L-alpha glycerylphosphorylcholine (GPC), a nutritional supplement, has been demonstrated to improve neurological function. However, a new study suggests that GPC supplementation increases incident stroke risk thus its potential adverse effects warrant further investigation. Here we show that GPC promotes atherosclerosis in hyperlipidemic Apoe−/− mice. GPC can be metabolized to trimethylamine N-oxide, a pro-atherogenic agent, suggesting a potential molecular mechanism underlying the observed atherosclerosis progression. GPC supplementation shifted the gut microbial community structure, characterized by increased abundance of Parabacteroides, Ruminococcus, and Bacteroides and decreased abundance of Akkermansia, Lactobacillus, and Roseburia, as determined by 16S rRNA gene sequencing. These data are consistent with a reduction in fecal and cecal short chain fatty acids in GPC-fed mice. Additionally, we found that GPC supplementation led to an increased relative abundance of choline trimethylamine lyase (cutC)-encoding bacteria via qPCR. Interrogation of host inflammatory signaling showed that GPC supplementation increased expression of the proinflammatory effectors CXCL13 and TIMP-1 and activated NF-κB and MAPK signaling pathways in human coronary artery endothelial cells. Finally, targeted and untargeted metabolomic analysis of murine plasma revealed additional metabolites associated with GPC supplementation and atherosclerosis. In summary, our results show GPC promotes atherosclerosis through multiple mechanisms and that caution should be applied when using GPC as a nutritional supplement. L-alpha glycerylphosphorylcholine (GPC) is a substrate for the synthesis of phosphatidylcholine (PC) and the neurotransmitter acetylcholine in the brain  choline (d9-choline), and d9-trimethylamine (d9-TMA) were purchased from Cambridge Isotope Laboratories . d6-choline  and  glycerylphosphorylcholine (d9-GPC) were chemically synthesized and purified in our lab, as previously reported [9]-betaine (d9-betaine) was purchased from C/D/N ISOTOPES INC . Rabbit polyclonal antibodies that recognize beta-GAPDH (#14C10), p44/42 MAPK (Erk1/2) (#4695S), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370S), p38 MAPK (#8690S), phospho-p38 MAPK (#9211S), NFκB p65 (# 8242S), and phospho-NFκB p65 (Ser536) (# 3033S) were purchased from Cell Signaling Technology . Horseradish peroxidase (HRP)–conjugated secondary antibodies that recognize rabbit IgG (#32460) was purchased from Thermo Fisher . All the solvents for mass spectrometry were purchased from Fisher Scientific , with HPLC grade and the other reagents purchased from Sigma, unless otherwise specified.dreported ,71. TMA added as internal standard.Five female and three male C57BL/6J mice were humanely euthanized and the intestine was cut into different segments: duodenum, jejunum, ileum, cecum, and colon. Each intestinal segment was weighed in a balance with a resolution of 0.1 mg and was cut into different pieces around 0.5–1 cm in length, where one half was used for incubation with dProteus mirabilis (ATCC 29906™), Escherichia fergusonii (ATCC 35469™), Lactobacillus acidophilus (ATCC 4356), and Escherichia coli strain Top10 (Invitrogen) were grown in LB medium in an incubator shaking at 250 rpm, 37 °C overnight. The bacterial suspension was spun down at 6000× g, 4 °C for 12 min and resuspended in ¼ volume of cold PBS for incubation with d9-GPC and d6-choline at a final concentration of 100 µM. P. mirabilis lysate was prepared by Microfluidizer , with lysozyme added in advance for incubation with d9-GPC and d9-choline.Apoe−/−) were weaned at 4 weeks of age and fed a standard chow diet , with 1.0% GPC added to the drinking water or regular drinking water. Mice were anesthetized with 300 mg/kg Ketamine + 30 mg/kg Xylazine prior to cardiac puncture for blood collection. The circulatory system was then perfused by intraventricular injection twice with 10 mL cold PBS prior to collection of the heart. Hearts were fixed and stored in 4% paraformaldehyde prior to frozen OCT sectioning, and the OCT section (6 µm thickness) was stained with Oil-Red-O and haematoxylin. Aortic root lesion area was quantified as the mean value of 6 sections [Apolipoprotein E knockout mice . The forward and reverse primers, cutC and 16S rRNA, were synthesized by Integrated DNA Technologies, Inc.  based on sequences reported in references [® Green Supermix  was used to conduct the PCR reaction in a 96-well plate, with 50 ng DNA added as template in each well. RT-PCR was done in StepOnePlus™ Real-Time PCR System (Applied Biosystems) with initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing and elongation at 60 °C for 1 min. The PCR products were confirmed by melting curves with denaturation ramp between 60 °C and 95 °C at 0.3 °C per minute, and the specificity of qPCR reaction was further confirmed by agarose gel electrophoresis. The 2−ΔΔCt method was used for the quantification of cutC gene with 16S rRNA gene as reference.RT-PCR of cecal bacteria encoding the choline TMA lyase ferences . iTaq™ U2 and H2.Cecal and fecal bacterial TMA lyase activity was determined by incubation of deuterium labeled chemicals with a structural formula containing TMA under anaerobic condition, as previously described . Incubat® dsDNA Assay Kit  according to the manufacturer’s instructions, and pooled and purified using the UltraClean® PCR Clean-up Kit  according to manufacturer’s instructions. High-throughput sequencing analysis of bacterial rRNA genes was performed on the purified, pooled sample using the Illumina MisSeq platform at the UCLA Genotyping and Sequencing Core, University of California, Los Angeles, as previously described [Bacterial DNA was extracted from mouse cecum using the PowerSoil DNA Isolation Kit . Amplification and sequencing of the V4 hypervariable region of the 16S rRNA gene was performed using the region-specific bacterial primers 515F and 806R. The PCR conditions consisted of an initial denaturation step of 94 °C for 3 min; 35 cycles of 94 °C for 45 s, 50 °C for 30 s, and 72 °C for 90 s, followed by 72 °C for 5 min. Replicate amplicons were quantified with Quant-iT™ PicoGreenescribed . De-multescribed , using eescribed . Qualityescribed  against Human fecal samples were collected from subjects participating in the Healthy Life in an Urban Setting (HELIUS) study, as previously reported . This stt test, Wilcoxon rank sum test, or one-way ANOVA was used to determine statistical significance where appropriate. Spearman’s or Pearson’s correlation was used to calculate the association between two variables. Plots of normalized concentration were drawn by the R program (version 4.0.5) with the ggplot2 package installed. Microbial composition at each taxonomic level was defined using the summarize taxa function in QIIME. Alpha diversity and beta-diversity (weighted UniFrac metrics) were calculated using MicrobiomeAnalyst [p < 0.05 was considered significant.Student’s eAnalyst ,81. LEfSeAnalyst  under theAnalyst  to extraeAnalyst . For all


Developing effective treatments for glioblastoma (GBM), a highly aggressive brain tumor that is resistant to current therapies, is an urgent medical need that can be addressed by the in-depth study of basic biology for the identification of relevant targets. The aim of the present study was to investigate the molecular mechanisms underlying the deregulation of protein synthesis associated with GBM progression and resistance to treatments. Our present work demonstrates the role of the RNA-binding proteins hnRNP H/F as key players in the control of protein synthesis in GBM through different overlapping mechanisms. Furthermore, our results show that hnRNP H/F potentiate cellular processes underlying the aggressive and resistant phenotype of GBMs, thus indicating hnRNP H/F as a potential target for therapeutic intervention.A-Raf kinase mRNA, which in turn modulates the MEK-ERK/MAPK signaling pathway. The underlying mechanism involves RNA G-quadruplex (RG4s), RNA structures whose modulation phenocopies hnRNP H/F translation regulation in GBM cells. Our results highlighted that hnRNP H/F are essential for key functional pathways regulating proliferation and survival of GBM, highlighting its targeting as a promising strategy for improving therapeutic outcomes. Deregulation of mRNA translation is a widespread characteristic of glioblastoma (GBM), aggressive malignant brain tumors that are resistant to conventional therapies. RNA-binding proteins (RBPs) play a critical role in translational regulation, yet the mechanisms and impact of these regulations on cancer development, progression and response to therapy remain to be fully understood. Here, we showed that hnRNP H/F RBPs are potent regulators of translation through several mechanisms that converge to modulate the expression and/or the activity of translation initiation factors. Among these, hnRNP H/F regulate the phosphorylation of eIF4E and its translational targets by controlling RNA splicing of the  Glioblastoma (GBM) is a grade IV brain tumor, which is one of the most aggressive primary brain tumors in humans . DespitemRNA translation is the most energetically consuming process in the cell, whose deregulation is widely recognized as a hallmark of cancer, supporting its development and progression ,6. BeingIn GBM cells, accumulating data has revealed that translational regulation is hijacked to promote tumor proliferation and/or develop resistance to treatments. Notably, the expression of several eIFs, including eIF3e ,19, 3c  and 5A , or RBPsA-Raf controls the Raf-MEK-ERK/MAPK pathway, resulting in eIF4E phosphorylation. This and other mechanisms affecting the expression of key translation factors allow hnRNP H/F to control global and selective translation to drive key functional pathways involved in GBM progression and response to treatments.Members of the hnRNP H/F family  are among the most deregulated RBPs in several types of cancers ,31, inclThe glioblastoma cells LN18, U251-MG and U87 were obtained from ATCC (CRL-2610), ECACC (#09063001) and SIGMA (#89081402-1VL), respectively. They were maintained in DMEM media containing 4.5 g/L glucose and supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin. Mycoplasma contamination was frequently assessed by PCR. Where indicated, cells were treated at 37 °C with cPDS , puromycin (Sigma P8833), cycloheximide (Sigma-Aldrich C7698), etoposide (Sigma-Aldrich E1383), with the indicated concentration and for the indicated time.TM RNAiMAX  according to the manufacturer’s instructions. In brief, cells were reverse-transfected with 2.5 nM of siRNAs which were synthesized by SIGMA. Cells were subsequently incubated at 37 °C for 72 h before harvesting and analysis. LN18 cells with stable silencing were generated by transduction of lentivectors , expressing shRNA control (SHC002), or shRNAs against hnRNP H and hnRNP F. All siRNA and shRNA sequences are available in siRNAs were transfected using the LipofectamineFor immunoblotting analysis, proteins were resolved on 12 or 7% denaturing polyacrylamide gels and were transferred to nitrocellulose membranes. The blots were blocked for 30 min with TBS-T-5% milk and then probed overnight with primary antibodies against hnRNP H/F , A-Raf full length , A-Raf short  p-ERK 1GAPDH reference mRNA, and relative levels of expression were quantified using the 2ΔΔCT method, where CT is cycle number at which the amount of amplified target reaches a fixed threshold. For RT-PCR, PCR conditions were 25 cycles of denaturing at 95 °C for 30 s, annealing for 20 s, and extension at 70 °C for 20 s. All primer sequences are available in A measure of 1 µg of total RNA was quantified using the Clariostar BMG (v.5.21 R4) combined with the MARS Clariostar Analysis Software (v.3.20 R2) and were reverse transcribed using the RevertAidH Minus First  following the manufacturer’s rules. qPCR analysis of cDNA (12.5 ng) was performed with the SybrGreen (KAPA KK4605) using the StepOne Applied Biosystems, Waltham, MA, USA (v2.2.2). Expression of indicated mRNAs was normalized to the 2, 0.5% NP40, 1 mM DTT) supplemented with 0.1% SDS. After centrifugation at 13,000 × g (4 °C) for 5 min, supernatant (nuclear soluble fraction) was transferred into a fresh tube. Cell extracts were treated with Benzonase (Millipore E1014) and DNase I  for 1 h at room temperature, and then precleared on protein-Sepharose beads for 1 h at 4 °C. BG4 , kindly provided by S. Balasubramanian) or hnRNP H/F  antibodies were incubated with 20 µL of slurry beads (washed and equilibrated in cell lysis buffer) for 1 h at 4 °C. A measure of 1 mg of cell extracts was added on beads and incubated on a wheel overnight at 4 °C. After five washes of the beads with cell lysis buffer, the immunoprecipitated proteins and RNAs were eluted in the NT2 buffer . Cell extracts were obtained as described in , except HPRT (reference mRNA) and compared with the RNA levels contained in the IgG control and input sample.Eluted samples from the immunoprecipitation were treated with proteinase K (Euromedex). RNAs were extracted with Phenol/Chloroform and resuspended in 10 µL of water. A measure of 4 µL was reverse transcribed using the RevertAidH Minus First (Thermo fisher) following the manufacturer’s indication. A 1/5 dilution of cDNA was used to analyze mRNA levels by qPCR with the SybrGreen . The quantities of mRNA contained in these mRNP complexes were then normalized to the quantity of g for 5 min and lysed in the lysis buffer ). Puromycin incorporation was detected using western Blot analysis.Cells were incubated with 10 µg/mL of puromycin (Sigma P8833) for 10 min at 37 °C. After 2 washes in ice-cold PBS, cells were scraped on ice in PBS, pelleted by centrifugation at 200 × 2, 1.5 mM KCl, 0.1 mg/mL CHX 20 U/mL RNaseOUT , 0.5% of Triton X-100, 0.5% sodium deoxycholate and 10 µL/mL of Protease Cocktail Inhibitor ]. A volume of lysate corresponding to 20 OD260nm was loaded on a continuous 5–50% sucrose gradient and subjected to ultracentrifugation at 222,228 × g in a SW41-Ti rotor for 2 h at 4 °C. Sucrose gradient was fractionated with an ISCO density gradient fractionation system  and the absorbance at 254 nm was registered. Fractions were flash-frozen immediately after fractionation and stored at −80 °C.Polysome profiling was performed as previously described . BrieflyGBM cells transfected with siRNAs or stably expressing shRNAs were harvested and counted with a coulter counter  every day for 7 days. After 72 h of siRNA transfection, cells were recovered and seeded in 6-well plates at different concentrations . Cells were incubated at 37 °C until colonies were visible with the naked eye (approximatively 10 days). Colonies were then fixed with 10% formalin for 10 min and incubated with 10% crystal violet oxalate  for 10 min at room temperature. Wells were washed with water until colonies were visible for counting. The plating efficiency was defined as the percentage of number of colonies formed over number of cells plated.6) were mixed with PBS/Matrigel (ratio 1:1) and subcutaneously injected in each flank of nude mice (n = 4 mice per condition). Tumor volumes were measured every day for 40 days. All procedures were performed in accordance with the guidelines set forth by the Johns Hopkins University Animal Care and Use Committee.Animal experiments were performed in the neurosurgical department of Johns Hopkins University in Baltimore. Briefly, LN18 cells . Subsequent analyses were performed using FlowJo software  After 72 h of siRNA transfection, LN18 cells were collected, washed in PBS and prepared for flow cytometry. For cell death, cells were incubated with 5 µL of AnnexinV-PE and 1 µL of SYTOXGreen solution at 1 µM for 10 min at room temperature and protected from light . For cell cycle analysis, cells were fixed with cold ethanol 70% and permeabilized with a solution of PBS/0.025% triton for 15 min on ice. Then, cells were labelled with propidium iodide (20 µg/mL) for 15 min at 37 °C with RNAse A at 10 µg/mL. Cells were kept on ice until the run on a flow cytometer  assays using nuclear extracts  pathway , we founA-Raf splicing and eIF4E phosphorylation  and transfected with control-, hnRNP H- and/or hnRNP F-specific siRNAs. Our data revealed that hnRNP H or hnRNP F depletion induced a significant 40% reduction in global translation rate comparable to the effect of downregulating the translational regulators hnRNP A1 and hnRNP I  Figure C,D. StriBased on the effect of hnRNP H/F on global translation C,D and cBased on the regulatory role of hnRNP H/F on metastasis-related mRNA expression A,B, and Since alterations in the mechanisms regulating translation affect cancer cell proliferation , we thenTo provide further insights into the effect of hnRNP H/F on GBM cell growth, we first assessed cell cycle changes in LN18 cells treated with either hnRNP H- and/or hnRNP F-specific or control siRNAs and followed propidium iodide (PI) staining using flow cytometry. Our data revealed that cells depleted for hnRNP H were accumulated in G2 phase (22.2% ± 2.9) compared with control cells (16.5% ± 0.7), while hnRNP F depleted cells accumulated in G1 phase (71.7% ± 3.9) compared with control cells (58.3% ± 0.7) . Of noteTo further confirm our in vitro data and explore whether the effect of hnRNP H/F on cell growth and proliferation finds an echo in the tumoral development in vivo, we developed a xenograft tumor model by subcutaneously injecting in each flank of a nude mouse, LN18 cells in which hnRNP H/F depletion was induced by shRNA. We found that downregulating hnRNP H and hnRNP F expression significantly and drastically decreased the volume of the tumors to such an extent that only few tiny tumors were recovered in the shRNA H condition D. AltogeRNA-binding proteins fine-tune gene expression by regulating sets of targets within specific post-transcriptional layers, in a coordinated manner, revealing their importance as a regulatory nexus to control cancer-related pathways ,13,14. BA-Raf mRNA in GBM  and assnstrated , our obsnRNP H/F , (ii) A-d an RG4  and  hninto RG4 , (iii) hinto RG4 —suggest While the role of hnRNP H/F in regulating alternative splicing is well established either at a genome wide scale ,42 or atA-Raf which impacts on translation regulatory pathways , respectively), and (2) A-Raf full length mRNA is overexpressed in GBM primary and recurrent solid tumors (mining of the TCGA splicing variants database), support a potential clinical importance of the hnRNP H/F–A-Raf–eIF4E axis in GBM, thus making hnRNP H/F a potential target for therapeutic intervention.Our work, in accordance with other findings , demonstTaken together, our data reinforce the notion that hnRNP H/F are an essential regulatory hub in GBM networks that induce a translational reprogramming affecting protein synthesis, either globally or specifically, and impact on GBM progression . Our wor


To validate the possible mechanism of GF, we used HepG2 cells with hydrogen peroxide treated oxidative stress and chronic exposure conditions via deteriorations of cellular SIRT1. Moreover, GF remarkably attenuated ECMs accumulations in transforming growth factor–β1-induced LX-2 cells relying on the SIRT1 existence. Taken together, GF attenuated liver fibrosis through AMPK/SIRT1 pathway as well as Nrf2 signaling cascades. Therefore, GF could be a clinical remedy for liver fibrosis patients in the future.Liver fibrosis, which means a sort of the excessive accumulation of extracellular matrices (ECMs) components through the liver tissue, is considered as tissue repair or wound-healing status. This pathological stage potentially leads to cirrhosis, if not controlled, it progressively results in hepatocellular carcinoma. Herein, we investigated the pharmacological properties and underlying mechanisms of Gardeniae Fructus (GF) against thioacetamide (TAA)-induced liver fibrosis of mice model. GF not only attenuated hepatic tissue oxidation but also improved hepatic inflammation. We further confirmed that GF led to ameliorating liver fibrosis by ECMs degradations. Regarding the possible underlying mechanism of GF, we observed GF regulated epigenetic regulator, Sirtuin 1 (SIRT1), in TAA-injected liver tissue. These alterations were well supported by SIRT1 related signaling pathways through regulations of its downstream proteins including, AMP-activated protein kinase (AMPK), p47 Liver fibrosis is provoked by chronic hepatic diseases and results from a disordered wound healing response . The patGardenia jasminoides Ellis) not only has been popularly applied to traditional medicine to treat hepatic disorders or to decrease various inflammation but also epidemically used as an excellent natural colorant. A total of 162 components have been separated and identified from GF [Gardeniae Fructus  to induce liver fibrosis of mice model, and our data elucidated new knowledge about anti-hepatofibrotic effects of GF. Additionally, we also provide the possible mechanisms GF which could mainly affect AMPK/SIRT1 pathway and/or Nrf2 signaling cascades on the liver fibrosis as well as chronic TAA exposure hepatic oxidative stress conditions. Sodium carbonate (Cat No. 222321), quercetin (Cat No. Q4950), dimethyl sulfoxide , silymarin , and thioacetamide (Cat No. 163678) were provided by Sigma Aldrich Co. . L-ascorbic acid was provided by Alfa Aesar . ®, Milford, MA, USA) using a reversed-phase C18 column (Phenomenex HPLC part: Luna 3μ C18(2) 100 Å, 150 mm × 4.6, Torrance, CA, USA). The mobile phase composition was as follows: solvent A (5% acetonitrile in deionized water with 0.05% formic acid), solvent B (100% acetonitrile with 0.05% formic acid). The gradient conditions; A:B = 90:10 (0 min) → 90:10 (2 min) → 80:20 (3 min) → 80:20 (6 min) → 0:100 (8 min) → 0:100 (10 min) → 90:10 (11 min) → 90:10 (14 min). The flow rate was 0.7 mL/min with UV absorption monitoring at 254 nm. The peak of geniposide was assigned by comparison of retention time and UV spectrum of authentic standard. Geniposide was detected as a major compound from the chromatogram of the extract. Representative UPLC chromatogram was illustrated in 2R) was calculated as 0.9994 . The GF used in this experiment was a medicinal herb produced in Goheung, Jellannam-do in 2019, and was supplied from Bonchowon . Dried GF (100 g) boiled with water (1 L) at 25 °C for 2 h. The powder of GF was a yield of 24.6% by weight and kept at −80 °C before use. GF power (1 mg) was dissolved in 2 mL of 100% methanol. The solution was centrifuged at 13,500 rpm for 3 min and the supernatant was collected for component analysis. We injected 3 μL of the sample into a Waters Acquity UPLC system (Watersn = 9 for each group): Normal, Control (TAA only), GF (200 mg/kg), and Silymarin (50 mg/kg), respectively. Mice in the normal group were intraperitoneally injected 0.9% normal saline orally administrated with distilled water (DW); the control group received TAA (i.p.) and DW ; the Silymarin group received TAA (i.p.) and Silymarin at 50 mg/kg/day (p.o.); the GF group received TAA (i.p.) and GF at 200 mg/kg/day (p.o.) for 8 weeks of experimental periods  were purchased from DBL . The mice were maintained at 22 ± 2 °C and controlled with a 12 h light/dark cycle and humidity (50 ± 5%). After 1 week of adaptation, a total of thirty-six mice were divided into 4 groups . The treatment drugs for liver fibrosis both GF (200 mg/kg/day) and Silymarin (50 mg/kg/day) were continuously administrated for 8 weeks 90 min prior to TAA injection. On the final day of the experiment, whole blood samples were collected from the abdominal vein and centrifuged at 4000 rpm for 10 min (at 4 °C), then stored in −80 °C freezer for further analyses. Liver tissues were removed immediately transferred 10% neutral formalin for the purpose of histological analysis and liquid nitrogen gas then stored at −80 °C for further biochemistry analysis, respectively.Liver tissue samples were fixed in 10% formalin then processed for embedding and sectioning at the Kyeongbook National University Hospital BMRI . Liver sections (3 μm thickness) were stained using hematoxylin and eosin (H&E), Masson’s trichrome (MT), and Sirius Red staining which were followed by the standard protocol. The images were captured using Olympus BX51 Microscope  and then analyzed using the I-Solution Lite software program . Liver enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by serum levels using a Transaminase CⅡ-Test . Serum ammonia was measured by ELISA kit  according to the manufacturer’s instructions.IHC analysis was performed for detecting Kupffer cells (F4/80), bile duct cells (CK-19), and Collagen type 3. We further completed IF analyses for observing 4-HNE, desmin, α-smooth muscle actin (α-SMA), and Collagen type 1, respectively . BrieflyMPO in serum was determined via a colorimetric kit of BioVision, Inc. . TBARS assay to evaluate malondialdehyde (MDA) was estimated according to the method of Mihara and Uchiyama .2 supplementary with 37 °C incubators, respectively.For validating possible mechanisms of GF on hepatic oxidation and fibrosis we cultured human blastoma cell line, HepG2, and hepatic stellate cells (HSCs) line, LX-2 cells, respectively. HepG2 cells were cultured in 10% fetal bovine serum (FBS) containing DMEM, and LX-2 cells were cultured 5% FBS contained DMEM under the condition of 5% CO2O2) during 6- and 24-h after pre-treatment with 50, 100, and 200 µg/mL of GF, then cells were washed with 10 mM PBS (pH 7.3) twice and fixed in 4% paraformaldehyde (PFA) or cell lysis buffers  according to each specific experimental condition. For hepatic oxidative stress condition, we treated 500 µM of hydrogen peroxide –β1 (10 ng/mL) for 18 h incubation after pre-treatment with GF (200 µg/mL) for 6 h and followed to the washing and fixing for further analysis, respectively. IHC or IF analysis using HepG2 cells or LX-2 cells, we used standard protocol. Briefly, the cells were washed with PBS  twice and fixed in 4% paraformaldehyde for 1 h at RT. Cells were washed with PBS twice and incubated with 0.3% Triton X-100 for 5 min at RT, then underwent PBS washing twice. Cells were treated 2.5% NHS which is known as blocking buffer for 1 h at RT and primary antibodies were treated then incubated at 4 °C overnight. After washing with PBS three times (each time for 10 min) at RT, we treated fluorescent conjugated secondary antibodies for IF analysis or HRP-conjugated secondary antibody for IHC, respectively.For IF analysis, we followed the further washing steps using twice PBS and one 0.05% PBST for 10 min, and IHC analysis we washed with PBS three times using PBS, respectively. After washing secondary antibodies for each experiment, the positive signals were detected by the development of AEC and performed counterstaining using hematoxylin. In the case of IF analysis, we completed the counterstaining by application of DAPI solution to the cells. Images were captured according to each experiment type as followed by the liver tissue sections.2, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, and freshly added 100 μM sodium vanadate, 1 mm PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin, respectively or NP-40 lysis buffer . For cytochrome c, COX IV, and GAPDH in either cytosolic or mitochondrial fraction, we obtained each fractionated sample using a commercially available kit .To obtain cell samples, we used the lysis buffer was composed of various compounds as follows; 50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, and 1250 μL protease inhibitor solution . The homogenates were incubated (4 °C for 20 min), and then 10% NP-40, respectively. After centrifugation with the above lysis buffer . Then the supernatant was collected as a cytosol sample, and the lysates were suspended with 20 mL ice-cold lysis buffer for intending to extract nucleus (50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 1% (v/v) glycerol, 0.1 mM PMSF, and 100 μL protease inhibitor solution, respectively) and suspended and incubated (4 °C for 30 min), then performed centrifugation . The supernatant was finally collected as a nuclear sample. Both cytosol and nuclear samples were stored at −80 °C for further experiments. To obtain cytosol samples of tissue, liver tissues were lysed with buffer consisting of follows; 0.1 mM EDTA, 10 mM HEPES (pH 7.8), 0.1 mM PMSF, 10 mM KCl, 1 mM DTT, 2 mM MgClw/v) skim milk solution for 1 h and visualized using ECL reagents of GE Healthcare . The bands were detected by Sensi-Q 2000 Chemidoc . Antibodies used in western blotting are shown in Samples containing 10–12 μg of protein were electrophoresed through 8–15% SDS-PAGE and transferred to a nitrocellulose membrane. Each membrane was blocked with 5% .t-test. Comparisons for more than two groups, we performed one-way or two-way ANOVA followed by Tukey’s post-hoc tests.All statistical data were expressed as mean ± SEM through the present manuscript by the performance of the Prism 9.2 software from GraphPad . Comparisons between two groups were performed using a two-tailed unpaired Student p < 0.001 for time-dependent manners of BW changes and among the groups, (p < 0.001 or 0.05 in p < 0.001), whereas GF 200 significantly decreased relative liver tissue mass as compared with the control group, but not Sily 50 . As expected, the control group significantly increased the positive signals of 4-HNE as compared with the normal group. Additionally, serum MPO activities in the control group were significantly increased as compared with the normal group (p < 0.001). This pathological alteration was well supported by abnormal oxidative stress status in both serum and hepatic protein levels of MDA, which is a marker of lipid peroxidation , whereas GF 200 significantly prevented from deterioration of two major antioxidant enzymes in the hepatic tissue against TAA mediated-hepatic tissue oxidation , while GF 200 significantly attenuated these positive signals as compared with the control group (p < 0.05 in p < 0.01); however, administration with GF 200 mg/kg exerted to decrease these pro-inflammatory cytokines as compared with the control group (p < 0.05 or 0.01 in p < 0.001); however, we also observed that GF 200 mostly normalized the above inflammatory target proteins’ abnormalities with statistical significance as compared with the control group , hepatic protein levels of IL-1β (p < 0.05), and phosphorated forms of NF-κB, IκBα, and p38 and iNOS protein levels which were evidenced by Western blot analysis . Representative ECMs molecules of IF or IHC against α-SMA, Collagen type 1, and Collagen type 3 were also abnormally enhanced through liver tissue by TAA injection (p < 0.001). These alterations, as our expectation, were significantly resolved in the GF 200 group as compared to the control group . Administration with GF 200 mg/kg not only significantly led to a decrease in TGF-β1 and TIMP-1, but also increase MMP-1 and MMP-13 as compared to the control group  in the control group as compared with the normal group (p < 0.05 or 0.01). The number of HSCs through the liver tissue was diminished in Sily 50 as compared with the control group , and H3K9Ac also well support the above alterations in hepatic protein levels of TAA-injected liver tissue by drastically increase manners compared to the normal group (p < 0.001). Administration with GF 200 mg/kg significantly normalized against these alterations in hepatic protein levels as compared with the control group (p < 0.01 for SIRT1 and H3K9Ac). Additionally, the up-streaming molecule of AMPKα, which is a p-LKB1 was significantly decreased in the control group as compared with the normal group, the GF significantly increased p-LKB1 as compared with the control group (p < 0.05). Regarding the molecular levels of the relationship between SIRT1 and AMPKα, we observed that AMPKα was drastically activated by the deterioration of SIRT1 (p < 0.05). We further observed that SIRT1 target oxidative stress-related proteins such as NOX2 and p47phox were significantly normalized by GF 200 against TAA-injected hepatic fibrosis (p < 0.05 or 0.01). Additionally, AMPKα targeted proteins which are NADPH activity relying on antioxidant enzymes Nrf2 and HO-1 were also deterred by TAA and remarkably recovered by GF 200 mg/kg -induced hepatocyte oxidation using HepG2 cells which is a human blastoma cell line. First, we intended to confirm the alterations of SIRT1 would be affected by time-dependent manner or not. Interestingly, we observed that chronic exposure of H2O2  significantly prevented the depletion of SIRT1 in cellular protein levels of the chronic exposure condition . Moreover, GF could block oxidative stress by the suppression of 4-HNE (p < 0.001). Along with this finding, the cell death signals were also normalized by GF by relieving apoptosis-related proteins, especially cytochrome c  GF showed strong antioxidant effects by enhancement of endogenous antioxidant components in the liver tissue especially recoveries of SOD and GPx. (2) GF could regulate Kupffer cell activation and their numbers by diminishing pro-inflammatory cytokines. (3) GF strongly led to inactivate HSCs activation by evidence of amelioration of ECM resolutions and pro-fibrogenic cytokines during liver fibrosis. The corresponded mechanisms for the above properties of GF may regulate AMPK/SIRT1 signaling pathway in hepatocytes and HSCs, especially focusing on the beneficial effects of SIRT1 in both cell types. Liver fibrosis is very a critical stage in the progression, which can decide to reserve normal stage or its advanced pathological stage such as liver cirrhosis or HCC, respectively. Nevertheless many trials to develop anti-hepatofibrosis therapeutics, there is no effective way existed in the world. From our study, we believe that GF would be one of the potential drug candidates to care for liver fibrosis. Therefore, further studies would be acquired to prove the safety and toxicity for clinical uses in the upcoming future.


Background Overactivation of human epidermal growth factor receptor 3 (HER3) triggers multiple intracellular pathways resulting in tumor cell survival. This Phase 1 study assessed the safety, efficacy, and pharmacokinetics (PK) of seribantumab, a fully human anti-HER3 monoclonal antibody. Methods Adult patients with advanced or refractory solid tumors were treated in six dose cohorts of seribantumab: 3.2, 6, 10, 15, or 20 mg/kg weekly, or 40 mg/kg loading dose followed by 20 mg/kg weekly maintenance dose (40/20 mg/kg) using a modified 3 + 3 dose escalation strategy with cohort expansion. Primary objectives were identification of a recommended Phase 2 dose (RP2D) and determination of objective response rate. Secondary objectives were assessment of safety, dose-limiting toxicities, and PK. Results Forty-four patients  were enrolled. Seribantumab monotherapy was well tolerated with most adverse events being transient and mild to moderate (grade 1 or 2) in severity; maximum tolerated dose was not reached. The highest dose, 40/20 mg/kg, was identified as RP2D. Best response was stable disease, reported in 24% and 39% of patients during the dose escalation and expansion portions of the study, respectively. Seribantumab terminal half-life was ≈100 h; steady state concentrations were reached after 3–4 weekly doses. Conclusions Seribantumab monotherapy was well tolerated across all dose levels. Safety and PK data from this study support further seribantumab investigations in genomically defined populations.Clinical trial registration NCT00734305. August 12, 2008. The human epidermal growth factor receptor  family includes four transmembrane receptor tyrosine kinases: epidermal growth factor receptor ERBB1), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4) ), abdominal pain, acute pancreatitis, malignant neoplasm progression, tumor pain, confusional state, renal failure, and hypotension (4% [1/25] each). Of these, the grade 4 confusional state, occurring in a patient receiving the lowest seribantumab dose, 3.2 mg/kg, led to treatment discontinuation. The etiology of the AE was uncertain, and it was considered possibly related to seribantumab due to the time course and therefore scored as a DLT. In this patient, confusion was noticed on Day 13 and on Day 15 the patient was admitted as an in-patient and seribantumab treatment was withheld. Confusion worsened over the following days, then resolved spontaneously on Day 26 without sequelae. No other DLTs were reported in this or other dose cohorts, including the highest dose studied, 40/20 mg/kg.The MTD was not reached in the dose escalation portion, and the 40/20 mg/kg dose was considered well tolerated and chosen for the dose expansion portion of the study.In the dose expansion portion of the study, the median duration of exposure was 7  weeks. All patients experienced at least one TEAE and the rate of grade ≥ 3 TEAEs was 50% (9/18) (Table All-cause SAEs occurred in 44% (8/18) of patients in the dose expansion portion. These included lower abdominal pain, acute pancreatitis, vomiting, disease progression, pyrexia, exertional dyspnea, pleural effusion, arrythmia, dehydration, flank pain, malignant neoplasm progression, hydronephrosis, and ureteric obstruction (6% [1/18] each). None of these events were deemed related to seribantumab.All-cause TEAEs in 22 patients receiving the seribantumab 40/20 mg/kg dose across the two study portions are summarized in Online Resource 1. All patients experienced at least one TEAE and the rate of all-cause grade ≥ 3 TEAEs was 45% (10/22). At least one TEAE related to seribantumab occurred in 77% (17/22) of patients. A total of 41% (9/22) of patients reported SAEs; 5% (1/22) of patients had a grade ≥ 3 TEAE related to seribantumab.trough]) increased over time in a dose-dependent manner.The PK of seribantumab following the first dose and subsequent doses was analyzed in serum samples Fig. . The areVolume of distribution for the seribantumab 40/20 mg/kg dose following the first dose was estimated at 4.42 L (95% CI: 2.67‒7.32) with no dose dependency observed Fig. F. FollowIn further PK modelling based on historical simulation data, a weight-based dosing of seribantumab 40 mg/kg or 20 mg/kg was determined to correspond to a fixed dose of 3 g or 1.5 g, respectively, when administered on a similar frequency, and was associated with comparable maximum, minimum and average steady state concentrations, and variability. Associations between sex, weight, and seribantumab clearance were found to be significant; however, no significant association was seen between weight and volume.Based on prior preclinical investigations of seribantumab, the maximal inhibition of tumor growth and HER3 is known to be achieved at seribantumab trough concentrations ranging from 100 to 400 mg/L . Specifitrough was estimated at approximately 200 mg/L. These findings are also consistent with the PK data from a previous Phase 1 study of seribantumab in combination with an oral pan-phosphoinositide 3-kinase (PI3K) inhibitor [In the PK analyses of this study, the 40/20 mg/kg dose was shown to result in serum trough concentrations remaining above 100 mg/L in the majority of patients treated . At least three treatment cycles were received by 20% and 40% of patients in the dose escalation and dose expansion portions, respectively. One patient in the dose expansion portion received 12 cycles of treatment.No tumors showed a CR or PR and, therefore, ORR was 0%. Best response of SD was observed in 24% (6/25) of patients in the dose escalation and 39% (7/18) of patients in the dose expansion portion (Table Median PFS estimate was 7.1 (95% CI: 4.7‒7.4) weeks in the dose escalation and 7.1 (95% CI: 6.6‒15.9) weeks in the dose expansion portion of the study. The longest median PFS of 14.1 (95% CI: 7.3‒19.1) weeks was observed in the 40/20 mg/kg cohort of the dose escalation. The longest individual PFS observed was 47.9 weeks in a patient with ovarian cancer who had a best response of SD and received 12 cycles of seribantumab treatment.The objectives of this Phase 1 study were to establish the RP2D and assess the safety, PK, and efficacy of seribantumab monotherapy in patients with advanced or refractory solid tumors. A total of 43 patients with > 10 different tumor types were treated with seribantumab at six dose levels.Most AEs observed in this study were transient and mild to moderate (grade 1 or 2) in severity. MTD was not reached at the seribantumab dose levels studied and there was no dose dependency to AEs or TEAEs. Only one DLT, grade 4 confusional state that was considered possibly related to seribantumab, occurred with the lowest dose (3.2 mg/kg). Seribantumab 40/20 mg/kg, the highest dose studied, was well tolerated and confirmed as the RP2D in the dose expansion portion of the study, supported by PK findings from prior preclinical and clinical studies , 10. OveTargeted therapies have changed the oncology treatment landscape in recent years, moving toward personalized medicine and customized treatment strategies for patients with cancers driven by genomic alterations . TargetiFusions of NRG1, the activating ligand of HER3, are rare genomic alterations found in 0.2%‒0.5% of all solid tumors and have been identified in > 10 unique tumor types , 16. SinThis expansion of knowledge has led to a rational adjustment in the seribantumab clinical development program, setting focus on solid tumors driven by NRG1 fusions , 21. DatWhen this Phase 1 study was performed, genomic biomarkers were rarely used to guide patient selection for targeted therapy. As a result, the patient population was heterogenous and included patients with tumors refractory to previous treatments, regardless of the primary site or histology. Additionally, although the tumors were initially analyzed for predefined biomarkers (data not reported), including HER3 protein and NRG1 mRNA levels, they were not analyzed for genomic alterations. Subsequent randomized Phase 1 and 2 studies identified detectable NRG1 levels as an important biomarker associated with seribantumab mechanism of action and potential clinical benefit when used in combination with standard of care agents , 22, butThe results presented here demonstrate that seribantumab monotherapy is generally well tolerated and associated with manageable AEs across multiple tumor types in a heavily pre-treated patient population. The safety and PK profile of seribantumab described here, combined with the demonstrated preclinical activity in tumor models with NRG1 fusions, support a tumor agnostic strategy for further development of seribantumab in patients with tumors driven by NRG1 fusions. These findings warrant further clinical investigations in these patients who currently have limited treatment options. A Phase 2 study  is ongoing and currently enrolling patients with advanced solid tumors harboring NRG1 fusions [


LPAR2) plays a role in carcinogenesis; however, the exact role of LPAR2 in tumors remains elusive. This study aims to explore the correlation between LPAR2 expression with tumor prognosis and immune infiltration in pan-cancers.Lysophosphatidic acid (LPA) and its receptors play a key role in regulating cancer progression. Upregulation of LPA receptor 2 , Tumor Immune Estimation Resource (TIMER), and UALCAN databases. The effects of LPAR2 on the clinical prognosis in pan-cancer were examined using the Kaplan–Meier plotter (KM plotter) as well as Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, and Human Protein Atlas (HPA) databases. Moreover, the R software program was applied for validation of expression and prognostic value of LPAR2 in tumor patients in the Cancer Genome Atlas (TCGA) dataset and the Gene Expression Omnibus (GEO) database. The relationship between the expression level of LPAR2 and the clinical and molecular criteria of head and neck squamous cell carcinoma (HNSC) and kidney renal clear cell carcinoma (KIRC) was analyzed using UALCAN, whereas the relationship between LPAR2 expression and prognosis in patients with HNSC and KIRC with different clinical characteristics was examined using the KM plotter. Furthermore, the correlation between LPAR2 expression and tumor immune infiltration was examined using TIMER. The correlation between LPAR2 expression and gene markers of tumor immune infiltrates was analyzed using TIMER and GEPIA. In addition, the cBioPortal for Cancer Genomics was used to calculate the mutations, methylations, and altered neighbor genes of LPAR2.The expression of LPAR2 was significantly correlated with the outcome of multiple types of cancer, especially HNSC and KIRC. Furthermore, high expression of LPAR2 was significantly associated with various immune markers in the immune cell subsets of HNSC and KIRC.The expression of LPAR2 plays significantly different prognostic roles in HNSC and KIRC possibly owing to its association with different immune markers. LPAR2 is correlated with tumor immune cell infiltration and is a valuable prognostic biomarker for HNSC and KIRC. However, further experiments are required to validate these findings.High expression of The online version contains supplementary material available at 10.1186/s41065-022-00229-w. LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, and LPAR6, which can be grouped into two subfamilies, namely, endothelial differentiation gene (EDG) family (LPAR1–3) and purinergic receptor family (LPAR4–6) [Lysophosphatidic acid  is a bioactive glycerophosphatidic acid, which is a naturally occurring lysophospholipid and is abundantly found in the human body , 5. LPA LPAR4–6) . LPARs cLPAR4–6) . The LPALPAR4–6) , 10. SevLPAR4–6) , 11. OwiLPAR4–6) . MultiplLPAR4–6) , 14.LPAR1 and LPAR3 in several tumors, studies on LPAR2 are limited [LPAR2 is aberrantly expressed in several tumors, including breast, colorectal, kidney, and pancreatic cancers [LPAR2 regulated cell–cell adhesion of neural crest cells by internalizing N-cadherin downstream of LPAR2 [LPAR2 is significantly associated with LPA-induced expression of interleukin (IL)-6 and IL-8, which promoted breast cancer progression [LPAR2 in tumors appears diverse and remains unclear [Although many studies have described the expression and function of  limited . Several cancers –19, and  cancers . A recenof LPAR2 . In addigression , 23. How unclear .LPAR2 and its relationship with pan-cancer prognosis using the Oncomine, TIMER, UALCAN, GEPIA, KM plotter and HPA databases, as well as expression and survival analysis of LPAR2 in the TCGA and GEO data was validated by R software. Furthermore, we examined the relationship between LPAR2 expression and the clinical and molecular criteria of HNSC and KIRC using UALCAN. Subsequently, we investigated the relationship between LPAR2 expression and the prognosis of patients with HNSC and KIRC with different clinical characteristics using the KM plotter. In addition, we analyzed the correlation between LPAR2 and tumor-infiltrating immune cells in the microenvironment of pan-cancer using TIMER and GEPIA. Lastly, we used the cBioPortal for Cancer Genomics online tool to analyze the alterations, mutations, methylations, and pathways of LPAR2. Therefore, in this study, we demonstrated a potential mechanism of action of LPAR2, examined the prognostic role of LPAR2 in HNSC and KIRC, and identified LPAR2 as a key factor in regulating the immune microenvironment of HNSC and KIRC. The overall design and workflow of this study is presented in Fig. In this study, we systematically investigated the expression of LPAR2 in pan-cancer and normal tissues using Oncomine, we found that LPAR2 expression was higher in bladder, brain and central nervous system (CNS), breast, colorectal, kidney, and lung cancers and lymphoma than in normal control tissues , breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), HNSC, KIRC, liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), and uterine corpus endometrial carcinoma (UCEC) than in the adjacent normal tissues  and thyroid carcinoma (THCA) than in the adjacent normal tissues , glioblastoma multiforme (GBM), HNSC, KIRC, kidney renal papillary cell carcinoma (KIRP), LIHC, LUAD, LUSC, PRAD, READ, STAD, and UCEC than in normal control tissues , STAD  and KIRC  , CESC , HNSC , TYHM , UCEC  and BRCA  , PRAD , and CHOL   and THYM   and other tumors , as well as with worse prognosis with KIRC (P < 0.0001) , KIRC  and MESO  , OV  and STAD     but with improved OS in patients with HNSC (P = 0.0023)  Fig.  J–K. TheLPAR2 expression plays significantly different prognostic roles in HNSC and KIRC, we used UALCAN and TCGA to examine the relationship between LPAR2 expression and the clinical characteristics of patients with HNSC and KIRC. For the criterion of tumor stage, we found that LPAR2 expression was significantly higher in patients with stage 1–4 HNSC than in patients in the control group (P < 0.001) ; however, there was no significant difference in LPAR2 expression between the Asian patients with HNSC and those in the control group (P > 0.05)  , 41–60 years (P < 0.001), 61–80 years, and 81–100 years (P < 0.001) , irrespective of tumor grade, HPV expression status, nodal metastasis status, and mutation status  Fig. D. For th05) Fig. C. In add01) Fig. B in the 01) Fig. A. These LPAR2 expression was upregulated in patients with tumor stages 3 and 4 (P < 0.001)  ; whereas there was no significant difference in LPAR2 expression between the Asian patients with KIRC and those in the control group (P > 0.05)  , 41–60 years (P < 0.01), and 61–80 years (P < 0.001) but not in the age group of 81–100 years (P > 0.05) ; nonetheless there was no significant difference between the mRNA expression of LPAR2 in patients with grade 1–2 KIRC and those in the control group (P > 0.05)   Fig. D. Howeve05) Fig. D. Simila05) Fig. C. In add01) Fig. B. MeanwhLPAR2 expression in HNSC and KIRC, we assessed the relationship between mRNA expression of LPAR2 and prognosis in patients with HNSC and KIRC with different clinical characteristics in KM plotter. Higher mRNA expression of LPAR2 was associated with better OS in HNSC tumor stage 2–4 . However, no significant correlation was observed between the mRNA expression of LPAR2 and OS in patients with HNSC tumor stage 1 (P > 0.05) ; however, no significant correlation was found between LPAR2 expression and OS in women with HNSC (P = 0.5)  but not in the Black/Asian patients (P > 0.05)  and grade 3     , tumor stage 3 , and tumor stage 4    but not in the Black/Asian patients (P > 0.05)  and women  with KIRC    Fig. A, C, D b05) Fig. B. In add05) Fig. I-J. LPARIRC Fig. G–H. In a41) Fig. K–M and i56) Fig. N–O.Fig. LPAR2 expression influenced the prognosis of patients with HNSC of high stage and grade. Upregulated expression of LPAR2 was beneficial to men with HNSC or patients with low LPAR2 mutation burden and was significantly associated with prognosis in White patients with HNSC and KIRC.These results suggested that LPAR2 expression and the degree of immune cell infiltration in HNSC and KIRC , B cell infiltration , and CD4 + T cell infiltration  but not with the infiltration of CD8 + T cells, macrophages, neutrophils, and DCs in patients with HNSC , B cell infiltration , CD4 + T cell infiltration (R = 0.242 P = 1.46e-07), neutrophil infiltration , and DC infiltration     Fig. A but not03) Fig. A. We fur45) Fig. B, and a 01) Fig. B. These LPAR2 is important for regulating immune cell infiltration in HNSC and KIRC, we assessed the relationship between LPAR2 expression and immune cell infiltration based on the immunological markers of HNSC and KIRC using the TIMER and GEPIA databases. In addition, we evaluated the relationship between LPAR2 expression and several immunological marker subsets, including total T cells, B cells, CD8 + T cells, tumor-associated macrophages (TAMs), monocytes, M1 and M2 macrophages, natural killer (NK) cells, neutrophils, DCs, T follicular helper (Tfh) cells, type 1 T helper (Th1) cells, Th2 cells, regulatory T cells (Tregs), Th17 cells, and exhausted T cells. All results were adjusted based on tumor purity. The results demonstrated a significant positive association between LPAR2 expression and B cell markers (CD19 and CD79A), M1 macrophage markers (INOS and IRF5), neutrophil markers (CD11b), Th2 markers (STAT6 and STAT5A), Tfh markers (BCL6), and T-cell exhaustion markers (CTLA4) in patients with HNSC , total T cell markers , B cell markers (CD19 and CD79A), monocyte markers (CD86 and CD115), TAM markers (CD68 and IL10), M1 macrophage markers (IRF5), M2 macrophage markers , neutrophil markers (CD11b and CCR7), NK cell markers (KIR2DL4), DC markers , Th1 markers , Th2 markers , Tfh markers (BCL6 and IL21), Treg markers , and T-cell exhaustion markers  , DC markers (BDCA-4), and Treg markers (STAT5B) in KIRC , T cells , B cells (CD19 and CD79A), monocytes (CD86 and CD115), TAMs (CD68), M1 macrophages (IRF5), M2 macrophages (VSIG4), neutrophils (CD11b and CCR7), DCs, Th1 cells , Th2 cells (STAT5A), Tfh cells (BCL6), Tregs , and exhausted T cells   datasets. LPAR2 mutations and amplifications were found in 3 of 528 patients with HNSC but not in 537 patients with KIRC , M1 macrophages (INOS and IRF5), neutrophils (CD11b), Th2 cells (STAT6 and STAT5A), Tfh cells (BCL6), and exhausted T cells (CTLA4) in HNSC  [https://www.ncbi.nlm.nih.gov/geo/)  [The UCSC Xena dataset was used to acquire TCGA expression and clinical information (etadata) . Datasetv/geo/)   and procv/geo/)  , 43. TheLPAR2 in pan-cancer were analyzed using the online cancer microarray database (Oncomine) (www.oncomine.org). The Student’s t-test was used to compare the mRNA expression of LPAR2 between normal and cancer samples. P-value was used to characterize significant differences. The fold change was 1.5, and the cut-off P-value was 0.0001.The expression data of 715 genes were obtained from 86,733 samples and the mRNA expression levels of https://cistrome.shinyapps.io/timer/) database comprises six tumor-infiltrating immune cell subsets [LPAR2 in various cancer types and investigate the relationship between LPAR2 expression and the degree of infiltration of specific immune cell subsets. Furthermore, differences in the survival of patients with cancer based on gene expression or immune cell infiltration were examined using KM survival analysis. Lastly, the correlation between the expression of LPAR2 and that of specific immune markers was examined.The Tumor Immune Estimation Resource (TIMER)  is an interactive web resource used for analyzing publicly available cancer omics data [LPAR2 in different cancer and normal samples using the TCGA data and investigate the relationship between LPAR2 expression and different clinical characteristics. In addition, the prognostic value of LPAR2 in pan-cancer and the relationship between LPAR2 expression and the prognosis of patients with different clinical characteristics were analysed.UALCAN (sortium) . In thishttp://kmplot.com/analysis/) is an online database, which contains microarray gene expression data and survival information derived from the European Genome-Phenome Archive, Gene Expression Omnibus (GEO), and TCGA. It is used to assess the influence of multiple genes on the survival rate in 21 cancer types in a large number of samples [LPAR2 in pan-cancer and investigate the relationship between LPAR2 expression and the prognosis of patients with different clinical characteristics.The KM plotter ( samples . In thishttp://gepia.cancer-pku.cn/index.html) uses standard processing pipelines to analyze the RNA-sequencing expression data of 8,587 normal samples and 9,736 tumors from the GTEx and TCGA projects [http://gepia2.cancer-pku.cn/#index) is an updated version of GEPIA [LPAR2 and pan-cancer prognosis as well as the relationship between the expression of LPAR2 and that of the markers of immune cell infiltration.GEPIA (projects . GEPIA2 of GEPIA . In thiswww.proteinatlas.org) was used to analyze the protein expression of LPAR2 in HNSC, KIRC, and normal tissues [LPAR2 in HNSC, KIRC, and their corresponding normal tissues. In addition, the relationship between the protein expression level of LPAR2 and the prognosis of patients with HNSC and KIRC was examined.The Human Protein Atlas (HPA) database  is used to analyze, visualize, and download cancer genomics datasets [LPAR2 analysis, which contained histopathological data of 528 patients with HNSC and 537 patients with KIRC. The genomic profiles were evaluated via the Genomic Identification of Significant Targets in Cancer (GISTIC) analysis and included the assessment of mutations, methylations, mRNA expression z-scores (RNA Seq V2 RSEM), protein expression z-scores (RPPA), and putative copy number alterations (CNAs). Co-expression was evaluated according to the instructions provided on cBioPortal.The cBioPortal for Cancer Genomics tool , and Addtional file 1: Table S1. Clinical characteristics of patients in HPA.Addtional file 2: Table S2. Clinical characteristics of patients with HNSC in HPA.Addtional file 3: Table S3. Clinical characteristics of patients with KIRC in HPA.Addtional file 4: Figure S1. Kaplan-Meier survival curves comparing the high and low expression of LPAR2 in different types of cancers in the Kaplan-Meier plotter databases(A-AH).Addtional file 5: Figure S2. Kaplan-Meier survival curves comparing the high and low expression of LPAR2 in different types of cancer in GEPIA databases(A-BB).Addtional file 6: Figure S3. Kaplan-Meier survival curves comparing the high and low expression of LPAR2 in different types of cancer in UACLAN databases(A-S).Addtional file 7: Figure S4. Kaplan-Meier survival curves comparing the high and low expression of LPAR2 in different types of cancer in TCGA databases(A-AG).Addtional file 8: Figure S5. Kaplan-Meier survival curves comparing the high and low expression of LPAR2 in HNSC and KIRC from GEO databases and the paired ROC curves of measuring the predictive value(A-D).


Dictyostelium, with findings providing important advances in the study of autophagy-related pathologies such as neurodegenerative diseases. Here, we utilize this model to analyze a role for MCFAs in regulating autophagy. We show that treatment with decanoic acid but not octanoic acid induces autophagosome formation and modulates autophagic flux in high glucose conditions. To investigate this effect, decanoic acid, but not octanoic acid, was found to induce the expression of autophagy-inducing proteins (Atg1 and Atg8), providing a mechanism for this effect. Finally, we demonstrate a range of related fatty acid derivatives with seizure control activity, 4BCCA, 4EOA, and Epilim , also function to induce autophagosome formation in this model. Thus, our data suggest that decanoic acid and related compounds may provide a less-restrictive therapeutic approach to activate autophagy.Ketogenic diets, used in epilepsy treatment, are considered to work through reduced glucose and ketone generation to regulate a range of cellular process including autophagy induction. Recent studies into the medium-chain triglyceride (MCT) ketogenic diet have suggested that medium-chain fatty acids (MCFAs) provided in the diet, decanoic acid and octanoic acid, cause specific therapeutic effects independent of glucose reduction, although a role in autophagy has not been investigated. Both autophagy and MCFAs have been widely studied in  Autophagy is a key homeostatic process required to degrade cytosolic proteins and organelles, with alterations in this pathway associated with a range of neurological disorders. Evidence suggests that impaired autophagy contributes to epileptogenesis ,2,3,4, aDietary therapies, such as ketogenic diets, are widely considered to be safe and inexpensive methods of activating autophagy. This process has been shown in the treatment of epilepsy ,11, wherDictyostelium [Dictyostelium as a well-established model organism in the field of epilepsy research [The therapeutic mechanism of the MCT diet has recently been proposed through activity of decanoic acid, initially from studies based in ostelium , but theostelium ,20. Multostelium , the inhostelium , the dirostelium ,23, and ostelium . Althougostelium . Howeverresearch ,28,29,30Dictyostelium and humans. In Dictyostelium, macroautophagy (hereafter referred to as autophagy) is required to liberate nutrients during starvation, as well as in the degradation of proteins, and in response to pathogen infection [Dictyostelium has been widely used as a model to study autophagy with findings from this organism providing important advances in the study of autophagy-related pathologies [Dictyostelium has demonstrated a link between the neurodegenerative disease, Chorea-acanthocytosis (ChAc), and autophagy [Dictyostelium have suggested a role for protein sequestration in neuronal malfunction [Dictyostelium highlights the value of this model to research this conserved cellular process.Macroautophagy, the most prevalent form of autophagy, is conserved between nfection ,32,33. Dhologies ,35,36,37utophagy , with fuutophagy . In addifunction ,41. The Dictyostelium in autophagy research, there are several well-established methods for assessing autophagy in this model. Fluorescence microscopy is widely employed to visualize autophagy structures in the cell, with Atg8 (microtubule-associated protein 1A/1B-light chain 3 (LC3) in mammals) tagged to GFP (GFP-Atg8) used extensively in Dictyostelium as a fluorescent marker [Dictyostelium an ideal model for the study of autophagy.Due to the widespread use of t marker . GFP-Atgt marker ,43. An it marker ,45. Anott marker . Autophat marker . Quantift marker ,47. One t marker . The avaDictyostelium, initially through a significant increase in the number of autophagosomes, visualized using live cell fluorescent microscopy to quantify GFP-Atg8 positive structures. We further demonstrate that decanoic acid increases autophagic flux and that decanoic acid treatment leads to an increase in the expression of two autophagy genes in wild type cells, suggesting a role for medium-chain fatty acids in inducing autophagy in Dictyostelium. We further assess the effects of fatty acid derivatives on autophagy in Dictyostelium to establish that a range of related fatty acid derivatives with established seizure control activity also function to induce autophagy in this model.In this study, we demonstrate that decanoic acid activates autophagy in 6 cells/mL in 2 mL of HL5 media  in six-well plates. Cells were treated with decanoic acid (60 μM), octanoic acid (60 μM), or DMSO control for 22 or 2 h to provide final treatment durations of 24 and 4 h. Autophagy inducer AR-12 , or DMSO control, was added, and shaking was continued for 1 h before addition of protease inhibitors  at a final concentration of 2.5-fold or HL5 control. Shaking was continued for 1 h before cells were removed  to tubes on ice. For live-cell imaging, cells were centrifuged for 3 min at 500 g at 4 °C and resuspended in KK2  and were imaged under a layer of 1% KK2 agar. Time in KK2 and time under agar were kept constant between each condition (20 min in KK2 and 3 min under agar). Cells were imaged on an Olympus IX71 wide-field fluorescence microscope. Images were captured using a Micropix camera, model Elite 2. For measurement of Atg8-positive structure size, cells were analyzed using ImageJ, and cell size was measured across the largest diameter. Thirty cells were analyzed per experiment.Cells expressing GFP-Atg8 were set up at a density of 1.33 × 10g at 4 °C and lysed in RIPA buffer (1 × 108 cells/mL) containing protease inhibitor , before mixing 1:1 in 2× laemmli buffer resulting in 5 × 107 cells/mL. Lysates were boiled in laemmli at 95 °C for 5 min before vortexing and centrifuging at max speed for 5 min. Samples (8 µL) were fractioned using a 12.5% gel and transferred to a PVDF membrane  by wet transfer using transfer apparatus according to the manufacturer’s protocols . After incubation with 5% BSA  in PBS  for 60 min, the membrane was incubated in 5% BSA in PBS with antibodies against GFP  at 4 °C overnight. Membranes were washed in PBST and incubated with a 1:10,000 dilution of odyssey goat anti-rat IR DYE 800  and 1 in 10,000 streptavidin  in 5% BSA in PBS for 1 h. Blots were washed in PBST and visualized using the Odyssey CLx imager .Cells expressing GFP-Atg8 were treated as described above. Following removal from the six-well plate, cells were centrifuged for 3 min at 500× 7 cells/mL and rotated for 1 h at 110 rpm. Cells were pulsed with 60 nM of cAMP  every 6 min for 4 h, in the presence or absence of the compound (60 µM decanoic acid or octanoic acid). Cells were basalated by the addition of caffeine  to 5 mM, with rotation at 200 rpm for 30 min, before being washed twice in cold DB buffer and resuspended to 2 × 107 cells/mL. For time-lapse imaging of PIP3 production, 360 µL chemotactically competent cells expressing PHcrac-GFP at 5 × 105 cells/mL were placed into a well of an eight-well chambered cover glass  was added to one sample at a final concentration of 100 µM) and left to adhere for 10 min. Cells were stimulated by adding cAMP, to a final concentration of 1 µM, and images were captured every 2 s for 1 min.Cells expressing PHcrac-GFP were made chemotactically competent as described . Briefly7 cells/mL were transferred to a beaker shaking at 150 rpm, and cAMP was added to a final concentration of 1 µM. At time points of 10, 20, 30, 60, and 120 s, 40 µL of cells were transferred into microfuge tubes containing 10 µL of 5 × SDS sample buffer and were heated to 95 °C for 5 min. The p-PKC (pan) antibody was used to detect the phosphorylation state of the activation loops of PKBR1 (T309) and PKBA (T278) [Chemotactically competent cells were prepared in the presence or absence of the compound (60 µM decanoic acid or octanoic acid) as described above, with the addition of PI3K inhibitor LY294002  to one sample at a final concentration of 100 µM for the final 15 min of the preparation. For cAMP stimulation, 0.5 mL of chemotactically competent cells at 2 × 10A (T278) . Brieflyatg8a and atg1, and the gapdh gene was used as an internal control. qPCR was carried out using SYBR® Green JumpStart Taq ReadyMix , and relative quantification was carried out using the 2−ΔΔCt method.Exponentially growing cells were treated for 1, 4, or 24 h with either decanoic acid (60 μM), octanoic acid (120 µM), or solvent (DMSO) control. RNA was extracted from these samples using the RNeasy mini kit  according to the manufacturer’s protocol. DNA was removed from the RNA using the DNA-free kit  according to the manufacturer’s protocol. cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit using oligo (dT)18 primers  according to the manufacturer’s protocol. qPCR was carried out using primers within the genes for t-tests were used to assess the significance between independent samples from two groups. The Kruskal–Wallis test with Dunn’s post hoc test was used to test significance between three or more independent groups of data. Statistical analysis was carried out using GraphPad Prism Software.Two-tailed non-parametric Mann–Whitney Dictyostelium. In these experiments, the incorporation of the autophagosome-associated Atg8 protein, linked to a fluorescent marker, was monitored, where GFP-Atg8-labeled autophagosome can be rapidly assessed (p ≤ 0.01)  B,C. This ≤ 0.01) B,D. StarDictyostelium during growth, in high glucose conditions. To do this, we quantified the average number of GFP-Atg8 structures following acute (4 h) or chronic (24 h) treatment with these fatty acids or with an autophagy inducer or a protease inhibitor. In these experiments, we chose a concentration of decanoic acid (60 µM) that reduces mTORC1 activity [p < 0.001) and 5.96 autophagosomes per cell following 24 h (p < 0.001) (p < 0.001) (We then investigated the effects of decanoic acid and octanoic acid on inducing autophagy in activity  during g< 0.001) E,F. Trea< 0.001) E,F. Trea< 0.001) E,F. Prot< 0.001) E,F. None< 0.001) E,G. Our Since our data suggest that decanoic acid treatment induces the formation of autophagosomes, consistent with the activation of autophagy, we continued by analyzing the effect of decanoic and octanoic acid on autophagic flux. This assay involves observing the autophagy-dependent cleavage of GFP-Atg8 present in autophagosomes, to form GFP, and the subsequent degradation of GFP. Autophagic flux can be activated by the addition of autophagy inducers to enhance both the cleavage of GFP-Atg8 and the breakdown of free GFP, or blocked by the presence of protease inhibitors .Dictyostelium through the measurement of free GFP and GFP-Atg8 levels following acute (4 h) or chronic (24 h) treatment. In these experiments, cells were treated with decanoic acid or octanoic acid (60 µM), or with an autophagy inducer  or a protease inhibitor [Dictyostelium.The effects of decanoic acid and octanoic acid on autophagic flux was analyzed in nhibitor  , that plays a key role in inhibiting autophagy through the activation of mTORC1, with a well-established role in regulating autophagy [3 levels in Dictyostelium. These experiments involved developing cells to an early aggregation stage and using a single pulse of cAMP (1 µM) to induce PI3K activity to produce PIP3 on the cell membrane, and measured this production using membrane localization of a fluorescent marker protein containing a pH domain linked to GFP [3 production) at around 10 s post induction. Pretreatment of cells for 60 min with either decanoic or octanoic acid (60 µM), a concentration that induced autophagy in these cells, did not significantly reduce PIP3 production visualized by PHcrac-GFP membrane localization (3 production (3 (area under the curve)  C. Strong3 production but also by a range of other signaling pathways independent of PI3K [3 [3, and inducing activation of PKBA and PKBR1 by a single pulse of cAMP (1 µM), cell samples were taken at intervals and analyzed by western blot  . This pr of PI3K , suggest of PI3K ,55,56, a PI3K [3 . Thus, wern blot . In the ern blot A for botern blot B. Quantiern blot C. Thus, atg1 [atg8a [atg1 and atg8a was not altered following 1 h decanoic acid treatment, was noticeable but not non-significantly increased after 4 h treatment, and was significantly upregulated following 24 h treatment (p ≤ 0.01 for both), consistent with a chronic induction of autophagy  [Dictyostelium  , with thic acid) ,20,61. Wostelium A. Initiaostelium B,C, thatostelium B,C. Noneostelium D. These Dictyostelium. In this model [atg1 and atg8a [Autophagy, required for the recycling of misfolded proteins and damaged organelles, is a key cellular process involved in a wide range of neurodegenerative diseases, where activation of the process has been suggested to provide therapeutic effects ,63. Hereis model , decanoind atg8a ,57,58,59Autophagy has a recognized role in epilepsy that was first established when rapamycin, an autophagy inducer, was shown to suppress seizures . SubsequDictyostelium are consistent with that shown in mouse models, where dietary medium-chain fatty acids restore suppressed autophagy [The role of fatty acids in regulating autophagy for therapeutic effect remains poorly understood. Our findings that show a decanoic acid-dependent activation of autophagy in utophagy . In regautophagy ,77,78 orutophagy ,80,81. Sutophagy ,83 with Dictyostelium [3, since this effect was also demonstrated for Epilim using in vitro and in vivo mouse models [Dictyostelium, decanoic acid did not significantly reduce maximum or total PIP3 levels, nor reduce the total activation of PKBA  [Dictyostelium, and additionally in rodent hippocampal slices and in iPSC-derived human astrocytes [atg1 and atg8), to activate autophagy. This mechanism is consistent with the structurally specific effect of decanoic acid (not octanoic acid) in both inhibiting mTORC1 and inducing autophagy, independent of the glucose restriction and insulin signaling associated with the classical ketogenic diets [Dictyostelium would provide additional evidence for the involvement of this pathway [Decanoic acid has recently been demonstrated to modulate a range of cellular processes, where some of these may underlie the effect shown here for autophagy induction. Our initial focus was on decanoic acid-dependent regulation of phosphoinositide signaling, first identified in ostelium , and pare models . In Dicton PIP3) , althougtrocytes . We propic diets ,85,86,87 pathway .Dictyostelium. Since activation of autophagy has been suggested as a potential treatment for epilepsy and a range of neurological and neurodegenerative disorders, we suggest that further investigation into the mechanism behind fatty-acid-induced autophagy activation could enhance our understanding of the benefits of medium-chain fatty acids in health and disease.In this paper, we have identified that the medium-chain fatty acid, decanoic acid, activates autophagy in


Trichophyton benhamiae complex are one of the most important causes of animal mycoses with significant zoonotic potential. In light of the recently revised taxonomy of this complex, we retrospectively identified 38 Trichophyton isolates that could not be resolved into any of the existing species. These strains were isolated from Iranian and Czech patients during molecular epidemiological surveys on dermatophytosis and were predominantly associated with highly inflammatory tinea corporis cases, suggesting possible zoonotic etiology. Subsequent phylogenetic (4 markers), population genetic (10 markers), and phenotypic analyses supported recognition of two novel species. The first species, Trichophyton persicum sp. nov., was identified in 36 cases of human dermatophytosis and one case of feline dermatophytosis, mainly in Southern and Western Iran. The second species, Trichophyton spiraliforme sp. nov., is only known from a single case of tinea corporis in a Czech patient who probably contracted the infection from a dog. Although the zoonotic sources of infections summarized in this study are very likely, little is known about the host spectrum of these pathogens. Awareness of these new pathogens among clinicians should refine our knowledge about their poorly explored geographic distribution.Pathogens from the IMPORTANCE In this study, we describe two novel agents of dermatophytosis and summarize the clinical manifestation of infections. These new pathogens were discovered thanks to long-term molecular epidemiological studies conducted in Czechia and Iran. Zoonotic origins of the human infections are highly probable, but the animal hosts of these pathogens are poorly known. Further research is needed to refine our knowledge about these new dermatophytes. Dermatophytes are one of the most common pathogens of animals and humans worldwide . Because2–7–Trichophyton species belongs to the Trichophyton benhamiae complex, which encompasses nine taxa. These are mostly animal pathogens whose hosts include pets, livestock, and free-living animals  in rural indigenous populations in the tropics , rabbits, and various rodents. It was shown that T. benhamiae is a complex of several zoophilic species which are partly different in their geographic distribution and host range , ribosomal DNA (rDNA), and tef1-α sequences generated during epidemiological surveys in Iran conducted between 2008 and 2019. One case of feline dermatophytosis was also identified. The cases are summarized in T. persicum isolates were identified by Iranian medical mycologists as T. verrucosum or Microsporum canis, based on morphology. Anamnestic data about the contact of patients with animals were usually missing. The only exception was the case of a 31-year-old woman (isolate S366) who lived in a rural area of Iran and reported contact with various farm animals, including cats and cattle.In total, 37 cases of dermatophytosis caused by T. persicum were reported especially from the area of Southern, Southwestern, and Eastern Iran  during 2012 to 2014. The last and larger group of cases occurred in the area of Eastern Iran (10 cases in Qaen) during 2017, together with a few cases traced mainly in Shiraz during 2017 to 2019.In general, the cases of dermatophytosis due to ern Iran . The firT. persicum manifested as tinea corporis  topically based on the initial diagnosis of bacterial pyoderma. There was no visible improvement at the 10-day follow-up.Trichophyton interdigitale according to the concept of Heidemann et al.  in combination with clotrimazole  was initiated based on the result of the mycological examination. The pustules disappeared in 2 weeks after starting the treatment. A cure was achieved 4 weeks after the onset of the treatment, with only slight erythema remaining. No recurrence has been observed to date.Later the patient mentioned that he owned a dog (German shorthaired pointer) that had been treated with antimycotics at a veterinary surgeon 2 months ago due to a lesion on its front paw above the ankle. Although the transmission of the infection from the dog cannot be confirmed due to the absence of an animal isolate, it is highly suspected.gapdh, tubb, and tef1-α sequences from members of the T. benhamiae complex  bootstrap was low (<70%). The relationships of these species with other taxa in the T. benhamiae clade are poorly resolved due to insufficient statistical supports, but the closest species are represented by T. japonicum and T. europaeum. The phylogenies based on the ITS rDNA region  and 4 loci (only isolates having full sequence data available) are shown for comparison in Fig. S1 and S2 in the supplemental material. The topologies of these phylogenetic trees are similar, with some differences in deep nodes that gained low statistical support in all phylogenies.The phylogenetic analysis resolved both new species within the á et al.  of the T complex . TrichopTrichophyton persicum and Trichophyton spiraliforme can be differentiated from each other by their ITS rDNA (2 substitutions in the ITS2 region), gapdh (12 substitutions), and tef1-α gene sequences (1 substitution and 1 indel). The ITS rDNA and gapdh loci can also differentiate both species from the other members of the T. benhamiae complex. The sequence of the tef1-α gene of T. spiraliforme is identical to those of T. europaeum and T. japonicum, and it shows a single substitution and indel compared to that of T. persicum. The two new species share identical tubb sequences that differ by two substitutions from those of all other members of the T. benhamiae clade. The tubb locus shows overall low discriminatory power in the T. benhamiae complex, and all remaining members of the T. benhamiae clade have identical tubb genotypes, as demonstrated previously (eviously .n = 37) and tef1-α (n = 8) sequences of T. persicum; specifically, there were three substitutions and one indel in the ITS region and a single substitution in tef1-α gene sequences. The remaining two loci were only available for the ex-type strains, and consequently, the level of intraspecific variability, along with the precise numbers of species-specific substitutions/indels useful for their differentiation, needs to be confirmed in future studies when more strains will be available. Given the importance of the ITS rDNA region for routine dermatophyte identification, we summarized all species-specific substitutions and indels between all species pairs from the T. benhamiae clade in Table S1.Some level of intraspecific variability was detected in the available ITS  is shown in MAT1-1-1 and MAT1-2-1 gene idiomorphs across the T. benhamiae complex. In general, both idiomorphs are unequally distributed in most species of the complex, and one idiomorph is frequently absent, suggesting clonal spread of the majority of species. Data from more strains are needed for MAT idiomorph distribution assessment in the two newly described species.The genotype network of the T. benhamiae var. benhamiae cluster 2 and T. benhamiae var. luteum. Both T. persicum and T. spiraliforme formed independent distant lineages. The analysis separated different subpopulations of T. benhamiae var. benhamiae. Cluster 2 of T. benhamiae var. benhamiae sensu Čmoková et al. . Trichophyton spiraliforme differs mainly by its restricted growth on potato dextrose agar (PDA). Slightly lower growth rates on PDA were also observed in some strains of T. europaeum, but not to such an extent. Trichophyton persicum grows restrictedly also but on all media tested, as observed in the ex-type strain. Limited data are available for several strains that were no longer viable and could not be used for detailed analyses. These strains  were all characterized by slow growth on Sabouraud dextrose agar (SDA) with chloramphenicol and cycloheximide. Very restricted growth on SDA at 37°C also differentiates T. persicum from other closely related zoophilic species.The growth rate of T. benhamiae clade species ; Iran, Qaen, tinea capitis (ectothrix), 5-year-old girl, 2017, M. J. Najafzadeh, ex-holotype living culture CCF 6543 = MJN-91.Vegetative hyphae smooth, septate, hyaline, 1 to 2.5 μm in diameter . Conidiophores poorly differentiated from vegetative hyphae and represented by conidiogenous hyphae, unbranched or with sparse lateral branches, usually densely septate. Microconidia are rare, borne sessile on hyphae or on short lateral protrusions of hyphae, irregularly shaped, usually clavate, ovoid, or pyriform, less commonly barrel shaped or irregular, occasionally released together with fragile lateral protrusions of hyphae, 2 to 6 (3.6 ± 0.7) by 1.5 to 3 (2 ± 0.3) μm. Macroconidia not observed. Chlamydospores present. Spiral hyphae not observed even after 21 days of cultivation. Sexual morph is unknown.Colonies on malt extract agar  in 7 days at 25°C attained 9 to 11 mm in diameter (ø = 11 mm), white (#F5F5F0) to pale orange yellow (#F3E5AB), raised in the center, velvety to cottony, edge entire, reverse light orange yellow (#F8DE7E) to vivid orange yellow (#F6A600). Colonies on SDA in 7 days at 25°C attained 10 to 12 mm in diameter (ø = 11 mm), white (#F5F5F0), pale orange yellow (#FFF587), or light yellowish brown (#E3D6A1); dark brown diffuse pigment produced into medium after 3 to 4 weeks of cultivation. Colonies on PDA in 7 days at 25°C attained 8 to 10 mm in diameter (ø = 8 mm), white (#F5F5F0) to pale orange yellow (#F3E5AB), raised, velvety or cottony, edge entire, reverse light orange yellow (#F8DE7E). Colonies on SDA in 7 days at 30°C attained 15 to 18 mm in diameter (ø = 17 mm) and at 37°C attained 5 to 8 mm in diameter (ø = 6 mm).Trichophyton persicum differs from zoophilic members of the T. benhamiae clade by its low growth rates on all media examined and restricted growth at 37°C. Other slow-growing species in this clade are the anthropophilic T. concentricum and zoophilic T. benhamiae var. luteum. The latter differs from T. persicum by a yellow colony reverse, the shape of the microconidia, and ecology. Trichophyton persicum most closely resembles T. eriotrephon by the variability in the shape of microconidia and restricted growth on 37°C. These species can be differentiated by conidium length, which is longer in T. persicum than in T. eriotrephon. The macromorphology of T. persicum may resemble that of T. verrucosum by slow growth and colony appearance, but the species differ in micromorphology, as T. verrucosum usually produces only chlamydospores and no microconidia. The only viable isolate examined by us exhibited a MAT1-2-1 mating type gene idiomorph.Trichophyton spiraliforme Cmokova, Kuklova & Hubka, sp. nov. MycoBank accession number MB839324; Etymology: referring to the abundant production of spiral hyphae.Holotype: PRM 954616, a dried herbarium specimen (isotype PRM 954617); Czech Republic, Hostouň near Prague, knee skin (tinea corporis profunda), 43-year-old man, 2017, J. Stará & I. Kuklová, ex-holotype living culture CCF 6259 = SK 4179/17.Vegetative hyphae smooth, septate, hyaline, 1 to 3.5 μm in diameter . Conidiophores both poorly differentiated from vegetative hyphae and represented by conidiogenous hyphae with sparse to numerous short lateral branches, occasionally well differentiated and branched in a pyramidal (grape-like) pattern. Microconidia abundant, pyriform to clavate, 2.5 to 4 (3 ± 0.4) × 2 to 2.5 (2 ± 0.2) μm. Macroconidia rare, borne laterally or terminally on hyphae, consisting of 5 to 8 cells (median = 5), 25.5 to 33.5 (26.8 ± 3.5) by 4.3 to 6 (5 ± 0.6) μm, elongated, cigar-shaped, clavate, with a tapering rounded apex and truncate base. Chlamydospores present. Spiral hyphae observed after 7 days of cultivation, abundantly present after 21 days. Sexual morph is unknown.Colonies on MEA in 7 days at 25°C attained 15 to 35 mm in diameter (ø = 21 mm), granular, white (#F2F3F4) to light yellow (#FAD6A5), flat, edge irregular, submerged, reverse pale orange yellow (#FAD6A5) to brilliant orange yellow (#FFC14F). Colonies on SDA in 7 days at 25°C attained 14 to 17 mm in diameter (ø = 15 mm), white (#F2F3F4), powdery, umbonate, edge irregular, submerged, reverse deep brown (#593319) in the center, light orange yellow (#FBC97F) in the marginal parts. Colonies on PDA in 7 days at 25°C attained 8 mm in diameter (ø = 8 mm), white (#F2F3F4) to light yellow (#FAD6A5), granular, occasionally with filamentous sectors, raised, reverse pale orange yellow (#FAD6A5) to brilliant orange yellow (#FFC14F). Colonies on SDA in 7 days at 30°C attained 25 to 27 mm in diameter (ø = 26 mm) and at 37°C attained 20 to 24 mm in diameter (ø = 22 mm).T. spiraliforme most closely resembles those of related species from the T. benhamiae complex  and T. mentagrophytes by colony texture and numerous relatively small microconidia borne on conidiophores branched in a grape-like pattern. It differs from the species mentioned by its restricted growth on PDA. The only isolate examined by us exhibited a MAT1-2-1 mating type gene idiomorph.The morphology of T. benhamiae complex  and T. mentagrophytes and Trichophyton rubrum complexes, is a significant clinical problem nowadays, since it can lead to treatment failure  are marginal infectious agents that contribute to the spectrum of all dermatomycosis agents only at rates of 0.5 to 5%. Based on long-term epidemiological data and the sequence data available in the GenBank database, T. persicum seems to be the dominant species from the clade in Iran, responsible for almost all cases and mostly restricted to a relatively small area in South, Southwestern, and Eastern Iran , Department of Botany, Charles University, Prague, Czech Republic, and dried herbarium specimens (holotypes and isotypes) were deposited into the herbarium of the Mycological Department, National Museum in Prague, Czech Republic (PRM).gapdh gene, encoding glyceraldehyde-3-phosphate dehydrogenase, was amplified with primers GPDF and GPDR , encoding a protein with an alpha domain motif, and primers TmHMG3S and TmHMG3R were used for the detection of the MAT1-2-1 idiomorph, encoding a regulatory protein with an HMG (high-mobility group) DNA-binding motif (−1) of DNA, 0.3 μl of both primers (25 pM ml−1), 0.2 μl of MyTaq polymerase, and 4 μl of 5× MyTaq PCR buffer . The PCR conditions followed the protocol described by Hubka et al. . The quality was evaluated by using a NanoDrop 1000 spectrophotometer. The ITS rDNA region (ITS1–5.8S–ITS2 cluster) was amplified using the primer set ITS1F and ITS4 , 72, a pand GPDR , a partiand Bt2b , and a pEF-DermR . The pring motif , 77. Thea et al. . PCR proa et al. . Automatgapdh, tubb, and tef1-α regions were performed using the FFT-NS-i option implemented in MAFFT online .Micromorphological characteristics were recorded at least 35 times for each feature and documented using an Olympus BX-51 microscope. The macromorphology of the colonies was documented on MEA , potato dextrose agar , and Sabouraud dextrose agar  at 25, 30, and 37°C. The colonies were documented using a Canon EOS 500D camera. Color names were determined using the ISCC-NBS centroid color charts (www.ncbi.nlm.nih.gov) under accession numbers MW936599 to MW936628, MW959139 to MW959143, MZ314457, MZ320340, MZ320335, and MZ320330. The alignments are available in the Dryad Digital Repository at https://doi.org/10.5061/dryad.59zw3r275. The names of newly described species were deposited in MycoBank (MB839323 and MB839324).The DNA sequences obtained in this study were deposited into the GenBank database (