Cell-mediated immunity is driven by antigenic peptide presentation on major histocompatibility complex (MHC) molecules. Specialized proteasome complexes called immunoproteasomes process viral, bacterial, and tumor antigens for presentation on MHC class I molecules, which can induce CD8 T cells to mount effective immune responses. Immunoproteasomes are distinguished by three subunits that alter the catalytic activity of the proteasome and are inducible by inflammatory stimuli such as interferon-γ (IFN-γ). This inducible activity places them in central roles in cancer, autoimmunity, and inflammation. While accelerated proteasomal degradation is an important tumorigenic mechanism deployed by several cancers, there is some ambiguity regarding the role of immunoproteasome induction in neoplastic transformation. Understanding the mechanistic and functional relevance of the immunoproteasome provides essential insights into developing targeted therapies, including overcoming resistance to standard proteasome inhibition and immunomodulation of the tumor microenvironment. In this review, we discuss the roles of the immunoproteasome in different cancers. The ubiquitin-proteasome system (UPS) is a multicomponent, multiprotein structure that catalyzes the proteolysis of unwanted, misfolded, and foreign proteins that have been covalently modified with ubiquitin molecules . SelectiThe immunoproteasome carries out proteasomal degradation of protein substrates for the MHC class I restricted antigen processing pathway ,8. TheseDue to its myriad functions, the immunoproteasome has become a focus in the investigation of the pathology of autoimmune conditions, cancer, inflammatory diseases, and neurodegenerative disorders . In tumoThe prominent role of the UPS in immunity has emerged over the last three decades. The genes for several components of the UPS, including TAP genes and 20S subunits, were found to be located within the genomic regions containing MHC class-II genes ,31,32. SPSMB9). β2i is also known as LMP10 or multi-catalytic endopeptidase complex-like-1 (MECL-1) and is encoded by the PSMB10 gene. β5i is alternatively called LMP7 and is encoded by the PSMB8 gene [PSME1) and β (PSME2) subunits forming a heteroheptamer. A homoheptameric variant of PA28, composed of only of one γ subunit, (PSME3) typically occurs in the nucleus [In the generation of the immunoproteasome, β1, β2, and β5 are replaced by more efficient IFN-γ inducible subunits, which are termed β1i, β2i, and β5i. β1i is also known as large multifunctional peptidase 2 (LMP2) and is encoded by the gene Proteasome Subunit Beta type 9  which fit better in the MHC class I cleft. These modifications also reduce the caspase-like activity of the β1i while promoting its chymotrypsin-like function. The changes in the β5i subunit increase the hydrophilic character of its catalytic site, generating a more favorable environment for peptide bond hydrolysis . While celopment . Differeelopment ,90,91.Antigen processing through proteasome catalyzed peptide splicing has also been reported to contribute to the immunopeptidome . These sThe PA28 regulatory complex may have the ability to associate with both the standard and the immunoproteasome; however, IFN-γ stimulus induces selective binding to the immunoproteasome complex . This asPSMB8 and other immunoproteasome subunits were reported to be incapable of producing pro-inflammatory cytokines such as IL-23, IFN-γ, IL-2, IL-4 and IL-10 [The immunoproteasome also directly influences T cell immunity independent of CD8 antigenic processing. Immunoproteasome-deficient T cells have been shown to have dramatically reduced expansion in response to viral infections, implying a direct role for the immunoproteasome in T cell maturation ,113. Mornd IL-10 ,116,117.nd IL-10 ,119. Hownd IL-10 ,121. Thend IL-10 . Throughnd IL-10 ,125,126.nd IL-10 ,132,133.The inducible immunoproteasome subunits are not exclusively expressed in immune cells, but also in other tissues at basal constitutive levels, such as colon, liver, lung, kidney, and small intestine epithelium mostly in the hybrid form as intermediate proteasome ,137,138.Neoplastic transformation is mediated by massive changes in cellular homeostasis. Induction of protein synthesis, a higher mutational burden, erroneous RNA splicing, and imbalanced redox environment due to metabolic changes all contribute to the production of misfolded or damaged proteins, requiring upregulation of protein turnover pathways ,145. ProPSMB8 gene, has a wide range of expression among different cancers, which have been evaluated in non-small cell lung carcinoma (NSCLC), renal cell carcinoma, glioma, colorectal cancer, triple-negative breast carcinoma (TNBC), laryngeal, and hypopharyngeal carcinoma [PSMB8 has been linked with poor prognosis. PSMB8 expression was found to be upregulated in all histological sub-types of renal cell carcinoma [PSMB8 expression in tumor tissue was associated with poor prognosis [PSMB8 are associated with more aggressive gliomas, and inhibition of PSMB8 was shown to reduce glioma cell proliferation and migration, as well to decrease glioblastoma tumor angiogenesis [PSMB8 correlated with lower overall survival does not hold for all types of neoplasms. In NSCLC patients, high expression of PSMB8 was frequently observed in cancers with more favorable outcomes [PSMB8 expression in TNBC tumor samples was associated with better disease-free outcomes, including in those with metastatic disease [The generation of MHC class-I peptides is an important facet of the maturation of cytotoxic T cells (CTLs). Given the central function of CTLs in mounting anti-tumor responses, immunoproteasome subunits induced by IFN-γ have been studied for their assumed role in cancer development. Amongst the three IFN-γ inducible β subunits, β5i has to date been most implicated in blood and solid malignancies. This subunit, encoded by arcinoma ,150,151.arcinoma . Similarrognosis . High leogenesis ,153. Howoutcomes . Likewis disease .PSMB8 in oncogenesis and disease progression seems to hinge on the fact that high levels of immunoproteasome expression can facilitate or impede tumor development in different contexts. For instance, the pro-tumorigenic role of PSMB8 in colorectal cancer is related to its role in colitis-induced chronic inflammation, which can drive neoplastic transformation of intestinal epithelium in the colon. Knockout of PSMB8 in mice was shown to prevent colitis-associated carcinogenesis [PSMB8-deficient mice were found to be resistant to chronic inflammation and neoplasia, with reduced expression of chemokines CXCL-1, CXCL-2, and CXCL-3. Upon induction of colitis, PSMB8−/− mice did not show macroscopic tumor development. The authors further attributed the pro-tumor effects of PSMB8 to reduced secretion of IL-17A in inflamed colons of PSMB8 deficient mice. The study proposed that IL-17A secretion was PSMB8-dependent via the NFκB signaling axis. The immunoproteasome has been shown to directly regulate NFκB signaling via direct proteolytic degradation of IκB, with knockdown of PSMB8 preventing the nuclear translocation of NFκB [PSMB8 serves as a promising treatment target for colorectal carcinomas. Supporting this, a study showed that ONX-914, an immunoproteasome inhibitor with a higher affinity for β5i subunit, suppressed tumor development in both preventive and therapeutic settings of colitis-induced carcinogenesis [The ambiguous role for ogenesis . PSMB8-d of NFκB ,118. In ogenesis .PSMB8 is context-dependent. PSMB8 deficiency has been shown to promote tumor growth in a mouse model of melanoma. It has been observed that PSMB8−/− mice implanted with B16 tumors have significant tumor growth and disease development [+ T cells in the draining lymph nodes and CTLs in the tumor microenvironment (TME) and decreased IFN-γ expression [PSMB8 in melanoma cell lines increased IFN-γ secretion, leading to efficient killing of tumor cells by tumor infiltrating CTLs. This seemed to be mediated through the presentation of more diverse and immunogenic HLA-1 peptides generated through overexpression of immunoproteasome subunits [However, as mentioned, deficiency of elopment . In the pression . This stsubunits .PSMB8 expressing NSCLC cells. STAT1, in a mutually inhibitory relationship with STAT3, was shown to be a major downstream signaling molecule, controlling IFN-γ related genes including immunoproteasome and antigen presentation machinery. Upon treatment with IFN-γ, the mesenchymal phenotype of the tumor cells was reversed and phosphorylation of STAT1 was increased. Immunoproteasome induction in the mesenchymal-like NSCLC cell lines was shown to generate an increased diversity and quantity of MHC class I peptides. When pulsed with these generated peptides, autologous CD8 T cells demonstrated robust effector responses against tumor cells in vitro [Thus, reduced expression of immunoproteasome subunits is a possible immune evasion mechanism deployed by tumor cells. In lung cancer, as in melanoma, higher expression of PSMB8 is associated with a more favorable prognosis, perhaps through increased immune surveillance ,157. In PSMB8 and PSMB9. Treatment of cisplatin-resistant tumor cells with proteasome inhibitors led to apoptosis induction, cell cycle arrest, and mitotic catastrophe. The authors propose that upregulation of immunoproteasome expression was a response to circumvent the cellular stress induced by cisplatin treatment [PSMB8, which can be rescued through exogenous IFN-γ, leading to resensitization [Immunoproteasomal subunit expression has also been reported to function as an indicator for treatment response and acquisition of chemoresistance. In both NSCLC and small cell lung cancer (SCLC), acquisition of cisplatin resistance correlated with increased expression of reatment . Sensitireatment . As menttization . The restization .PSMB8 expression, with cells exhibiting UPS-driven apoptosis in response to immunoproteasome ablation [PSMB8 and PSMB9 associated with better response to anti-PD-1 and anti-CTLA-4 treatment [A similar finding was noted in breast cancer. In TNBC, sensitivity to proteasome inhibitor treatment strongly correlated with high ablation . To mainreatment .PSMB8 expression in tumor cells not just reprograms the cellular pathways within the cell but also affects the tumor microenvironment. In highly invasive glioblastoma, a nexus of cellular communication is maintained between tumor cells, endothelial cells, and the extracellular matrix to allow increased angiogenesis. PSMB8 expression was reported to regulate this cellular communication. Elevated expression of PSMB8 was found in resected glioblastomas, and inhibition of PSMB8 reduced the migration and invasion of tumor cells in vitro. Endothelial cells demonstrated similar reduced migratory and tubulogenic properties when co-cultured with conditioned media taken from PSMB8-inhibited glioblastoma cell cultures. This interaction seemed to be mediated through reduced expression of vascular endothelial growth factor-A (VEGF-A) by tumor cells and integrin expression by endothelial cells. This was supported by a mouse model, which demonstrated that PSMB8 inhibition decreased tumor vessel formation [PSMB8 in cancer, regulation of PSMB8 has been reported through microRNAs, with miR-451a shown to target PSMB8 in prostate and thyroid cancer to prevent tumor cell proliferation and invasion [ormation . Howeverormation . In addiinvasion ,163.PSMB9 and PSMB10, are less described in cancer. β2i, or MECL1, encoded by PSMB10, has been reported to be downregulated in metastatic breast carcinoma, NSCLC, and acute promyelocytic leukemia however its functional relevance in tumor development is yet to be determined [PSMB10 as a genetic risk factor for chronic myelogenous leukemia (CML) [PSMB9, was found to be reduced in breast cancer, renal cell carcinoma, APL, and NSCLC while elevated in melanoma and ovarian cancer [PSMB8 regarding association with overall survival. Higher expression of PSMB9 in melanoma tumors has been linked with better patient outcomes while lower expression levels in NSCLC cells exhibited better prognosis [PSMB9. Higher APM scores, and higher PSMB9 expression, correlated with better responses for immune checkpoint therapy (ICB) in both NSCLC and melanoma with improved overall survival [The roles of the other two catalytic subunits, encoded by termined ,164. A ria (CML) . The β1in cancer ,168,169.rognosis ,157. A rPSMB8 in colon and lung cancer [The regulation of catalytic βi-subunits in cancer is brought about by several mechanisms. As described, NFκB, mTOR, and STAT1 have been shown to regulate the expression of g cancer ,157,158.g cancer . As descPSMB8/9 along with antigen presentation machinery proteins. This was found to be associated with increased time until recurrence time and increased CD8 T cell infiltration [PSMB8 genomic regions in mucinous type epithelial ovarian cancers, which correlated with increased susceptibility to proteasome inhibitors [Apart from transcriptional control, immunoproteasome subunits are also regulated epigenetically. Hypomethylation of 6p21.3 CpG islands in high-grade serous epithelial ovarian carcinoma upregulates ltration . Low methibitors . Epigenehibitors ,171,172.Even though immunoproteasome can process varied kinds of protein substrates, association with PA28 plays an important role in the generation of CTL-specific epitopes, by alerting conformation of the α rings ,175,176.PSME1 gene) was found in tumor biopsies with its presence correlated with poorer overall survival in patients, and was designated as a reliable biomarker to monitor tumor relapses and treatment [PSME1 were associated with better overall survival [PSME genes showed independent and often contrasting pathways, for instance, PSME1 expression was positively correlated with increases in cell adhesion, apoptosis, and NFκB and Wnt signaling pathways while PSME2 was negatively correlated with the same [PSME3 seemed to share features of PSME1 regulation, with correlation to NFκB and Wnt signaling pathways.In ovarian cancer, the C-terminal fragment of PA28  has approved three proteasome inhibitors, with the first being bortezomib for multiple myeloma . BortezoWhile bortezomib possesses β5i inhibitory activity, effective achievable intratumoral concentration, drug resistance, and off-target effects prevent immunoproteasomal inhibition . CarfilzUnlike the previous epoxyketone-derived IPIs, UK-101 was reported to selectively inhibit the catalytic β1i subunit and showed robust activity against prostate cancer in both in vitro and in vivo studies ,209. AnoPSMB8 identified as having particular centrality [The dysregulation of immunoproteasome expression in cancer is a well-known phenomenon, and the underlying molecular mechanisms of this have been revealed to be diverse and context-dependent. The immunoproteasome itself has diverse functions with outputs in a wide range of cellular processes. These effects may explain why immunoproteasome induction and inhibition have contradictory roles in different cancers, which mirrors the context-dependent role of inflammation in cancer. However, given the appropriate tumoral and clinical context, the immunoproteasome remains an attractive target, with ntrality ,155,157.ntrality ,180. Sma


O-glucoside and cyanidin 3-O-rutinoside. They are widespread in nature, being present in considerable amounts in red fruits and red vegetables. Overall, the present review intends to discuss the most recent findings on the potential health benefits from the daily intake of anthocyanin-rich foods, as well as their possible pharmacological mechanisms of action. However, before that, some emphasis regarding their chemical structure, dietary sources, and bioavailability was done.In recent years, the consumption of natural-based foods, including beans, fruits, legumes, nuts, oils, vegetables, spices, and whole grains, has been encouraged. This fact is essentially due to their content in bioactive phytochemicals, with the phenolic compounds standing out. Among them, anthocyanins have been a target of many studies due to the presence of catechol, pyrogallol, and methoxy groups in their chemical structure, which confer notable scavenging, anti-apoptotic, and anti-inflammatory activities, being already recommended as supplementation to mitigate or even attenuate certain disorders, such as diabetes, cancer, and cardiovascular and neurological pathologies. The most well-known anthocyanins are cyanidin 3- In the past few years, it is widely accepted that the daily intake of medicinal herbs, fruits, and vegetables provides a wide array of benefits to human health . This faConsidering that nature-based products have been used since ancient times to treat several disorders, such as colds, pain, gastrointestinal aches, and hypertension, among others, it is not surprising that this tendency continues to increase worldwide . A clearAmong the phenolic compounds, anthocyanidins and their conjugated acyl-glycosylated or glycosylated forms, called anthocyanins, are both members of the flavonoids and an interesting class of water-soluble vacuolar pigments . They arThis comprehensive review aimed to assess and elucidate the impact of anthocyanins and anthocyanin-rich foods on human health. For that, the first part of the manuscript focuses on a description regarding the chemical structure and function as well as main dietary sources of anthocyanins. Afterwards, the bioavailability and metabolism after intake are mentioned. Finally, we summarized and discussed the most recent literature regarding the main therapeutic effects of anthocyanins on different disorders.Phenolic compounds are secondary metabolites produced by plants to protect them against pathogens and predators, ultraviolet radiation, climate conditions, and acidified soils, acting also as attractants for pollinators, antifeedants, and phytoalexins ,26. TheyConcerning the flavonoids , they reFocusing on anthocyanidins, their B ring is joined to the C ring through carbon 2 and the sugar residue of anthocyanins is typically found conjugated at carbon 3 ,33 Figu. Further2O2), water, and/or sulphites ,11041,11In general, both native anthocyanidins and their conjugated forms are found in plasma and urine; nevertheless, the intact ones are more rapidly absorbed in the stomach (20–25%) and detected in plasma a few minutes after their ingestion . This evβ-glucosidase enzymes release the aglycone of the coloured compounds, increasing their hydrophobic character, thus facilitating their entrance by passive diffusion in epithelial cells [Notwithstanding, the major absorption rate is observed in the gut . Therefoal cells . Glycosial cells ,111,114;al cells . Particual cells ,109.α-galactosidase, β-D-glucuronidase, β-D-glucosidase, and α-rhamnosidase), which break down the glycosidic bonds and catalyse them into smaller phenolic compounds  or simple phenolic acids, such as ρ-hydroxybenzoic, homovanillic, phenylpropionic, protocatechuic, syringic, and vanillic acids, to simplify their absorption by colonic mucosa and consequently increase their availability [ρ-coumaric, protocatechuic and vanillic acids, and phenolic conjugates  [Unabsorbed anthocyanins reach the colon and are hydrolysated within 20 min−2 h by colonic bacteria  ,118. Actinobacteria, Bifidobacteria, and Lactobacilli [Additionally, the cleavage of glycosidic bonds also enhances the beneficial effects exhibited by anthocyanins . In factobacilli ,111. Theobacilli ,119.Knowing that the incorporation of phenolic compounds into foods and pharmaceutical products is a challenge, due to their instability and susceptibility to degradation, during processing and storage, various delivery systems have been developed . Among tTherefore, and in order to enhance their bioefficacy and stability, and thus prevent their rapid degradation, anthocyanins are mainly encapsulated with liposomes, nanocomplexes of alginate and chitosan, and gel emersions . In factFurthermore, bioengineering-based, targeted drug delivery approaches using biodegradable PLGA@PEG nanoparticles revealed more notable results in both in vivo and in vitro Alzheimer’s disease models than anthocyanins alone, namely, lower levels of oxidative stress and neuroinflammatory hallmarks ,122. FurThe low ingestion of fruits and vegetables causes around 1.7 million deaths worldwide, being related to 14% of gastrointestinal malignancies, 9% of stroke, and 11% of coronary artery disorders . TherefoIn order to attenuate the emerging resistance of microorganisms to antibiotics over time, natural products have gained much attention since they are rich in many metabolites with antimicrobial, antifungal, and anti-parasitic effects. In fact, these activities are part of the plants’ defense mechanism against pathogens and infections during their development and growth. Among these phytochemicals, anthocyanins already showed capacity to reduce the replication and growth of some Gram-negative and Gram-positive bacteria and parasites ,125.Escherichia coli and Salmonella, exhibiting minimum inhibitory concentration (MIC) values varying from 10 to 400 mg/mL, and also to reduce the growth of Desulfovibrio spp. and Enterococcus pathogenic bacteria by increasing the abundance of probiotics, such as Akkermansia and Bifidobacteria [Listeria monocytogenes, Staphylococcus aureus, and Vibrio parahaemolyticus, displaying MIC scores of 0.27, 0.21, and 0.030 mg/mL, respectively, after 24 h of exposure. Furthermore, it has also been reported that 100 µM cyanidin 3-O-glucoside can also inhibit the expression and secretion of Helicobacter pylori toxins by suppressing the SecA transcription pathway, thereby making the proteins’ exportation difficult [O-rutinoside and cyanidin 3-O-rutinoside at 1% did not reveal any inhibitory effect, enriched fractions of berries at concentrations ranging from 0.1 to 1% showed capacity to inhibit the yeasts Saccharomyces cerevisiae and Rhodotorula rubra, and the bacteria Bacillus cereus, Salmonella typhimurium, and Lactococcus lactis cocci growth [Pseudomonas aeruginosa, a microorganism associated with biofilm-mediated infections, exhibiting an MIC value of 2 mg/mL [S. aureus, E. coli, and P. aeruginosa bacteria [Acinetobacter baumannii and Proteus mirabilis pathogenic microorganisms over 24 h of treatment [Anthocyanins already revealed an ability to stop the replication of two common foodborne pathogens, bacteria . These rbacteria , who alsifficult . Even thi growth . Mulberr 2 mg/mL . Similarbacteria . More rereatment .Trichinella spiralis infection, a causative agent of human trichinellosis, by enhancing the CD3+, CD4+, and erythrocytes cells, promoting platelet aggregation and decreasing CD8+ splenocyte cells when compared to T. spiralis-infected mice that did not receive the extract [Trypanosoma brucei parasites in a murine model, after 21 days of treatment, by delaying the establishment of the trypanosomes and increasing the glutathione and aconitase-1 levels in the brain compared to the untreated group [A dose of 0.225 µg/mL of cornelian cherry fruits showed potential to stimulate murine immune response during  extract . Besidesed group . Additioed group . In a general way, the antimicrobial, antifungal, and anti-parasitic activities of the anthocyanins are mainly due to their capacity to react with the DNA, proteins, and sulfhydryl groups and interfere with AKT, ATPase, and superoxide dismutase activities, which, in turn, decrease the citric acid cycle and microbial metabolism, and inhibit microbial enzymes ,132. The2O2 and lipid peroxide. Since synthetic antioxidants have various adverse effects on health, there is a trend towards relying on antioxidants from natural products. Currently, the notable antioxidant properties of anthocyanins are well-described, and it is mainly due to their conjugated rings and hydroxyl groups [Free radicals are a product of natural metabolism; however, their accumulation becomes toxic to cells and trigger many reactions, such as the oxidation of cellular components  and lipid peroxidation, accelerating aging processes, and eliciting the occurrence of many chronic diseases, such as neurodegenerative and cardiovascular disorders, cancer, atherosclerosis, and ulcerative colitis . These rl groups ,134,135.O-glucoside at concentrations between 100 and 200 µM showed potential to protect human keratinocyte HaCaT cells against ultraviolet-A radiation, preventing DNA fragmentation and the release of hydrogen peroxide (H2O2) [Many in vitro assays already demonstrated the antioxidant potential of anthocyanin derivatives and foods rich in them . Focusine (H2O2) .50) value of 47.44 µg/mL, being three times more active than ascorbic acid (IC50 = 179.69 µg/mL); it also protects human erythrocytes against haemoglobin oxidation and prevents haemolysis damage induced by peroxyl radicals, in a concentration-dependent manner . Positive correlations (r > 0.4) between anthocyanin content and antioxidant assays have been reported [25) score of 16.58 µg/mL) and protect human adenocarcinoma Caco-2 cells against oxidative stress induced by tert-butyl hydroperoxide [2+/L (extract concentrations between 10 and 200 µg/mL) [50 = 4.1 µg/mL) [50 values lower than 50 µg/mL [Regarding the anthocyanin-enriched fractions from natural products, extracts of blackberries, blueberries, strawberries, sweet cherries, and red raspberries at 10 µM displayed the potential to inhibit human LDL oxidation, having been two times more effective than an ascorbic acid control . The colreported . Additioperoxide . Blackbe0 µg/mL)  and also0 µg/mL) . After 21 µg/mL) . Blackbe50 µg/mL . Concerning in vivo trials, delphinidin (1 mg/0.1 mL DMSO/mouse) showed capacity to protect mouse skin against UV-B radiation, preventing apoptosis after 8 h of exposure . Additioα-carotene (+7%), and vitamin C (+23%) levels [2O2 compared to the control, by reducing oxidative damage by 18% after 24 h of their consumption [In humans, the ingestion of fresh strawberry fruits  by 13 healthy volunteers revealed the capacity to increase the plasma ferric-reducing antioxidant power (+3.1%), ) levels . Besides) levels . Further) levels . A rando) levels . More resumption . The samsumption . Anthocysumption ,150,151.These considerable health-promoting properties are intimately linked to the capacity of anthocyanins to increase the glutathione levels by restoring or raising the activity of exogenous antioxidant enzymes and by activating the genes responsible for coding these enzymes. Besides, it is also due to their ability to inhibit NADPH and xanthine oxidases and modify arachidonic metabolism and mitochondrial respiration, and hence, reduce the formation of free radicals and reactive species, protecting cell components from damage . Furtherα, interleukin (IL)-6, and 1β) and nitric oxide radicals [Anthocyanins also possess anti-inflammatory capacity. Inflammatory conditions happen in response to pathogens that were not removed  or due to an inadequate long-term response to stimuli . They are characterized by oedema, redness, pain, fever, function losses, and larger amounts of pro-inflammatory cytokines -radicals . TherefoO-glucosides already exhibited potential to reduce the C-reactive protein levels by 77% in human liver cancer HepG2 cells, and vascular cell adhesion molecule-1 secretion in endothelial cells by 47%, at concentrations of 50 µg/mL compared to the non-exposed cells group [β, and TNF-α in lipopolysaccharide (LPS)-induced RAW264.7 macrophages at concentrations of 80 µg/mL [β and IL-6, and to suppress AP-1 signalling and nuclear factor kappa B (NF-kB) pathways. Additionally, it was also verified that they can decrease IKK, IkBa, p65, and JNK phosphorylation, avoid p65 nuclear translocation in LPS/IFN-γ-stimulated RAW 264.7 macrophage cells [α, IL-8, and Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES) at doses between 10 and 25 µg/mL in human bronchial epithelial BEAS-2B normal cells treated with LPS [α pro-inflammatory markers, and increase the IL-10 concentration in RAW264.7 macrophage cells stimulated with LPS over 24 h of exposure [Among the anthocyanins, cyanidin and delphinidin 3-ls group . Additio80 µg/mL . Anthocyge cells , and inhge cells . Similarge cells , and purge cells . Besideswith LPS . These rexposure . β, and TNF-α inflammatory cytokines in mice brain [2O2 damage, essentially by inhibiting IκBα phosphorylation, NF-κB activation, and IL-6 production, over 24 h of exposure [Enriched fractions of anthocyanins from raspberries already showed anti-inflammatory effects in dextran sulphate sodium-induced acute colitis in mice. Animals ingested 20 mg/kg of the extract for 10 days. At the end of the study, blood samples were collected, and mice were sacrificed for histological assessment. The results were compared in the control group; i.e., rats only received a normal diet. The obtained data revealed that a diet rich in anthocyanins contributed to improve colitis damage, by enhancing pro-inflammatory markers reduction and controlling weight . Similarce brain . Anotherexposure .O-glucoside and delphinidin 3-O-glucoside, daily for 24 weeks showed lower levels of C-reactive protein, vascular cell adhesion molecule-1, and plasma IL-1β levels  when compared to the untreated group. No changes were detected regarding TNF-α concentrations, which indicate that the intervention was safe [Hibiscus sabdariffa diluted in water showed lower monocyte chemoattractant protein-1 (−23.2%) levels, an important biomarker considering the evaluation of inflammatory disorders, after 3 h of their consumption compared to the placebo group [β, and TNF-α concentrations of−22, −7, −9 and −4%, respectively. These data were observed 4 h after the intake relative to the placebo group [Regarding human studies, 150 hypercholesterolemic subjects who ingested 320 mg anthocyanin capsules, rich in cyanidin 3-was safe . These dwas safe . Furtherbo group . Additiobo group . In anotbo group . α and IL-6 molecules and increments in anti-inflammatory IL-10 cytokines [Besides the mentioned, anthocyanins also facilitate muscle recovery after intense exercise, attenuating its damage and inflammation ,150,151.These effects are closely linked to the ability of anthocyanins to prevent CD40 activation, a member of the tumour necrosis factor receptor superfamily . AdditioCancer causes more deaths than strokes and coronary disorders and it is characterized by uncontrolled cell growth and proliferation . Anthocy50 value of 667.84 µg/mL), causing necrosis at concentrations superior to 400 µg/mL, after 24 h of treatment [Myrtaceae family also showed an ability to reduce the proliferation of human colon adenocarcinoma HT-29 cells, mainly by arresting the G2/M phase, hence causing cell apoptosis in comparison with the control group over 24 h of treatment [50 values of 45 and 149 µg/mL, respectively, without toxicity to MCF-10A normal breast cells [50 values of 200 and 400 µg/mL, respectively [The capacity of anthocyanins to inhibit the growth of metastatic cells is not surprising. Indeed, the enriched fraction of anthocyanins from sweet cherries already showed an ability to inhibit human adenocarcinoma Caco-2 cells growth  in rats with induced oesophageal carcinoma showed an ability to reduce cancer growth by 22% over 20 weeks of treatment, when compared to the control group . Adding Vitis coignetiae Pulliat also showed the potential to inhibit the tumorigenicity in mice infected with Hep3B human hepatocellular carcinoma cells  [Furthermore, a recent study revealed that anthocyanins from blueberry extracts showed the capacity to prevent the formation and growth of colorectal cancer in azoxymethane/dextran sodium sulphate-treated Balb/c mice, which ingested a diet containing 10% of the extract during 9 weeks of treatment, compared with the control . Anthocyper day) .O-glucosides also exhibited the capacity to reduce Lewis lung carcinoma cells in mice [On the other hand, berry anthocyanins displayed anticarcinogenic effects on lung cancer progression in rat xenograft models (reductions around 40%) treated with a diet supplemented with 7.5%, after 6 weeks of the intervention . The sup in mice .In addition, the administration of delphinidin  in athymic nude mice implanted with PC3 cells resulted in significant inhibition of prostate tumour growth, essentially by reducing the expression of NF-κB/p65, Bcl2, Ki67, and PCNA after 8 weeks of treatment . An anthBesides, the daily administration of anthocyanin-rich extracts from black rice  in mice xenografted with human tumour models showed the ability to reduce breast cancer growth, causing reductions around 41% , mainly Sorbus aucuparia (5 mL/kg for 11 days) in rats showed potential to reduce melanoma [Regarding skin cancer, the administration of anthocyanins extracted from melanoma . Furthermelanoma .Although there are no relevant studies regarding the anti-cancer effects on humans, it was already verified that the consumption of 20 g of black raspberry powder for between 2 and 4 weeks (three times per day) by Barrett’s oesophagus patients, a precursor lesion for oesophageal tumour, can reduce the proliferation rates and CD 105-stained blood vessels, and increase apoptosis in colon tumours . The ability of anthocyanins to inhibit the initiation and development of a tumour is closely associated with their ability to increase antioxidant defences, exert anti-inflammatory actions, and interfere with the ERK, JNK, PI3K/Akt, MAPK, and NF-κB pathways, as well as being associated with their regulated proteins and influence on the estrogenic/antiestrogenic levels ,183. FurKnowing neurological pathologies are closely related to oxidative stress and inflammatory levels, it is not surprising that the consumption of secondary metabolites from plants, such as anthocyanins, can reduce their occurrence.2O2 levels and apoptotic protease caspase-3/7 activity in BV-2 microglia cells in about 20% relative to untreated cells. They also showed an ability to diminish anti-glycation and anti-Aβ A aggregation [O-glucoside, cyanidin 3-O-sophoroside, delphinidin 3-O-glucoside, and malvidin 3-O-glucoside at 1 µM, also revealed an ability to protect dopaminergic neurons from the primary cells of Parkinson’s disease against rotenone toxicity, after 72 h of treatment, essentially by attenuating mitochondrial dysfunction [Anthocyanin-enriched fractions extracted from berries at 20 µg/mL already showed capacity to reduce nitric oxide, reactive oxygen, and carbonyl species, as well as the Hregation . Furtherfunction .2+ perturbations, losses of mitochondrial integrity, and Bax accumulation [1-42 plaques and neuro-apoptotic markers  by inhibiting the ρ-JNK/NF-κB/ρ-GSK3β pathway in mice brain [Besides, it was already reported that anthocyanins (ingestion of 100 mg/kg for 10 months) can restore the ion pump activities in rats experimentally demyelinated with ethidium bromide, by diminishing the proinflammatory mediators’ secretion and oxidative-stress levels and restoring the IL-10 levels . Additiomulation . Kim et ce brain .Concerning human assays, a 12-week randomised study involving 49 older adults with mild-to-moderate dementia who drank 200 mL anthocyanin-rich cherry juice showed improvements in verbal fluency, learning, memory, and cognition when compared to the untreated group . Similarβ-amyloid concentrations, and to suppress NF-kB, the Nf E2-related factor-2 (Nrf-2) signalling pathway, COX, and caspase activities [As mentioned before, the neuroprotective effects showed by anthocyanins are mainly due to their ability to cross the blood–brain barrier and protect neurons and glia cells from oxidative damage induced by reactive species and free radicals, reduce the inflammatory cytokines and tivities . Furthertivities .α-amylase and α-glucosidase carbohydrate-hydrolysing enzymes; and, consequently, restore the glucose levels, incentive insulin secretion, and pancreatic β-cells proliferation [Diabetes mellitus, with type 2 standing out, affects several people worldwide, being associated with obesity, oxidative stress, inflammatory events, and cardiovascular risk . Given tferation ,199.O-glucoside already showed potential to inhibit pancreatic α-amylase activity, revealing an IC50 value of 24.4 µM [α-glucoside action, showing to be three times more efficient than the acarbose control, which is the current pharmaceutical drug used to treat type 2 diabetes, but whose intake cause several gastrointestinal side effects [Cornus kousa displayed anti-angiogenic effects in the pre-adipocyte cell line 3T3-L1s, suppressing lipogenesis and adipogenesis in comparison with untreated cells after 8 days of exposure [Among the anthocyanins, cyanidin 3- 24.4 µM . Anthocy effects . Besidesexposure .O-rutinoside (30 mg/kg) also revealed an ability to alleviate postprandial hyperglycaemia and inhibit intestinal α-glucosidase in normal rats over three hours of intervention, as compared with the control group [Hibiscus sabdariffa L. (300 mg/kg) in obese-hypercholesterolemic rats revealed the capacity to reduce their weight, as well as to improve their lipid profile and liver enzymes action over 3 weeks of treatment in comparison to the group that did not receive the diet [The oral administration of acarbose combined with cyanidin 3-ol group . Besidesol group . On the ol group . Furtherthe diet . Blueberthe diet . These rthe diet ,205.Regarding human studies, it was already reported that twenty dyslipidaemic children and adolescents who daily ingested 50 g of cornelian cherry fruits, during 6 weeks, showed better lipid, apolipoprotein and vascular inflammation profiles and lower levels of LDL and triglycerides, intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 . Higher levels regarding apolipoprotein A (+11.6%) and a diminished content of apolipoprotein B (−13.6%) were also verified .The anthocyanins potential to be used as novel therapeutic agents against diabetes and obesity is fundamentally attributed to their chemical structure, which gives them inhibitory effects on the carbohydrate-hydrolysing enzymes’ action and capacity to interact in a competitive and/or non-competitive mode with the enzymes’ substrate and create hydrophobic bonds with these enzymes, thereby discontinuing their act and retard carbohydrate absorption ,200. FurCardiovascular pathologies are the principal cause of morbidity and mortality worldwide. They are intimately associated with the adoption of unhealthy behaviours, such as smoking, excessive alcohol intake, and other risk factors, including metabolic syndrome . SeveralO-glucoside and delphinidin 3-O-glucoside at concentrations of 50 µM showed the ability to reduce in vitro platelet aggregation and lysosome secretion, essentially by reducing β-thromboglobulin, serotonin ATP, platelet factor 4, CD63, and transforming growth factor β1 secretion after 40 min of exposure [O-glucoside can stimulate adiponectin expression and improve flow-mediated dilation in human adipose tissues and human aortic endothelial cells treated with 50 µM of cyanidin 3-O-glucoside after 24 h of exposure [Particularly, cyanidin 3-exposure . Moreoveexposure . Blueberexposure .Moreover, 50 µg/mL of anthocyanin-enriched fractions from strawberries already showed capacity to reduce the triglycerides and low-density lipoprotein (LDL) contents around 17% and 23%, respectively, in HepG2 cells after 24 h of treatment . AdditioConcerning in vivo trials, it was already reported that blueberry anthocyanin-enriched extracts can attenuate cyclophosphamide-induced cardiac damage in rats, treated with 80 mg/kg during four weeks by ameliorating the arterial blood pressure, heart rate, and activities of the heart enzymes, and improving cardiac dysfunction, left ventricular hypertrophy, and fibrosis. Additionally, they also showed the potential to prevent cardiomyocyte apoptosis . O-galactoside and peonidin 3-O-galactoside contents were found [β-thromboglobulin, soluble P-selectin, platelet factor 4, and transforming growth factor β1 levels in hypercholesterolaemic patients for 24 weeks of treatment as compared with the baseline [O-glucoside and cyanidin 3-O-glucoside concentrations [Furthermore, 31 pre-hypertensive men who ingested a single dose of 640 mg anthocyanins daily for 4 weeks revealed higher levels of high-density lipoprotein (HDL) concentrations and lower concentrations of triglycerides . Moreovere found . Similarre found . Furtherre found . Beyond re found , and alsre found . Additiobaseline . These ptrations . Similartrations . The contrations . On the trations .Therefore, the regular consumption of anthocyanin-rich foods and beverages can contribute to reduce the risk of cardiomyopathies, coronary problems, and ischemia ,33. ThesIn vivo and in vitro evidence also revealed that anthocyanins can improve the eyes and thus vision, owing to their ability to increase blood circulation in retina capillaries and the production of retinal pigments, which, in turn, improve night vision and protect eyes from oxidative damage, diabetic retinopathy, and molecular degeneration .2O2, by increasing the activity of heme-oxygenase-1 and glutathione S-transferase-pi antioxidant enzymes after 24 h of exposure [Anthocyanins isolated from blackberries at 100 µg/mL showed potential to protect human adult retinal pigment epithelial ARPE-19 cells against oxidative stress induced by Hexposure . Similarexposure  regardinO-rutinoside at 10 µM already showed an ability to relax the bovine ciliary smooth muscle through activation of the endothelin-1 receptor and NO/cGMP pathway, inhibiting myosin light chain phosphorylation, hence causing relaxation [Concerning in vivo studies, delphinidin 3-laxation . Additiolaxation .Focusing on human assays, a double-blind, placebo-controlled, crossover study involving healthy volunteers revealed that the daily intake of six capsules rich in blackcurrant anthocyanins at a concentration of 50 mg can improve dark adaptation, video display terminal work-induced transient refractive alteration, and asthenopia symptoms  in comparison to the placebo . In anotGiven that, it was already reported that intact anthocyanins can pass through the blood-aqueous barrier and blood-retinal barrier in rats and rabbits, being widely distributed in ocular tissues . This evAnthocyanins are the coloured compounds largely found in nature, for which evidence indicates that their regular consumption offers several health benefits to human health, mainly due to their ability to reduce free radicals, reactive species, and pro-inflammatory markers. These abilities can counteract oxidative stress levels, avoid the development of inflammatory processes, and protect human organs and cell components against damage, and thus confer protection at distinct levels. Thus, anthocyanins’ structure, biochemistry, and encapsulation have been deeply studied in order to increase their use, stability, and consequent bioavailability and action. Until now, and although more in vivo and clinical trials are needed, evidence suggests that anthocyanins are promising candidates for the engineering of new pharmaceutical drugs, and can be used as an alternative or as an adjuvant therapy capable to attenuate or prevent the occurrence of many disorders, including diabetes, cancer, and cardiovascular and neurological pathologies. In fact, their use in pharmaceutical products, nutraceuticals, foods, and as food colourants is increasing worldwide.


Acute lymphoblastic leukemia (ALL) is a malignant disease most commonly diagnosed in adolescents and young adults. This study aimed to explore potential signatures and their functions for ALL.Differentially expressed mRNAs (DEmRNAs) and differentially expressed long non-coding RNAs (DElncRNAs) were identified for ALL from The Cancer Genome Atlas (TCGA) and normal control from Genotype-Tissue Expression (GTEx). DElncRNA–microRNA (miRNA) and miRNA–DEmRNA pairs were predicted using online databases. Then, a competing endogenous RNA (ceRNA) network was constructed. Functional enrichment analysis of DEmRNAs in the ceRNA network was performed. Protein–protein interaction (PPI) network was then constructed. Hub genes were identified. DElncRNAs in the ceRNA network were validated using Real-time qPCR.A total of 2,903 up- and 3,228 downregulated mRNAs and 469 up- and 286 downregulated lncRNAs were identified for ALL. A ceRNA network was constructed for ALL, consisting of 845 lncRNA-miRNA and 395 miRNA–mRNA pairs. These DEmRNAs in the ceRNA network were mainly enriched in ALL-related biological processes and pathways. Ten hub genes were identified, including SMAD3, SMAD7, SMAD5, ZFYVE9, FKBP1A, FZD6, FZD7, LRP6, WNT1, and SFRP1. According to Real-time qPCR, eight lncRNAs including ATP11A-AS1, ITPK1-AS1, ANO1-AS2, CRNDE, MALAT1, CACNA1C-IT3, PWRN1, and WT1-AS were significantly upregulated in ALL bone marrow samples compared to normal samples.Our results showed the lncRNA expression profiles and constructed ceRNA network in ALL. Furthermore, eight lncRNAs including ATP11A-AS1, ITPK1-AS1, ANO1-AS2, CRNDE, MALAT1, CACNA1C-IT3, PWRN1, and WT1-AS were identified. These results could provide a novel insight into the study of ALL. Acute lymphoblastic leukemia (ALL) is a malignant disease most commonly diagnosed in adolescents and young adults, especially in patients younger than 15 years. Despite significant improvements in the management of ALL, the long-term survival rate of ALL patients, especially adult patients, remains low . TherefoLncRNA is a type of RNA longer than 200 nucleotides. Dysregulated lncRNA as tumor suppressor genes or oncogenes plays a key role in a variety of biological processes, such as cell proliferation, apoptosis, migration, and invasion. Increasing studies are focusing on the role and mechanism of lncRNA in the occurrence and development of ALL . For insThe development of transcriptome analysis and RNA sequencing technology is increasing the possibility of identifying lncRNAs that may be involved in the pathogenesis of ALL. Moreover, further studies on the function of abnormally expressed lncRNAs may help understand the pathogenesis of ALL and provide important insights for the treatment of ALL. In this study, we comprehensively analyzed DElncRNAs and DEmRNAs in bone marrow samples of ALL. A ceRNA network was constructed for ALL on the basis of DElncRNA–miRNA and miRNA–DEmRNA pairs. DEmRNAs in the ceRNA network were significantly associated with ALL-related biological processes and pathways. Among DElncRNAs in the ceRNA network, eight lncRNAs including ATP11A-AS1, ITPK1-AS1, ANO1-AS2, CRNDE, MALAT1, CACNA1C-IT3, PWRN1, and WT1-AS were validated by Real-time qPCR, which could become potential diagnostic and therapeutic targets of ALL.1, which were derived from the IlluminaHiSeq RNA-Seq platform. All the data from three phases together, including 12 cases of phase 1, 468 cases of phase 2, and 14 cases of phase 3 were enrolled in the study. There were 321 (64.98%) males, 172 (34.82%) females, and 1 unknown (0.02%). The age distribution of the ALL group is as follows: 403 cases of 0–14 years old and 91 cases of ≥ 14 years old. All data of normal tissue samples were obtained from 407 whole blood in the Genotype-Tissue Expression (GTEx) database2. There were 265 (65.11%) males and 142 (34.89%) females in the control group. The age distribution of the control group is as follows: 34 cases of 20–29 years old, 34 cases of 30–39 years old, 72 cases of 40–49 years old, 130 cases of 50–59 years old, 132 cases of 60–69 years old, and 5 cases of 70–79 years old. Complete description of the multiple ethnicity groups, the biospecimen procurement methods, and sample fixation was provided in the GTEx official annotation. Differential expression analyses between ALL samples and normal samples were carried out using the EdgeR package in R . mRNAs and lncRNAs with adjusted p < 0.05 and | log 2fold change (FC)| ≥ 2 were considered as DEmRNAs and DElncRNAs. Volcano plots and heatmaps were generated using the ggplot2 and packages in R, respectively.LncRNA and mRNA RNA-seq data of 494 bone marrows with ALL  were retrieved from TCGA repositoryage in R . The obt3 that provides > 10,000 lncRNAs  analysis of DEmRNAs in the ceRNA network was carried out using Database for Annotation, Visualization, and Integrated Discovery (DAVID) , includi7 (minimum required interaction score > 0.4)  databasee > 0.4) . Further–ΔΔCT method. Differences between the two groups were analyzed using Student’s t-test. p-value < 0.05 was considered statistically significant.Bone marrow samples were isolated from 25 ALL patients and 15 healthy participants and red blood cells were removed. Total RNA was extracted from bone marrow samples and then was stored at −80℃. Extracted samples were lysed using 1 ml of Trizol and placed for 5 min on ice. RNA concentration and purity were determined using a NanoDrop UV spectrophotometer. Then, RNA was reverse transcribed into cDNA. Primer sequences of ATP11A-AS1, ITPK1-AS1, ANO1-AS2, CRNDE, MALAT1, CACNA1C-IT3, PWRN1, and WT1-AS were designed and synthesized by Shanghai Shengong Biological Engineering Co., Ltd. . The primer sequences are listed in p-value < 0.05 and | log2 fold change (FC)| ≥ 2, 2903 up− and 3228 downregulated mRNAs were identified for ALL, as shown in the volcano plot . Furthermore, PWRN1 and SMAD3 were significantly correlated (r = 0.521493415 and p = 4.32E−08).Correlation analysis between hub genes and DElncRNAs was performed by corrplot package. The significant correlations between DElncRNAs and hub genes are shown in n = 25) compared to normal samples (n = 15) by Real-time qPCR.Among all DElncRNAs in the ceRNA network, the most significant difference between 10 lncRNAs  in the ceRNA network and WT1-AS (which was closely related to hub gene FZD7) was selected for verification. However, as the primers of AC009093.1, C17orf77, and ITCH-IT1 for RQ-PCR were not ideal, the remaining eight lncRNAs were validated. As In this study, we constructed a ceRNA network for ALL based on DElncRNA–miRNA and miRNA–DEmRNA relationships. Among all DElncRNAs in the ceRNA network, eight lncRNAs were validated in ALL bone marrow samples using Real-time qPCR. These lncRNAs might become potential biomarkers for ALL.via targeting the Wnt pathway  patients and BCP-ALL cell lines . Functionally, CRNDE upregulated CREB expression by suppressing miR-345-5p, thus promoting cell proliferation and reducing cell apoptosis in BCP-ALL . A largeIX4 axis . HoweverHowever, there are several limitations in this study. First, since there was no normal control of ALL in TCGA database, data of 407 whole blood in the GTEx database were obtained as control. Given that ALL primarily affects younger individuals, the age distribution of the control group is not ideally matched with the ALL from TCGA database, which may cause confounding. Second, the sample size of this study is small, and larger clinical samples should be used to verify these lncRNAs. In addition, this study lacks functional experiments. In future research, we will further the function and clinical value of these lncRNAs in ALL.In our study, a ceRNA network was constructed for ALL. Among all DElncRNAs in the ceRNA network, eight lncRNAs were validated in ALL bone marrow samples using Real-time qPCR, which might provide a novel insight into the further study of ALL.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Institutional Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University (2019-KY-194). The study was conducted in accordance with the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.WW designed the project, proposed the research concept, and wrote the manuscript. WW, CL, and FWa constructed the bioinformatic analysis, constructed the graphic images and data charts, and performed the statistical processing. CW, FWu, XL, and SG performed the experiments. All authors read and approved the manuscript and agreed to be accountable for all aspects of the research in ensuring that the accuracy and integrity of any part of the work are appropriately investigated and resolved.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.


Using various computational tools, we interrogated the role of MXD3 in tumor immune infiltration, immune evasion, tumor progression, therapy response, and prognosis of cohorts from various cancer types. Our results indicated that MXD3 was aberrantly expressed in almost all The Cancer Genome Atlas (TCGA) cancer types and subtypes and was associated with the tumor stage, metastasis, and worse prognoses of various cohorts. Our results also suggested that MXD3 is associated with tumor immune evasion via different mechanisms involving T-cell exclusion in different cancer types and by tumor infiltration of immune cells in thymoma (THYM), liver hepatocellular carcinoma (LIHC), and head and neck squamous cell carcinoma (HNSC). Methylation of MXD3 was inversely associated with messenger (m)RNA expression levels and mediated dysfunctional T-cell phenotypes and worse prognoses of cohorts from different cancer types. Finally, we found that genetic alterations and oncogenic features of MXD3 were concomitantly associated with deregulation of the DBN1, RAB24, SLC34A1, PRELID1, LMAN2, F12, GRK6, RGS14, PRR7, and PFN3 genes and were connected to phospholipid transport and ion homeostasis. Our results also suggested that MXD3 expression is associated with immune or chemotherapeutic outcomes in various cancers. In addition, higher MXD3 expression levels were associated with decreased sensitivity of cancer cell lines to several mitogen-activated protein kinase kinase (MEK) inhibitors but led to increased activities of other kinase inhibitors, including Akt inhibitors. Interestingly, MXD3 exhibited higher predictive power for response outcomes and overall survival of immune checkpoint blockade sub-cohorts than three of seven standardized biomarkers. Altogether, our study strongly suggests that MXD3 is an immune-oncogenic molecule and could serve as a biomarker for cancer detection, prognosis, therapeutic design, and follow-up.MAX dimerization (MXD) protein 3 (MXD3) is a member of the MXD family of basic-helix-loop-helix-leucine-zipper (bHLHZ) transcription factors that plays pivotal roles in cell cycle progression and cell proliferation. However, there is insufficient scientific evidence on the pathogenic roles of MXD3 in various cancers and whether MXD3 plays a role in the immuno-oncology context of the tumor microenvironment, pathogenesis, prognosis, and therapeutic response of different tumors through certain common molecular mechanisms; thus, we saw a need to conduct the present  The list of the 33 TCGA cancer types, their histology, and body location is provided in the https://www.gtexportal.org/home/). The GTEx Expression dataset (V8.0) is composed of 17,382 samples from 30 organs (54 tissues), donated by 948 healthy individuals. The workflow of this study is shown in The clinical data of cancer cohorts were obtained from The Cancer Genome Atlas (TCGA) Program of the National Cancer Institute  T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells (DCs). We also analyzed correlations of MXD3 expression with tumor infiltration of four immunosuppressive cell types that are known to promote T cell exclusion, viz., myeloid-derived suppressor cells (MDSCs), cancer-associated fibroblasts (CAFs), M2 subtype of tumor-associated macrophages (M2-TAMs), and regulatory T (Treg) cells across 39 TCGA cancer types. A correlation analysis was conducted using the purity-corrected partial Spearman's rho value and statistical significance (p < 0.05). We used GraphPad Prism Software (vers. 8.0.0 for Windows) for data visualization. Heatmaps were used to visualize infiltration levels of immune cells across 33 TCGA cancers. In addition, we used the QUERY module the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to evaluate the effect of genetic and epigenetic alterations of MXD3 on dysfunctional T-cell phenotypes We used the TIMER2 server to analyze correlations between MXD3 expression and infiltration of six immune cell types, including B cells, cluster of differentiation 8-positive (CD82.5https://www.oncomine.org/resource/login.html) p value of < 10−04, a multiple of change of < 2, and gene rank of 10%. In addition, we used the cBioPortal for Cancer Genomics  to assess gene mutation co-occurrence patterns between MXD3 signatures and other proteins across 10,953 patients from TCGA Pan-Cancer Atlas Studies p value of < 10−10, and a q-value of < 10−10.We used the ONCOMINE database , disease-free survival (DFS), and disease progression-free survival (PFS) of the cohorts. For survival analyses of differentially expressed MXD3 between cancer cohorts, we set the median expression as the expression threshold to split the patient samples into MXD3 high- and low-expression groups, with the hazard ratio (HR), 95% confidence interval (CI), and log-rank test 2.8p < 0.05.We used the GPS-Prot algorithm to conduct the PPI network analysis of MXD3, while the OPENTARGET platform was used to conduct a gene-disease network analysis of MXD3 based on genetic associations. Furthermore, we used MXD3 coexpressed and co-mutated genes to conduct PPI and enrichment analyses. The enrichment analysis of the Kyoto Encyclopedia of Genes (KEGG) pathways and gene ontologies (GOs) were conducted using the Enrich server 2.950)) to 265 small molecules from the GDSC database.In order to analyze the effect of MXD3 on therapeutic responses, we used the ROC plotter server to analyze associations of MXD3 transcriptome levels with therapeutic responses in breast, glioblastoma, colorectal, and ovarian cancer patients 2.10We accessed the gene prioritization of MXD3 across two parameters, including the response to immune checkpoint blockade (ICB) therapy and gene-knockout phenotypes in CRISPR screens. The z-score in the Cox-PH regression was used to evaluate the effect of gene expression on patient survival in ICB treatment cohorts. The normalized log(multiple of change [FC]) in CRISPR screens was employed to evaluate the effect of gene knockout-mediated lymphocyte-induced tumor death in cancer models 2.11https://dtp.cancer.gov/databases_tools/compare.htm) to identify potent anticancer inhibitors of MXD3. The PDB file of the crystal structures of MXD3 (PDB: 6JKK) was obtained from the Protein Data Bank (https://www.rcsb.org/) while the mol2 file of the three-dimensional (3D) structure of the identified compounds (S764609 and S764582) were obtained using the Avogadro molecular builder and visualization tool vers. 1. XX (http://avogadro.cc/) https://pymol.org/edu/?q=educational/). All PDB files were converted to PDBQT files using AutoDock Vina  We used the public COMPARE module of the National Cancer Institute (NCI)-COMPARE program algorithm  > H (His)] A. To cha2 phases D. A gene2 phases E associa2 phases F.Fig. 2M3.2p < 0.05) overexpressed in tumors of all TCGA cancer types compared to their corresponding adjacent normal tissues, except for kidney chromophobe (KICH), pancreatic adenocarcinoma (PAAD), and skin cutaneous melanoma (SKCM) (We explored the oncogenic role of MXD3 across the TCGA pan-cancer database and found that mRNA levels of MXD3 were significantly (a (SKCM) . Howevera (SKCM) B. Furthea (SKCM) C. Howevea (SKCM) D. Altoge3.3r = 0.42 ∼ 0.77, all p < 0.05), while LICH  and HNSC  showed week correlations between MXD3 expression levels and tumor infiltration of the six immune cell types, while HNSC-human papillomavirus-positive (HPVpos) showed good correlations of MXD3 expression levels with tumor immune infiltration only of B cells  and CD8 + T cells . All other cancer types showed significant negative associations  or no significant association (p > 0.05) between MXD3 expression levels and tumor infiltration of the six immune cell types  showed significant positive correlations of MXD3 expression with infiltration of six immune cell types . THYM showed a strong correlation , PAAD, PCPG, PRAD, SARC, SKCM, STAD, THCA, and UCEC; tumor infiltration of Tregs in BRCA, BRCA-LumA, BRCA-LumB, BRCA-Basal, CESC, DLBC, ESCSA, HNSC, HNSC-HPVpos, KICH, KIRC, KIRP, LICH, LUAD, PAAD, PCPG, PRAD, sarcoma, stomach adenocarcinoma (STAD), testicular germ cell tumor (TGCT), and THYM; tumor infiltration of CAFs in CESC, lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), KIRP, LGG, TGCT, and THCA; and tumor infiltration of M2-TAMs in BRCA-LumA, CESC, cholangiocarcinoma (CHOL), DLBC, GBM, MESO, PCPG, THYM and uterine corpus endometrial carcinoma (UCEC) B.We evaluated the biomarker relevance of MXD3 by comparing it with standardized biomarkers based on their predictive power of response outcomes and OS of ICB sub-cohorts. Interestingly, we found that MXD3 alone had an area under the receiver operating characteristic curve (AUC) of > 0.5 in 12 of the 23 ICB sub-cohorts C. MXD3 eOur results also demonstrated that high expression levels of MXD3 were associated with worse programmed death 1 protein (PD1) outcomes in glioblastoma (ICB_Zhao2019_PD1), in kidney renal clear cell carcinoma (ICB_Miao2018_ICB), PD1 in melanoma (ICB_Riaz2017_PD1), CTLA4 in melanoma (ICB_Nathanson2017_CTLA4), and ACT in melanoma (ICB_Lauss2017_ACT) but achieved good PD-ligand 1 (LI) therapeutic outcomes in bladder (ICB_Mariathasan2018_PDL1) cancer cohorts. At the same time, analysis of gene-knockout phenotypes from genetic screens revealed that MXD3-knockout was a strong influencer of lymphocyte-mediated tumor killing in MC38 colon cancer (Kearney2018_NK_20) and K562 leukemia (Pech2019_NK_E:T = 2.5) models D.3.4r = 0.1 ∼ 0.48, all p < 0.05, Analysis of the promoter methylation status revealed that MXD3 is hypermethylated in KIRP. At the same time, it is hypomethylated in various cancer types, including LICH, LUAD, lung squamous cell carcinoma (LUSC), SARC, TGCT, UVM, BRCA, colon adenocarcinoma (COAD), cervical squamous cell carcinoma, and endocervical adenocarcinoma (CESC), CHOL, HNSC, KIRC, and UCEC A. These 3.5r ≥ 0.99) with expression levels of several other genes, including DBN1, RAB24, SLC34A1, PRELID1, LMAN2, F12, GRK6, RGS14, PRR7, and PFN3 , docetaxel, RDEA119 (a MEK inhibitor), PD-0325901 (a MEK inhibitor), and bleomycin but led to increased activities of I-BET-762, WZ3105, QL-XI-92, KIN001-102, GSK690693, GSK1070916, NPK76-II-72–1, QL-X-138, and navitoclax in various cancer cell lines A. Furthe3.750 concentrations (-log10) of −4.87 ± 0.19 and 4.73 ± 0.22 μM for S764609 and S764582, respectively  to identify potent anticancer drugs whose specific mechanism of action involved the inhibition of MXD3 signaling. Surprisingly, out of the over 88,000 pure compounds reported for anticancer activities against panels of NCI cancer cell lines, we identified only two synthetic compounds, (5E)-5-[(4-ethylphenyl) methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one (S764582) and 7-nitro-N-(2-phenylphenyl)-2,1,3-benzoxadiazol-4-amine (S764609), that specifically targeted the MXD3 signaling pathway. These compounds were respectively reported to inhibit the growth of 58 and 59 cancer cell lines, representative of leukemia, melanoma, prostate, renal, ovarian, CNS, NSCLC, and colon cancers with GIectively A. In ordectively B.Fig. 8I4Our results revealed that MXD3 is closely related to the occurrence and development of various systemic diseases and cancers. Studies also reported functional links between MXD3 and clinical diseases, especially tumors MXD3 gene suggested distinct conclusions for different tumors, as we observed that MXD3 predicted worse survival of PRAD, MESO, LGG, KICH, UVM, LUAD, KIRC, ACC, THYM, HNSC, LICH, and GBM cohorts. Our findings are consistent with previous clinical and preclinical studies, which demonstrated that MXD3 was highly expressed in neuroblastoma and medulloblastoma cells Cancers of different types and subtypes exhibited distinct genetic heterogeneity and clinical features In the present study, we failed to detect a correlation between MXD3 expression and survival prognosis of BLCA, BRCA, CESC, CHOL, COAD, DLBC, ESCA, KIRP, LAML, LUSC PRAD, THYM, UCEC, or UCS patients; hence, the current TCGA clinical data-based evidence cannot support the role of MXD3 overexpression in the clinical prognosis of those cancers. We, therefore, postulated the possibility that high MXD3 expression levels in those cancer types are a byproduct of dysregulated signaling without prognostic significance for tumor cells. However, in contrast, as discussed below, our further analysis suggested the involvement of MXD3 genetic and epigenetic alterations in regulating the tumor immune microenvironment and worse clinical prognoses of cohorts of those cancers.The TME and tumor immune evasion are correlated with cancer prognoses and therapeutic The second mechanism of tumor immune evasion is the T-cell exclusion mechanism The accumulation of genetic alterations drives the progression of normal cells through hyperplastic and dysplastic stages to invasive cancer and, ultimately, metastatic disease r ≥ 0.99) between the MXD3 expression level and expression levels of DBN1, RAB24, SLC34A1, PRELID1, LMAN2, F12, GRK6, RGS14, PRR7, and PFN3 in cancers. Our search for co-occurring genetic alterations also revealed that these same genes exhibited very high (>80%) enrichment with MXD3 in the frequency and pattern of genetic alterations, suggesting that these genes are functional partners associated with the oncogenic role of MXD3 in various cancer types.Interestingly, genetic alterations of MXD3 were associated with worse prognoses in the context of OS, DFS, and PFS. These findings, together with the overall expression profiles of MXD3 in primary tumors and pathological and metastatic stages, suggest its involvement at all stages of tumor progression, and hence it can serve as an attractive target for cancer therapies. However, cancer development and progression cannot be attributed to a single gene, as co-occurrences of gene alterations are frequently observed and conjoin with the primary genetic driver as co-drivers to promote tumor progression and limit therapeutic responses Furthermore, we integrated MXD3 with these co-functional partners across all tumors for enrichment analyses and identified enrichment of “inorganic ion homeostasis,” “regulation of phospholipid transport,” “chemokine signaling,” and “complement and coagulation cascades” in the MXD3 network-mediated pathogenesis of various cancers. Alterations in iron metabolism are among metabolic and immunological hallmarks of cancer Antibodies against PD-1 or PDL-1 effectively treat a variety of cancers and improve prognoses Our analysis also revealed that glioblastoma, ovarian, and breast cancer patients with higher MXD3 expressions were resistant to chemotherapies. In accordance with our observations, previous studies reported that MXD3-knockdown resulted in apoptosis of neuroblastoma cells and enhanced the therapeutic efficacy of chemotherapies against a neuroblastoma cell line 5DBN1, RAB24, SLC34A1, PRELID1, LMAN2, F12, GRK6, RGS14, PRR7, and PFN3 and were connected to phospholipid transport and ion homeostasis. Collectively, our study suggests that MXD3 could serve as a biomarker for cancer detection, prognosis, therapy design, and follow-up.In conclusion, our results suggest that MXD3 plays pivotal pathogenic roles in the immuno-oncology context of the TME, prognoses, and therapeutic responses through different mechanisms involving T-cell exclusion, tumor infiltration of immune cells in THYM, LICH, and HNSC. Genetic alterations and epigenetic modifications of MXD3 were associated with poorer prognoses. Oncogenic features of MXD3 were concomitantly associated with deregulation of 6SY Wu, and KC Lin helped with data collection; BL wrote the manuscript; ATH Wu and CZ Wu designed and oversaw the study.7ATH Wu was supported by research grants by Taipei Medical University and the Ministry of Education, Taiwan (DP2-110–21121-03-C-09 and DP2-110–21121-01-H-03–03). ATH Wu and SY Wu were funded by the Lotung Poh-Ai Hospital research grant (grant no. 11002). KC Lin and CZ Wu were funded by Wan Fang Hospital, Taipei Medical University research grant (110-phd-04).8Not applicable.9Not applicable.10The raw data supporting the conclusions of this article are available upon reasonable request.Not applicable.Szu-Yuan Wu: Conceptualization, Data curation, Resources, Software, Writing - review & editing. Kuan-Chou Lin: Conceptualization, Data curation, Software, Writing – original draft. Bashir Lawal: Conceptualization, Data curation, Software, Writing – original draft, Writing - review & editing. Alexander T.H Wu: Conceptualization, Funding acquisition, Resources, Supervision, Visualization, Writing – original draft, Writing - review & editing. Ching-Zong Wu: Conceptualization, Funding acquisition, Software, Supervision, Visualization, Writing – original draft.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.


In vitro culture media are being developed to understand how host site-specific nutrient profiles influence microbial pathogenicity and ecology. To mimic the cystic fibrosis (CF) lung environment, a variety of artificial sputum media (ASM) have been created. However, the composition of these ASM vary in the concentration of key nutrients, including amino acids, lipids, DNA, and mucin. In this work, we used feature-based molecular networking (FBMN) to perform comparative metabolomics of Pseudomonas aeruginosa, the predominant opportunistic pathogen infecting the lungs of people with CF, cultured in nine different ASM. We found that the concentration of aromatic amino acids and iron from mucin added to the media contributes to differences in the production of P. aeruginosa virulence-associated secondary metabolites.IMPORTANCE Different media formulations aiming to replicate in vivo infection environments contain different nutrients, which affects interpretation of experimental results. Inclusion of undefined components, such as commercial porcine gastric mucin (PGM), in an otherwise chemically defined medium can alter the nutrient content of the medium in unexpected ways and influence experimental outcomes. Pseudomonas aeruginosa, the primary bacterial opportunistic pathogen infecting the lungs of people with CF, contributes to lung function decline by growing at high density within biofilms that are recalcitrant to the innate immune response and antibiotic therapy  have been created that aim to replicate the nutrient availability of the CF lung (9–Cystic fibrosis (CF) is a genetic disease characterized by the accumulation of thick and viscous bronchial mucus, which provides a nutrient-rich environment for the growth of opportunistic pathogens . Pseudom therapy –4. Whiletherapy ––9. To stF lung 9–. The varin vivo. Soothill ASM was modified by the addition of 250 mg/liter of each of the 20 canonical amino acids to generate Romling ASM, as amino acids are present in CF sputum  –17. Thes–F sputum . The ASMF sputum . To reflF sputum . DerivedF sputum . CordwelF sputum .N-acetylglucosamine (GlcNAc), and dioleoylphosphatidylcholine (DOPC) at concentrations similar to those measured from CF sputum to create SCFM2 , purines, folate, branched-chain amino acids, and tryptophan. These results suggested that these metabolites are more available in CF sputum than SCFM2. Therefore, SCFM2 was further modified by the addition of p-aminobenzoic acid, NAD+, adenine, guanine, xanthine, and hypoxanthine to generate SCFM3 . Importantly, due to their roles in virulence-associated phenotypes, altered secondary metabolite production by P. aeruginosa due to compositional differences likely influences the outcome of microbe-microbe interactions, virulence assays, and comparative assessment of experiments performed in different ASM. Additionally, the influence of crude complex nutrients, like mucin, on experimental outcomes may not be due to the nutrient itself but, rather, other constituents of the mixture.Herein, we describe the application of comparative metabolomics to lations. –17. PAO1ations. –, 33. We P. aeruginosa phenotype, resulting in a tan-green macrostructure at the air-liquid interface, with large open pores within the structure giving way to turbid growth toward the bottom of the well. Surprisingly, the composition of Cordwell ASM induced a phenotype similar to those observed in Romling and Winstanley ASM; PAO1 grew as a dense brown biofilm without clear structural features. Biofilms in SDSU ASM grew as a light-green structure with tendrils near the center of the well that reached the surface of the medium. For SCFM-series ASM, growth in SCFM1 was turbid and bright green, which was noticeably distinct from biofilms in SCFM2 and SCMF3, which were comparable in structure with film-like growth at the air-liquid interface, aggregates on the bottom of the well, and a marked blue color.To assess the effect of ASM composition on growth, strain PAO1 was cultured in ASM . Samplesin vitro studies  was measured at ∼1010 CFU/ml. As biofilm disruption was affected by culture viscosity and aggregation, it is impossible to distinguish whether the difference in growth between batches is due to ASM composition or processing of samples.PAO1 growth in ASM was enumerated as CFU per milliliter . While gP. aeruginosa secondary metabolites have authentic commercial standards, most must be identified using other methods, such as spectral matching to metabolite databases. Therefore, to quantify and identify the secondary metabolites produced by PAO1 in ASM, we performed feature-based molecular networking (FBMN) on untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics data . Of those features, 80 (16%) had spectral matches to the GNPS libraries with 30 (6%) to phenazine, quinolone, rhamnolipid, and pyochelin reference spectra. Of the five phenazines produced by PAO1, 1-hydroxyphenazine (1-HP), pyocyanin (PYO), phenazine-1-carboxamide (PCN), and phenazine-1-carboxylic acid (PCA) were quantified  37). Fr. Fr37). Pseudomonas quinolone signal; PQS), and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) , 2-heptyl-3-hydroxy-4-quinolone (e (HQNO)  38, 39), 39Pseude (HQNO) . Twenty-P. aeruginosa rhamnolipids consist of 1 to 2 rhamnose units bound to 3‐ alkanoic acids (HAAs), yielding mono- and di-rhamnolipids, respectively , the nonribosomal peptide cytoplasmic precursor to pyoverdines (Table S4)  . Althougis study .To determine which molecular features differentiated the metabolome of PAO1 in different ASM, the feature table was analyzed using principal-component analysis. From the initial results , the clustering of samples in the scores plot was due to batch effects, primarily driven by the abundance of benzethonium in batch 1 samples. Benzethonium is an ingredient of bactericidal surface disinfectants. As this compound was introduced during sample processing, it was removed from the data set, and the data were reanalyzed.A principal-component model was created with five principal components. Extracts of replicate cultures grown in the same ASM clustered together within the two-dimensional space created by principal component 1 (PC1) and principal component 2 (PC2) of the scores plot, which explained 48.6% and 21.5% of the variation between samples, respectively . Four sam/z 336.1201; C18H19NO4 [M+Na]). PYO contributed positively to the separation of both PC1 and PC2, suggesting that its abundance influenced the clustering of SCFM2 and SCFM3 samples from the other ASM  on the feature table. However, the number of features that had statistically different levels between ASM cultures were too numerous to investigate further (Data Set S2). Alternatively, we quantified the total levels of the phenazines, quinolones, rhamnolipids, and siderophores .Total phenazine levels were lowest in Soothill ASM samples . Proport9-HQ), and db:NHQ (C9:1-HQ) as the most abundant forms . Soothill and the SCFM-series ASM cultures had similarly low levels of total quinolones , with HHQ, NHQ . The other HHAs incorporated include C10-C12:1 (16% [Soothill ASM] to 22% [Romling ASM]) and C10-C12 (10% [ASMDM ASM] to 20% [SDSU ASM]).Total rhamnolipid levels were similar in all ASM cultures, except for SCFM2 and SCFM3 . PAO1 inIn the FBMN, the pie chart representation of relative metabolite abundance indicated that pyochelin and pyoverdine levels were highest in SCFM1 cultures. Quantification of total siderophore levels across all ASM samples confirmed this observation ; Fig. S7P. aeruginosa secondary metabolites were identified and quantified via FBMN (Table S6). The addition of PGM to SCFM1 was sufficient to alter the total levels of the phenazine, rhamnolipid, and siderophore secondary metabolite families . Gross morphology of the biofilms was visualized using a stereomicroscope, and growth was enumerated as CFU per milliliter. Addition of PGM increased the opacity of the cultures and led to a log fold increase of PAO1 growth in PGM-SCFM1 compared to SCFM1 . families . PAO1 prfamilies . While tA mucin suspension was subjected to untargeted LC-MS/MS metabolomics analysis and inductively coupled plasma MS (ICP-MS) to quantify metals. Molecular networking analysis of PGM lipophilic extracts revealed the presence of amino acids, small peptides, and lipids (Data Set S5). ICP-MS analysis revealed that, in addition to other metals (Table S6), a 0.5% (wt/vol) solution of commercial PGM contains approximately 30 μM iron . Additioin vitro, many ASM have been created with various levels of important nutrients, such as amino acids, mucin, DNA, and iron.  activate the quinolone receptor PqsR, driving autoinduction of their biosynthesis and subsequent production of virulence factors associated with the quinolone quorum-sensing pathways  system  system . The simP. aeruginosa produces two siderophores to acquire environmental iron: high-affinity pyoverdine and low-affinity pyochelin. Their production levels are regulated by the ferric uptake regulatory (Fur) system, which senses the amount of bioavailable environmental iron , at least in part, drove changes in levels of PCA, rhamnolipids, and siderophores between SCFM1 and SCFM2 cultures , 76, 77.ruginosa , 54, 78.in vitro , protein-bound iron sources , and bioactive lipids. Additionally, comprehensive characterization of CF samples from patients at different disease stages would support the development of SCFM formulations that emulate the distinct nutrient profiles associated with disease progression and treatment.The Web of Science Core Collection  was searched for papers with the “TOPIC” terms “sputum medium,” “sputum media,” “cystic fibrosis medium,” “cystic fibrosis media,” “sputa medium,” “sputa media,” “CF medium,” or “CF medium” in the title abstract or keyword fields on 10 May 2020. All terms were searched as written, including quotation marks. The results of this search were analyzed using the Web of Science tools “Create Citation Report” and “Analyze Results.” The Analyze Results tool was used to download data corresponding to Web of Science Categories. Google Scholar (Alphabet) was used to quantify the number of citations for the 9 most commonly used ASM formulations .Miller Luria broth  was prepared according to the manufacturer’s instructions. ASM formulations were prepared using the media components, concentrations, and commercial sources in Data Set S1 at the reported pH. ASM were prepared as reported, with the following modifications: no antibiotics were included, 0.1% (wt/vol) agar was excluded from the Cordwell formulation, and porcine gastric mucin type III  was used in all ASM where mucin was a component –20. Prio–P. aeruginosa strain PAO1  was inoculated from a streak plate into 5 ml LB and incubated overnight at 37°C with shaking at 220 rpm  in polystyrene 24-well plates. Each medium was evaluated in its own 24-well plate (12 control wells and 12 growth wells per medium). The media were inoculated, disrupted, and extracted in three batches, comprised of 2 to 4 ASM formulations and a parallel LB plate as a control. Batch 1 consisted of LB, Soothill, SDSU, and Cordwell ASM, batch 2 consisted of LB, Romling, Winstanley, ASMDM, and SCFM1 ASM, and batch 3 consisted of LB, SCFM2, and SCFM3 ASM. All plates were covered and incubated statically at 37°C for 72 h. After incubation, gross phenotypes of P. aeruginosa cultures  was added to each well, and aggregates were mechanically disrupted by pipetting using wide-bore pipette tips (USA Scientific), followed by water bath sonication (Branson) for 10 min and a secondary mechanical disruption by pipetting using wide-bore pipette tips. Disrupted samples were aliquoted for measurements of growth (CFU per milliliter and pH , and P. aeruginosa [PA] standard mix data), MSV000086723 , and MSV000087153 (PGM lipophilic extracts).All mass spectrometry data, including the raw files, mzXML files, metadata tables, MZmine settings, and Cytoscape files are available via MassIVE as follows: MSV000086721 (LB and ASM formulations media control,


Neurofibromatosis type 1 (NF1) is an autosomal dominant disease diagnosed with the presentation of café-au-lait macules, skinfold freckling, iris Lisch nodules, neurofibromas, osseous lesion, and optic gliomas. Mediastinal mass as the first presentation of NF1 is very rare, with a frequency of about 2.7%. Here, we present a rare case of NF1 in a 3-year-old boy admitted with respiratory distress and superior vena cava syndrome. Neurofibromatosis type 1 (NF1), or Von Recklinghausen disease, is an autosomal dominant disorder caused by mutation in the NF1 gene, located at chromosome 17 .The cardinal signs of this disease are café-au-lait spots, neurofibromas of any type, freckling in the axillae or groin, optic glioma, Lisch nodules, and dysplasia of the sphenoid or long bones. Also, familial history of NF1 in first-degree relatives is another central marker for diagnosis .Orthopedic problems, including scoliosis, osteopenia, and osteoporosis, hypertension, precocious puberty, learning disabilities, such as attention deficit hyperactivity disorder (ADHD), and seldom seizure are the rare presentations or complications of this disease . In lessA 3-year-old boy was admitted with severe respiratory distress and tachypnea after upper respiratory tract infection. He had dyspnea, cough, orthopnea, respiratory rate about 43 /min, and mild plethora of the face.He was the only child of family, and his parents were not relatives. He was born through normal vaginal delivery, and his Apgar score was 10 at birth and 5 minutes after birth. He had no developmental delay, but his weight and height were under 3 percentiles of growth, which was also evident on physical examination. We did not find significant data in his past medical history.On physical examination, we found a 5 x 4 cm mass on the left supraclavicular area and neck. The mass was firm and non-tender. Blood pressure was normal. Skin examination revealed multiple café-au-lait spots .Laboratory data showed hemoglobin (Hb) 10.9 g/dL, WBC 13300 /µL, neutrophil 29%, eosinophil 7%, monocyte 8%, lymphocyte 56%, platelet count 262000/µL, urea 21mg/dL, creatinine 0.6 mg/dL, uric acid 3.9 mg/dL, and lactate dehydrogenase (LDH) 754 U/L. Beta-HCG was 0.66 m IU/ml and alpha fetoprotein was 1.01 IU/ml.Chest radiograph was obtained, which showed a large mediastinal mass , and cheCorticosteroids were started because of respiratory distress and superior vena cava syndrome. After three days, the patient became stable, and incisional biopsy was taken from the neck mass, indicating proliferation of spindle cells within wire-like collagen fibrils in loose background, in favor of neurofibromatosis .Neurofibromatosis type 1 (NF1) is an autosomal dominant disease and about one-half of cases are familial . The disIntrathoracic neurofibromatosis mostly arises from ganglia or nerves of sympathetic trunk and rarely from the trachea or esophagus , 9. MediSymptoms and signs of mediastinal neurofibromatosis vary by location, tumor size, and tumor extension to other organs. Superior vena cava syndrome may result due to the impairment of venous return. Dyspnea is a presentation of airway compression or may results from phrenic nerve involvements and hemidiaphragm paralysis .The first imaging modality, chest radiography, shows a homogenous or heterogenous mass, and computed tomography scan technique may detect more details. Magnetic resonance imaging (MRI) in neurofibromatosis type 1 shows areas of T2 hyperintensity, and it seems to be more useful for the diagnosis of NF1-related malignant tumors . A sensiElongated spindle-shaped cells and pleomorphic fibroblast-like cells are the main histologic findings in NF1 . MoleculFinally, other differential diagnoses must be considered in any patient with signs and symptoms similar to NF1, like NF1-like syndrome, familial café-au lait spots (FCALS), and segmental NF1 . Our patDespite the rarity of NF1 as a mediastinal mass, this diagnosis should be considered as a differential diagnosis, especially when one or more other signs and symptoms are present.