Activation of the Wnt pathway has been linked to colorectal cancer (CRC). Previous reports suggest that Wnt3a can activate p38. Besides, p38α feeds into the canonical Wnt/β-catenin pathway by inhibiting GSK3β through phosphorylation. Recently, we identified p38α as a new druggable member of β-catenin chromatin-associated kinase complexes in CRC.Min/+ mice). The role of p38α in β-catenin transcriptional activity was assessed by pharmacological inhibition with ralimetinib.The functional relationship between p38α and β-catenin was characterized in CRC cells, patient-derived CRC stem cells, patient-derived tumor intestinal organoids, and in vivo models (C57BL/6-APCWe used the GSK3β inhibitor TWS-119, which promotes the activation of Wnt signaling, to uncouple p38α nuclear/cytoplasmatic functions in the Wnt pathway. Upon GSK3β inhibition, nuclear p38α phosphorylates β-catenin at residues S111 and T112, allowing its binding to promoter regions of Wnt target genes and the activation of a transcriptional program implicated in cancer progression. If p38α is pharmacologically inhibited in addition to GSK3β, β-catenin is prevented from promoting target gene transcription, which is expected to impair carcinogenesis.p38α seems to play a dual role as a member of the β-catenin destruction complex and as a β-catenin chromatin-associated kinase in CRC. This finding may help elucidate mechanisms contributing to human colon tumor pathogenesis and devise new strategies for personalized CRC treatment.The online version contains supplementary material available at 10.1186/s13578-023-01175-4. APC) gene on chromosome 5q (90%) or by CTNNB1 activating mutations on chromosome 3p (5%). One of the hyperproliferating cells may then give rise to a small adenoma by clonal expansion. Subsequently, the occurrence of mutations in the RAS pathway  is the main malignant tumor affecting the gastrointestinal tract and the third most common cancer in both men and women , 2. CRC se death .o and Gαq as well as the downstream phosphoprotein Dishevelled, thereby inhibiting GSK3β activity. Stabilized β-catenin then translocates into the nucleus and forms complexes with members of the T cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors, exerting its oncogenic role by activating the transcription of many regulatory genes, such as c-Myc [Activation of the Wnt pathway has been linked to CRC; indeed, abnormalities of chromosome 5q have been recognized as early events in the carcinogenic process leading to sporadic and hereditary CRCs . In the as c-Myc .Recent advances suggest the existence of a functional relationship between the Wnt and p38 MAPK signaling pathways.Currently, p38 is believed to act as a tumor suppressor in normal cells at the onset of cellular transformation, while it seems to function as an oncogene after the acquisition of a malignant phenotype , 12. OurVarious studies have suggested that p38α plays a role in chemoresistance by supporting survival or delaying ongoing cell death processes –18. Indep38α is relatively inactive in its non-phosphorylated form and becomes rapidly activated upon phosphorylation of two Thr-Gly-Tyr motifs , 20. Phoq and Dishevelled, which ultimately promotes transient activation of p38 [Recent findings identified crosstalk between the p38 and Wnt pathways. Indeed, Wnt ligands have been reported to be able to activate p38. In totipotent mouse F9 teratocarcinoma cells, Wnt3a binds to Frizzled and activates Gαn of p38 . Moreoven of p38 . On the n of p38 . Recentln of p38 .Despite this evidence, the exact role played by p38α in the Wnt signaling pathway remains unclear. Elucidating the underlying molecular mechanisms might be beneficial for designing new therapeutic strategies to counter chemoresistance and improve treatment response in CRC patients.HCT-116 and HT-29 cells were purchased from ATCC and cultured in DMEM  with 10% FBS  and 100 IU/mL penicillin–streptomycin .l-glutamine , 10 ng/mL bFGF , 20 ng/mL EGF , B27 supplement , and N-2 supplement . Patient-derived CRC organoids were generated using IntestiCult™-SF Organoid Growth Medium (Human)  according to the manufacturer’s instructions.CRC-stem cells (CRC-SCs) were isolated from CRC patients and propagated as tumorspheres. Briefly, human colon tissue fragments were obtained in accordance with the ethical standards of the Institutional Committee on Human Experimentation after informed consent. Tumor samples were subjected to mechanical and enzymatic dissociation using the human Tumor Dissociation Kit  according to the manufacturer’s instructions. For magnetic separation, cells were labeled with CD44 microbeads  24–48 h after dissociation using the Miltenyi Biotec cell isolation kit . CRC-SCs were maintained in DMEM/F12 Advanced  supplemented with 6 mg/mL glucose , 2 mM 2 incubator.All cell lines were tested to be mycoplasma-free . All cell cultures were performed in a 37 °C and 5% COHCT-116 cells were transfected with 50 nM validated siRNAs (Ambion) directed against (p38α) MAPK14 using HiPerfect reagent  according to the manufacturer’s instructions.Min/+ mice (n = 24) treated with azoxymethane (AOM) , respectively. Four-month-old APCMin/+ male mice were administered with AOM (14 mg/kg body weight) once a week for 5 weeks; one month later, they were subjected to daily intraperitoneal injections of the p38α inhibitor SB202190  (0.05 μmol/kg body weight) or DMSO for 14 days and then sacrificed. Body weight was recorded daily. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that comply with national and international laws and policies.For in vivo studies, normal and adenocarcinoma colon mucosa tissues were obtained from C57BL/6 mice (n = 12) and from APC2O. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 min. Antigen retrieval was conducted in 10 mM sodium citrate buffer (pH 6.0) for 30 min. Sections were incubated overnight with anti-p38α  or anti-β-catenin  primary antibodies. Then, they were incubated with secondary biotinylated antibodies and subsequently with streptavidin–biotin-peroxidase . Samples were developed with DAB and mounted with permanent mounting media. Negative controls were used in each experiment. p38α and β-catenin immunoreactivity was evaluated through a semiquantitative approach by two independent pathologists. Ten fields with an equal area were selected for analysis at 40 × magnification. Protein expression was assessed with ImageJ software and reported as a positivity percentage.Tissue specimens were fixed in 4% buffered formalin, embedded in paraffin, and sectioned at 4 mm thickness. Sections were dewaxed and rehydrated in dHTWS-119 (S1590) and ralimetinib (S1494) were purchased from SelleckChem. Wnt3a (H17001) and SB202190 (S7067) were purchased from Sigma-Aldrich.Nuclear and cytoplasmic fractions were obtained by using the Nuclear Extraction Kit  according to the manufacturer’s instructions. Immunoblots were carried out as previously described [Primary antibodies: anti-p38α MAPK (9228), anti-Lamin B1 (12586S), anti-PDI (2446S), anti-β-catenin (9562S), anti-c-Myc (9402), anti-p-β-catenin (9561), anti-p-GSK3β (35480), anti-GSK3β (9315), and anti-Vinculin (13901) all from Cell Signaling Technologies. Secondary antibodies: Rabbit IgG HRP and Mouse IgG HRP . Densitometric evaluation was performed using ImageJ software.Cells were seeded on glass coverslips, fixed in 4% paraformaldehyde, and permeabilized using 0.01–0.1% Triton X-100. Coverslips were incubated with the indicated primary antibodies and then with Alexa Fluor 488  and 647  secondary antibodies; nuclei were counterstained using DAPI . Slides were sealed using Vectashield Mounting Medium . Images were acquired using a Zeiss fluorescence microscope. Primary antibodies: p38α , β-catenin , c-Myc , and FLAG .Total RNA was isolated using TRIzol reagent  according to the manufacturer’s instructions. To avoid possible DNA contamination, RNA was treated with DNase-1 . RNA purity was confirmed by spectrophotometry and RNA integrity by agarose gel electrophoresis. cDNA was synthesized by retro-transcribing 1 μg of total RNA using the iScript cDNA Synthesis Kit  according to the manufacturer’s instructions. Real-time PCR primers were designed using Primer Express software. PCR assays were performed in 96-well optical reaction plates using a QuantStudio 3 machine (Thermo Fisher Scientific). Each assay was carried out in triplicate wells. Baseline values of amplification plots were set automatically and threshold values were kept constant to obtain normalized cycle times and linear regression data. The following reaction mixture per well was used: 5 μL of SYBR Green PCR Master Mix , 1.2 μL of primers at a final concentration of 100 nM, 2.3 μL of RNAse-free water, 1.5 μL of cDNA. For all experiments, the following PCR conditions were used: denaturation at 95 °C for 30 s, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C. Quantitative normalization of cDNA in each sample was performed using β-actin as an internal control. Relative quantification was done using the ddCT method. Primer sequences are available upon request. Results are representative of at least three independent experiments.SDM_ beta-catenin _S111A_FW 5'-P-GCAGATCCCAGCTACACAGTT,SDM_ beta-catenin _S111A_RV 5'-P-ATGCCCTCATCTAATGTCTCA,SDM_ beta-catenin _T112A_FW 5'-P-GATCCCATCTGCACAGTTTGATG.SDM_ beta-catenin _T112A_ RV 5'-P- TGCATGCCCTCATCTAATG.The plasmids described in this manuscript were generated with specific primers, as previously described . Site-diThe pGEX-4T-3 GST-beta-catenin Wild-Type, pGEX-4T-3 GST-beta-catenin S111A, and pGEX-4T-3 GST-beta-catenin T112A constructs were generated by cloning the relevant β-catenin fragments from human beta-catenin pcDNA3, human beta-catenin pcDNA3-S111A, and human beta-catenin pcDNA3-T112A, respectively, into the pGEX-4T-3 GST Expression Vector , using the BamHI and NotI enzymes.g for 20 min at 4 °C. Recombinant protein expression was confirmed by SDS-PAGE. GST-Fusion proteins were purified using Pierce Glutathione Magnetic Agarose Beads  according to the manufacturer’s instructions. GST-fused proteins were evaluated and quantified by SDS-PAGE.BL21(DE3) Competent Cells  transformed with different constructs were grown in Luria Broth medium with ampicillin  and induced with 200 nM IPTG when they reached the optical density of 0.6 (A600) at 37 °C, overnight. Cells were then collected by centrifugation, and pellets were lysed with B-PER lysis buffer . The lysate was centrifuged at 20,000×Cells were collected and homogenized in lysis buffer  supplemented with protease and phosphatase inhibitors. Coupling was performed between Dynabeads Protein A or G  and antibodies in 100 μL of 0.01% Tween20-1X PBS for 45 min at room temperature on a rocking platform. Cell lysates were immunoprecipitated with antibody-bead complexes. Immunocomplexes were washed extensively and boiled in Laemli sample buffer and subjected to SDS-PAGE and immunoblot analysis. Input corresponds to 10% of the lysate. IgGs were used as negative controls. Primary antibodies: p38α , β-catenin , GSK3β , APC , Axin1 , FLAG-M2 . Rabbit IgG HRP  was used as a secondary antibody and revealed using the ECL-plus chemiluminescence reagent . Results are representative of at least three independent experiments.2 resin for phospho-enrichment. Then, the flow-through was treated with C18 Spin Tips & Columns  for desalting. The samples enriched for phospho-peptides and the desalted flow-through were further purified with SP3 and then analyzed by nLC-ESI–MS/MS on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) with a 32 min gradient. Samples were run in technical duplicate, in a positive mode with electrospray ionization. Data acquisition and processing were performed with Analyst TF . Data were analyzed using the Proteome Discoverer, Mascot, and Scaffold setting software. The parameter settings of data processing were as follows: DataBase = Uniprot_CP_Human_2020_GST_CTNB1 ; Enzyme = Trypsin ; Modifications = Acetyl (Protein N-term), Carbamidomethyl (C), Oxidation (M), Phosphorylation (STY); Peptide Thresholds: 95.0% minimum; Protein Thresholds: 99.0% minimum; 2 peptides/protein minimum.Mass spectrometry analysis was performed by the Cogentech SRL service. Gel bands were subjected to reduction with dithiothreitol (DTT) , alkylation with iodoacetamide (IAA) , and digestion with trypsin . Peptides were loaded onto a TiOChromatin isolated from HT-29 and HCT-116 cells was subjected to chromatin immunoprecipitation using the MAGnify Chromatin Immunoprecipitation System  according to the manufacturer’s instructions. Briefly, cells were cross-linked in 1% formaldehyde  for 10 min. Cross-linking was blocked with 0.125 M glycine  for 5 min; then, cells were washed with 1 × PBS and lysed in lysis buffer. Chromatin was sonicated to a fragment length of about 200–500 bp and immunoprecipitated with 1 µg of anti-p38α , anti-β-catenin , rabbit IgGs, FLAG-M2 , or mouse IgGs. Quantitative real-time PCR was performed using SYBR Green IQ reagent  with the CFX Connect detection system (Bio-Rad Laboratories). Primer sequences are available upon request. Results are representative of at least three independent experiments.The analysis of p38α kinase activity was performed using the ADP-Glo Kinase Assay  according to the manufacturer’s instructions. 10 ng of p38α active protein  were assayed in a kinase reaction buffer with 500 ng of human recombinant β-catenin protein , 150 μM ATP, and varying concentrations  of ralimetinib . A total of 500 ng of p38 peptide substrate was used as a control. The generated luminescence was measured using a SPECTROstar Omega microplate reader (BMG Labtech).ddPCR was performed using 1 ng of RNA extracted from the HCT-116 CRC cell line or 20 ng of RNA extracted from patient-derived CRC-SC tumorspheres using the Purelink RNA Micro Kit  and retro-transcribed to cDNA using the SuperScript VILO cDNA Synthesis Kit  according to the manufacturer’s instructions. Reactions were prepared using the ddPCR Supermix for Probes Kit  according to the manufacturer’s instructions. 20 µL of each reaction mix were converted into droplets with the QX200 Droplet Generator (Bio-Rad Laboratories). The droplets were transferred to a 96-well plate, sealed, and cycled in a C100 Thermocycler (Bio-Rad Laboratories) under the following cycling protocol: 25 °C for 3 min, 95 °C for 10 min followed by 40 cycles of 94 °C for 30 s and 60 °C for 1 min, a post-cycling step of 98 °C for 10 min, and infinite hold at 4 °C. The plate was then transferred into a QX200 Reader (Bio-Rad Laboratories).For patient-derived CRC organoids, 10 ng of RNA were extracted using the Purelink RNA Micro Kit  according to the manufacturer’s instructions. The ddPCR assay was prepared using the One-Step RT-ddPCR Advanced Kit for Probes  according to the manufacturer’s instructions. 20 µL of each reaction mix were converted into droplets with the QX200 Droplet Generator (Bio-Rad Laboratories). The droplets were transferred to a 96-well plate, sealed, and cycled in a C100 Thermocycler (Bio-Rad Laboratories) under the following cycling protocol: 25 °C for 3 min, reverse transcription at 50 °C for 60 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s and 55 °C for 1 min, a post-cycling step of 98 °C for 10 min, and infinite hold at 4 °C. The plate was then transferred into a QX200 Reader (Bio-Rad Laboratories).c-Myc: ddPCR Gene Expression Assay, MYC, Human, Homo sapiens .β-catenin: ddPCR Gene Expression Assay, β-catenin, Human, Homo sapiens .The data were analyzed using Bio-Rad QX Manager 1.2 Standard Edition. Probes were as follows:5 HT-29 cells were co-transfected with 1 µg of p38 and 1 µg of β-catenin plasmids, using the Neon transfection system  according to the manufacturer’s instructions, and plated onto 24-wells. After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector  and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid  or the FOPFlash negative control  using Lipofectamine 3000  and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM). After 24 h in culture, cells were harvested and both firefly and Renilla luciferase activity was measured in triplicate according to the manufacturer’s instructions. Firefly luciferase activity was normalized against Renilla luciferase activity and fold increase in TOPFlash-compared to FOPFlash-transfected cells was determined.To assess the transcriptional activity of β-catenin in HT-29 cells, we took advantage of the TOP/FOP reporter system using a dual-luciferase kit . On the day of the seeding, 6 × 10Activation of p38α and nuclear β-catenin is associated with many cancers, and several genes that are regulated by these signaling pathways are crucial for cancer development and progression. Recently, we demonstrated that p38α acts as a β-catenin chromatin-associated kinase involved in tumor proliferation, metastatic dissemination, and chemoresistance . In ordeIn order to confirm the results of Thornton and colleagues  in our CMin/+ mice. This murine model, identified in 1990 in an ethylnitrosourea mutagenesis screen in C57BL/6 mice, represents a very useful tool to study CRC. The genetic basis for the intestinal phenotype is a T-to-A transversion at nucleotide 2549 of the mouse APC gene resulting in the truncation of the APC protein at amino acid 850. For this reason, the Wnt/β-catenin pathway is overactive in these mice, which develop multiple intestinal neoplasia (Min) after spontaneously losing the heterozygous wild-type APC allele in intestinal epithelial cells [Min/+ mice were administered with a potent carcinogen, AOM (14 mg/kg body weight), once a week for 5 weeks to induce epithelial cell transformation from aberrant crypt foci to adenoma and malignant adenocarcinoma. One month later, animals were subjected to daily intraperitoneal injections of the p38α inhibitor SB202190 (0.05 μmol/kg body weight) or dimethyl sulfoxide (DMSO) for 14 days and then sacrificed , followed by inhibition of p38α with ralimetinib, in HT-29 CRC cell lines. HT-29 cells were serum-starved to retain β-catenin in the cytoplasm and then switched to a serum-supplemented medium or a medium containing Wnt3a and TWS-119. Immunoblot analysis showed that β-catenin and p38α expression was barely detectable in the nucleus under serum starvation, while it increased substantially after serum supplementation  , the addThese data were further confirmed by immunofluorescence staining Fig. : Fig. S3TCF7 and LEF1 themselves  are required for β-catenin and p38α physical interaction. Our results showed that this interaction was not affected in HCT-116 cells overexpressing FLAG-β-catenin-S111A or FLAG-β-catenin-T112A as compared to FLAG-β-catenin-WT-transfected cells Fig. A. Subseqion Fig. C. Moreovc-Myc expression in patients-derived CRC-SCs and patient-derived tumor intestinal organoids  decreased significantly upon treatment with ralimetinib  and elicit transcriptional activation. Like other transcription factors, β-catenin functions are tightly regulated by protein phosphorylation and dephosphorylation [Emerging evidence indicates the existence of a functional relationship between p38 MAPK and the Wnt signaling pathway. Specifically, Thornton and colleagues investigated the cytoplasmic activity of p38α, revealing its ability to inhibit GSK3β by phosphorylation . Moreovecyclin D1 and c-Myc genes [c-Myc was observed in patient-derived CRC-SCs and tumor intestinal organoids upon treatment with ralimetinib.T112 is a key phosphorylation site in β-catenin N-terminus and is specifically targeted by 3-phosphoinositide-dependent protein kinase 1 (PRKD1) and casein kinase II (CKII) , 40. CKIyc genes . In addiyc genes . In CRC This study uncouples p38α cytoplasmic and nuclear functions in the Wnt signaling pathway in CRC cells. Our results identified two β-catenin residues that are phosphorylated by p38α and seem to play an important role in β-catenin transcriptional activation. Considering the role of β-catenin activation in CRC tumorigenesis, our data suggest that p38α pharmacological inhibition is a promising approach to develop new therapeutic strategies for colorectal carcinogenesis.The translational relevance of these findings is further strengthened by the fact that ralimetinib proved effective in CRC model systems such as patient-derived CRC-SCs and tumor intestinal organoids. Of note, ralimetinib displayed a tolerable safety profile, with evidence of antineoplastic activity, in a recent phase I clinical trial in patients with advanced or metastatic cancer (ID NCT01393990). Moreover, it has already been tested in combination with other agents in a phase I study in CRC patients (ID NCT02860780).Taken together, the data presented in this paper may help design prospective clinical trials of tailored interventions for CRC based on p38α inhibition.Additional file 1: Figure S1. p38α pharmacological inhibition or genetic ablation modulates p-GSK3-β levels. (A-B) Immunoblotting analysis of p-β-catenin (S33/37/T41)  and p-GSK3β (T390)  in HCT-116 CRC cells treated or not with the p38α inhibitor ralimetinib (10 μM) for 48 h (A) or silenced by genetic ablation for p38α (B). CTRL = Control. Results are representative of at least three independent experiments. Figure S2. Immunohistochemistry quantification of p38α and β-catenin protein expression in a CRC mouse model. Immunohistochemistry quantification of p38α and β-catenin protein expression in colon tissue from C57BL/6 and AOM-APCMin/+ mice treated as indicated in Fig. #P < 0.05 vs. DMSO. Figure S3. Quantification of p38α and β-catenin immunofluorescence data. Quantification of the immunofluorescence analysis of p38α and β-catenin co-staining experiments shown in Fig. #P < 0.05 vs. DMSO. Figure S4. Analysis of β-catenin genetic ablation efficiency. (A) Quantification results of the digital droplet PCR (ddPCR) assay (copies/µL) of c-Myc mRNA expression as processed by QuantaSoft. HCT-116 CRC cells were silenced by genetic ablation for β-catenin and then treated or not with the p38α inhibitor ralimetinib (10 μM) for 24 h. The error bars represent the maximum and minimum Poisson distribution for the 95% confidence interval generated by QuantaSoft. (B) Immunoblotting analysis of c-Myc protein amount in HCT-116 CRC cells treated as in A. β-actin was used as a loading control. Results are representative of at least three independent experiments. S33/37/T1 (phosph


N-isopropyltryptamine, is presented, as well as its crystal structure and the structures of its three synthetic precursors.The synthesis of the norpsilocin derivative, 4-hydroxy- N-isopropyltryptamine (4) or 4-HO-NiPT (systematic name: 3-{2-[(propan-2-yl)amino]ethyl}-1H-indol-4-ol), C13H18N2O, was synthesized in three steps from 4-benzyloxyindole (1) (systematic name: 4-phenoxy-1H-indole), C15H13NO. (1) was treated with oxalyl chloride and isopropylamine to produce N-isopropyl-4-benzyloxy-3-indoleglyoxylamide (2) {systematic name: 2-[4-(benzyloxy)-1H-indol-3-yl]-2-oxo-N-(propan-2-yl)acetamide}, C20H20N2O3. (2) was reduced to generate 4-benzyloxy-N-isopropyltryptamine (3) or 4-HO-NiPT, which was characterized as its chloride salt 4-benzyloxy-N-isopropyltryptammonium chloride (3a) (systematic name: {2-[4-(benzyloxy)-1H-indol-3-yl]ethyl}(propan-2-yl)azanium chloride), C20H25N2O·Cl. Finally the benzyl group of (3) was removed via hydrogenation to generate 4-HO-NiPT. The crystal structures of the title compound and all three synthetic precursors are presented.The title compound, 4-hydroxy- Psilocin is an agonist of the serotonin 2A (5-HT2A) receptor; this activity is believed to be responsible for producing a head-twitch response (HTR) in murine models, as well as subjective ‘psychedelic’ effects in human subjects as well as other potentially beneficial biological and clinical results , the monomethyl derivative of psilocybin, and aerugeniscin , its trimethyl variant. Like psilocybin, both baeocystin and aeruginascin are 4-phosphoryloxytryptamines, which are hydrolized to their analogous 4-hydroxytryptamines: norpsilocin (C11H14N2O) and 4-hydroxy-N,N,N-trimethyltryptamine respectively.Psilocybin and psilocin are both natural products, found in over 200 species of ‘magic mushrooms’  has been examined and shown to be a full agonist of the 5-HT2A receptor  the pharmacology of analogous monoalkyl tryptamines  is relatively unknown. Accordingly, the importance of these compounds within the context of the overall polypharmacological ‘magic mushroom’ experience is not understood. In an effort to explore the proprieties of monoalkyltryptamines, we previously reported the structural characterization, cellular, and behavioral data for baeocystin and norpsilocin , replaces the methyl group of norpsilocin with an isopropyl group. The only previous literature report of this molecule is as a metabolite of the new psychoactive substance (NPS) 4-acetoxy-N,N-diisoproptyltryptamine (4-AcO-DiPT) from 2022  contains a single molecule °. The benzene ring of the benzyloxy group is near perpendicular from the indole ring with a plane to plane twist of 89.73 (6)°.The asymmetric unit of 4-benzyloxyindole, Cle Fig. 1. The indN-2-propyl-α-oxo-4-(phenylmethoxy)-1H-indole-3-acetamide, C20H20N2O3 (2) contains two molecules of the indole-amide ° and a C26—O6—C34—C35 angle of −178.2 (4)°. The benzene rings of the benzyloxy groups are slightly twisted from the indole rings with plane-to-plane  twists of 19.98 (16) and 21.45 (16)°, respectively. The amide arms are slightly turned away from the indole rings with a C7—C8—C9—C10 angle of 157.3 (4)° and a C27—C28—C29—C30 angle of −160.8 (4)°. The amine groups are in anti conformations with C8—C9—C10—N2 = 165.2 (4)° and C28—C29—C30—N4 = 174.3 (4)°. The isopropyl groups are also in anti conformations with a C9—C10—N2—C11 angle of −178.1 (4)° and a C29—C30—N4—C31 angle of 178.5 (4)°. In the asymmetric unit, the indole–amide molecules are linked by an N—H⋯O hydrogen bond between the indole nitrogen and the carbonyl oxygen of the amide group.The asymmetric unit of de Fig. 1. The indN-isopropyltryptammonium chloride, C20H25N2O+·Cl− (3a) contains two tryptammonium cations (identified by atoms N1 and N3) and two chloride anions °; in the N3 cation, the benzyloxy group is turned nearly perpendicular to the indole ring with a C26—O2—C34—C35 angle of 85.8 (2)°. In both cations, the ethylamino arms are turned away from the indole rings, with a C7—C8—C9—C10 angle of 73.5 (2)° and a C27—C28—C29—C30 angle of 72.3 (2)°. The amine groups of the arm are both in anti conformations with C8—C9—C10—N2 = −153.55 (16)° and C28—C29—C30—N4 = −172.94 (14)°. On the contrary, the isopropyl groups are in syn conformations with a C9—C10—N2—C11 angle of −59.7 (2)° and a C29—C30—N4—C31 angle of −54.9 (2)°. In the asymmetric unit, the tryptammonium cations and chloride anions are linked by N—H⋯Cl hydrogen bonds arising from the indole nitrogen atoms.The asymmetric unit of 4-benzyloxy-ns Fig. 1. The indN-isopropyltryptamine C13H18N2O (4) contains a single tryptamine molecule  beyond normal van der Waals contacts. The molecules of (2) are linked by N—H⋯O hydrogen bonds, generating infinite chains along the [100] direction between indole N atoms and the O atoms of the amide carbonyl groups (Table 13a) are linked into infinite chains propagating along [010] by N—H⋯Cl hydrogen bonds between the indole nitrogen atoms and the chloride anions and the ammonium N atoms and the Cl− ions (Table 24) are held together in infinite chains along the [010] direction by N—H⋯O hydrogen bonds between the indole NH groupings and the hydroxide O atoms (Table 31)–(4) are shown in Fig. 2There are no significant intermolecular interactions in . There are three other monoalkyltryptamines reported in the literature, the natural product baeocystin  are indole-3-yl-N-isopropylglyoxalylamide : Single crystals of (1) were grown from the vapor diffusion of diethyl ether into a methylene chloride solution of a commercial sample (Biosynth).N-isopropyl-4-benzyloxy-3-indoleglyoxylamide (2): to a solution of (1)  in diethylether (50 ml) was added oxalylchloride  dropwise at 273 K. The resulting mixture was stirred for 6 h at 273 K, and 2-propylamine  was added dropwise. The mixture was warmed to room temperature and stirred overnight. Solvent was removed in vacuo, and the resulting residue was purified on a silica gel column (methylene chloride/methanol) to afford the product as a yellow oil . Single crystals of (2) suitable for X-ray diffraction studies were grown by vapor diffusion of diethyl ether into a methylene chloride solution.4-Benzyloxy-N-isopropyltryptamine (3): To a suspension of (2)  in tetrahydrofuran (12 ml) was added borane–tetrahydrofuran complex  dropwise at 273 K. The reaction was then heated at reflux overnight. The resulting yellow solution was cooled to room temperature and quenched with hydrochloric acid (2.0 M), then heated at 273 K for 2 h. The mixture was cooled to room temperature and ammonium hydroxide was added until the pH exceeded 8. The mixture was extracted with methylene chloride and the organic layer was washed with water and brine, dried over sodium sulfate, and solvent was removed in vacuo. The residue was purified by silica gel column chromatography (methylene chloride/ammonia-methanol solution) to afford (3) as a yellow solid . The compound was treated with hydrochloric acid (1.0 M in diethylether) and filtered to yield the chloride salt (3a). Single crystals of (3a) suitable for X-ray diffraction studies were grown from the vapour diffusion of diethylether into a methanol solution.1H NMR : δ 8.11 , 7.49 , 7.41 , 7.35 , 7.08 , 6.99 , 6.96 , 6.57 , 3.11 , 2.93 , 2.77–2.71 , 0.99 .4-Hydroxy-N-isopropyltryptamine (4): To a solution of (3)  in methanol (4.0 ml) was added palladium on carbon (30 mg) and palladium hydroxide on carbon (30 mg). The mixture was stirred for 3 h under an atmosphere of hydrogen. The resulting black suspension was filtered and washed with methanol. Solvent was removed in vacuo and the resulting residue was purified by silica gel chromatography (methylene chloride/ammonia methanol solution) to afford (4) as an off-white solid . Single crystals of (4) suitable for X-ray diffraction studies were grown from the slow evaporation of an acetone solution. 1H NMR : δ 7.88 , 7.05 , 6.90–6.79 , 6.59 , 3.02 , 2.96 , 2.91–2.83 , 1.12 . 13C NMR : δ 151.9 (ArC), 138.7 (ArC), 123.5 (ArC), 120.8 (ArC), 118.5 (ArC), 114.0 (ArC), 107.0 (ArC), 102.7 (ArC), 49.5 (AkC), 48.0 (AkC), 27.6 (AkC), 22.1 (AkC). MS (ESI) calculated for C13H16N2O: 218.1; found: 218.9 [M + 1].6.1), H1, H2, H3A and H4A in compound (2), H1, H2A, H2B, H3A, H4A and H4B in compound (3a) and H1, H1A and H2 in compound (4) were found from difference-Fourier maps and were refined isotropically. DFIX restraints were used on all of these hydrogen atoms  with N—H distances of 0.87 (1) Å for the indole N atoms, 0.90 (1) Å for the ethylamino N atoms, 0.95 (1) Å for ethylammonium N atoms, and 1.00 (1) Å for the O–H distance. Isotropic displacement parameters were set to 1.2 Ueq of the indole N atoms and 1.5 Ueq of the parent ethylamino N atoms and the parent oxygen atom. All other hydrogen atoms were placed in calculated positions (C—H = 0.93–0.98 Å). Isotropic displacement parameters were set to 1.2 Ueq (C) or 1.5 Ueq (C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989023002098/hb8057sup1.cifCrystal structure: contains datablock(s) 1, 2, 3a, 4, global. DOI: 10.1107/S2056989023002098/hb80571sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989023002098/hb80572sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989023002098/hb80573asup4.hklStructure factors: contains datablock(s) 3a. DOI: 10.1107/S2056989023002098/hb80574sup5.hklStructure factors: contains datablock(s) 4. DOI: Click here for additional data file.10.1107/S2056989023002098/hb80571sup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023002098/hb80572sup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023002098/hb80573sup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023002098/hb80574sup9.cmlSupporting information file. DOI: 2246617, 2246618, 2246619, 2246620CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Background: Carbonylated proteins (CPs) serve as specific indicators of increased reactive oxygen and nitrogen species (RONS) production in cancer cells, attributed to the dysregulated mitochondrial energy metabolism known as the Warburg effect. The aim of this study was to investigate the potential of alpha-ketoglutarate (aKG), 5-hydroxymethylfurfural (5-HMF), and their combination as mitochondrial-targeting antioxidants in MTC-SK or NCI-H23 cancer cells. Methods: MTC-SK and NCI-H23 cells were cultured in the absence or presence of varying concentrations (0–500 µg/mL) of aKG, 5-HMF, and the combined aKG + 5-HMF solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of MTC-SK and NCI-H23 cells. Results: The mitochondrial activity of MTC-SK cells exhibited a concentration- and time-dependent reduction upon treatment with aKG, 5-HMF, or the combined aKG + 5-HMF. The half-maximal inhibitory concentration (IC50%) for mitochondrial activity was achieved at 500 µg/mL aKG, 200 µg/mL 5-HMF, and 200 µg/mL aKG + 66.7 µg/mL 5-HMF after 72 h. In contrast, NCI-H23 cells showed a minimal reduction (10%) in mitochondrial activity even at the highest combined concentration of aKG + 5-HMF. The CP levels in MTC-SK cells were measured at 8.7 nmol/mg protein, while NCI-H23 cells exhibited CP levels of 1.4 nmol/mg protein. The combination of aKG + 5-HMF led to a decrease in CP levels specifically in MTC-SK cells. The correlation between mitochondrial activity and CP levels in the presence of different concentrations of combined aKG + 5-HMF in MTC-SK cells demonstrated a linear and concentration-dependent decline in CP levels and mitochondrial activity. Conversely, the effect was less pronounced in NCI-H23 cells. Cell growth of MTC-CK cells was reduced to 60% after 48 h and maintained at 50% after 72 h incubation when treated with 500 µg/mL aKG (IC50%). Addition of 500 µg/mL 5-HMF inhibited cell growth completely regardless of the incubation time. The IC50% for 5-HMF on MTC-CK cell growth was calculated at 375 µg/mL after 24 h incubation and 200 µg/mL 5-HMF after 72 h. MTC-SK cells treated with 500 µg/mL aKG + 167 µg/mL 5-HMF showed no cell growth. The calculated IC50% for the combined substances was 250 µg/mL aKG + 83.3 µg/mL 5-HMF (48 h incubation) and 200 µg/mL aKG + 66.7 µg/mL 5-HMF (72 h incubation). None of the tested concentrations of aKG, 5-HMF, or the combined solution had any effect on NCI-H23 cell growth at any incubation time. Caspase-3 activity increased to 21% in MTC-CK cells in the presence of 500 µg/mL aKG, while an increase to 59.6% was observed using 500 µg/mL 5-HMF. The combination of 500 µg/mL aKG + 167.7 µg/mL 5-HMF resulted in a caspase-3 activity of 55.2%. No caspase-3 activation was observed in NCI-H23 cells when treated with aKG, 5-HMF, or the combined solutions. Conclusion: CPs may serve as potential markers for distinguishing between cancer cells regulated by RONS. The combination of aKG + 5-HMF showed induced cell death in high-RONS-generating cancer cells compared to low-RONS-generating cancer cells. The content of carbonylated proteins may serve as a stable marker of ROS in the dysregulated mitochondrial energy synthesis of cancer cells, known as the Warburg effect [Plasma protein carbonylation is involved in various types of cancer, such as hematological malignancies, colorectal cancer, gastric cancer, and medullary thyroid carcinoma . Howeverl source . This isg effect . While c2+, O2, and ascorbate-dependent 2-oxoglutarate-dependent dioxygenases (2-OGDDs), which play a role in epigenetic regulation, such as DNA demethylation by Jumonji C domain-containing lysine demethylases (KDM 2–7) and translocation hydroxylases (TET 1–3) [In recent years, alpha-ketoglutarate (aKG), an endogenous intermediate metabolite of the Krebs cycle, has gained attention as a potential novel anticancer agent . Alpha-kTET 1–3) ,7,8. TheTET 1–3) . In cellTET 1–3)  such as TET 1–3) . The comIn contrast to normal cells, where high oxidative stress levels lead to apoptosis, cancer cells take an advantage of an oxidative milieu. Cancer cells build up a complex environment including stroma components which enhance oxidative stress, resulting in tumor growth. Experimental and clinical trials showed the key role of oxidative stress in several tumor aspects, thus influencing the characteristics of tumor cells. Intracellular pathways and the potential of DNA mutations that control the cell proliferation, the cell survival, the cell motility, and cell invasiveness, as well as the stromal components, are affected by oxidants. A dysregulation in these pathways strongly affects cancer development, cancer dissemination, inflammation, tissue repair, and neo-angiogenesis .A new study from Hanahan and Weinberg defined two new “hallmarks of cancer” depending on oxidative stress: the tumor microenvironment and the metabolic reprogramming of cancer cells. Intratumoral hypoxia and/or infiltration of CAFs should be treated with a disruption of the Warburg metabolism in cancer and stromal cells, as well as a reconfiguration through the antioxidative strategy of the pentose phosphate pathway. Pharmacological approaches could be medicaments which affect the lactate shuttle and inhibitors of glycolysis in combination with inhibitors of autophagy. Autophagy is a compensation mechanism of nutrient-poor cancer cells. In further studies, the effects of all biochemical reactions of ROS in cancer cells should be investigated .Medullary thyroid carcinoma (MTC) cells, which are neoplasms of the endocrine system, account for 10% of all thyroid cancers and are mainly caused by mutations in the RET proto-oncogene from para-follicular C-cells. Oxidative stress factors, such as carbonyl groups , and reduced antioxidative regulating enzymes have been associated with the risk of MTC in patients . The detNon-small-cell lung cancer (NSCLC) has a high incidence rate and is often resistant to chemotherapy and radiotherapy. Pro-oxidative treatment of NCI-H23 cells with vincenin-2, a pro-oxidant flavonoid, has been shown to increase carbonylated proteins and activate apoptotic genes, leading to cell death. Additional radiation further increases carbonylated proteins in NCI-H23 cells, promoting cell death . PachymiThe aim of this study was to investigate the antiproliferative and antitumoral effects of aKG and 5-HMF, which are associated with reduced mitochondrial activity, increased apoptosis, and a decrease in oxidative stress factors such as carbonylated membrane proteins in MTC-SK and NCI-H23 cells.® Cell Culture; Corning Incorporated, 14831 New York, NY, USA).The following materials were used: media—RPMI-1640, FBS, DMEM, and antibiotic–antimycotic solution ; reagents—Cytofix–Cytoperm permeabilization Kit , FITC active caspase-3 apoptosis kit , and WST-1 cell proliferation reagent ; chemicals—alpha-ketoglutarate , 5-hydroxymethyl-furfurale , guanidine-HCl, butyl-hydroxy-toluene , and di-nitro-phenyl-hydrazine (DNPH) ; flasks  were provided by Prof. Dr. Pfragner at the Medical University of Graz. In the MTC-SK cell line, the predominant findings were terminal chromosomal rearrangements most frequently concerning chromosome 11p, i.e., the locus of the calcitonin and calcitonin gene-related peptide genes and the H-ras oncogene, and a characteristic instability of the centromeric region of chromosome 16 and somatic pairing of the homologous chromosomes 16 [® Cell Counter . Aliquots were taken for further experiments.In line with our previous study , we used6 cells/mL was centrifuged (400× g) after incubation for 72 h without any substances (negative control), with 4 or 0.4 µM CPT (positive control), and with 250 µg/mL aKG + 83.3 µg/mL 5-HMF or 500 µg/mL aKG + 166.7 µg/mL 5-HMF. The cells were resuspended in 0.5 mL of JC-1 solution  and incubated for 15 min with 5% CO2 at 37 °C, followed by two washing steps using the assay buffer . Afterward, the cells were resuspended in 1 mL of assay buffer and analyzed by FACS. The aggregate dye produced the extinction of the monomer at 535 nm and the aggregate at 475 nm.A population of 2.0 × 10g for 10 min. Afterward, the cells were washed for several times using cold PBS, and 500 µL of Cytofix–Cytoperm was added on ice at −20 °C for 20 min. After incubation, the cells were labeled with FITC-tagged antibodies, and the examination of the caspase activity at 495 nm was performed using the washing step.Caspase-3-activated apoptosis was investigated as described in our previous study . The cel2. The calculation of the protein concentrations was performed at 562 nm (Spectra Max Photometer Pro 384) as soon as the plates reached room temperature. Furthermore, to calculate  the carbonylated proteins, the samples were diluted to 4 mg/mL protein. About 1 mg of protein was precipitated by pipetting 250 µL of the 4 mg/mL protein solution with 250 µL of 20% trichloric acid (TCA) solution. All precipitated samples (1 mg) were centrifuged at 5000× g for 3 min, and the supernatant was removed. Pellets (1 mg) were dissolved in 10 mM DNPH containing 6 M guanidine pH 2.5 and incubated for 45 min at room temperature. Protein damage was measured spectrophotometrically with the protein carbonyl assay [The cell lysate’s protein concentrations were estimated using the BSA test kit. For triple estimations, 25 µL of diluted albumin standards and cell lysates were transferred into microtitration plates. Afterward, BCA reagent solution (50:1) was added, and the microtitration plate was closed using a top cover. The plates were incubated for 30 min using a plate shaker at 37 °C and 5% COt-tests where appropriate and indicated. Linear regression and exponential regression curves were calculated on the basis of Pearson regression . All values are given as the mean and standard deviation. Statistical significance was set at p < 0.05, with high significance considered at p < 0.01.Group comparisons were made using n = 5; p < 0.01), 375 µg/mL aKG , and 500 µg/mL aKG  compared to the control . There was a 29% reduction in proliferation using 500 µg/mL aKG compared to the control signal, and it was also significant lower compared to 375 µg/mL aKG. After 72 h, the inhibiting effect of aKG on the cell growth became more effective. While the usage of 125 µg/mL aKG showed no significant effect  compared to the control , 500 µg/mL aKG showed 37.2% inhibition , followed by 375 µg/mL aKG , 250 µg/mL aKG , and 200 µg/mL aKG . Linear regression , with the concentrations of 375 µg/mL reducing growth to 23,234 ± 3197 cells  and 500 µg/mL reducing growth to 21,023 ± 1543 cells . After 72 h, the inhibitory effect of 5-HMF on cell proliferation was generally more effective and resulted in a decreased cell growth of MTC-SK cells using different 5-HMF concentrations; a 38% reduction was measured using the lowest 5-HMF concentration of 125 µg/mL  compared to the control , followed by 200 µg/mL 5-HMF , 250 µg/mL 5-HMF , 375 µg/mL 5-HMF , and 500 µg/mL 5-HMF . Correlation of the cell growth with 5-HMF concentrations during incubation was obtained for 0, 125, 200, and 250 µg/mL 5-HMF with an exponential increase. The usage of 375 and 500 µg/mL 5-HMF caused no cell growth. The calculated IC50% of 5-HMF was 200 µg/mL after 72 h incubation.5-HMF: On NHI-H23 5-HMF showed no significant effect on cell growth using several concentrations B.n = 5; p < 0.01) showed a significant reduction in cell proliferation for MTC-SK cells compared to the control , and there was no significant difference compared to the control signal .Combination of aKG + 5-HMF: n = 5; p < 0.01) with a reduction of nearly 30%, to 200 µg/mL aKG + 66.7 µg/mL 5-HMF  with a reduction of 47%, to 250 µg/mL aKG + 83.3 µg/mL 5-HMF  with a reduction of 47%, to 375 µg/mL aKG + 125.4 µg/mL 5-HMF  with a reduction of 53%, and to 500 µg/mL aKG + 166.7 µg/mL 5-HMF  with a reduction of 60%. No difference in cell growth was detected between the highest concentration of the combined solution after 48 h and the starting conditions.Each combined aKG + 5-HMF solution showed a significant reduction in the MTC-SK cell growth after 48 h. The control  showed a significantly higher cell growth compared to 125 µg/mL aKG + 41.7 µg/mL 5-HMF  compared to the control , followed by 375 µg/mL aKG + 125.4 µg/mL 5-HMF , 250 µg/mL aKG + 83.3 µg/mL , 200 µg/mL aKG + 66.7 µg/mL 5-HMF , and 125 µg/mL aKG + 41.7 µg/mL 5-HMF .After 72 h incubation, we obtained similar results compared to 48 h incubation in a concentration-dependent manner. The highest reduction in cell growth, 69%,was estimated using 500 µg/mL aKG + 166.7 µg/mL 5-HMF , followed by 200 µg/mL aKG + 66.7 µg/mL 5-HMF aKG + 0.52 µM 5-HMF (k = 7.33), and 250 µg/mL aKG + 83.3 µg/mL 5-HMF (k = 3.95). The normal cell growth of MTC-SK cells showed a linear regression of 18.4, which was more than four-fold higher than with 250 µg/mL aKG + 83.3 µg/mL 5-HMF. Additionally, the IC50% was calculated for the combined 250 µg/mL aKG + 83.3 µg/mL 5-HMF solution, resulting in a lower concentration compared to the IC50% of the single components, e.g., aKG (500 µg/mL) and 5-HMF (250 µg/mL).n = 5; p < 0.05) and 500 µg/mL aKG + 166.7 µg/mL 5-HMF  significantly increased the cell growth of NCI-H23 compared to the control . The same was detected after 72 h with 125 µg/mL aKG + 41.7 µg/mL 5-HMF  compared to the control . Interestingly, highest concentrations of aKG + 5-HMF showed no cell proliferation compared to starting conditions.Using NCI-H23 cells, aKG + 5-HMF showed less effects on cell growth. After 48 h, the combined solutions with concentrations of 375 µg/mL aKG + 125.4 µg/mL 5-HMF ; using 200 µg/mL aKG the mitochondrial activity decreased to 83.2 ± 3.1% , while the concentration of 250 µg/mL aKG led to a decrease to 70.0 ± 3.2%, , 375 µg/mL aKG led to a decrease to 61.2 ± 2.1%, , and 500 µg/mL aKG led to a decrease to 50.1 ± 1.8% . The lowest concentration of 125 µg/mL aKG  had no effect on the mitochondrial activity of MTC-SK cells.aKG: Aliquots of the cell growth experiments were used to measure mitochondrial activity. n = 5). The concentration of 125 µg/mL aKG showed a significant reduction in mitochondrial activity to 71.1 ± 3.1%, , 200 µg/mL aKG showed a significant reduction to 69.3 ± 4.2%, , 250 µg/mL aKG showed a significant reduction to 63.1 ± 1.7%, , 375 µg/mL aKG showed a significant reduction to 55.2 ± 1.8%, , and 500 µg/mL aKG showed a significant reduction to 44.1 ± 1.8% .After 48 h incubation, all used aKG concentrations decreased the mitochondrial activity compared to the control . Significantly lower levels of the mitochondrial activity were measured using 125 µg/mL aKG , 200 µg/mL aKG , 250 µg/mL aKG , 375 µg/mL aKG , and 500 µg/mL aKG . The IC50% of aKG was calculated to be around 500 µg/mL aKG regardless of the incubation time.After 72 h incubation with aKG, the same effects were estimated compared to the results after 48 h. The control signal was detected at 100.0 ± 2.9%, , 250 , and 375 µg/mL  compared to the control (100.0 ± 8.4%) and after 72 h between the control signal (100.0 ± 2.0%) and 200 µg/mL aKG .NCI-H23 cells treated with aKG showed a significant decrease in the mitochondrial activity after 24 h incubation for 200  compared to the control . There was a stabilization at the same level of the effect with 125 µg/mL 5-HMF as when using 200 µg/mL 5-HMF , 250 µg/mL 5-HMF , and 375 µg/mL 5-HMF . The usage of 500 µg/mL 5-HMF showed a significant decrease in the mitochondrial activity to 70.2 ± 9.8% , compared to 200 µg/mL 5-HMF.5-HMF: n = 5; p < 0.01), compared to the control , followed by 375 µg/mL 5-HMF , 250 µg/mL 5-HMF , 200 µg/mL 5-HMF , and 125 µg/mL 5-HMF .A 48 h incubation of MTC-SK cells with various 5-HMF concentrations generated a higher effect on the mitochondrial activity compared to 24 h. The highest significant decrease was measured using 500 µg/mL 5-HMF  compared to the control , followed by 375 µg/mL 5-HMF , 250 µg/mL 5-HMF , 200 µg/mL 5-HMF , and the lowest concentration of 125 µg/mL 5-HMF . The IC50% was calculated for 48 and 72 h incubation to be around 200 µg/mL 5-HMF.After 72 h incubation of different 5-HMF concentrations, the inhibitory effect on mitochondrial activity was slightly better compared to 48 h incubation. A nearly 80% reduction using 5-HMF was obtained using 500 µg/mL 5-HMF . Increasing the incubation time to 48 h 5-HMF reduced the mitochondrial activity significantly between the control  and 125 , 200 , 250 , 375 , and 500 µg/mL . After 72 h, no improvement using several 5-HMF concentrations compared to 48 h incubation was detected. The mitochondrial activity of all used 5-HMF concentrations was reduced significantly compared to the control : 125 , 200 , 250 , 375 , and 500 µg/mL .Combination of aKG + 5-HMF: n = 5; p < 0.01) compared to the control (100 ± 2.1% n = 5), whereas 200 µg/mL aKG + 66.7 µg/mL 5-HMF decreased it to 91.5 ± 4. 5% , 250 µg/mL aKG + 83.3 µg/mL 5-HMF decreased it to 86.2 ± 2.2% , 375 µg/mL aKG + 125.4 µg/mL 5-HMF decreased it to 79.8 ± 2.2% , and 500 µg/mL aKG + 166.7 µg/mL 5-HMF decreased it to 77.2 ± 2.1% .Within 24 h incubation, the different aKG + 5-HMF concentrations reduced the mitochondrial activity significantly in a dose-dependent manner: The lowest concentration of 125 µg/mL aKG + 41.7 µg/mL 5-HMF decreased the mitochondrial activity significantly to 92.2 ± 2.5% , whereas 200 µg/mL aKG + 66.7 µg/mL 5-HMF decreased it to 57.2 ± 2.2% , 250 µg/mL aKG + 83.3 µg/mL 5-HMF decreased it to 55.9 ± 3.2% , 375 µg/mL aKG + 125.4 µg/mL 5-HMF decreased it to 53.2 ± 2.8% , and 500 µg/mL aKG + 166.7 µg/mL 5-HMF decreased it to 48.2 ± 2.4% , which also resulted in an IC50% after 48 h incubation.Within 48 h incubation using 125 µg/mL aKG + 41.7 5-HMF, there was a significant reduction in the mitochondrial activity to 64.2 ± 2.2% , 200 µg/mL aKG + 66.7 µg/mL 5-HMF , 250 µg/mL aKG + 83.3 µg/mL 5-HMF , 375 µg/mL aKG + 125.4 µg/mL 5-HMF , and 500 µg/mL aKG + 166.7 µg/mL 5-HMF  compared to the control . The IC50% after 72 h was reached by the addition of 200 µg/mL aKG + 66.7 µg/mL 5-HMF to MTC-SK cells.After 72 h incubation using the different aKG + 5-HMF combinations, there was a sustained reduction in the mitochondrial activity. A significant reduction in the mitochondrial activity was obtained for 125 µg/mL aKG + 41.7 µg/mL 5-HMF , showed a significant increase in caspase-3 activity compared to the control (0 µg/mL aKG). No caspase activity was recorded using 0–500 µg/mL aKG on NCI-H23 cells . Increasing the 5-HMF concentration to 200 µg/mL led to a significantly higher caspase-3 activity of 29.2 ± 0.8%  compared to the control, followed by 250 µg/mL 5-HMF, 34.2 ± 1.9% , 375 µg/mL 5-HMF, 42.9 ± 0.8% , and 500 µg/mL 5-HMF, 59.6 ± 2.4% . No caspase activation was measured using all 5-HMF concentrations on NCI-H23 cells  compared to the control  after 72 h incubation, followed by 250 µM aKG + 83.3 µg/mL 5-HMF , 375 µg/mL µM aKG + 125.7 µg/mL 5-HMF , and 500 µg/mL aKG + 1666.7 µg/mL 5-HMF .The combination of aKG + 5-HMF E: Both sThe usage of several combined aKG + 5-HMF solutions showed no significant effect on NCI-H23 cells (7F).n = 3; p < 0.01) compared to the control , followed by 375 µg/mL aKG + 125.4 µg/mL 5-HMF  and 500 µg/mL aKG + 166.7 µg/mL 5-HMF . The usage of several combined aKG + 5-HMF solutions showed no significant effect on NCI-H23 cells (23 cells B.2 = 1. The highest concentration (500 µg/mL aKG + 166.7 µg/mL µM 5-HMF) showed the lowest protein modification per mg cell membrane protein and the lowest mitochondrial activity, whereas the lowest concentration (125 µg/mL aKG + 41.7 µg/mL 5-HMF) showed a high oxidative modified protein level with a high cell growth. The mitochondrial activity of MTC-SK in absence of any substance was 2.5-fold lower compared to the highest used aKG + 5-HMF solution.Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular cells . It accoFamilial MTC syndromes include multiple endocrine neoplasia (MEN) types 2A and 2B, and familial MTC, which are inherited in an autosomal dominant manner. Children with any of these syndromes have a 100% risk of developing MTC . TherefoThe updated guidelines recommend ultrasound evaluation of thyroid nodules and measurement of serum thyroid-stimulating hormone (TSH) for diagnosing MTC. On the basis of the results, particularly the nodule size, a fine needle aspiration biopsy (FNAB) is advised . In someIn the case of incomplete tumor resections, external beam radiation therapy can be considered (EBRT) .3, total thyroidectomy and bilateral central neck dissection are recommended. In cases where MTC tumors are ≤1 cm, total thyroidectomy is sufficient. Consideration of total neck dissection depends on calcitonin levels higher than 200 pg/mL [For MTC patients with a tumor size ≥1 cm00 pg/mL .In metastatic MTC, vandetanib and cabozantinib are approved by regulatory agencies for administration. The National Comprehensive Cancer Network (NCCN) recommends involving patients with symptomatic or progressing MTC in clinical trials with targeted therapies, immunotherapy, small-molecule kinase inhibitors, or decarbazine (DTIC)-based chemotherapy with EBRT. Biophosphonate and denosumab treatment is recommended for bone metastases .While surgery remains the standard treatment for MTC, there is growing interest in using medication such as tyrosine kinase inhibitors (TKIs), e.g., vandetanib, cabozantinib, selpercatinib, and pralsetinib . These TIn the context of RET-mutant medullary thyroid carcinoma (MTC), it is recommended to use selpercatinib as a kinase inhibitor targeting various forms of rearranged during transfection (RET), including wildtype RET, multiple mutated RET isoforms, VEGFR1, and VEGFR3. Pralsetinib has shown selective activity against RET alterations .Preclinical studies suggest the involvement of not only RET but also VEGFR, EGFR, and MET in the tumorigenesis of MTC .In this study, we investigated the effects of alpha-ketoglutarate (aKG) and 5-hydroxymethylfurfural (5-HMF) on MTC cells, focusing on their antioxidative, antiproliferative, and anticarcinogenic properties.The individual compound aKG is primarily involved in the Krebs cycle to prevent hypoxia and processes such as the activation of HIFα, upregulation of transcription genes, and neo-angiogenesis, which play crucial roles in the cancerogenesis of solid tumors . HIF-1, Further investigations using radiolabeled carbon-marked aKG showed delayed cell penetration. However, the use of derivatized (esters) and radiolabeled aKG demonstrated better penetration into the cells. Nonetheless, the effect of inhibiting HIF-1 in mutated SDHA fibroblasts was lower with derivatized aKG compared to nonderivatized aKG. Exogenously administered aKG to tumor cells showed a highly dose-dependent reduction in cell growth, mitochondrial activity, and caspase activation in osteosarcoma cells , colon a14C): Long et al. [14C-labeled aKG was washed out after cell separation before measurement, resulting in a lack of radioactivity signal. Recent research has also shown that aKG reacts with peroxynitrite (ONOOH) to generate succinate and carbon dioxide at physiological pH in media and cells [(i) Decarboxylation of carbon dioxide  pH in medium: No chemical reaction of ONOOH or Han cells .(iii) Tumor cells: Due to elevated turnover rates for energy generation, the pH conditions in tumor cells are more acidic (lactate metabolism) compared to normal cells. Under these conditions, aKG is more stable and can effectively penetrate tumoral cells and their mitochondria.(iv) Increasing incubation time results in a decrease in pH. While some studies have shown the antitumoral effect of aKG using only a single incubation time point, we investigated the effects after 24, 48, and 72 h. The best effect of exogenously administered aKG on inhibiting cell growth and mitochondrial activity in MTC-SK cells was observed after 72 h using the highest concentration of aKG . Similar+H+ (−0.32 volts), FADH2 (−0.22 volts), ubiquinone (0.06 volts), or succinate (0.3 volts), aKG has a higher reduction capacity (−0.38 volts). Carbonylated proteins (CP) have been established as reliable markers for determining oxidative stress levels in cells. The isolated CP content in membranes of MTC-SK is 3–4 times higher compared to unaffected fibroblasts, and similar results have been observed in Jurkat cells [(v) Oxidative stress: It is widely accepted that cancer cells generate more radicals  compared to normal healthy cells. Detecting oxidative stress in cultured cells is challenging due to the stability and reactivity of these species. Most radicals interact with various organic substances, including monomeric  or polymeric  components, leading to modified substances. However, aKG is one of the few organic substances that decomposes to succinate and carbon dioxide in the presence of radicals without prior modification. Glucose, for example, converts to malondialdehyde, polyunsaturated fatty acids produce free carbonyls , tyrosine results in nitro-tyrosine, and guanosine forms the mutagenic compound 2-hydroxy-guanosine in the presence of radicals. Compared to NADHat cells , but to In conclusion, aKG is a potent antioxidative substance that counteracts the cell growth of cancer cells through its multifunctional reactions, particularly involving the HIF-1 mechanism. 5-HMF also possesses antioxidative potential, an upregulating effect on antioxidative enzymes, an inhibiting effect on free radicals , and an antitumoral effect ,33. HoweAlthough 5-HMF has a decreased ability to reduce reactive oxygen and nitrogen species (RONS), it is more effective than aKG in preventing the nitration of tyrosine residues on proteins. Additionally, 5-HMF upregulates antioxidative acting enzymes and has an inhibiting effect on free radicals . TherefoBoth aKG and 5-HMF effectively reduce the content of CP in isolated membrane proteins in a dose-dependent manner, indicating that energy metabolism-generated oxidative stress and disrupted oxidative turnover lead to apoptosis in cancer cells, as shown in The antioxidative, antiproliferative, and anticancerogenic effects of aKG + 5-HMF, as previously administered to cancer patients, were confirmed in MTC-SK cells, as well as in previous studies on leukemic cells. Further studies are needed in cell lines with high proliferating properties and high energy rates to confirm these modes of action. Hopefully, future clinical trials in cancer patients will verify the remarkable results observed in in vitro cell studies, such as those conducted in non-small-cell lung cancer patients, and explore the potential of aKG + 5-HMF as an additive metabolic therapy in cancer treatment.


Escherichia coli O93:−:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D 1H, 13C-HSQC, and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~¼ was 2-O-acetylated, whereas ~¼ did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-β-d-Manp-(1→3)-β-d-Manp2Ac6Ac-(1→4)-β-d-GlcpA-(1→3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.The structure of the O-antigen from the international reference strain  Escherichia coli is a bacterium that belongs to the family Enterobacteriaceae and it is part of the colonic flora of humans, where the bacterium and the host mutually benefit from each other. However, clinical syndromes that result from pathogenic strains include enteric/diarrheal disease, urinary tract infection, and sepsis/meningitides (ngitides .E. coli make Shiga toxin(s) known as Stx1, one of the most potent bacterial toxins described to date (E. coli strains. Collectively these bacteria are referred to as Shiga toxin producing E. coli (STEC) and this pathogen is a potential health risk as it may cause, in particular, hemolytic uremic syndrome (HUS) resulting in kidney failure. The prevalence of STEC in healthy adults in Japan was investigated by collecting close to 3 million samples during a 2-year period and a large number of serogroups was identified carrying stx genes, among others, E. coli O93  pathotype  were isolated from ducks in China during an 8-year period, where E. coli O93 was the predominant serogroup  expressed with a nonrandom modal chain-length distribution. For comparison, the rough LPS from E. coli CWG303 ΔwaaG , analyzed and putative functions of the gene products have been proposed and annotated -like . Two genes coding for glycosyltransferases (GTs) were identified, viz., wfdV and wfdW, after galF and wexC, respectively. Analysis and identification of conserved regions revealed that wfdV belongs to GT family 2 that acts through an inverting mechanism whereas a corresponding analysis showed that wfdW belongs to GT family 1, which also acts through an inverting mechanism. The function of the product coded by the gene denoted by orf5 has not yet been identified.The part of the genome of nnotated  The pres pathway . The genAT)-like . A gene nic acid  . As there are not any other genes in the sequence between galF and gnd the sugar residues in the RU should contain d-GlcNAc and d-Man as well as maybe d-GlcA; sugar residues coded outside the O-antigen cluster by housekeeping genes, e.g. d-Glc and d-Gal, may also be constituents of the O-polysaccharide.The Wzx/Wzy-dependent pathway employs WecA to make Und-d-GlcNAc  as the i-GalpNAc , are absE. coli O-antigen structures, their NMR chemical shifts and function of GTs used in the biosynthesis of the O-polysaccharides -d-GlcpNAc, which is consistent with the fact that WfdV is proposed to operate via an inverting mechanism and that UDP-d-GlcpA with an α-anomeric configuration should then be the donor molecule as the product has the β-anomeric configuration.Databases storing information on polysaccharide structures facilitate comparison among entries, which can be highly beneficial when unknown structures are to be elucidated. ECODAB stores (for each serogroup) available information from literature on charides . Importaoli O161 . weiG co1H NMR spectrum of the PS–OH sample showed four resonances from anomeric protons and the sugar residues corresponding to these were denoted A–D by order of descending proton chemical shift  NMR spectrum of the O93 PS–OH showed a total of 27 cross-peaks consisting of 4 cross-peaks in the spectral region for anomeric atoms , and correlations from 1H,1H-TOCSY NMR spectra were used. The computer program CASPER can determine polysaccharide structure using NMR data in conjunction with some additional information such as sugar components -d-GlcpNAc in conjunction with the above described unassigned NMR data (Supporting information).To determine the structure of the PS–OH, spectral data from a 1D mponents . The inf13C NMR chemical shift data -α-d-GlcNAc--β-d-Man-(1→ and →3)-β-d-Man--β-d-GlcA- and a major population (denoted by a double-prime), respectively, which were not present in the material after treatment under dilute basic conditions, fully consistent with the presence of O-acetyl groups as substituents on the sugar residue(s) of the O-antigen polysaccharide. The 1H,13C-meHSQC NMR spectrum showed characteristic chemical shift displacements as a result of O-acetylation, inter alia, for a 6-O-acetylation resulting in cross-peaks between δC 63.88 and the two protons at δH 4.37 and 4.46 (δC6 ~2 and ΔδH6 ~0.5 (δH/δC 5.53/69.97 and δH/δC 5.55/69.87 (C2′ and C2″ resulting in a →3)-β-d-Man2Ac- to δH 4.37 and δH 4.46 (H6 of C″) were observed in the 1H,1H-TOCSY acquired with a mixing time of 120 ms -β-d-Man2Ac6Ac--β-d-Man2Ac6Ac- were observed -β-d-Manp and ϕH,ψH ≈ +25°, +60° for that of β-d-Manp-(1→3)-β-d-Manp2Ac6Ac were adopted. The orientation of the carbonyl group then is such that the anisotropic shielding can affect B2″ and the A1″ protons, consistent with the significant upfield chemical shift displacement of these -β-d-Glcp-(1→3)-α-d-GlcpNAc-(1→2)-β-d-Manp-(1→3)-β-d-ManpNAc-(1→ and α-d-GlcpNAc-(1→2)-β-d-Manp4,6(S)Pyr-(1→3)-d-ManpNAc, respectively  from ectively , which aE. coli O93 O-antigen , in relation to the determined structure of the BRU of the O-antigen from E. coli O93 calls for the presence of a third GT . The function of the gene product from orf5 was still uncharacterized and the information deposited by PP-undecaprenyl acceptor to result in a tetrasaccharide-PP-undecaprenyl entity, which subsequently is translocated from the cytoplasmic side of the inner membrane to the periplasmic side by Wzx where the oligosaccharide will be polymerized by the Wzy–Wzz complex to give the O-antigen polysaccharide.The analysis of function of gene products, from E. coli O93 revealed putative functions of proteins that were encoded in this part of the genome -d-GlcpNAc, and was consistent with the presence of UDP-glucuronic acid as the donor molecule and Und-PP-d-GlcNAc as the acceptor molecule in conjunction with the proposed GT family acting by an inverting mechanism.The initial analysis of the gene cluster related to the biosynthesis of the O-antigen polysaccharide from e genome . This inO-acetylation pattern of the O-antigen polysaccharide, which has tetrasaccharide RUs, →2)-β-d-Manp-(1→3)-β-d-Manp2Ac6Ac-(1→4)-β-d-GlcpA-(1→3)-α-d-GlcpNAc- of the BRU. From 3D models of the different O-antigen populations it is evident that the O-acetyl groups can form exposed epitopes on the polysaccharide backbone, but whether the dual substitution pattern on a single sugar residue in the RU renders the bacterium any advantages as a pathogen during infection remains to be established.To gain deeper insight into the structure of the O-antigen polysaccharide the bioinformatics information was coupled with an NMR analysis. Using the computer program CASPER, structure proposals were generated in a highly efficient way without the need to carry out NMR chemical shift assignments; the top-ranked proposal was subsequently confirmed to be correct by means of additional NMR experiments. Detailed NMR analysis was employed to identify the Escherichia coli reference strains of O93 (E. coli O93:−:H16) was obtained from SSI Diagnostica A/S . Bacterial culture, LPS extraction and preparation of lipid-free polysaccharides (PS) were carried out as previously described (O-deacetylated polysaccharide (PS–OH) was prepared by treatment of the PS with aq. NaOH (0.5 M) at 20 °C for 18 h followed by extensive dialysis against deionized water using Spectra/Por 4 Membrane Tubing with a MWCO of 12–14 kDa (fisher scientific).escribed . O-deace4 in aq. ammonia (1 M) at ambient temp for 30 min and acetylation at 100 °C for 30 min using Ac2O and pyridine (1:1). The resulting mixture of alditol acetates was analyzed by gas–liquid chromatography (GLC) whereby components were separated on a PerkinElmer Elite-5 column using a temperature program of 150 °C for 2 min, 1 °C⋅min−1 up to 173 °C, followed by 8 °C⋅min−1 to 220 °C with hydrogen as the carrier gas (25 psi). The column was fitted to a PerkinElmer Clarus 400 gas chromatograph equipped with flame ionization detectors. The retention times of the derivatives were compared with those of authentic standards as references.Hydrolysis of the PS was carried out using 2 M TFA at 120 °C for 30 min followed by reduction with NaBH2O and examined by NMR spectroscopy at 70 °C as solutions in 99.96% D2O (10 mg in 0.55 mL) with a trace amount of NaN3 and was used for 1D 13C NMR, 2D 1H,13C-HMBC, 1H,13C-H2BC and 1H,13C-BS-CT-HMBC, 1H,13C-HSQC, and 1H,13C-CT-CE-HSQC experiments. A PS–OH sample purified by gel-permeation chromatography was used for further analysis employing 1D 1H and diffusion-filtered 1H NMR experiments, F2 coupled 1H,13C-HSQC, 1H,13C-HSQC, 1H,1H-TOCSY, and 1H,1H-NOESY NMR experiments (<1 mg in 0.55 mL) acquired at 60 °C. NMR spectra were recorded using a Bruker Avance III 700-MHz spectrometer equipped with 5-mm TCI (1H/13C/15N) Z-Gradient (53.0 G cm−1) CryoProbe or a Bruker Avance III 600-MHz NMR spectrometer equipped with a 5-mm TXI (1H/13C/15N) Z-gradient (55.7 G cm−1) inverse probe. Chemical shifts are reported in ppm using internal sodium 3-trimethylsilylpropanoate-2,2,3,3-d4  for 1H NMR and external 1,4-dioxane 10% in D2O (δC 67.40) for 13C NMR as references. Chemical shift differences were obtained by comparison with NMR data of the corresponding monosaccharides  during mixing time with a strength of 40% of the maximum; both types of experiments used 2,048 × 256 data points in the F2 and F1 dimensions, respectively. For the PS material, a 1H,1H-NOESY experiment, with suppression of effects from zero-quantum coherence  by The bioinformatics analysis using the genome of pression  and prevSI_CASPER_inputs_O93_cwac069Click here for additional data file.


Whether intratumoral CD4+ T cells target HPV16 E2 and if HPV16-E2-specific immunity contributes to better clinical outcome is unknown. In a prospective HPV16+ OPSCC cohort, we regularly detect HPV16-E2-specific CD4+ and CD8+ intratumoral T cells, albeit at lower frequencies than the co-infiltrating HPV16-E6/E7-specific T cells. These HPV16-reactive T cells produce multiple cytokines when activated, indicating their polyfunctionality. Importantly, their combined intratumoral presence predicts superior survival, emphasizing the value of HPV16-E2-specific T cells in anti-tumor immunity and suggests its use as a target antigen for immunotherapy.Tumor-infiltrating HPV16-E2-specific CD8  •+ OPSCC may contain T cells reactive to the viral proteins E6, E7, and E2HPV16•These HPV16-reactive T cells display a polyfunctional type 1 cytokine profile•Patients with intratumoral T cells to all 3 proteins display superior survival The infiltration of human papillomavirus (HPV)-induced oropharyngeal cancer by tumor-reactive T cells is important for treatment response. Santegoets et al. describe that in addition to E6 and E7, a third HPV protein, called E2, is targeted by tumor-infiltrating T cells and that these cells contribute to better clinical outcome. Oropharyngeal squamous cell carcinoma (OPSCC) accounts for 0.5% of all cancer cases with ∼98,000 new cases each year.,,+ OPSCC.,,,,+ T cells in OPSCC.,+ T cells, whether E2-specific T cells contribute to a better clinical outcome, and how this response compares to T cell reactivity against the oncogenes E6 and E7. The latter is of particular interest since we recently demonstrated a strong role for CD4+ and CD8+ E6- and/or E7-reactive T cells+ OPSCC tumors.Early studies on HPV-driven carcinogenesis in cervical cancer (CxCa) revealed a multistep process of persistent HPV infection with progression from pre-cancerous to malignant lesions. In this process, the episomal DNA of HPV becomes integrated into the host genome,+ and CD8+ T cells in our previously described prospective HPV16+ OPSCC cohort with over 10 years of follow-up,Here, we studied the presence and potential clinical impact of intratumoral HPV16 E2-specific CD4+ T cells could be detected in 21 of 35 IL-2 expanded tumor-infiltrating lymphocyte (TIL) cultures of HPV16+ OPSCC patients. In 19 cases, E2-reactivity coincided with E6/E7-reactive CD4+ T cells, in 2 cases, only E2-reactivity was found, and in 6 cases, only a response to E6/E7 was found  analysis followed by FlowSOM consensus metaclustering revealed that HPV16-reactive T cells are capable of producing multiple cytokines up to a total of 5 simultaneously  with fast production of TNFα and GM-CSF to full-blown activation with additional production of IFNγ, IL-2, and CCL4  with fast production of TNFα to full-blown activation with CD154 expression and production of GM-CSF, IFNγ, IL-2, and CCL4 .Further information and requests for resources and reagents should be directed to the lead contact, Professor Dr. Sjoerd H. van der Burg  and cervical carcinoma (CxCa). Patients with histologically confirmed OPSCC were included in a study investigating the circulating and local immune response in patients with head and neck cancer (P07-112) and women with histologically proven cervical carcinoma were included in the CIRCLE study investigating cellular immunity against anogenital lesions. Patients were included after signing an informed consent.In this study, a total of 61 TIL batches from OPSCC patients were analyzed. This number was based on availability of expanded TIL samples of HPV16+ tumors and autologous monocytes and/or EBV-transformed B-lymphoblastoid cells (EBV-B).,,Blood samples were drawn in sodium-heparin collection tubes  prior to surgery. PBMC were isolated by ficoll density centrifugation, washed in PBS, frozen in 90% FCS (PAA laboratories) supplemented with 10% DMSO (Sigma) and stored in liquid nitrogen until use. Tumor biopsies were obtained from the operating theater and handled as described previously.,,E2-p1: 1–30, 16–45, 31–60, 46–75, 61–90, 76–105; E2-p2: 91–120, 106–135,121–150, 136–165, 151–180, 166–195; E2-p3: 181–210, 196–225, 211–240, 226–255, 241–270, 256–285; E2-p4: 271–300, 286–315, 286–315, 301–330, 316–345, 331–365. The HPV16 E6 and E7 peptides consisted of 22-mer peptides with 14 amino acids overlap that were put together into 1 pool: E6: 1–22, 11–32, 21–42, 31–52, 41–62, 51–72, 61–82, 71–92, 81–102, 91–112, 101–122, 111–132, 111–132, 121–142, 131–152, 137–158 and E7: 1–22, 11–32, 21–42, 31–52, 41–62, 51–72, 61–82, 71–92, 77–98. ICS was performed with antibodies directed against CD3 (UCHT1), CD4 (RPA-T4), TNFα (MAb11), GM-CSF (BVD2-21C11), IL-2 , CD8 (HIT8a), CD137 (4B4-1), CD154 (TRAP1), IFNγ (B27) and CCL4  following overnight stimulation with SLP-loaded autologous monocytes or EBV-B cells in the presence of 10 μg/mL Brefeldin A (Sigma-Aldrich). Acquisition was done on an LSRII fortessa (BD biosciences). Specific cytokine-producing T cells were identified by manual gating using FlowJo software V10.8.1. Cells were gated for live, single cells, CD3, CD4 and CD8 expression. Activated CD4+ and CD8+ cells were selected based on CD137 and/or CD154 expression and further analyzed for TNFα, GM-CSF, IFNγ, IL2 and CCL4 expression. To this end, cells were first gated for TNFα and GM-CSF, yielding TNFα+GM-CSF-, TNFα+GM-CSF+, TNFα-GM-CSF+ and TNFα-GM-CSF- populations. Next, these four populations were gated for IFNγ and IL2 , followed by subsequent gating on CD4 or CD8 and CCL4. Cytokine gates were set on total CD4+ and CD8+ T cells (reference plots). A gating example for this sequential gating is depicted in −and CD8-activated T cell populations as above) following stimulation with E2, E6/E7-loaded autologous monocytes or EBV B cells. Next, the newly formed FCS files containing these HPV16 E2, E6 and/or E7-reactive T cells were used for analysis by UMAP, FlowSOM and wanderlust trajectory analysis using OMIQ. The obtained different cell populations were visualized and quantified in OMIQ and after exporting the counts, graphs were generated using Graphpad prism V9.5.1 .To determine the specificity of the tumor infiltrating T cells in HPV16+ OPSCC, cultured TIL batches were analyzed for the presence HPV16 E6 and/or E7-reactive T cells using a combined [3H]-thymidine-based proliferation and cytokine production assay as described previously.Statistical was performed using. Differences between frequencies of E2 and E6/E7-reactive T cells were compared using a two-tailed paired T-test. Differences in frequencies of reactive T cells between OPSCC and CxCa patients were compared using the Mann-Whitney test. Differences in survival were calculated with the non-parametric log rank Mantel-Cox test. Grouping was done based on the detection of an intratumoral HPV16-specific T cell response -positive) or no detectable HPV16-specific immune response . A patient was scored as IR-positive when E2, E6 and/or E7-specific T cells were detected by antigen-specific proliferation and/or cytokine production (cytokine bead array/ICS). Differences were considered significant when p < 0.05. Statistical details of experiments such as n value, information on grouping for survival analysis and level of statistical significance  can be found in the figures and/or figure legends.


Autophagy protein 5 (ATG5) regulates airway epithelial cell autophagy, immune response, and inflammation, which is involved in asthma progression. This study aimed to evaluate ATG5 levels and its clinical roles in adult asthma patients.Totally, 200 adult asthma patients and 100 healthy controls (HCs) were enrolled in this case-control study. Subsequently, serum ATG5 was measured by enzyme-linked immunosorbent assay.P < 0.001). In asthma patients, ATG5 was positively related to male gender (P = 0.022), a family history of asthma (P = 0.035), eosinophil count (P < 0.001), and immune globulin E (P < 0.001), while it was negatively correlated with forced expiratory volume in 1 s (FEV1)/forced vital capacity (P < 0.001) and FEV1 (Predicted) (P < 0.001). Meanwhile, ATG5 was inversely associated with T helper (Th) 1 cells (P = 0.008), while it was positively linked with Th2 cells (P < 0.001), Th2/Th1 ratio (P < 0.001), interleukin (IL)-4 (P = 0.002), and IL-4/interferon-γ ratio (P = 0.015). Additionally, ATG5 was positively correlated with tumor necrosis factor-α (P < 0.001), IL-1β (P = 0.001), IL-6 (P = 0.003), and IL-17 (P = 0.029). Notably, ATG5 was elevated in asthma patients at exacerbation compared to those at remission [median (interquartile range): 53.6 (37.6–90.0) vs. 35.6 (28.2–51.5) ng/mL] (P < 0.001). It was also noteworthy that ATG5 was positively linked with exacerbation severity in asthma patients (P = 0.005).ATG5 was increased in asthma patients compared with HCs [median (interquartile range): 44.2 (31.7–77.8) vs. 23.2 (16.7–39.2) ng/mL] (Serum ATG5 is related to increased Th2/Th1 ratio, inflammation, exacerbation risk and severity in adult asthma patients, which serves as a candidate marker for the management of asthma. However, further validation is still needed.The online version contains supplementary material available at 10.1186/s13223-023-00821-3. Asthma is characterized by airway obstruction, bronchial hyper-responsiveness, and airway inflammation, resulting in a persistent cough, dyspnea, and chest tightness , 2. Gene+ (CD4+) T helper (Th) cells, airway inflammation, airway hyperresponsiveness, and airway remodeling  and IgE [median (IQR): 151.5 (79.0-287.0) vs. 36.5 (23.3–51.8) IU/mL] were higher in adult asthma patients than those in HCs (both P < 0.001). Furthermore, the mean levels of FEV1/FVC (mean ± SD: 71.8 ± 7.8% vs. 84.1 ± 3.6%) and FEV1 (Predicted) (mean ± SD: 81.9 ± 8.1% vs. 99.5 ± 6.3%) were lower in adult asthma patients than those in HCs (both P < 0.001). Additionally, in adult asthma patients, the median (IQR) values of Th1 cells, Th2 cells, and the Th2/Th1 ratio were 14.0 (11.4–17.2)%, 15.3 (12.6–19.1)%, and 1.2 (0.8–1.5), respectively. More specific clinical characteristics of all participants were described in Table The adult asthma patients included 89 (44.5%) females and 111 (55.5%) males with a mean age of 28.9 ± 8.0 years. Meanwhile, the HCs included 40 (40.0%) females and 60 (60.0%) males with a mean age of 30.1 ± 8.2 years. There was no difference in age (P < 0.001) : 44.2 31.7–77.8) vs. 23.2 (16.7–39.2) ng/mL]           or allergen-specific immunotherapy (P = 0.016) compared to those without during the enrollment. Meanwhile, ATG5 was not different in patients with or without corticosteroids, anti-histamines, and other treatments during the enrollment   Fig. A. Howeve4 Fig. A.rs=-0.238, P = 0.008)             : 53.6 (37.6–90.0) vs. 35.6 28.2–51.5) ng/mL] (.5 ng/mL]1/FVC and FEV1 (Predicted) in adult asthma patients.ATG5, as one of the essential proteins regulating autophagy, is considered to overexpress and mediate immune responses in asthma , 15, 20.+ T cells [Allergen-activated Th2 cells release cytokines, promoting airway eosinophil inflammation, airway hyperresponsiveness, and airway remodeling, which play a central role in the development of asthma , 21. Not T cells , 22, whi T cells –25. SignIt is reported that ATG5 also participates in airway inflammation in asthma patients to a certain extent . TherefoAsthma has a long course, and a potential marker is needed to observe the asthma exacerbation risk and severity to control the progression of asthma . Our stuThere were some limitations in our study: (1) The specific mechanisms of ATG5 regulating Th1 cells, Th2 cells, and inflammatory cytokines in adult asthma patients were required to explore in future studies. (2) Our study did not include an age and sex-matched disease control group. Future research should enroll disease controls to further comprehensively verify the potential of ATG5 as a marker in adult asthma patients. (3) Although the sample size in our study was relatively large, in order to have a clearer conclusion on the clinical significance of ATG5 in adult asthma patients, a larger sample size was needed in further studies. (4) Future studies should consider collecting more direct ATG5 samples  to assess its correlation with clinical indexes in asthma patients. (5) Our study did not evaluate the changes in ATG5 in asthma patients before the exacerbation, and further studies should consider assessing the changes in ATG5 to verify its potential as a candidate marker in asthma patients.In conclusion, serum ATG5 is positively correlated with the Th2/Th1 ratio, proinflammatory cytokines, and exacerbation in adult asthma patients, which may have important clinical significance for the management of asthma.Below is the link to the electronic supplementary material.Supplementary Fig. 1: Changes of ATG5 from M0 to M3 in adult asthma patients after treatment. ATG5 (skewed distributed continuous data) was decreased continually from M0 to M3 in adult asthma patients after treatment (Friedman test)Supplementary Table 1: Comparison of ATG5 in patients with different treatments during the enrollment


These dimers are connected by O—H⋯O hydrogen bonds into chains along the a-axis direction, forming R22(16) ring motifs. Further O—H⋯O interactions involving the ethanol solvent molecule connect the chains into a three-dimensional network. In addition, C—I⋯π interactions are observed. The intermolecular interactions in the crystal structure were quantified and analysed using Hirshfeld surface analysis.The title compound, C The carbon atoms in the arylhydrazone molecule are magnetically non-equivalent as a result of limited rotation around the C—N bond, thus the NH group is locked and becomes ‘asymmetric’, which translates into diastereotopic protons and carbons in the title compound.In the crystal of the title compound, pairs of molecules are linked by O—H⋯O and N—H⋯O hydrogen bonds, forming dimers with CrystalExplorer17.5   of 5-amino-2,4,6-triiodoisophthalic acid were dissolved in 15 mL of water, and the solution was cooled in an ice bath to 273 K, then 69 mg (1 mmol) of NaNO2 were added followed by 0.2 mL of HCl, and mixed for 1 h. The temperature of the mixture should not exceed 278 K.Azocoupling: NaOH  was added to a mixture of 1 mmol (66 mg) of malononitrile with 5 mL of water. The solution was cooled in an ice bath, and a suspension of 3,5-bis(methoxycarbonyl)benzenediazonium chloride (prepared according to the procedure described above) was added in two equal portions under vigorous stirring for 1 h. The precipitate was filtered off, recrystallized from methanol and dried in air. Crystals suitable for X-ray analysis were obtained by slow evaporation of an ethanol solution.The title compound: Yield 79% , yellow powder soluble in DMSO, methanol, ethanol and DMF. Analysis calculated for C13H9I3N4O5: C 22.90, H 1.33, N 8.22; found: C 22.87, H 1.30, N 8.18 %. ESI–MS: m/z: 636.88. IR (KBr): 3123 ν(NH), 2937 ν(NH), 2233 ν(CN) and 1707 ν(C=N) cm−1. 1H NMR : δ 1.02–1.06 , 3.42–3.47 , 7.25 and 7.32  and 11.21 . 1H, in 13C{1H} NMR : δ 18.56 (CH3), 56.78 (CH2), 85.37, 89.06 and 94.93 (3C–I), 96.80 (C=N), 109.71 and 109.91 (CN), 149.79 and 150.13 (CCOOH), 162.97 (C–NH), 169.48 and 169.79 (C=O).6.Uiso(H) values assigned as 1.2Ueq or 1.5Ueq(methyl only) of the parent atoms, with C—H distances of 0.97 (methylene) and 0.96 Å (methyl). The remaining hydrogen atoms bound to nitrogen and oxygen were located in difference-Fourier maps and refined with fixed positional thermal displacement parameters and with Uiso(H) values assigned as 1.2Ueq(NH) or 1.5Ueq(OH) of the parent atoms. One reflection, (001), affected by the incident beam-stop was omitted in the final cycles of refinement.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698902300676X/tx2072sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902300676X/tx2072Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902300676X/tx2072Isup3.cmlSupporting information file. DOI: 2286318CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: