims to include a thorough assessment of banana and its phytochemicals for cancer prevention and therapy with a focus on cellular and molecular mechanisms of action.The available research studies on anticancer activities of banana extracts, fractions and pure compounds were collected using various scholarly databases, such as PubMed, ScienceDirect, and Scopus, based on predetermined selection criteria.Various banana extracts, fractions, and phytoconstituents, including ferulic acid, protocatechualdehyde, 2-pentanone, 4-epicyclomusalenone, cycloeucalenol acetate, and chlorogenic acid, have been shown to exhibit cancer preventative and anticancer activities in breast, cervical, colorectal, esophageal, hepatic, oral, prostate, and skin cancers. Bioactive components present in bananas have exhibited antiproliferative, cell cycle arrest-inducing, apoptotic, anti-adhesive, anti-invasive, and antiangiogenic effects through modulation of diverse, dysregulated oncogenic signaling pathways.Based on the critical analysis of available literature, banana products and phytoconstituents show enormous potential for future development of drugs for cancer prevention and therapy. However, more mechanistic studies and well-designed clinical trials should be performed to establish its efficacy. While the precise cause for cancer initiation is still unclear, the most important contributing variables for this condition are toxins, pollution, radioactive substances, oncogenic viruses , Arthsastra (250 BC), and Chilappthikaram (500 AD), suggesting the fruit’s importance and demonstrating its ancient use in India. The genus name Musa was chosen to commemorate Roman physician and botanist Antonius Musa (63 BC-14 AD)  . Historiof India . At presof India .The banana is a perennial herb that looks like a tree .M. acuminate plants have been used to treat non-communicable as well as transmissible diseases, especially in Asia and Africa. It has been extensively used by indigenous people as both food and medicine  genotype of M. acuminata and M. balbisiana which have an epicatechin content of 114.44 mg/100 g dry weight basis and gallocatechin content of 591.41 mg/100 g dry weight basis. In addition to catechin compounds, protocatechuic acid, gallic acid, 7-O-neohesperoside naringenin, and hydroxycinnamic acids have additionally been identified in banana pulp  (2), vanillic acid (3), caffeic acid (4), ellagic acid (5), 13-octadecanoic acid (6), palmitic acid (7), oleic acid (8), linoleic acid (9), methyl palmitate (10), methyl oleate (11), methyl linoleate (12), stearic acid (13), carvacrol (14), pentadecanoic acid (15), palmitoleic acid (16), benzoic acid (17), tannic acid (18), cinnamic acid (19), ferulic acid (20), delphinidin-3-rutinoside (21), cyanidin-3-rutinoside (22), petunidin-3-rutinoside (23), peonidin-3-rutinoside (24), malvidin-3-rutinoside (25) (26), gallic acid (27), protocatechualdehyde (28), hydroxycinnamic acid (29), caffeoylquinic acid (30), quercetin (31), proanthocyanidin (32), catechin (33), gallocatechin (34), epicatechin (35), procyanidin (36), (+) catechin hydrate (37), apigenin (38), myricetin (39), kaempferol (40), endo-β-1,3-glucanase (Ban-Glu) (41), and α-tocopherol (42).A triterpenoid, namely cycloartenol (43), was extracted from the dichloromethane extract of ripe pulp of various banana cultivars  Figure 2mic acid , caffeoyuercetin , proanthBan-Glu) , and α-toartenol , was extultivars .Musa spp., namely, campesterol (44), stigmasterol (45), β-sitosterol (46) (47) (48) is present in ripe banana fruits (49) and epi-sesamin (50), have also been isolated and identified in the methanol extract of banana peel  Figure 4rol (46) . The met46) 47) . In addi . In add46) (47)  is prese51), and vitamin A (retinol) (52) are the most abundant antioxidants present in the banana pulp and peel (Musa spp. biomasses as well (53 and 54) (55) has additionally been found in bananas. Several additional novel compounds, such as 2-(4-hydroxyphenyl)-naphthalic anhydride (56), and methyl 2-benzimidazolecarbamate (57), were reported to be present in banana   . Zeaxantretinol)  are the 58), serotonin (59) and histamine (60) have all been detected in bananas and their by-products , 2-phenylethylamine (62), putrescine (63), cadaverine (64), and tyramine (65), are also present in banana , 5-(hydroxymethyl)-2-furancarboxyaldehyde (67), cis-9- hexadecenal (68), (S) (+)-naproxene (69) (70), Anigorufone (71), 2-pentanone (72), and N-acetylserotonin (73)  Figure 5nin (73) , 56–59. ene (69) , AnigoruM. acuminata) flower is used to prepare a popular Sri Lankan dish as a curry, boiled or deep-fried salad (M. acuminata) extracts in preclinical trials  revealed that M. acuminata flower extract was safe as well  showed potent nephroprotective activity in gentamicin-induced nephrotoxicity in mice. Cellulose nanofibers isolated from the banana peel (M. paradisiaca) exhibited no cytotoxicity against Caco-2 cell line  also did not induce any cytotoxicity in RAW 264.7 murine macrophage cell lines, which was evident from the presence of 70% viable cells   which isin vitro  peel and pulp was observed to be cytotoxic and blocked the proliferation of MCF-7 cells (Regarding poptosis . Anotherpoptosis . Additio00 μg/mL . Additio-7 cells .M. acuminate lectin (MAL) from the phloem exudates of M. acuminate pseudostem also demonstrated antitumor activity in Swiss albino mice bearing Erhlich carcinoma cells. Mechanistic studies showed initiation of apoptosis through the arrest of cell cycle progression at G2/M phase via stimulation of caspase-3, caspase-8 and caspase-9 with increased poly (ADP-ribose) polymerase (PARP) cleavage. It further triggered apoptosis through the phosphoinositide 3-kinase (PI3K)/Akt  signaling pathway, which inhibited the expression level of phosphorylated Akt (pAkt). MAL also blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK)  .M. paradisiaca) exhibited anticancer activity against HeLa cervical cancer cell line. The extract inhibited cellular proliferation and induced apoptosis, which was evident by the appearance of breaking up of the nuclei associated with significantly increased caspase-9 activity. The extract also blocked the cell cycle progression at the G0/G1 phase as well  peel and pulp exhibited in vitro anticancer activity against HCT-116 colon carcinoma cell line. It was observed that the peel and pulp extract arrested cell growth by inducing cytotoxicity and blocked the proliferation of HCT-116 cells -initiated cyclin D1 suppression. It also downregulated the transcriptional level of the cyclin D1 gene and reduced the expression level of cyclin-dependent kinase 4 (CDK4). Additionally, PCA attenuated the enzymatic activity of HDAC and reduced the expression of HDAC2, but not HDAC1, thereby inducing cell cycle arrest at the G1 to S phase in both the cell lines tested  and p38 mitogen-activated protein kinase (MAPK) proteins and cleavage of PARP (2) production and cyclooxygenase-2 (COX-2) protein expression in tumor necrosis factor-α (TNF-α)-stimulated colon cancer cells (HT29).The hexane fraction of banana  was used as a dietary supplement to evaluate its anticancer potency in 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in male Swiss mice over a 12-week experimental period. In all treated groups, it reduced the number of aberrant crypt foci (a colon cancer biomarker) in the colorectal mucosa, suggesting anticarcinogenic efficacy although the detail mechanism of action was not reported  and increased reactive oxygen species (ROS) level  pseudostem and rhizome demonstrated cytotoxicity against HepG2 cell lines. Crude chloroform and acetone extracts of the banana pseudostem and rhizome exhibited substantial cytotoxicity against the cell line tested (An anticancer study on S) level . Anothere tested . BioactiM. paradisiaca) soft piths (BSPs) exhibited potent cytotoxic and antiproliferative activity against the human oral squamous cell carcinoma (OSCC) cell line (HSC-4) (Ethyl acetate sub-fraction of the ethanol extract of banana ( (HSC-4) .in vitro. The banana flower extract at a concentration of 2 mg/mL reduced the viability of BPH-1 cells through cell-cycle arrest at the G1 phase. Moreover, it reduced the expression level of cyclin D1 and cyclin-dependentkinase6 (Cdk6) and elevated the expression level of p53 and p27   and p27 . It furtactivity .M. cavendish green peel hydroalcoholic extract demonstrated antiproliferative and cytotoxic activity against A-375 human malignant melanoma cells at a concentration of 100 μg/mL  has gained a lot of interest, other parts of the banana plant, such as the leaf, flower, and stem, have also been investigated for anticancer purposes. In addition to banana extracts and fractions, some phytoconstituents, including ferulic acid, protocatechualdehyde, 2-pentanone, 4-epicyclomusalenone, cycloeucalenol acetate, and chlorogenic acid, have been shown to exhibit cancer preventative and anticancer therapeutic abilities. Cancer preventive studies are limited, although two such study is reported where consumption of banana can reduce the incidence of esophageal cancer . Different aspects, such as banana processing and variety, often affect bioavailability and accessibility.in vitro experiments. Breast, cervical, colorectal, esophageal, hepatic, oral, prostate, and skin cancers are only a few of the cancer types affected by banana and its phytochemicals. The reviewed literature reveals the promise that banana and its phytochemicals can be used in chemotherapy for different forms of cancer. The role of banana and its phytoconstituents on breast cancer and colon cancer have been studied extensively in both in vitro and in vivo research. Findings from in vivo studies on other cancer types and clinical situations are sparse. With encouraging preclinical data, mechanistic investigations on anticancer actions of the components of banana are warranted. The translation impact of available research findings is restricted by the lack of well-designed, prospective clinical studies and safety evaluation of banana extracts and constituents in humans. The potential pharmacokinetic constraints of banana phytochemicals highlight the need to establish efficient and well-regulated delivery mechanisms for optimized delivery systems against various malignancies. More experiments on novel molecular targets and signaling pathways of banana bioactive materials, in addition to well-controlled clinical trials, will increase the therapeutic potential of this popular and medicinal fruit for cancer prevention and treatment. According to the selective toxicity tests, bananas and their main components are safe. However, more research is to be carried out to see whether the same favourable safety profile occurs in human subjects, and to determine which banana secondary metabolites may be cytotoxic, if any. Moreover, banana-derived products may be utilized as an adjuvant to various chemotherapeutic drugs (which have many adverse side effects) for a variety of cancer subtypes.Despite the vast amount of research that has been performed and documented over the last few decades, the bulk of the findings cited in this review are focused on in vitro studies, more in vivo mechanistic experiments should be performed. Provided the positive anticancer findings presented in this study, randomized clinical trials involving banana phytochemicals should be carried out. However, further research into the anticancer ability of other important phytochemicals found in bananas is warranted. Characterization of different phytochemicals found in bananas that function alone or synergistically with other compounds or established drugs to have cancer ameliorative or protective effects is also required. In conclusion, based on our in-depth analysis of the existing literature, banana extracts and the isolated phytoconstituents found in banana present as promising medicinal agents for cancer prevention and these agents could also be developed as multi-targeted drugs for cancer pharmacotherapy.Our systematic study and review of limitations also identify various future research paths. Although numerous bioactive banana compounds have been identified, further research into the anticancer ability of these phytochemicals found in bananas is required. Furthermore, since the bulk of research is limited to The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.Conceptualization: AM. Literature search and collection: SBo, SBa, and PD. Writing—original draft preparation: AM, SBa, and ES. Writing—review and editing: AM, AA, and AB. Supervision: AM. Project administration: AB. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cidea/Cidec ratio along with an increased number and reduced size and area of LDs. In addition, combination treatment reduced expression levels of endoplasmic reticulum stress-related genes . Pema and Tofo treatment significantly improved survival rates and reduced the number of tumors in the liver compared to the NASH control group. These results suggest that SPPARMα and SGLT2 inhibitor combination therapy has therapeutic potential to prevent NASH-HCC progression.Ballooning degeneration of hepatocytes is a major distinguishing histological feature of non-alcoholic steatosis (NASH) progression that can lead to cirrhosis and hepatocellular carcinoma (HCC). In this study, we evaluated the effect of the selective PPARα modulator (SPPARMα) pemafibrate (Pema) and sodium-glucose cotransporter 2 (SGLT2) inhibitor tofogliflozin (Tofo) combination treatment on pathological progression in the liver of a mouse model of NASH (STAM) at two time points . At both time points, the Pema and Tofo combination treatment significantly alleviated hyperglycemia and hypertriglyceridemia. The combination treatment significantly reduced ballooning degeneration of hepatocytes. RNA-seq analysis suggested that Pema and Tofo combination treatment resulted in an increase in glyceroneogenesis, triglyceride (TG) uptake, lipolysis and liberated fatty acids re-esterification into TG, lipid droplet (LD) formation, and  Non-alcoholic steatosis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD), which is closely linked to type 2 diabetes and metabolic syndrome ,2,3. NASLipid droplets (LDs) are storage organelles that store neutral lipids such as triglycerides (TGs) and sterol esters during excess energy states and serve as a reservoir of energy supplies during the fasting state ,7,8. ImpPemafibrate (Pema) is the first clinically available selective PPARα modulator (SPPARMα); it is used to improve dyslipidemia and reduce macrovascular and microvascular complications ,17,18,19Sodium-glucose cotransporter-2 (SGLT2) inhibitors are a class of lower blood glucose drugs that increase urinary glucose excretion by inhibiting glucose reabsorption at the proximal tubule in the kidney ,24,25. RPema and Tofo were kindly provided by Kowa Co., Ltd. . Streptozotocin (STZ) was purchased from Sigma-Aldrich  and Arabic gum from Wako Pure Chemical Industries .STAM mice were generated as previously described . Pathogen = 20 each). C57BL/6J mice with normal chow were assigned to the normal group (n = 8). Pema and/or Tofo were mixed in the diet and administered to each group. The study protocol was approved in accordance with the relevant guidelines and regulations of the Animal Care and Use Committee of Tokyo New Drug Research Laboratories, Kowa Company, Ltd. .Male STAM mice were purchased from SMC Laboratories  at 5 weeks of age and were fed HFD32. C57BL/6J normal mice were purchased from Japan SLC  and fed a normal diet, CE-2. All mice were housed at 23 ± 3 °C and 55 ± 15% relative humidity (RH) under a 12-h light/dark cycle (07:00–19:00 h) and provided with food and water ad libitum. At 6 weeks of age, STAM mice were divided into four groups based on body weight: control, Pema , Tofo  ,29, and Serum total cholesterol (TC), TG, glucose, non-esterified fatty acids (NEFA), AST, ALT, phospholipids (PL), and creatinine (CRN) levels were determined using a Labospect 003 autoanalyzer .2O2 in methanol. Obtained liver sections were treated with the anti-ER-TR7  antibodies overnight at 4 °C. After treatment with secondary antibodies, the substrate reaction was performed using 3,3′-diaminobenzidine  solution.A histological study was performed as previously described . For immAccording to Kleiner et al. , the NAFFor genome-wide transcriptome analysis, RNA-sequencing (RNA-Seq) was performed as previously described . BrieflyPpia mRNA was used as an independent control. All primers used for qPCR are listed in qPCR was performed as previously described ,31,32. Phttps://imagej.nih.gov/ij/download.html (accessed on 7 August 2016) was applied. H&E staining images were opened with Image J software and converted into grayscale (8 bit). Then, the lipid drop areas were extracted by using the threshold . After eliminating blood vessels, LD areas were analyzed and quantified using the “Analyze particles” function. Quantified LD area data were firstly obtained by pixel, and then they were converted into μm2 . LD diameter was also analyzed. Data were shown as the mean values from three different images of each animal. A histogram was created with Microsoft Excel spreadsheet software.LD evaluation was performed as previously described . For hepp < 0.05, ** p < 0.01. In the survival study, data were analyzed using the multiple log-rank test and Cox proportional hazard model.All data are presented as the mean ± SEM. Homogeneity invariance was evaluated by Bartlett’s test followed by parametric or non-parametric Dunnett’s multiple comparison test (two-sided). * To investigate the effects of Pema and Tofo combination on NASH development in the STAM mouse model, each drug and combination was administered for three weeks. STAM mice showed significant hyperglycemia and hypertriglyceridemia; higher phospholipid, FGF21, and AST levels; lower body weight; and higher liver weight, compared to normal C57BL/6J mice . Pema siH&E staining clarified that STAM control mouse liver owned liver nodules, macro- and micro-vesicular lipid accumulation, inflammatory cell infiltration, and ballooning degeneration of hepatocytes, unlike normal mouse liver C. Pema-tAlthough Pema, Tofo, and combination treatment resulted in decreased macrovesicular steatosis, this reduction was not reflected in the steatosis and Oil Red O staining scores . To veriPdk4 gene expression level, indicating that it mediates the inhibition of glucose oxidation and preferential activation of FAO  and the monoacylglycerol pathway are key players in TG synthesis A. The STTo better understand the effect of Pema and Tofo combination treatment on steatosis in STAM mice, we measured the TG concentration in the liver A. The STAgpat6, Dgat1, and Acat1), formation , budding (Fitm2 and Bscl2), stabilization (Plin4 and Plin5), lipolysis , expansion , and fusion (Cidea and Cidec). Among these genes, the Pema and Tofo combination induced Cidea expression. Recently, Sans et al. suggested that hepatic CIDEA and CIDEC correlated negatively and positively, respectively, with steatohepatitis and liver injury in mice, as well as steatosis and NASH in obese humans [Cidea and Cidec, and combination treatment strongly induced Cidea gene expression, thereby increasing the Cidea/Cidec ratio. These results may contribute to the reduction in LD size and stimulation of lipolysis by combination treatment.LDs consist of an inner core of neutral lipids, including TG and sterol esters, a phospholipid monolayer, and LD-associated proteins (LDAPs) ,7,8. Bece humans . In addie humans . Pema, TIre1a, Perk, and Atf6), chaperones (Grp78 and Pdi1a), antioxidant defense-regulated transcription factor (Nrf2), IRE1α interaction protein form apoptosis mediator complex (Traf2), and proapoptotic BCL-2 protein family (Bak1 and Bax). Pema and Tofo combination significantly reduced Ire1a, Grp78, Xbp1, and Phlda3 expression levels , budding (Fitm2 and Bscl2), and fusion (Cidea and Cidec) proteins. Although the biological role of LD diversification has not been clarified yet, increased numbers of small LDs may explain the protection against lipotoxicity [Fitm2, is an evolutionarily conserved fatty acyl-CoA diphosphatase that maintains the ER structure, protects against ER stress, and enables normal lipid storage in LDs [Cidea was the most highly induced gene among LDAPs, and the Cidea/Cidec ratio was significantly increased by the Pema and Tofo combination. These results suggest that the combination of Pema and Tofo promotes fatty acid catabolism via lipolysis and β-oxidation while promoting re-esterification of excess fatty acids and LD biogenesis, thereby preventing lipotoxicity.In addition, we found that Pema and Tofo combination significantly increased LD number, reduced LD size, and improved macrovesicular steatosis. Consistent with the increased number of LDs, Pema and Tofo combination also induced expression of genes involved in LD inner core lipid synthesis and formation , chaperones (Grp78 and Pdi1a), antioxidant defense-regulated transcription factor (Nrf2), IRE1α interaction protein form apoptosis mediator complex (Traf2), and proapoptotic BCL-2 protein family (Bak1 and Bax) genes were observed in the liver of STAM mice. Although numerous reports have indicated that the ER stress response plays a key role in NASH development, it is unknown which UPR sensor signaling contributes to the development of this disorder [Ire1a, Grp78, Xbp1, and Phlda3 expression levels in STAM mouse livers. Among the UPR sensors, IRE1 is the most evolutionarily conserved, implying that it plays a crucial role in cell fate determination under ER stress conditions. It has been indicated that IRE1 is capable of inducing cell fate by two distinct pathways through XBP1-mediated gene regulation and interaction with TNF receptor-associated factor 2 (TRAF2) to initiate the apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) signaling cascades [Hepatic steatosis has a risk of steatohepatitis, fibrosis, cirrhosis, liver failure, and HCC, and it was reported that dysregulation of microbial metabolites such as aromatic and branched-chain amino acid (AAA and BCAA) or of iron metabolism are related to liver fat accumulation and facilitate steatosis ,47; howedisorder ,40. Pemacascades . Howevercascades . Similarcascades . Howevercascades . In addicascades . TherefoMttp was induced, insulin and SREBP1c target gene of Gck was reduced, and Pck1, which is negatively regulated by insulin, was induced. From several reports and our observations, this model may not completely reflect the human NASH liver metabolic state and may be a model in which the effect of SGLT2 inhibitors is more likely to be effective. Thus, additional studies using other NASH models with obesity and insulin resistance are warranted to evaluate the effect of the Pema and Tofo combination treatment on human NASH development.Epidemiological studies have shown that NASH is closely linked to type 2 diabetes and metabolic syndrome ,2,3. HowIn addition, although Pema and Tofo combination treatment significantly induced fatty acid catabolism, fatty acid re-esterification, and LD biogenesis; impeded the IRE1α-XBP1-PHLD3A pathway; and alleviated ballooning degeneration of hepatocytes, the precise underlying mechanism is still largely unknown. It is well known that hepatocytes are not equally responsible for liver metabolism, and the existence of so-called metabolic zonation based on oxygen tension has been proposed ,60. For In conclusion, the combination of SPPARMα and SGLT2 inhibitor treatment prevented ballooning degeneration of hepatocytes and HCC progression. Our global gene expression analysis gives evidence of the liver protective effect of the combination therapy by inhibiting the lipotoxicity-induced IRE1α-XBP1-PHLD3A pathway. Taken together with our previous report that SPPARMα treatment prevents NASH development by reducing myeloid cell recruitment without reducing hepatic TG content , the com


Nuclear factor erythroid 2-related factor (Nrf2) is a transcriptional activator of the cell protection gene that binds to the antioxidant response element (ARE). Therefore, Nrf2 protects cells and tissues from oxidative stress. Normally, Kelch-like ECH-associated protein 1 (Keap1) inhibits the activation of Nrf2 by binding to Nrf2 and contributes to Nrf2 break down by ubiquitin proteasomes. In moderate oxidative stress, Keap1 is inhibited, allowing Nrf2 to be translocated to the nucleus, which acts as an antioxidant. However, under unusually severe oxidative stress, the Keap1-Nrf2 mechanism becomes disrupted and results in cell and tissue damage. Oxide-containing atmospheric environment generally contributes to the development of respiratory diseases, possibly leading to the failure of the Keap1-Nrf2 pathway. Until now, several studies have identified changes in Keap1-Nrf2 signaling in models of respiratory diseases, such as acute respiratory distress syndrome (ARDS)/acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and asthma. These studies have confirmed that several Nrf2 activators can alleviate symptoms of respiratory diseases. Thus, this review describes how the expression of Keap1-Nrf2 functions in different respiratory diseases and explains the protective effects of reversing this expression. Reactive oxygen species (ROSs) have free radicals, including superoxide anions and hydroxyl radicals, and active non-radical oxygen species, including hydrogen peroxide and nitroperoxide, which are generated by the partial reduction in oxygen . In brieNuclear factor erythroid 2-related factor (Nrf2) is a transcription factor involved in oxidative stress response. Nrf2 plays an important role in the regulation of ARE-mediated expression of phase II detoxifying antioxidant genes, including heme oxygenase 1 (HO-1), superoxide dismutase 1 (SOD1), glutathione (GSH), UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), and N-acetyltransferases (NATs) ,9,10. KeDuring respiration, humans inhale oxygen-containing air that enters the airways, passes through the bronchi, and eventually reaches the alveoli . The inhARDS and ALI can result in respiratory failure and are conditions that cause inflammation in the lungs as well as dyspnea and cyanosis . ARDS ha−/− mice also showed higher macrophages and protein concentrations in bronchoalveolar lavage fluid (BALF) than in Nrf2+/+ mice. In addition, the activity of NQO1 required to inhibit the production of radical species was also dependent on Nrf2 [Although the ARDS pathological mechanism is not fully identified, it has been steadily reported that ARDS is closely related to oxidative stress and inflammation. TLR4 signaling by PAMPs, such as lipopolysaccharide (LPS), regulates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and NADPH oxidase 4 (NOX4), causing overproduction of ROSs and recruitment of inflammatory cells into the lungs and leading to lung damage ,29. Amon on Nrf2 . Recentl on Nrf2 ,35. Acti on Nrf2 ,37. Inte on Nrf2 . In addi on Nrf2 . In TLI- on Nrf2 . Similar on Nrf2 . Synthet on Nrf2 . Althoug on Nrf2 . Naphtha on Nrf2 . Moreove on Nrf2 . It has  on Nrf2 . Recentl on Nrf2 . Several on Nrf2 ,49. In a on Nrf2 ,51,52.COPD is a respiratory disease currently listed as the fourth leading cause of death. Its major manifestations include gradual airflow restriction, emphysema, and chronic bronchitis . The pat−/− mice showed higher COPD sensitivity than wild-type (WT) mice [−/− models [−/− PPE-induced COPD model has been shown to weaken lung type and inflammation [−/− mice [−/− mice. Nrf2 expression was also higher in the Wnt activators treatment group than in the non-treatment group [Direct or indirect CS exposure can cause oxidative stress and lung inflammation. The expression of Nrf2 protects against oxidative stress caused by excessive exposure to oxidizers in CS. High expressions of Nrf2 and related genes, such as HO-1 and GCLC, have been identified in blood mononuclear cells of COPD patients. In addition, patients with low forced expiratory volume in 1 s (FEV1) generally have higher Nrf2 and HO-1 expressions than patients with higher FEV1 . Among CWT) mice . In addi− models . Transplammation . Adminisammation ,61. Inteammation . RAGE ha−/− mice . The abs−/− mice . Recentl−/− mice . In recent group . This suBrassica oleracea extract, has been previously reported to inhibit oxidative stress by promoting activation of Nrf2 in various disease models, including COPD [Inula helenium L. reduced inflammatory cytokines, levels, and MDA , also known as an indication of oxidative stress, and activated Nrf2/HO-1 pathway [These studies show that research is continuously underway to improve the treatment of COPD by activating Nrf2. Sulforaphane (SFN), a ing COPD ,66,67. Hing COPD . It has ing COPD . Flavonoing COPD . Treatme pathway .IPF is a type of chronic interstitial disease characterized by a progressive decline in pulmonary function. It has a median survival of approximately 3–5 years after the diagnosis . CurrentIn general, IPF has been known as one of the aging-related diseases, primarily occurring in patients aged over 60 years . Aging nIt has been indicated that bleomycin-induced pulmonary fibrosis showed suppression of Keap1-Nrf2 in vivo . In anotFurthermore, rapamycin, an mTOR inhibitor, has frequently been investigated as a regulator of the Nrf2 signaling pathway in various cell types ,93. In pAsthma is a chronic inflammatory disease characterized by reversible airflow obstruction, airway bronchial hyperresponsiveness to a variety of allergens or infectious organisms, leading to shortness of breath, cough, and wheezing . The patThe Keap1-Nrf2 pathway is a major antioxidant defense mechanism that protects the lung against oxidative stress. Lungs are constantly exposed to various harmful substances, such as CS, dust, PM, and even viruses and bacteria, which may cause oxidative stress. Since oxidative stress has been closely related to human respiratory diseases, antioxidant systems, including Keap1-Nrf2, have been become an important therapeutic target for various respiratory diseases. The emerging role of the Keap1-Nrf2 regulation, indicating that the enhancement of defense mechanism attenuates inflammatory response, oxidative stress, fibrosis, and cellular death and ultimately has protective effects against the development of diseases in experimental models in


Glycosaminoglycans (GAGs) including chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate, except for hyaluronan that is a free polysaccharide, are covalently attached to core proteins to form proteoglycans. More than 50 gene products are involved in the biosynthesis of GAGs. We recently developed a comprehensive glycosylation mapping tool, GlycoMaple, for visualization and estimation of glycan structures based on gene expression profiles. Using this tool, the expression levels of GAG biosynthetic genes were analyzed in various human tissues as well as tumor tissues. In brain and pancreatic tumors, the pathways for biosynthesis of chondroitin and dermatan sulfate were predicted to be upregulated. In breast cancerous tissues, the pathways for biosynthesis of chondroitin and dermatan sulfate were predicted to be up- and down-regulated, respectively, which are consistent with biochemical findings published in the literature. In addition, the expression levels of the chondroitin sulfate-proteoglycan versican and the dermatan sulfate-proteoglycan decorin were up- and down-regulated, respectively. These findings may provide new insight into GAG profiles in various human diseases including cancerous tumors as well as neurodegenerative disease using GlycoMaple analysis. Glycosaminoglycans (GAGs) are linear polysaccharides consisting of repeating disaccharide units. Among these are heparin, heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS), which are covalently bound to core proteins, forming proteoglycans (PGs) . HyaluroO–, where GlcA, Gal, and Xyl stand for glucuronic acid, galactose, and xylose, respectively  is removed by 2-O-phosphoxylose phosphatase encoded by PXYLP1  (N-acetylglucosaminyltransferase (GlcNAcT)-I encoded by exostosin-like 2 (EXTL2) or EXTL3  and GlcNAcT-II, which are catalyzed by a HS polymerase enzyme hetero-complex composed of exostosin 1 (EXT1) and EXT2   is utilized as the substrate for the sulfation reaction (PAPSS1 or PAPSS2 (SLC35B2 and SLC35B3 (Heparan sulfate consists of repeating disaccharide units of (GlcNAc) , and Glc(GlcNAc) . The inior EXTL3 . Thereafand EXT2 . EXTL1 aand EXT2 . The Glcor NDST4 . HS C5-ed (IdoA) . The disreaction . PAPS isr PAPSS2 . PAPS is SLC35B3 , 2006.N-acetylgalactosamine β1–]n and [–4IdoAβ1–3GalNAcβ1–]n, respectively, is also attached to a linker tetrasaccharide, GlcA-Gal-Gal-Xyl, on a serine residue of specific core proteins -I encoded by CSGALNACT1 or CSGALNACT2 , whereas HAS3 synthesizes a markedly shorter hyaluronan (∼1 × 106 Da)  . HA is a 106 Da) . During  106 Da) .The abundance of GAGs and their pattern of sulfation are dynamically altered, affecting GAG–protein interactions  during a1 (2 (2[TPM + 0.001]) in Xena were converted to TPM values (2 (TPM + 1). The Wilcoxon matched-pairs signed rank test was used to compare gene expression levels between primary tumor and normal tissues.Expression data of GAG-related genes  in 37 hu1 . TPM val1  and PG g1  from hea1 , a UDP-Xyl and UDP-GlcNAc transporter (SLC35B4), and a UDP-Gal transporter (SLC35A2) were included. PAPSS1 and PAPSS2, CANT1, and IMPAD1/BPNT2 encoding PAPS synthases 1 and 2, calcium-activated nucleotidase 1/UDP-diphosphatase, and 3′-phosphoadenosine 5′-phosphate 3′-phosphatase, respectively, were also listed. Furthermore, genes encoding regulators of GAG biosynthesis such as maintenance of Golgi-resident glycosyltransferases (GOLPH3) (SPPL3) (ZNF263) (KDM2B)  , a Golgi (SPPL3) , a trans(ZNF263) , and an  (KDM2B)  were als (KDM2B) .To evaluate GAG biosynthesis capability in human tissue, we used RNA-Seq data from 37 human tissues, obtained from the Human Protein Atlas . TPM is XYLT1, XYLT2, FAM20B, B4GALT7, B3GALT6, B3GAT3, EXT1, EXT2, EXTL2, EXTL3, CHSY1, CHSY3, CHPF, CHPF2, CSGALNACT1, DSE, and DSEL, clustered together  were correlated with poor prognosis in cancer patients (fibronectin (FN1), brevican (BCAN), versican (VCAN), perlecan (HSPG2), and several laminins were high in glioblastomas compared with in the normal brain  was selectively expressed in neuroblastoma  and decorin (DCN), was upregulated in pancreatic cancer  expression in pancreatic neuroendocrine tumor correlates with poor clinical outcomes (lumican (LUM) and biglycan (BGN) has been reported in pancreatic cancers (syndecan 1 (SDC1) mRNA level tended to increase the mortality rate due to pancreatic cancer (neurocan (NCAN), which showed almost no expression, were increased in pancreatic carcinoma  in malignant prostate tissues is significantly upregulated compared with that in benign tissues , bamacan (SMC3), and collagen type IX alpha2 (COL9A2) and downregulations of GPC1, GPC2, HSPG2, CSPG4, and DCN were detected (In primary prostate tumors, han half . In addiin chain . These rin chain , suggest tissues . A signie cancer . The GPCe cancer . In our detected .CSGALNACT1 and DSE in both the adrenal gland and thyroid gland tumors compared with that in normal tissues (Finally, we estimated GAG changes in the adrenal and thyroid glands during tumorization. GlycoMaple analysis predicted decreased levels of CS and DS, because of the downregulation of  tissues . To dateIn this study, we analyzed the expression levels of GAG biosynthetic enzymes as well as PGs in various normal and tumor tissues. GAG biosynthetic levels differed, depending upon the tissue functions and requirements. Analyses of the amount and sulfation pattern of GAGs are not easy to perform and require relatively abundant starting materials. The estimation of glycan structures based on gene expression data could be performed easily from small amounts of samples, and is useful to discover properties of cells of interest and obtain clues to structural changes among cell types. We applied GlycoMaple analysis to visualize the expression of genes involved in GAG biosynthesis and PG levels and to estimate glycan changes based on gene expression. Comprehensive analysis using gene expression levels in human tissues revealed new findings showing that expression of some genes required for GAG biosynthesis are regulated in a similar manner. For example, expression of genes required for the biosynthesis of GAG backbones including CS, DS, HS, and HA may be similarly regulated. GlycoMaple estimation showed that GAG biosynthetic patterns and core proteins of PGs were markedly changed during tumor progression in some tissues, which correlated with previous research. The process of CS formation would be upregulated during the formation of many tumor types . HoweverThe original contributions generated for this study are included in the article/MF and Y-FH conceptualized and designed the study and wrote a draft of the manuscript. Y-FH conducted analyses and validated the results. SM strengthened the background of GAG. All authors checked 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.


This case report is presented to improve our understanding of the atypical immunophenotype of hairy cell leukemia.A 58-year-old woman presented to our department with fatigue for >10 days.The patient was diagnosed with an increased proportion of abnormal lymphocytes in peripheral blood and bone marrow smear, positive for CD11c, CD19, CD20, CD22, CD25, CD123, CD200, and Kappa, partial expression of CD23, but no expression of CD103, positive for BRAF V600E mutation.Cladribine combined with rituximab achieved complete remission of minor residual disease negativity.Hairy cell leukemia is rare, and the diagnosis and differential diagnosis should be made by combining the patient's medical history, clinical manifestations, immunophenotype, gene detection, and other means. Purine nucleoside analogs are the first-line treatments. The HCL immunophenotype was characterized by strong expression of CD19, CD20, CD22, and CD200. CD5, CD23, CD10, and CD27 in hair cells are usually negative or weakly expressed, whereas CD11c, CD103, CD123, and CD25 are positive.2A 58-year-old woman presented to our department on November 30, 2020, due to fatigue for >10 days. The patient had no significant medical history. Physical examination findings were anemia appearance, no yellow stain on sclera, no petechiae on skin and mucous membrane, no enlargement of superficial lymph nodes of the body, no touch of liver and spleen under ribs.9 cells/L, neutrophil count of 30.6%, lymphocyte count of 61.0%, monocyte count of 0.1%, red blood cell count of 2.81 × 1012 cells/L, hemoglobin (of) 95 g/L, platelet count of 68 × 109 cells/L. Lymphocytes accounted for 62% of the blood smears and 23% of the bone marrow smears. Abnormal cells accounted for 64% of bone marrow smears, with different cell sizes, large amounts of cytoplasm, gray blue, and hairy processes . The patient's symptoms disappeared, and his spleen returned to normal size and was in good condition.Routine blood examination revealed a white blood cell (WBC) count of 1.5 × 103 It is recognized as a separate disease entity by the World Health Organization (WHO2008) and the 2016 Revised World Health Organization Classification of Lymphatic neoplasms.,4HCL was first reported in 1958 as a malignant tumor with pancytopenia and splenomegaly. However, in China, the proportion of pancytopenia, bone marrow dry aspiration, and combined myelofibrosis in HCL patients is relatively low.HCL is a rare chronic b-lymphocyte proliferative disease, accounting for about 2% of all leukemias, with a lower incidence in Asians than in Europeans and Americans. Bone marrow is the most common organ involved and is prone to peripheral blood and spleen invasion. HCL presents an indolent course of disease, with insidious onset and unknown etiology. There were more males than females, and the male to female ratio was about 4:1. The incidence was mainly in the elderly, with a median age of 63 years, and was rare in the young. The disease is often characterized by pancytopenia, especially monocytopenia, anemia, and splenomegaly. Liver and superficial lymph node enlargement is less common. Blood smear and bone marrow smear were visible to the “hair cell.” This type of cell is small B cells, slightly larger than the normal size of lymphocytes. The nucleus was round or oval, chromatin was rough, cytoplasm was rich, cytoplasm was gray-blue, and there were pseudopod-like hairy protrusions. Bone marrow pathology can show characteristic fried egg-like changes. Bone marrow puncture is often “dry aspiration” and reticular fibrous hyperplasia can be seen on biopsy. 1. The disease progresses slowly and can be relieved after treatment; however, it is difficult to cure and can be transformed into an invasive disease. 2. It is more common in middle-aged and elderly individuals. 3. All expressed mature B cell-associated antigens, but CD5 and CD10 were negative. 4. Most of the heteromorphic cells were small to medium sized lymphocytes. HCL has specific immunophenotype and molecular markers. In immunophenotype, HCL cells were positive for CD11c, CD25, CD103 and CD123 in addition to expressing mature B cell associated antigens. CD23, CD5, and CD10 are usually negative. An immune score system was established according to whether CD11c, CD103, CD123, and CD25 were expressed, and each expression marker was 1 point. 98% of HCL immunity scores were 3 or 4, whereas other HCL-like diseases were 0 or 1. BRAF V600E has a high mutation rate in classical HCL and is a molecular marker for the diagnosis of HCL. Tiacci et al conducted parallel sequencing of the entire exome of 242 patients with leukemia. BRAF V600E mutation was found in 47 patients with HCL. None of the 195 other patients with peripheral B-cell lymphoma or leukemia carried the BRAFV600E mutation, including 38 patients with splenic marginal zone lymphoma or splenic B-cell lymphoma/leukemia, which were not classified. In 2008, the WHO non-classifiable splenic B-cell lymphoma/leukemia included diffuse splenic red myeloid small B-cell lymphoma (SDRPSBCL) and HCL-V.The morphology of hair cells in the peripheral blood or bone marrow of patients is an important indication and basis for the diagnosis of the disease. However, the proportion of hair cells varies and not all are typical. It is not a specific manifestation of HCL and needs to be differentiated from other HCL-like diseases, including variant hair cell leukemia (HCL-V), splenic marginal zone lymphoma (SMZL), and diffuse splenic red myeloid small B-cell lymphoma. These 4 types of diseases are chronic B-cell proliferative diseases, and villous cells can be seen in the bone marrow and peripheral blood. They showed common clinical characteristics:HCL-V is a rare proliferative disease in mature B cells. It is characterized by spleen enlargement and “hair cells” in the peripheral blood and bone marrow. Compared with HCL, HCL-V is a more aggressive disease. The median survival time was shorter, and the response to purine analogs was worse. HCL-V has increased peripheral blood lymphocytes, including CD20 (+), CD22 (+), CD25 (−), CD123 (−), CD11c (−), CD103 (+/−), and BRAF V600E mutation (−). Expression of mature B cell surface antigen, may express DBA44, CD38, CD23(−), CD25(−), CD103 (−), CD123 (−), and CD11c positive rate is about 50%.,11SMZL accounts for <2% of all lymphatic system malignancies. The age of onset was >60 years, and the incidence was the same in both men and women. The main manifestation is splenomegaly, and most patients may involve the peripheral blood and bone marrow. There are no characteristic molecular markers, and approximately 30% of SMZLs have KLF2 mutations. which belongs to splenic B-cell lymphoma/leukemia, not classified. SDRPSBCL affects people >40 years of age, mainly involving the spleen, bone marrow, and peripheral blood. Leukopenia and thrombocytopenia may be present, and splenomegaly is present in all patients, with occasional skin manifestations. Its immunophenotype is CD20 (+), CD23 (−), CD25 (−), CD123 (−), CD103 (−), and CD11c positive rate is about 87%. About 24% of SDRPSBCL had BCOR mutations or deletions, 2% had BRAF G469A mutation, and no BRAF V600E mutation.In 2008, the WHO established SDRPSBCL as an independent disease entity,Thus, an accurate diagnosis of HCL or other diseases similar to the above can be established using complete immunophenotypes and molecular markers Table .,9,12 and CD103 (−) rarely appeared in classical HCL. Chen et al found CD103 (−) in only 2 patients by flow cytometry in 35 cases of classical HCL, among which 1 patient was accompanied by CD10 (+) and none by CD23 (+). In the largest series of cases reported by Yanying et al, no CD103 (−) cases were detected in 25 classical HCL cases in China. Shao et al reported that 169 classical HCL patients were all CD103 (−).,9 Therefore, bone marrow pathological immunohistochemistry and genetic testing for BRAF V600E should be performed for the accurate diagnosis of classical HCL in cases with atypical immunophenotypes, especially CD103 (−) cases.The patient we reported with classical HCL showed atypical immunophenotype (CD103 negative and CD23 positive) on both flow cytometry tests. However, bone marrow pathology and gene tests were positive for BRAF V600E, which supported the diagnosis of classical HCL. In classical HCL, the positive rate of CD23 ranged from 17% to 62.5%, Although complete response (CR) rates are high, ranging from 80% to 90%, there is often a positive MRD that leads to relapse and repeated treatment. MRD can be cleared by combination therapy with rituximab. Cladribine followed by rituximab (1 month after cladribine) showed 100% CR rates for both initial treatment HCL and relapsed HCL, with 5-year failure-free survival rates of 95% and 100%, respectively. Seventy-six percent of the initial treated HCL patients had negative MRD, and 64% of relapsed HCL patients had negative MRD. However, the optimal timing for rituximab is unknown. A randomized controlled phase 2 study found that nearly all patients (97%) receiving cladribine with concurrent rituximab achieved CR with negative MRD, which was significantly better than cladribine alone (24%). After a median follow-up of 96 months, 94% of patients in the cladribine with concurrent rituximab treatment group still maintained negative MRD CR, whereas the negative MRD CR rate in the cladribine delayed (≥6 months after clatribin) rituximab group was low (67%). Whether negative MRD CR means that HCL patients do not require additional treatment or cure still requires long-term follow-up monitoring. For this patient, we chose cladribine with concurrent rituximab treatment and achieved MRD (−) CR 1 month later.Patients with asymptomatic HCL can be observed temporarily without treatment. For HCL that requires treatment, purine nucleoside analogs such as cladribine or pentostatin monotherapy have been the standard first-line treatment for HCL for 30 years.4Classical HCL has no specific immunophenotypes. In addition to clinical features and morphological characteristics of cells, complete immunophenotypic analysis  and molecular detection (BRAFV600E mutation) can be differentiated from other HCL-like diseases and achieve accurate diagnosis. A combination of purine nucleoside analogs and rituximab resulted in a higher MRD (−) complete response rate.Contribution: Conceptualization by Hong-li Zhao, Hong-hua Cui, Wei-zhang Shen; Investigation by Hong-li Zhao, Hong-hua Cui, Li-fang Jin, Meng Zhao, Wei-zhang Shen; Writing–original draft by Hong-li Zhao, Hong-hua Cui, Li-fang Jin, Meng Zhao, Wei -zhang Shen; Writing–review and editing by Hong-li Zhao and Wei-zhang Shen.Conceptualization: Hong-li Zhao, Hong-hua Cui, Li-fang Jin, Meng Zhao, Wei-zhang Shen.Investigation: Hong-li Zhao, Hong-hua Cui, Li-fang Jin, Meng Zhao, Wei-zhang Shen.Writing – original draft: Hong-li Zhao, Hong-hua Cui, Li-fang Jin, Meng Zhao, Wei-zhang Shen.Writing – review & editing: Wei-zhang Shen.


N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinal damage in rats and determine the mechanisms of its protective effects. Male Sprague–Dawley rats (7 to 9 weeks old) were used in this study. Following intravitreal injection of NMDA (200 nmol/eye), the number of neuronal cells in the ganglion cell layer and parvalbumin-positive amacrine cells decreased, whereas the number of CD45-positive leukocytes and Iba1-positive microglia increased. Metformin attenuated these NMDA-induced responses. The neuroprotective effect of metformin was abolished by compound C, an inhibitor of AMP-activated protein kinase (AMPK). The AMPK activator, AICAR, exerted a neuroprotective effect in NMDA-induced retinal injury. The MEK1/2 inhibitor, U0126, reduced the neuroprotective effect of metformin. These results suggest that metformin protects against NMDA-induced retinal neurotoxicity through activation of the AMPK and MEK/extracellular signal-regulated kinase (ERK) signaling pathways. This neuroprotective effect could be partially attributable to the inhibitory effects on inflammatory responses.Metformin, an anti-hyperglycemic drug of the biguanide class, exerts positive effects in several non-diabetes-related diseases. In this study, we aimed to examine the protective effects of metformin against  Metformin, an anti-hyperglycemic drug of the biguanide class, controls blood glucose levels by increasing insulin sensitivity and enhancing glucose uptake in the liver, and is widely used in the treatment of type 2 diabetes mellitus . In addiPrevious studies have demonstrated several beneficial effects of metformin in various central nervous system disorders ,5,6. In N-methyl-D-aspartic acid (NMDA) receptors contributes to the neurotoxic effect of glutamate [Neuronal cell loss associated with glutamate-induced neurotoxicity is involved in the pathogenesis of several ocular diseases including glaucoma  and diablutamate ,18. Neurlutamate ,20,21. Tlutamate ,24,25,26n = 4, vs., 91 ± 3 cells/mm in the metformin group, n = 4, p = 0.8088; unpaired t-test)  had no significant change in cell number in the ganglion cell layer (GCL) compared to controls  A. In con t-test) B,C.n = 5, vs. 43 ± 3 cells in the NMDA + metformin group, n = 6, p = 0.0031; unpaired t-test, Next, we investigated the protective effect of metformin against retinal damage using cross-sections stained with neuron-specific markers, anti-NeuN, and anti-parvalbumin (PV) antibodies. Many NeuN-positive cells were observed in the GCL, and some were also found in the inner nuclear layer (INL)  higher in the NMDA + metformin group  than in the NMDA + vehicle group . These results suggest that metformin exerts a protective effect on amacrine cells and on RGCs.Next, we examined the effect of metformin (20 nmol) on NMDA-induced loss of PV-positive cells. In control retinas of the saline + vehicle group, PV-positive cells were observed in the inner part of the INL and their processes were found in the IPL (We examined whether (1) the protective effect of metformin against NMDA-induced retinal injury is mediated through activation of the AMPK system, and (2) activation of AMPK protects against NMDA-induced retinal injury. The co-administration of the AMPK inhibitor, compound C, with metformin abolished the neuroprotective effect of metformin A,B. As oTo determine whether the neuroprotective effect of metformin was mediated through the MEK/ERK signaling pathway, we examined the effect of U0126, an inhibitor of MEK1/2, on NMDA-induced cell loss in the GCL. The pharmacological inhibition of the MEK/ERK signaling pathway attenuated the neuroprotective effect of metformin .n = 5, vs. 799 ± 60 cells in the NMDA + metformin group, n = 6, p = 0.0002; unpaired t-test; the number of Iba1-positive cells; 1279 ± 122 cells in the NMDA + vehicle group, n = 5, vs. 917 ± 85 cells in the NMDA + metformin group, n = 6, p = 0.0034; unpaired t-test) is enhanced in Müller cells after intravitreal NMDA injection ,24,25,262+ influx, leading to endoplasmic-reticulum stress [Our present results suggest that the activation of the MEK/ERK signaling pathway contributes to the neuroprotective effect of metformin against NMDA-induced retinal neuronal cell loss. However, overactivation of NMDA receptors induces the pathologic increase of the Cam stress , oxidatim stress , and mitm stress . TherefoIn summary, our results indicated that metformin protects against NMDA-induced retinal neurotoxicity through the activation of AMPK and that this effect is probably mediated through the activation of the MEK/ERK signaling pathway in the retina. This neuroprotective effect could be partially attributable to the inhibitory effects on inflammatory responses. Glutamate-induced neurotoxicity may be associated with ocular neurodegenerative changes observed in glaucoma  and diabA total of 133 male Sprague–Dawley rats  were used in this study. The rats were housed under controlled conditions of temperature (22 ± 2 °C) and luminosity (12 h light/12 h dark). They were fed with a standard diet , and water was given ad libitum.n = 15), NMDA + metformin (1 nmol) (n = 8), NMDA + metformin (10 nmol) (n = 8), and NMDA + metformin (20 nmol) (n = 13). Under general anesthesia with pentobarbital sodium , metformin  or vehicle , mixed with 200 nmol of NMDA  at a total volume of 5 µL, was injected into the vitreous cavity of one eye of rats. As a control, the same volume of saline was injected into the vitreous cavity of the other eye. In a separate experiment (n = 4), the effects of intravitreal injection of metformin (20 nmol) and saline (5 µL) on the retina were compared.We first examined the effect of metformin on NMDA -induced neuronal cell loss in the retina. To assess the effect of metformin on the damage to retinal neurons, using retinal cross-sections stained with hematoxylin and eosin, 44 rats were divided into four groups as follows: NMDA + vehicle (n = 6), metformin (20 nmol) (n = 6), NMDA + vehicle (n = 5), and NMDA + metformin (20 nmol) (n = 6). One day after injection, rats were deeply anesthetized with pentobarbital sodium and systemic perfusion was performed with the fixative solution  in phosphate buffered saline  via the aorta. Their eyes were enucleated and processed for immunohistochemistry according to the methods described subsequently (see “4.4. Immunohistochemistry”).In addition to retinal ganglion cells (RGCs), several amacrine cell types express NMDA receptors in the retina ,36; intrn = 6), NMDA + metformin (20 nmol) (n = 6), and NMDA + metformin (20 nmol) + compound C  (n = 6). NMDA, metformin, and compound C were dissolved in dimethyl sulfoxide . In the second series of experiments, we examined the effect of AICAR (Tocris Bioscience), an activator of AMPK, on NMDA (200 nmol)-induced retinal neuronal cell loss in the GCL. In this experiment, 17 rats were divided into three groups as follows: NMDA + vehicle (n = 5), NMDA + AICAR (20 nmol) (n = 6), and NMDA + AICAR (50 nmol) (n = 6). AICAR was dissolved in saline.Next, we examined whether (1) the protective effect of metformin against NMDA-induced retinal injury is mediated through activation of the AMPK system, and (2) activation of AMPK protects against NMDA-induced retinal injury. In the first series of experiments, we examined the effect of compound C, an inhibitor of AMPK, on the effect of metformin on the NMDA (200 nmol)-induced retinal neuronal cell loss in the GCL. In this experiment, 18 rats were divided into three groups as follows: NMDA + vehicle (n = 7), NMDA + U0126  (n = 5), NMDA + metformin (20 nmol) (n = 10), and NMDA + metformin (20 nmol) + U0126 (1 nmol) (n = 5). NMDA, metformin, and U0126 were dissolved in DMSO. The U0126 dose was selected on the basis of the results of our previous studies [Activation of the MEK/ERK signaling pathway contributes to the RGC survival in various models of retinal neurodegeneration ,24,25,26n = 6), metformin (20 nmol) (n = 6), NMDA + vehicle (n = 5), and NMDA + metformin (20 nmol) (n = 6). The immunohistochemical staining of retinal cross-sections was performed as described in the “4.4. Immunohistochemistry” section.We previously demonstrated that leukocyte infiltration and microglial activation in the retina occurred one day after intravitreal injection of NMDA in rats ,38,39,40In Experiments 2, 3, and 4, intravitreal injections were administered using the technique described in Experiment 1, wherein the total injection volume used was 5 µL per eye.Histological evaluation was performed according to the methods described in our previous studies ,37,38,40Immunohistochemical staining of retinal cross-sections was performed according to the methods described in our previous studies ,38,39,40Two images of the region of the mid-peripheral retina, approximately 1 mm adjacent to the optic nerve on both sides, were obtained from each retinal section. RGC and amacrine cell damage were assessed by counting the NeuN-positive cells in the GCL and PV-positive cells in the INL, respectively. To assess the inflammatory response, the numbers of CD45- and Iba1-positive cells in the GCL and IPL of each retina were counted. The cell number was normalized to the total counting area of the GCL and IPL in each retinal section.All data are presented as the mean ± standard error (SE). An unpaired t-test and a Kruskal–Wallis test, followed by Dunn′s multiple comparison test, were used to compare values between the two groups and among more than two groups, respectively . A P value of less than 0.05 was considered to indicate a statistically significant difference.