MT triggered the activity and expression of antioxidant enzymes, which in turn decreased hydrogen peroxide (H2O2) and superoxide anion (O2⋅–) accumulation caused by chilling stress. Meanwhile, MT attenuated the chilling-induced decrease, in the net photosynthetic rate (Pn) and promoted photoprotection for both photosystem II (PSII) and photosystem I (PSI), regarding the higher maximum quantum efficiency of PSII (Fv/Fm), actual photochemical efficiency (ΦPSII), the content of active P700 (ΔI/I0), and photosynthetic electron transport. The proteome analysis and western blot data revealed that MT upregulated the protein levels of PSI reaction center subunits , PSII-associated protein PsbA (D1), and ribulose-1,5-bisphosphate carboxylase or oxygenase large subunit (RBCL) and Rubisco activase (RCA). These results suggest that MT enhances the chilling tolerance of cucumber through the activation of antioxidant enzymes and the induction of key PSI-, PSII-related and carbon assimilation genes, which finally alleviates damage to the photosynthetic apparatus and decreases oxidative damage to cucumber seedlings under chilling stress.Chilling adversely affects the photosynthesis of thermophilic plants, which further leads to a decline in growth and yield. The role of melatonin (MT) in the stress response of plants has been investigated, while the mechanisms by which MT regulates the chilling tolerance of chilling-sensitive cucumber remain unclear. This study demonstrated that MT positively regulated the chilling tolerance of cucumber seedlings and that 1.0 μmol⋅L For warm climate crops, chilling is considered as a considerable challenge among all abiotic stresses. The previous study showed that low-temperature stress mainly affects light energy utilization and photosynthetic efficiency by disrupting electron transport chains in chloroplasts and mitochondria, leading to reactive oxygen species (ROS) accumulation . ROS of 1O2) and superoxide in the thylakoid membrane  is a new type of hormone-like substance. It is a small molecule indole that was first discovered in the bovine pineal gland in –1 MT significantly promoted the activities of SOD, APX, GR, monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR), which are involved in the ascorbate-glutathione cycle and accelerated the scavenging of ROS under chilling stress  is an important vegetable crop that is cultivated worldwide. However, chilling stress severely affects cucumber productivity and quality  and 25–31°C or 13–21°C day or night temperature under a 13-h photoperiod. At the one-leaf-stage, the seedlings were transferred to black plastic containers  with 1/2 Hoagland nutrient solution and then 0 μmol⋅L–1, 0.3 μmol⋅L–1, 0.6 μmol⋅L–1, 1.0 μmol⋅L–1, 1.5 μmol⋅L–1, and 2.0 μmol⋅L–1 MT were added until cucumber seedlings were at the two-leaf stage. Twenty-four hours later, all seedlings were exposed to low temperatures (8°C/5°C day or night) and the seedlings treated with 0 μmol⋅L–1 MT (H2O) were used as the control. Then, leaf samples were taken at 0, 1, 3, and 5 days from 4 to 5 plants (n = 3) for the following analyses.Cucumber (Jinyou 35) seeds  were germinated on petri dishes (8.6 cm in diameter) with moisture filter paper in the dark at 28°C for 1 day, and then germinated seeds were sowed to the nutritional box (8.0 cm in diameter and 8.0 cm in height) filled with base material in a solar greenhouse. The seedlings were routinely managed. The conditions were as follows: maximum of 800–1000 μmol mLeaf area was determined as described previously .Malondialdehyde (MDA) content was measured using the thiobarbituric acid (TBA) colorimetric method . Electro2O2) was clearly visible with an inverted fluorescence microscope using the H2O2 fluorescent probe 2’, 7’-dichlorodihy drofluorescein diacetate (H2DCFDA)  according to 2O2 fluorescent probe, pH 7.5) for 30 min under dark conditions at 25°C. After rinsing with the HEPES-NaOH buffer three times (15 min each time), the H2O2 production was visual in the form of green coloration (excitation at 488 nm and emission at 522 nm) under an inverted fluorescence microscope . Cellular superoxide anion (O2⋅–) was detected using dihydroethidium (DHE)  as described by 2⋅– (excitation at 490 nm and emission at 520 nm) was clearly visible with the inverted fluorescence microscope.Cellular hydrogen peroxide . The O2⋅– production rate was determined using the method presented by The quantitation of Hg at 4°C for 20 min. The supernatant was reserved at 4°C and prepared to measure the activities of antioxidant enzymes (Cucumber leaves (0.5 g) were ground using 3 ml phosphate buffer  and centrifuged the resulting extract at 12,000  enzymes . SOD act–2⋅s–1), CO2 concentration (360–380 mg⋅L–1), and leaf temperature (25°C ± 1°C) from the beginning to end.The gas-exchange parameters were assayed with a photosynthetic instrument  and controlled PFD  with a computer-operated PAM control system .O–J = (Ft-F0)/(FJ-F0); △VO–J = VO–J-VO–J (control); number of reaction centers per unit area mRC/CS = (ABS/CSm)/(ABS/RC); efficiency of electron moving beyond QA (ψ0) = ET/TR = 1-VJ; the capacity of electron transport from PSII to PSI φE0 = ET0/ABS = (1-F0/Fm) × ψ0 (0 was measured according to 0 = (I0-Im)/I0 .The Chlorophyll a Fluorescence Transient (OJIP) curve and 820 nm transmission were assayed according to the method of Fm) × ψ0 . To meas3, 5 mM EDTA⋅Na2, and 5 mM MgCl2) was added. After centrifugation at 2,000 g for 15 min, 5 × loading buffer  was added and then boiled at 100°C for 15 min. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel was prepared to separate proteins. Antibodies specific to the PsbA (D1), PSI reaction center subunit II (PsaD), ribulose-1,5-bisphosphate carboxylase or oxygenase (Rubisco) large subunit (RbcL), and Rubisco activase (RCA) proteins  were used to detect D1, PsaD, RbcL, and RCA and followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG antibody . The eECL Western Blot Kit  was used to detect immune responses, and the ChemiDoc™ XRS imaging system  was used to record the chemiluminescence.To extract total protein, 0.2 g leaf samples were ground with liquid nitrogen and extraction buffer . An equal volume of Tris-balanced phenol was added and centrifuged at 5,500 t-test p-value < 0.05.Dithiothreitol was added to the protein solution to make a final concentration of 5 mM and then reduced at 56°C for 30 min. Then, iodoacetamide was added to a final concentration of 11 mM and incubated at room temperature for 15 min in darkness. The urea concentration of the sample was diluted to less than 2 M. Trypsin was added in a 1:50 mass ratio (trypsin: protein), and enzymatic hydrolysis was performed overnight at 37°C. Trypsin was added at a mass ratio of 1:100 (trypsin: protein) again, and enzymatic hydrolysis was continued for 4 h. After trypsin digestion, the peptides were desalted with a Strata X C18 SPE column (Phenomenex) and then vacuum freeze-dried. The peptides were dissolved in 0.5 M triethylammonium bicarbonate (TEAB) and labeled according to the instructions of the Tandem Mass Tag (TMT) kit. The peptides were separated with high pH reverse-phase high-performance liquid chromatography (HPLC) using a Thermo Betasil C18 column , dissolved with mobile phases A (0.1% formic acid), and separated by an EASY-nLC 1000 UPLC system. Mobile phase A was aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile solution containing 0.1% formic acid. Liquid phase gradient setting was as follows: 0–43 min, 6–22% phase B; 43–56 min, 22–30% phase B; 56–58 min, 30–80% phase B; 58–60 min, 80% phase B, and the flow rate maintained at 300 nL/min. The peptides were separated by a UPLC system and then injected into a capillary ion source for ionization and analyzed by times TOF Pro mass spectrometry. The ion source voltage was set at 1.4 kV, and the peptide parent ions and their secondary fragments were detected and analyzed using TOF. The scanning range of secondary mass spectrometry was set to 100–1700 m/z. The data acquisition mode was parallel accumulation serial fragmentation (PASEF) mode. Secondary mass spectrometry data were retrieved using the Maxquant search engine (v1.6.5.0), and peptide length was analyzed with mass spectrometry. Finally, bioinformatics analysis was performed. The quantitative proteins were identified and analyzed with threshold value of differential expression change of 1.2-fold and a statistically tested g and 4°C for 15 min, and the supernatant was retained. Then, 0.5 ml isopropyl alcohol was added to the supernatant, mixed gently, and incubated for 10 min at room temperature. The sample was centrifuged at 10,000 g and 4°C for 10 min to remove the supernatant, and then 1 ml of 75% ethanol was added to the precipitate. Oscillation and centrifugation were performed at 7,500 g and 4°C for 5 min. The supernatant was removed, and the precipitation was dried at room temperature. The precipitate was dissolved in 100 μL RNA solution. The resulting total RNA was reverse transcribed according to the instructions of HiScript® IIIRT SuperMix for qPCR (+gDNA wiper) . Genomic DNA was removed using 4 × gDNA wiper Mix at 42°C for 2 min. Then, 5 × HiScript III qRT SuperMix containing all the components required for reverse transcription was mixed with RNA and could be used immediately for PCR at 37°C for 15 min and then 85°C for 5 s. Relative gene expressions were analyzed by real-time quantitative PCR (RT-qPCR) using ChamQ™ Universal SYBR® qPCR MasterMix  according to the instructions. The predenaturation was performed at 95°C for 30 s. Then, 40 cycles were performed at 95°C for 10 s and 60°C for 30 s. Finally, 95°C for 15 s and 60°C for 60 s and 95°C for 15 s acquired fusion curves. The cucumber β-actin gene (Gene ID: Solyc11g005330) was used as an internal reference gene. The primers were designed and synthesized by BGItech and were shown in Cucumber leaves were ground thoroughly with liquid nitrogen, and total RNA was extracted with TransZol reagent . First, 0.2 g ground powder was added 1 ml TransZol and then mixed. After standing for 5 min, 0.2 ml chloroform was added followed by shaking vigorously for 15 s and then standing for 3 min at room temperature. The solution was centrifuged at 10,000 p < 0.05.The experimental design was a completely randomized block design. The data are presented as the mean ± standard deviation (SD) of three to five replicates. Analysis of variance was conducted using DPS software. Duncan’s multiple range test (DMRT) was applied to statistical analysis among treatments, and the standard for significant difference was –1 MT, respectively. As shown in 2O showed leaf-wilting and spots at advanced stages of the chilling stress. Furthermore, we examined the change in growth and lipid peroxidation of cucumber seedlings under low-temperature intensity. An increase in the chilling injury index, EL, and MDA content in cucumber seedlings treated with H2O was observed along with an increase in stress duration  increased under chilling stress. Additionally, we found that MT significantly relieved the decrease in Pn and Gs induced by chilling stress.The generation of oxidative stress is closely related to photosynthetic capacity, so we studied the effect of MT on photosynthesis. To determine whether MT could improve the photosynthetic capacity, we examined the changes in gas-exchange parameters in cucumber seedlings treated with H2O- and MT-treated seedlings. Under normal conditions, no significant differences were observed in higher maximum quantum efficiency of PSII (Fv/Fm) and actual photochemical efficiency between H2O and MT treatments. However, when exposed to chilling at 8°C or 5°C for 1 and 3 days, H2O and MT treatments showed dramatic decreases in Fv/Fm and ΦPSII in cucumber seedlings, but the decrease in MT was notably lower than that of H2O treatment  is damaged under stress. The K-point is a specific marker of photoinhibition of the PSII donor side. To observe the K-point, the O-J segments of the OJIP curves before and after chilling stress were standardized  larger subunit protein level by MT  partner repository with the dataset identifier PXD029134.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Website: The authors declare that the experiments were performed in compliance with the current laws of China.XZ performed most of the experiments, analyzed the data, and completed the first draft. HB designed the research and edited the study. YF, TJ, XL, and XA worked together with XZ to accomplish the experiment. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Clonorchis sinensis (CsCatD2). In this study, we performed all-atomic molecular dynamics simulations by applying different systems for the ligand-free/bound forms under neutral and acidic conditions to investigate the pH-dependent structural alterations and associated functional changes in CsCatD2. CsCatD2 showed several distinctive characteristics as follows: (1) acidic pH caused major conformational transitions from open to closed state in this enzyme; (2) during 30–36-ns simulations, acidic pH contributed significantly to the formation of rigid β-sheets around the catalytic residue Asp219, higher occupancy (0% to 99%) of hydrogen bond than that of Asp33, and enhanced stabilization of the CsCatD2-inhibtor complex; (3) neutral pH-induced displacement of the N-terminal part to hinder the accessibility of the active site and open allosteric site of this enzyme; and (4) the flap dynamics metrics, including distance (d1), TriCα angles (θ1 and θ2), and dihedral angle (ϕ), account for the asymmetrical twisting motion of the active site of this enzyme. These findings provide an in-depth understanding of the pH-dependent structural dynamics of free and bound forms of CsCatD2 and basic information for the rational design of an inhibitor as a drug targeting parasitic CatD.Cathepsin D  family peptidases of parasitic organisms are regarded as potential drug targets as they play critical roles in the physiology and pathobiology of parasites. Previously, we characterized the biochemical features of cathepsin D isozyme 2 (CatD2) in the carcinogenic liver fluke  Clonorchis sinensis. This parasite is prevalent in far East Asian countries, including China, Korea, and Northern Vietnam, and it is reported to infect approximately 35 million people worldwide . The effect of either acidic pH or inhibitor-binding, and both effects on the correlated motions of CsCatD2stigated . Overall at pH 7 A, the co at pH 4 D but sli at pH 4 B and CsC at pH 7 C. Interebound interacts with the inhibitor under different pH conditions, hydrogen bond and hydrophobic interactions were investigated because hydrogen bonds between inhibitor and surrounding residues are crucial for stabilizing the complex. Noncovalent interactions changed with time during the 50-ns simulation. In particular, acidic pH significantly contributed high hydrogen bond occupancy of the whole simulation time at the Asp33 of 94 to 99% and Asp219 of 0 to 91%  between the flap tip and hinge residue, TriCα angles, θ1 (Gly79–Asp33-Met297) and θ2 (Gly79–Asp219–Met297), which account for the opening and closing of the active site, and dihedral angle ϕ (Gly79–Asp33–Asp219–Met297) are responsible for the twisting motion  to semi-open (12.6 Å) state in CsCatD2free; and from semi-closed (9.7 Å) to closed (8.7 Å) state in CsCatD2bound. Compared to the initial structures, the acidic pH and bound inhibitor steered the dihedral angle ϕ and reduced the TriCα angles (θ1 and θ2) simultaneously. Both the distance d1 and TriCα angles are likely to decrease. For instance, the dihedral angle ϕ of CsCatD2free at pH 7 shifted from +2.1° to −4.7° at acidic pH, to −8.0° for the bound ligand, and to −3.8° for both factors. These results collectively provide evidence of the “twisting” of the binding pocket, which can be seen by the asymmetrical movement of the pocket by the bound inhibitor and acidic pH.The dynamics of the flap structure of CsCatD2 were analyzed. Based on the average structures obtained through a 50-ns MD simulation, each parameter was measured B. The co Soliman . It was d1 was calculated based on the snapshots obtained at 0, 10, 20, 30, 40, and 50 ns during the 50-ns MD trajectories of all systems , CsCatD2free at pH 4 (12.66 ± 0.10), CsCatD2bound at pH 7 (10.52 ± 0.46), and CsCatD2bound at pH 4 (8.69 ± 0.04) (d1 showed semi-open conformation (11.30 Å at pH 7 and 11.29 Å at pH 4) in CsCatD2free, whereas d1 indicated a semi-closed conformation (9.20 Å at pH 7 and 9.66 Å at pH 4) in CsCatD2bound. Toward the end of the 50-ns MD simulation, CsCatD2free showed only two open states at pH 4 and then transitioned to a semi-closed state (10.60 Å) at 50 ns. Although the bound forms showed a similar trend at both pH conditions, CsCatD2bound had a lower minimum d1 of 7.74 Å at pH 4 than the d1 (8.74 Å) at pH 7 at 50 ns, revealing three semi-closed conformations or three closed states. Notably, the acidic pH induced the distance d1 of CsCatD2free close to the level of CsCatD2bound at pH 7 around the simulation times  (The time-series distance  systems . The mea ± 0.04) . At the d 46 ns) .ϕ in CsCatD2free at pH 4 (free at pH 7 (average ϕ: 2.29 ± 0.43) showed a few small negative values of ϕ on an intermittent basis, whereas CsCatD2free at pH 4 (average ϕ: −4.68 ± 0.43) made the preferable twisted conformation as represented by a majority of negative values of ϕ. Acidic pH induced opposite patterns of ϕ in CsCatD2free during 40–50 ns via conformational shifts to the semi-open state (ranging from 16.24 to 5.59) at pH 7 and semi-closed state (from −18.36 to –6.54) at pH 4. The bound forms had negative values of dihedral angles ϕ, −7.56 ± 0.38 at pH 7 and −3.78 ± 0.19 at pH 4, throughout the entire simulation time. Among the TriCα angles θ1 and θ2  of flap dynamics were correlated with each other to account for the discrepancies in the open or closed states of the binding cleft.A similar trend was also observed at the dihedral angle  at pH 4 B. CsCatD1 and θ2 C,D, the free and CsCatD2bound were constructed using Yet Another Scientific Artificial Reality Application (YASARA) Structure v21.6.17 [free and CsCatD2bound based on the experimental X-ray-characterized structures of free cathepsin D (PDB code: 5n7n) and inhibitor-bound cathepsin D (PDB code: 5n7q) from the tick I. ricinus [https://swift.cmbi.umcn.nl/gv/dssp/; accessed on 10 August 2021).The complete mRNA sequence of CsCatD2 was retrieved from GenBank (Acc. No.: GU433605)  and the v21.6.17  as descrv21.6.17 . A hybri ricinus . The qua ricinus , ERRAT [ ricinus , and prohttps://pymolwiki.org/index.php/Cealign; accessed on 10 August 2021). The TM-score, calculated by TM-align, indicates the fold similarity between the two structures. All structure graphics were prepared using PyMOL .Structural superpositions were carried out using TM-align  with defAll MD simulations were performed using a pre-installed “md_runfast.mcr” macro file within the YASARA Structure v21.6.17  for bothd1 (Gly79–Met297) between the flap tip and hinge residue, TriCα angles θ1 (Gly79–Asp33–Met297) and θ2 (Gly79–Asp219–Met297) for the conformational changes of the active site, and the dihedral angle ϕ (Gly79–Asp33–Asp219–Met297) for the twisting motion of the binding pocket. The sim format was converted into xtc format using “md_convert.mcr” macro file and, subsequently, into dcd format using Wordom v0.22-rc3 [Subsequently, snapshots were analyzed using the customized “md_analyze.mcr” and the built-in “md_analyzeres.mcr” macro files within the YASARA package for the RMSD, RMSF, Rg, SASA, and flap parameters, such as the distance 0.22-rc3 . DCCM an0.22-rc3  of R StuC. sinensis, which can cause food-borne parasitic infection and cholangiocarcinoma. The structure–inhibition relationships and validated computational simulations determined in this study will provide important insights into the binding mechanisms of parasitic CatD to potential inhibitors during the pH shift. Especially, acidification of 30–36-ns simulation showed significant contribution to establishment of rigid β-sheets and higher occupancy of hydrogen bonds near the catalytic residue Asp219, thereby enabling increased stabilization of the CsCatD2-inhibtor complex. Further, in vitro and in vivo experiments are strongly needed to confirm the structure–function and inhibition.Parasitic CatD homologues have gained significant attention for being attractive targets for therapeutic drug design as CatD plays a critical role in the degradation of blood to facilitate the survival of the parasite. Here, we report the pH-dependent structure and flap dynamics of CsCatD2 from the blood-feeding parasite


However, the mechanisms underlying the response of wheat seedling roots to changes in NH4+/NO3– ratio remain unclear. In this study, we investigated wheat growth, transcriptome, and proteome profiles of roots in response to increasing NH4+/NO3– ratios . High NH4+/NO3– ratio significantly reduced leaf relative chlorophyll content, Fv/Fm, and ΦII values. Both total root length and specific root length decreased with increasing NH4+/NO3– ratios. Moreover, the rise in NH4+/NO3– ratio significantly promoted O2– production. Furthermore, transcriptome sequencing and tandem mass tag-based quantitative proteome analyses identified 14,376 differentially expressed genes (DEGs) and 1,819 differentially expressed proteins (DEPs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that glutathione metabolism and phenylpropanoid biosynthesis were the main two shared enriched pathways across ratio comparisons. Upregulated DEGs and DEPs involving glutathione S-transferases may contribute to the prevention of oxidative stress. An increment in the NH4+/NO3– ratio induced the expression of genes and proteins involved in lignin biosynthesis, which increased root lignin content. Additionally, phylogenetic tree analysis showed that both A0A3B6NPP6 and A0A3B6LM09 belong to the cinnamyl-alcohol dehydrogenase subfamily. Fifteen downregulated DEGs were identified as high-affinity nitrate transporters or nitrate transporters. Upregulated TraesCS3D02G344800 and TraesCS3A02G350800 were involved in ammonium transport. Downregulated A0A3B6Q9B3 is involved in nitrate transport, whereas A0A3B6PQS3 is a ferredoxin-nitrite reductase. This may explain why an increase in the NH4+/NO3– ratio significantly reduced root NO3–-N content but increased NH4+-N content. Overall, these results demonstrated that increasing the NH4+/NO3– ratio at the seedling stage induced the accumulation of reactive oxygen species, which in turn enhanced root glutathione metabolism and lignification, thereby resulting in increased root oxidative tolerance at the cost of reducing nitrate transport and utilization, which reduced leaf photosynthetic capacity and, ultimately, plant biomass accumulation.Wheat growth and nitrogen (N) uptake gradually decrease in response to high NH Triticum aestivum L.) is one of the most important cereal foods worldwide. The tremendous increase in wheat crop productivity over the past several decades has made China the largest wheat consumer and producer in the world , resulting in root growth inhibition , 4-coumarate-CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) genes, resulting in an increase in lignin biosynthesis, enhancing stress resistance  identify the effects of different NH4+/NO3– ratios on plant and root growth at the seedling stage, (ii) investigate the wheat root transcriptome and proteome profiles related to glutathione metabolism and lignin biosynthesis in response to different NH4+/NO3– ratios, and (iii) provide new insights into the wheat root response to N for the improvement of N fertilizer application based on a scientific N management strategy.Glutathione metabolism is one of the defense mechanisms that play a vital role in plant resistance to oxidative stress . Plant gsistance . Althoug3 for 30 min, rinsed 5 × with sterile water, and germinated on wet filter paper in the dark at 25°C for 2 days. Germinated seeds were sowed in plastic pots  filled with perlite. The pots were placed on plastic trays .Experiments were carried out at the experimental station of Shandong Agricultural University, Tai’an, China . The Jimai 22 (JM22) winter wheat cultivar was grown under controlled conditions. Variations in temperature, illumination intensity, and relative humidity are shown in 4+/NO3– ratio treatments with a 6-mM total N concentration were tested in a controlled climate chamber experiment. Treatments included: 100/0 (Na), 75/25 (Nr1), 50/50 (Nr2), 25/75 (Nr3), and 0/100 (Nn). Ca(NO3)2, NH4Cl, or NH4NO3 were used to set the ratios. The NH4+/NO3– ratio preparation method is shown in –1 Ca2+, 5 mmol L–1 KCl, 4 mmol L–1 MgSO4, 1 mmol L–1 KH2PO4, 50 μmol L–1 Fe-ethylenediaminetetraacetic acid (EDTA), 0.5 μmol L–1 H3BO3, 0.74 μmol L–1 MnSO4, 0.27 μmol L–1 ZnSO4, 0.001 μmol L–1 CuSO4, 0.001 μmol L–1 CoCl2, 0.005 μmol L–1 Na2MoO4, and 0.025 μmol L–1 KI; and pH 6.5. The nutrient solutions were supplemented once a day (1.5 L/tray). Wheat growth was evaluated from sowing (0 days) to 42 days after sowing (DAS). We observed that the first leaf emerged through the coleoptile at 7 DAS and unfolded at 14 DAS. The morphological characteristics of 42-day-old wheat plants under Nn treatment showed that four leaves were unfolded, and the fifth leaf was visible ].4/K2SO4, w/w: 1/15) and digested in 10 ml H2SO4 at 420°C for 2 h and cooled. Distillation was used as a FOSS Kjeltec 8200 analyzer . The boric acid is used as the receiving solution. The solution was titrated with sulfuric acid. The N content was calculated according to the following formula:The dried samples were pulverized, and their N content was measured using the semi-micro Kjeldahl method . The powsample is the volume of used sulfuric acid for the sample (ml), Vblank is the volume of used sulfuric acid for blank (ml), N of acid represents normality of the used acid (mol L–1), and W is the sample dry weight (g).Where VEight intact fully expanded leaves of each treatment at 42 DAS were selected to measure relative chlorophyll content and the chlorophyll fluorescence analysis using the MultispeQ device  according to the method described by –1 DW).Root growth was evaluated by image analysis. Eight wheat plants of each treatment were sampled at 42 DAS. Roots were rinsed 3× with sterile water. Root sample images were captured with an EPSON V750 scanner  at a resolution of 300 dpi. The images were analyzed in WinRHIZO  which provided the total root length (TRL), root number (RN), and root average diameter (RAD). The root length and root biomass data were used to calculate the specific root length . The reactions were incubated at room temperature for 20 min, and 9.5 ml of 8% NaOH solution was added. After cooling the tube to room temperature, the optical density (OD)410 value was measured.Roots of 10 wheat plants under each treatment were sampled from 14 to 42 DAS at 7-day intervals. The fresh root nitrate content was determined using the salicylic acid method, as described by 2 and added 5 ml of 10 mM formic acid to extract NH4+. The root homogenates were centrifuged at 10,000 × g (4°C) for 15 min. Supernatants were transferred to 5 ml polypropylene tubes with 0.45-μm organic ultrafiltration membranes and centrifuged at 50,000 × g (4°C) for 10 min. A total of 100 ml of OPA reagent was prepared by combining 0.2 M potassium phosphate buffer (composed of equimolar amounts of potassium dihydrogen phosphate and potassium monohydrogen phosphate), 3.75 mM OPA, and 2 mM 2-mercaptoethanol, 1 day before use. Prior to adding 2-mercaptoethanol, the solution pH was adjusted to 7.0 using 1 M NaOH and filtered through two layers of filter paper. Extract (10 μl) was added to 3 ml of OPA reagent, and color was allowed to develop for 30 min in the absence of light (25°C) before sample absorbance was measured at 410 nm.Ammonium concentrations in roots were measured using the O-phthalaldehyde (OPA) fluorometric method, as described by 2–) content and production rate were determined according to the method described by g for 10 min. About 2 ml supernatant was added to 0.5 ml of 50 mM phosphate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine hydrochloride. After 20 min at 25°C, the mixture was added to 1 ml of 58 mM sulfanilamide and 1 ml of 7 mM α-naphthylamine, and let at 30°C for 30 min. After this period, the absorbance was measured at 530 nm.Wheat roots under each treatment were sampled from 14 to 42 DAS at 7-day intervals. Anion radical  and incubated at 70°C for 1 h. After cooling to room temperature, 0.9 ml of 2 M NaOH was added to terminate the reaction, then mixed and added 0.1 ml of 7.5 M hydrochloride. Finally, 4 ml acetic acid was added to each tube. The solution absorbance was measured at 280 nm using a spectrophotometer. The lignin contents were calculated according to the following linear calibration curve:Wheat roots of each treatment were sampled from 14 to 42 DAS at 7-day intervals. Root lignin contents were determined according to a modified method described by –1 FW) = (ΔA280 − 0.0068) ÷ 0.035 × V × 10–3 ÷ W × T.Lignin content  , which were processed by the same method used for plant samples.Where ΔAWheat roots of each treatment were sampled from 14 to 42 DAS at 7-day intervals. About 0.5 g root samples with three replicates from each treatment were ground to a powder in liquid N, and extracted with pH 7.0 phosphate buffer. The activities of PAL (EC:4.3.1.24), 4CL (EC:6.2.1.12), CCR (EC:1.2.1.44), CAD (EC:1.1.1.195), caffeic acid 3-O-methyltransferase , and peroxidase  were measured using enzyme activity assay kits .Roots were sampled in each treatment group at 42 DAS, immediately frozen in liquid N, and stored at −80°C until the RNA extraction and proteome analysis. Total RNA was extracted using the TRIzol reagent . Total RNA quantity and purity were determined using a NanoDrop and Agilent Bioanalyzer 2100 system (Agilent Technologies). Approximately 10 μg of total RNA representing a specific adipose type was subjected to isolation of poly (A) mRNA with poly T oligo-attached magnetic beads . Following purification, mRNA was fragmented into small pieces using divalent cations at high temperatures. Cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol by the manufacturer for the mRNA-Seq Sample Preparation Kit ; the average insert size for the paired-end libraries was 300 bp (±50 bp). Finally, we performed paired-end sequencing on an Illumina HiSeq 4000 at the LC Sciences, San Diego, CA, United States, following the protocol recommended by the vendor.1. Clean data were obtained by removing the low-quality and repeat reads. Sequence quality was verified using the FastQC software2, including Q20 , Q30 , and guanine-cytosine (GC) content. We aligned the sample reads to the wheat reference genome of the International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v1.0 (IWGSC 2018) using HISAT2. Mapped reads from each sample were assembled using the StringTie software. The value for fragment per kilobase of transcript per million mapped reads (FPKM) was calculated to quantify its expression abundance and variations using the StringTie software (version v1.3.3)3. DESeq2 software4 was used to analyze differentially expressed genes (DEGs) selected with the parameter false discovery rate (FDR) < 0.05, and an absolute fold change of ≥2. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to reveal the functional enrichment of DEGs.The adaptor reads were removed from raw data using the Cutadapt softwareg for 10 min at 4°C. The supernatant was transferred into a new tube, and 4× volumes of cold ammonium acetate/methanol were added, followed by incubation at −20°C overnight. Then, samples were centrifuged at 16,000 × g for 10 min at 4°C, the supernatant was discarded, cold methanol was added, and the mixture was incubated at − 20°C for 1 h. After centrifugation at 16,000 × g for 10 min at 4°C, the sediment was collected, resuspended in cold acetone, washed two times, and air-dried. Samples were dissolved in 0.6 ml lysis buffer , and centrifuged at 20,000 × g for 10 min at 4°C. The supernatant was collected, and the protein concentration was quantified using the Bio-Rad Protein Assay Kit  following the instructions of the manufacturer. Then, 30 μg of protein from each sample was used to determine the quality of the proteins using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Root samples were ground under liquid N, and 0.8 g of the powder obtained was transferred to a 5-ml centrifuge tube, homogenized with 1 ml lysis buffer, and placed on ice for 10 min. Tris–phenol (1 ml) was added to the tube, homogenized, and placed again on ice for 10 min. Samples were then centrifuged at 16,000 × g for 10 min at 4°C, the sediment was collected, resuspended in cold 80% acetone, washed two times, and air-dried, followed by the addition of 300 μl of 0.1 M triethylammonium bicarbonate (TEAB) to the proteins and sonication on ice for 5 min. Trypsin (20 μg) was added to the samples and incubated at 37°C overnight. After incubation, 1% trifluoroacetic acid (TFA) was added to the mixture to terminate the reaction. The peptides were desalted using a C18 SPE column  and vacuum-dried prior to reconstitution in 0.5 M TEAB and processing according to the protocol of the manufacturer for tandem mass tag (TMT) kit . Tryptic peptides were fractionated by high-pH reverse-phase high-performance liquid chromatography (HPLC) using a Waters XBridge Shield C18 RP column . Peptides were separated into 60 fractions, combined into 20 fractions, and vacuum centrifugation-dried. The fractions were dissolved in 0.1% formic acid, centrifuged at 20,000 × g for 2 min at 4°C, and the supernatant was transferred to a sampler vial. Liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis was performed by Micrometer Biotech Company .About 500 μg of protein from each sample was transferred to a new tube, adjusted to an equal volume with lysis buffer, and incubated at 30°C for 30 min. After cooling to room temperature, 30 mM iodoacetamide (IAM) was added, and the mixture was incubated at room temperature for 45 min in the dark, before adding 5× volumes of cold acetone, followed by incubation at −20°C overnight. After centrifugation at 20,000 × Triticum aestivum UniProt database . At least one unique peptide with an FDR of ≤1% was required for analysis of the protein identification and quantification data. For analysis of differentially expressed proteins (DEPs), only proteins with quantitative information from at least two biological replicates were used. The average of three biological replicates was used to indicate final protein abundance; finally, proteins showing an average protein-abundance change significantly greater than 1.3-fold  were defined as DEPs.Acquired MS/MS spectra were searched against the 2– content, lignin content, and lignin biosynthesis-related enzyme activity were processed in DPS v. 7.05 . Multiple comparisons were performed after a preliminary F-test. The means were tested with a least significant difference test, and the significance was set at the probability level of 0.05. Graphs were plotted using OriginPro 2017 .Data for leaf chlorophyll content and chlorophyll fluorescence parameters, TRL, RAD, SRL, N content, O4+/NO3– ratio affected shoot and root biomass and morphological traits of wheat seedlings  (Fv/Fm) and the steady-state efficiency (ΦII) decreased with increasing NH4+/NO3– ratios  reduced TRL and SRL by 77.98% and 48.59%, respectively, compared with the Nn treatment. Meanwhile, RAD increased with increasing NH4+/NO3– ratios. Indeed, the RAD of the Nr1 treatment increased by 280.21% compared to Nn.TRL and SRL decreased significantly as the NHless RAD . In contTranscriptome sequencing generated from 3.80 to 5.50 million raw data. After quality control, over 97% of the reads with Q30 were saved for further analysis. On average, more than 70% of these reads were specific to wheat and uniquely aligned to the reference genome .n treatment, increasing the NH4+/NO3– ratio significantly increased the number of DEGs and DEPs  and 1,471 DEPs (848 upregulated and 623 downregulated) were detected in the Na/Nn group. The results of the Venn diagrams showed that 1,108 upregulated DEGs and 1,372 downregulated DEGs, and 128 upregulated DEPs and 182 downregulated DEPs were unique to Na/Nn  and 6,726 (downregulated) DEGs were identified in the four pairwise comparisons listed in and DEPs , 4A. A tto Na/Nn , 4B. Fura/Nn transcriptome comparison group  and phenylpropanoid biosynthesis (ko00940)  were unique to Nr3/Nn  . ConcomiNn group . Four pao Nr3/Nn . Additioa/Nn (TraesCS1A02G095800) was an AP domain-containing protein (EC:3.4.11.1). Although only two proteins (A0A3B6K9P2 and Q8RW00) were GST, eight uncharacterized genes  had GST molecular function, accordingly with the GO enrichment analysis. Finally, A0A3B6MRF8 may play a role in zinc-ion binding, whereas A0A3B6JL78 has POD activity.The four comparison groups identified a total of 236 DEGs and 53 DEPs related to the glutathione metabolism pathway 00480) , 8. Thir0 , 8. Tha/Nn . AdditioTraesCS2D02G581200 was defined as 4CL (EC: 6.2.1.12). Seven upregulated genes were associated with shikimate O-hydroxycinnamoyl transferase . Fourteen upregulated genes and one downregulated gene were associated with CCR (EC: 1.2.1.44). One up and two downregulated genes were associated with COMT (EC: 2.1.1.68). Four upregulated and two downregulated genes were associated with CAD (EC: 1.1.1.195). Furthermore, 19 upregulated and 28 downregulated genes were associated with a POD (EC: 1.11.1.7). Based on the protein annotation, two PKS_ER domain-containing proteins (A0A3B6NPP6 and A0A3B6LM09) were obtained, whose amino acid sequences were aligned to those of the corresponding proteins in the wheat genome. Results of the phylogenetic tree constructed showed that both A0A3B6NPP6 and A0A3B6LM09 belong to the CAD subfamily  were identified in the four comparison groups , 10, andubfamily . Six uprproteins .a/Nn, and one DEG and one DEP were unique to Nr1/Nn (TraesCS3D02G344800 and TraesCS3A02G350800) were involved in ammonium transport (AMT) in the Na/Nn, Nr1/Nn, and Nr2/Nn comparison groups  was downregulated in the Nr3/Nn group but upregulated in the Na/Nn, Nr1/Nn, and Nr2/Nn comparison groups  were identified in all four comparison groups , 12. Sevo Nr1/Nn . Addition groups . In the n groups . Five DEa and Nr1 treatments gradually increased but sharply decreased from 35 to 42 DAS  was better for wheat growth. Conversely, the results of our study were not in line with this finding. In this study, we conducted experiments with different NH4+/NO3– ratio treatments to investigate the effects of the NH4+/NO3– ratio on wheat seedling growth. We found that the NO3–-N treatment resulted in the highest wheat growth. As the NH4+/NO3– ratio increased, significant growth inhibition occurred , which can oxidize cytosolic NADPH and transfer an electron across the cell membrane to generate O2– in the cytosol and cell wall  were upregulated under the high NH4+ conditions   was at or near saturation at 0.5 mM N, but it was still taking up nitrate at 10 mM nitrate  . This conditions . Howevermmol–1 N . A recen nitrate . In our d NRT2.4 , 12. Thi– ratios . Correla content . Based o4+/NO3– ratios reduced leaf chlorophyll content, Fv/Fm, and ΦII values in the ammonium sensitive wheat, indicating that a high NH4+/NO3– ratio reduces leaf photosynthetic capacity. In addition, increasing the NH4+/NO3– ratios caused O2– generation in the roots and increased the activity of enzymes involved in lignin biosynthesis, leading to root growth inhibition. Under a high NH4+/NO3– ratio, we identified upregulated DEGs and DEPs related to glutathione metabolism and phenylpropanoid biosynthesis in roots, which may contribute to an increase of root tolerance to oxidative damage. Furthermore, increasing the NH4+/NO3– ratios downregulated DEGs and DEPs related to N metabolism and nitrate transport, leading to a decrease in root N content. These results show that high NH4+/NO3– ratios in young wheat seedlings were unfavorable for root N uptake and transport due to excess lignification of the root cell wall.In the present study, the increment in the NHhttps://figshare.com/articles/dataset/_/16826116.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: DY conceived and designed the experiments, analyzed the data, prepared figures and tables, authored and reviewed drafts of the manuscript, and approved the final draft. JZ, CB, and LL performed the experiments. ZW contributed reagents, materials, analysis tools, and authored and reviewed drafts of the manuscript. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Camelus dromedarius) is well-adapted to the desert environment with the ability to tolerate increased internal body temperatures rising daily to 41–42°C during extreme hot. This study was undertaken to assess whether in vitro incubation of camel blood at 41°C, simulating conditions of heat stress, differently alters cell vitality, phenotype, and function of leukocytes, compared to incubation at 37°C (normothermia). Using flow cytometry, the cell vitality (necrosis and apoptosis), the expression of several cell markers and adhesion molecules, and the antimicrobial functions of camel leukocytes were analyzed in vitro. The fraction of apoptotic cells within the granulocytes, lymphocytes, and monocytes increased significantly after incubation of camel whole blood at 41°C for 4 h. The higher increase in apoptotic granulocytes and monocytes compared to lymphocytes suggests higher resistance of camel lymphocytes to heat stress. Functionally, incubation of camel blood at 41°C for 4 h enhanced the phagocytosis and ROS production activities of camel neutrophils and monocytes toward S. aureus. Monocytes from camel blood incubated at 41°C for 4 h significantly decreased their expression level of MHC class II molecules with no change in the abundance of CD163, resulting in a CD163high MHC-IIlow M2-like macrophage phenotype. In addition, heat stress treatment showed an inhibitory effect on the LPS-induced changes in camel monocytes phenotype. Furthermore, in vitro incubation of camel blood at 41°C reduced the expression of the cell adhesion molecules CD18 and CD11a on neutrophils and monocytes. Collectively, the present study identified some heat-stress-induced phenotypic and functional alterations in camel blood leukocytes, providing a paradigm for comparative immunology in the large animals. The clinical relevance of the observed changes in camel leukocytes for the adaptation of the camel immune response to heat stress conditions needs further in vitro and in vivo studies.The dromedary camel ( Camelus dromedarius) are economically important arid-desert-adapted livestock with a relatively high capability of tolerating extreme hot conditions  compared to normal temperatures (37°C).(Camelus dromedarius) aged between 10 and 14 years. The animals were housed and fed in free stalls at the Camel Research Center, King Faisal University, Al-Ahsa, Saudi Arabia. Blood was obtained by venipuncture of the external jugular vein (vena jugularis externa) into vacutainer tubes containing EDTA . To avoid the impact of environmental heat stress on the current in vitro study, the experiments were carried out in October 2020 when the camels were not exposed to heat stress conditions. The average daily temperature during the sampling period ranged between a minimum temperature of 18°C and a maximum temperature of 27°C. The Ethics Committee at King Faisal University, Saudi Arabia approved all experimental procedures and management conditions used in this study (Permission number: KFU- KFU-REC/2020-09-25).Blood samples were collected from five apparently healthy female dromedary camels in vitro heat treatment of camel whole blood was performed as previously described (6 cells/mL) in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 10 mg/mL streptomycin  were incubated either at 37 or 41°C for 4 h.The escribed  with somn = 5) were diluted (1:2) with RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 10 mg/mL streptomycin  and were stimulated with 1 μg/mL LPS from E.coli  for 4 h at 37 or 41°C. After incubation, blood samples were diluted (1:4) with phosphate buffer saline (PBS) and centrifuged at 10°C for 10 min at 1,000 × g. After removing the supernatant, the cell pellet was resuspended in PBS for further analysis.Whole blood stimulation with lipopolysaccharide (LPS) was performed as previously described . Brieflyg for 10 min without break. After careful removal of blood plasma, the erythrocytes were lysed by adding 5 mL distilled water for 20 s and subsequent addition of 5 mL double concentrated PBS to restore tonicity. After centrifugation at 500×g for 10 min with break, the cell pellet was resuspended. The erythrolysis was repeated  until the complete removal of red blood cells (clear white pellet of leukocytes). Subsequently, the cells were suspended in 10 mL PBS and washed two times (250×g and 100×g for 10 min each) and finally adjusted to 5 × 106 cells/mL in RPMI medium.Separation of camel leukocytes was done after hypotonic lysis of erythrocytes . Blood s6 cell/mL in RPMI cell culture medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 10 mg/mL streptomycin . In each well, 100 μL JC-1 solution  was added and the plates were incubated for 15 min in a humidified atmosphere (5% CO2) at 37°C. After two washing steps with PBS, cells were suspended in 200 μL PBS, transferred to flow cytometer tubes, and acquired with the BD Accuri C6 flow cytometer. In apoptotic cells (with low MMP), JC-1 forms monomers, which emit in the green fluorescence channel (FL-1) at 525 nm upon excitation at the 488 nm. In normal cells (with high MMP), JC-1 forms aggregates, which display an orange fluorescence .Cell viability of blood leukocytes was analyzed using the dye exclusion assay . Separatstaphylococcus aureus (S. aureus) bacteria  were labeled with fluoresceinisothiocyanate  according to manufacturer instructions. FITC-conjugated and heat-killed S. aureus bacteria were washed thoroughly, suspended in RPMI medium (2 × 108 bacteria/mL), and stored in single-use aliquots at −80 °C. Leukocytes separated from heat-stressed whole blood or heat-stressed leukocytes were plated in 96 well-plates (1 × 106/well) in duplicates and incubated with FITC-conjugated S. aureus (30 bacteria/cell) for 30 min . Control samples were incubated without bacteria. After incubation, samples were washed twice with PBS and analyzed by flow cytometry. Phagocytic activity of monocytes and neutrophils was defined as the percentage of green fluorescing cells among viable cells. Mean green fluorescence intensity (MFI) of phagocytosis-positive cells was measured as an indicator for the number of bacteria phagocytosed by each cell.Heat killed 6/well in RPMI medium) were incubated in duplicates for 20 min  with heat-killed S. aureus (30 bacteria/cell) in the presence of the ROS-sensitive dye dihydrorhodamine (DHR)-123 . After incubation, labeled cells were washed with PBS and the relative amount of generated ROS was determined by flow cytometry  after the acquisition of 100 000 events (gated leukocytes).ROS generation was measured in 96-well round-bottom microtiter plates  as previously described . Leukocy5) were incubated  with unlabeled primary monoclonal antibodies (mAbs) specific for the cell markers CD14, CD163, and MHC-class II or with directly labeled mAbs to the cell adhesion molecules CD11a and CD18 (The expression of the monocytes markers CD163 and major histocompatibility complex (MHC) class II molecules as well as the cell adhesion molecules CD11a and CD18 was analyzed using membrane immunofluorescence test as previously described . Separatp-value was <0.05.Statistical analysis was carried out using the software Prism (GraphPad software version 5). Results were presented as means ± S.E. of the mean (SEM). Differences between the means were tested using the one-factorial analysis of variance (ANOVA) in combination with the Bonferroni's method for a pairwise comparison of the means. Results were considered statistically significant if the p > 0.05) after incubation of blood at 37 or 41°C for 1, 2, or 4 h (p < 0.05) increase in the fraction of apoptotic granulocytes (20.3 ± 1.4 % compared to 3.5 ± 0.4 % after 2 h incubation at 41°C), lymphocytes (7.9 ± 0.6 % compared to 3.7 ± 0.6 % after 2 h incubation at 41°C), and monocytes (13.4 ± 2.0 % compared to 2.8 ± 0.3 % after 2 h incubation at 41°C). In addition, the fractions of apoptotic cells within the granulocytes and monocytes populations, but not the lymphocytes population, were significantly higher in blood incubated for 4 h at 41°C compared to blood incubated at 37°C for the same time (p < 0.05). For camel granulocytes, monocytes, and lymphocytes, the fraction of apoptotic cells did not change significantly (p > 0.05) after incubation of whole blood at 37°C for 1, 2, or 4 h (5 cells/well), 4 h-incubation of separated leukocytes at 37°C (4.8 ± 0.1 × 105 cells/well) or 41°C (4.7 ± 0.1 x 105 cells/well) did not induce a significant change in the number of viable PI-negative cells.The analysis of cell necrosis using the DNA sensitive dye propidium iodide  revealed, or 4 h . The ide, or 4 h . Incubat, or 4 h . Incubat, or 4 h . When th, or 4 h . The perp > 0.05) after incubation of whole blood at 37°C (p < 0.05) higher fraction of phagocytosis-positive cells (for neutrophils and monocytes) and higher (p < 0.05) phagocytosis capacity (only for neutrophils) in blood incubated for 4 h at 41°C than in blood incubated at 37°C for the same time (p < 0.05) in the phagocytosis activity of monocytes (56.8 ± 4.2% vs. 45.8 ± 2.6% for cells incubated at 37°C), no significant (p > 0.05) change was observed in the phagocytosis activity of neutrophils (52.1 ± 2.8% vs. 48.8 ± 2.7% cells incubated at 37°C) compared to cells incubated at 37°C.For both neutrophils and monocytes and for all incubation times , neither the percentage of phagocytosis-positive cells nor the phagocytosis capacity (number of bacteria phagocytosed by each cell as measured by the MFI of phagocytosis-positive cells) showed a significant change ( at 37°C . The comame time . Althougp > 0.05) after 1, 2, or 4 h of incubation of whole blood at 37°C. For blood samples incubated for 4 h at 41°C, neutrophils and monocytes produced significantly (p < 0.05) more ROS compared to the ROS response after 1 or 2 h of incubation at the same temperature (p < 0.05) higher ROS response in neutrophils and monocytes, when blood was incubated at 41°C compared to 37°C (The ROS response of both neutrophils and monocytes did not change significantly (perature . In addi to 37°C . For sep to 37°C .p < 0.05) lower value after 4 h of incubation  in comparison to CD163 levels after 1 h  and 2 h  of incubation at the same temperature (p < 0.05) higher expression level after 4 h of incubation  in comparison to 1 h  and 2 h  incubation time (p < 0.05) higher CD163 expression levels after 4 h incubation at 41°C  compared to 37°C   . After 46 ± 181) .To evaluate the impact of heat stress on the response of monocytes to stimulation with lipopolysaccharide (LPS), the expression levels of the monocytic markers CD163 and MHC class II molecules were compared between blood samples stimulated at 37 and 41°C for 4 h. For both heat treatments (37 and 41°C), LPS stimulation for 4 h resulted in decreased expression levels of CD163 with increased expression levels of MHC class II molecules . While tp > 0.05) after 1 or 2 h incubation of whole blood at 37 or 41°C (p < 0.05) decrease in the expression level of CD18 on neutrophils and monocytes when the blood was incubated at 41°C and only on monocytes when the blood was incubated at 37°C. In addition, after 4 h incubation at 41°C, monocytes showed a lesser abundance of CD18 (p < 0.05) compared to 37°C (p < 0.05) after 4 h compared to 1 and 2 h incubation (p < 0.05) lower levels compared to cells incubated for 2 h at the same temperature or for 4 h at 37°C .Dromedary camels are known for their well-adaptation to the extreme hot desert environment, with the capability to elevate their body temperature to 41–42°C during severe hot , 5, 6. IAs the heat-stress-induced changes were mainly observed after 4 h incubation of whole blood at 41°C and to exclude a possible impact of the separation procedure used for leukocyte separation from heat-stressed blood on the obtained results, leukocytes were firstly separated from blood and were then subjected to heat stress for 4 h. The comparable effects of heat treatment on separated leukocytes and whole blood samples, regarding leukocyte vitality, phagocytosis, ROS production, and cell adhesion molecules expression indicate no significant impact of the cell separation method on the results of the current study.in vitro heat stress .JH designed the work, collected the samples, performed the experiments, and prepared the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our previous studies have proved that 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) is a novel proliferation-promoting protein. The overexpression of HSD17B4 promotes hepatocellular carcinoma (HCC) cell proliferation. Vitamin K2 (VK2), a fat-soluble vitamin, has the function of promoting coagulation and can inhibit the progression of liver cancer. A previous study demonstrated that VK2 could bind to HSD17B4 in HepG2 cells. However, the mechanism of VK2 in inhibiting HCC cell proliferation is not clear. In this study, we investigate whether VK2 can inhibit the proliferation of HCC cell induced by HSD17B4 and the possible mechanism. We detected the effect of VK2 on HSD17B4-induced HCC cell proliferation, and the activation of STAT3, AKT, and MEK/ERK signaling pathways. We measured the effect of HSD17B4 on the growth of transplanted tumor and the inhibitory effect of VK2. Our results indicated that VK2 directly binds to HSD17B4, but does not affect the expression of HSD17B4, to inhibit the proliferation of HCC cells by inhibiting the activation of Akt and MEK/ERK signaling pathways, leading to decreased STAT3 activation. VK2 also inhibited the growth of HSD17B4-induced transplanted tumors. These findings provide a theoretical and experimental basis for possible future prevention and treatment of HCC using VK2. VK2 is a fat-soluble vitamin; in addition to promoting blood coagulation, it has antitumor effects. VK2 has been shown to induce cancer cell apoptosis and suppress cancer growth and differentiation in various types of cancer cells. Recently, some studies have shown that VK2 has anticancer effects on hepatocellular cancer, breast cancer, and leukemia –4. Natur17β-Hydroxysteroid dehydrogenase 4 (HSD17B4) is a ubiquitous oxidoreductase in mammals, with the highest levels in the liver , 14. OurIn this study, the inhibitory effect of VK2 on HCC was proved by cell and transplanted tumor experiments. We found that VK2 directly binds to HSD17B4, but does not affect the expression of HSD17B4, to inhibit the proliferation of HCC cells by inhibiting the activation of Akt and MEK/ERK signaling pathways, leading to a decrease in STAT3 activation. These findings provide a theoretical and experimental basis for possible future prevention and treatment of HCC with VK2.2, and saturated humidity. The cell seeding density was 3 × 104 cells/cm2. After 24 h of culture, the cells were cultured in RPMI-1640 medium without FBS for another 24 h. The HSD17B4 expression plasmid was transfected 24 h before VK2 administration.The human hepatocellular carcinoma cell line HepG2  was cultured with RPMI-1640 medium (Gibco Invitrogen Corp.) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin. The cells were cultured in a cell incubator at 37°C, 5% CO6) were seeded in a six-well plate and cultured for 24 h at 37°C and 5% CO2. Cells were transfected when the cells have grown to about 70% confluence. The cells were washed twice with serum-free RPMI-1640, and 1 ml serum-free Opti-MEM I  was added to each well. The DNA–Lipofectamine 2000 complex was prepared according to the manufacturer’s instructions . A total of 6 μg transfection plasmid and 30 μl Lipofectamine reagent were added to each reaction. The empty vector control is indicated by “−, ” and the HSD17B4 plasmid is highlighted with a “+” below. Cells were placed in an incubator at 37°C for 6 h. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% FBS for 24 h, and the transfection efficiency was determined by Western blotting.HSD17B4 cDNA was cloned into the pLL3.7 vector  . HepG2 cThe specific small interfering RNA (siRNA) against HSD17B4 (siHSD17B4), containing 5′-GUACCUUUGUAUUUGAGGAdTdT-3′ and 5′-UCCUCAAAUACAAAGGUACdTdT-3′, and the non-specific siRNA (siNC), containing 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′, all were purchased from Sigma . TransfeCell proliferation assays were performed using a 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-etrazolium, inner salt (MTS) cell proliferation assay kit (Promega) according to the manufacturers’ recommendations. The cells were treated with MTS for 3 h before the termination of HSD17B4 plasmid incubation. The OD readings were performed to determine the number of viable cells at 490 nm. All groups were evaluated in an average of three separate wells per experiment.−ΔCt (Ct sample −Ct control). The primer pairs are shown in According to the instructions, total RNA was isolated from cells using TRIzol reagent (Invitrogen). SuperScript Reverse Transcription Kit (Invitrogen) was used to synthesize cDNA, which was used as a template for real-time quantitative PCR using the SYBR Green PCR Master Mix Kit (TaKaRa). Quantitative real-time PCR was performed using the Rotor-Gene 3000 detection system (Gene Biosystems). The PCR cycle consisted of an initial denaturation step at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 55°C for 30 s, and extension at 72°C for 20 s. As an internal control, 18S rRNA was used for RNA template standardization. All PCRs were performed in triplicate. The relative expression level of mRNA was 2g for 10 min, or cytoplasmic protein was obtained by centrifugation at 12,000 g for 20 min. The supernatant was used for testing. The same amount of proteins (100–200 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with 5% skim milk powder for 1 h at room temperature and incubated overnight at 4°C with primary antibodies: rabbit anti-HSD17B4 (Homemade), 1:2,000 ; rabbit anti-cyclin D1 , 1:1,000; rabbit anti-PCNA , 1:1,000; mouse anti-β-actin , 1:1,000; rabbit anti-p-STAT3 , 1:500; rabbit anti-STAT3 , 1:500; rabbit anti-p-Akt , 1:500; rabbit anti-Akt , 1:500; rabbit anti-p-MEK , 1:500; rabbit anti-MEK , 1:500; rabbit anti-p-ERK , 1:500; and rabbit anti-ERK , 1:500. After incubation with appropriate fluorescent secondary antibodies at room temperature for 1 h, the anti-antigen complexes were imaged using chemiluminescence Plus Protein Western blot assay kit (Santa Cruz Biotechnology). The expressions of various genes were expressed in relative grayscale, and β-actin was used as the protein loading control.Cells are lysed in lysis buffer A for cytoplasmic protein extraction, and then, the cells were used for nucleoprotein extraction in lysis buffer B . After tHepG2 cells with HSD17B4 overexpression were treated with different concentrations of VK2 for 48 h. The cell lysates interacted with anti-HSD17B4 antibody; the precipitate was collected for the determination of the content of VK2 that bound to HSD17B4 in cells. Subsequently, a cell lysates assay for anti-VK2 antibody was performed by commercial ELISA kit (BlueGene Biotech) according to the manufacturer’s instructions. The optical density (OD) was measured at 450 nm. The results were expressed as pg/ml according to a calibrator curve.in vivo, 2 × 106 transduced HepG2 cells were injected into BALB/c nude mice aged 4 weeks. Six weeks post-inoculation, the animals were sacrificed, and the volume of the tumor was recorded.In order to test the inhibitory effects of VK2 on tumor growth The results were expressed as mean ± standard deviation (SD). Statistical differences among groups were analyzed using one-way ANOVA. At the p < 0.05 level, the difference was considered statistically significant. All statistical analyses were performed using SPSS 13.0 software.To test the effect of VK2 on HCC cells proliferation induced by HSD17B4, we measured the activation of cells by MTS assay. VK2 (50 μM) showed a time-dependent inhibition on the proliferation of HepG2 cells with HSD17B4 overexpression  expression . To deteTo further investigate the mechanism by which VK2 suppressed proliferation induced by HSD17B4 overexpression in HepG2 cells, we detected the activation of STAT3 pathway that was directly related to proliferation of HCC cells. HSD17B4 overexpression increased the phosphorylation level of STAT3, and VK2 not only suppressed phosphorylation of STAT3 on cells that were transfected with the empty plasmids but also suppressed their increased activation by induced HSD17B4 overexpression .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


KDM1A is overexpressed in human DLBCL tissues and negatively related to overall survival rate of DLBCL patients. ZY0511, a novel and potent LSD1 inhibitor developed by our group, inhibited the proliferation of human DLBCL cells. ZY0511 interacted with LSD1, induced methylation level of histone 3 lysine 4 and histone 3 lysine 9 in DLBCL cells. Mechanistically, transcriptome sequencing results indicated that ZY0511 induced the genes enrichment significantly related to cell cycle, autophagy, and apoptosis signaling pathways. Further study confirmed that ZY0511 blocked cell cycle at G0/G1 phase and expression of CDK4 and cyclin D1. ZY0511 decreased mitochondrial membrane potential and induced apoptosis, which can be reverted by a pan-caspase inhibitor, Z-VAD-FMK. Moreover, ZY0511 treatment significantly increased autophagy-associated marker proteins and autophagosomes formation in DLBCL cells. In vivo xenograft experiments confirmed that intraperitoneal administration of ZY0511 significantly suppressed SU-DHL-6 xenograft tumor growth in vivo. In conclusion, our findings identify that ZY0511 inhibits DLBCL growth both in vitro and in vivo via the induction of apoptosis and autophagy, and LSD1 inhibitor might be a promising strategy for treating DLBCL.Lysine-specific demethylase 1  is an attractive agent for treatment of cancer. However, the anti-tumor effect of LSD1 inhibitors against diffuse large B-cell lymphoma (DLBCL) and the underlying mechanism are still unclear. Here, we report that The online version contains supplementary material available at 10.1007/s12032-021-01572-0. Diffuse large B-cell lymphoma (DLBCL) is the most aggressive class of non-Hodgkin lymphomas (NHLs) . The staKDM1A is an epigenetic regulatory factor [Epigenetic dysregulation plays a crucial role in the initiation and development in human DLBCL , 4. In 2y factor . LSD1 rey factor . LSD1 isy factor , 8. Thery factor . Furthery factor . Conditiy factor . Above sIn the last decades, numerous LSD1 inhibitors, including TCP, ORY-1001, ORY-2001, CC-90011, INCB059872, and IMG-7289 are undergoing clinical trials for treatment of AML, myelodysplastic syndromes (MDS), myeloproliferative neoplasms, SCLC, relapsed Ewing sarcoma, myelofibrosis, essential thrombocythemia, multiple sclerosis, mild to moderate Alzheimer’s disease [50 value of 1.7 nM [KDM1A was highly expressed in human DLBCL tissues and associated with the poor survival of DLBCL patients. By interacting with LSD1 in DCBCL cells, ZY0511 inhibited cell proliferation both in vitro and in vivo. Mechanistically, ZY0511 blocked cell cycle at G0/G1 phase, induced apoptosis, and induced autophagy via inactivation of mTOR/p70S6K signaling pathways. Our findings reveal that ZY0511 might be a promising treatment strategy for treating DLBCL.In 2016, our group developed a potent LSD1 inhibitor, ZY0511, which potently and selectively inhibited LSD1 activity with an ICf 1.7 nM . Here, wThe main antibody information was listed below: LSD1 , H3 , H3K4me , H3K4me2 , H3K9me , H3K9me2 , phospho-p53 (Ser 15), phospho-mTOR (Ser 2448), p70 S6K , phospho-p70 S6k (Thr 421/Ser 424), phospho-S6 (Ser235/236), Beclin-1 , Bax , Cleaved Caspase-3 , GAPDH , Ki67 , mTOR , p53 , Caspase-8 , CDK4 , CDK6 , Cyclin D1 , LC3B , p62/SQSTM1 , S6 , PCNA.GSK2879552, 3MA, chloroquine (CQ), and Z-VAD-FAM were purchased from Selleckchem . ZY0511 was synthesized in laboratory as described previously .2.The human DLBCL cell lines including SU-DHL-4, SU-DHL-6, SU-DHL-10, and Farage were purchased from American Type Culture Collection . The DLBCL cells were cultured in RPMI 1640 medium containing 20% fetal bovine serum  at 37 °C and 5% COThe human DLBCL cells were lysed with RIPA lysis buffer containing protease inhibitor and phosphatase inhibitors. Next, the bicinchoninic acid (BCA) method was used to quantify the protein concentrations. Proteins were isolated by SDS-PAGE gel, and then transferred onto polyvinylidene fluoride membranes . After blocking with 5% skimmed milk, the membranes were incubated with the specific antibodies at 4 °C overnight. The membranes were washed with TBST buffer three times and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies at room temperature for 1 h. At last, the protein levels were visualized using Chemiluminescent HRP Substrate  and signals were detected using chemiluminescence imaging system.5 cells/mL) and treated with DMSO or ZY0511 at the final concentration of 200 μM at 37 °C for 1 h. The cells were harvested and then washed with pre-chilled PBS for twice, followed by resuspension in pre-chilled PBS containing protease inhibitor. The cell suspension was aliquoted into PCR tubes (1.5 × 106 cells/tube) and heated at different temperatures  for 3 min, followed by placed at room temperature for 3 min. Next, the sample was subjected to three freeze (in liquid nitrogen)—thaw cycles (at room temperature), and the supernatant was obtained by centrifugation at 20,000×g for 20 min at 4 °C. Subsequently, loading buffer was added, and then proteins were denatured at 100 °C for 10 min. Finally, the level of LSD1 was detected by western blot assay. Western blot signals based on densitometry method were quantified by Fiji and CETSA curves in intact cells were graphed by GraphPad Prism 8 software .The DLBCL cells were seeded (2 × 104 cells/well), 48 h (4 × 104 cells/well), 72 h (2 × 104 cells/well), and 96 h (1 × 104 cells/well), respectively. Then, 20 μL MTT (5 mg/mL) was added to form formazan. Optical density was measured using a microplate reader . The IC50 values were calculated by GraphPad Prism 8 software using XY modeling.An MTT  experiment was conducted to assess the proliferation inhibitory rate of ZY0511 against DLBCL cells. The DLBCL cells were treated with various concentrations of ZY0511 in 96-well plates for 24 h (8 × 105 cells/mL) and treated with ZY0511 at the final concentrations of 0.5, 1, 2 μM for 24 h. The DLBCL cells were grown in medium containing EdU, which is a thymidine analogue labeled cells in the proliferation phase for 2 h. Then, the EdU positive cells were stained with Cell-Light EdU Apollo488 In Vitro Flow Cytometry Kit according to Ribobo’s instructions . Cells were measured by flow cytometry  and analyzed using NovoExpress software .The DLBCL cells were seeded . PrimeScript™ RT reagent Kit with Gdna Eraser  was used to synthesize cDNA. RNA quality was evaluated by Real-Time system  using the SYBR® Green Supermix . Primers were listed in Table S1.P < 0.05 (Student’s t-test) were used to filter significant probe sets for each biological sample (n = 3). The gene counts of each transcript were calculated and DESeq2 was used for normalization to determine differentially expressed genes.RNA-seq experiment was conducted using a profiler service provided by Novogene Co. Ltd . After 48 h of treatment with DMSO or ZY0511 (1 μM), total RNA was purified from SU-DHL-4 cells and stored in TRIzol . A fold-change > 1.5, FDR  < 0.05, and 5 cells/mL) and treated with ZY0511 at the final concentrations of 0.5, 1, 2 μM for 24 h. Then, the DLBCL cells were collected and fixed with 70% ethanol solution at 4 °C overnight. Fixed cells were stained with propidium iodide according to KeyGEN BioTECH’s instructions . Cells were measured by flow cytometry  [The DLBCL cells were seeded   and anal5 cells/mL) and treated with ZY0511 at the final concentrations of 0.5, 1, 2 μM for 48 h. Then, the DLBCL cells were harvested and stained with Annexin V-PE and 7-AAD according to BD’s instructions , followed by apoptosis analysis by flow cytometry [The DLBCL cells were seeded (2 × 10ytometry .5 cells/mL) and treated with ZY0511 at the final concentrations of 0.5, 1, 2 μM for 48 h. Then, the DLBCL cells were harvested and incubated with tetraethyl benzimidazolyl carbocyanine iodide (JC-1) according to Beyotime’s instructions , followed by ΔΨm assay by flow cytometry analysis.The DLBCL cells were seeded (2 × 105 cells/mL) and treated with ZY0511 (2 μM) and/or Z-VAD-FMK (50 μM) for 48 h. After resuspending the DLBCL cells, 20 μL cell suspension was added 20 μL mixture solution of AO which stains living cells to green and PI which stains dead cells to red. A cell counter was used to determine the cell number and the percent of live cells and dead cells.The DLBCL cells were seeded  for 1 h. Then, slides were incubated with primary antibody overnight at 4 °C, followed by incubating with Cy3 or FITC-conjugated secondary antibody for 1 h at room temperature with shaking [5 cells/mL) and treated with ZY0511 at the final concentration of 2 μM for 48 h. The treated cells were fixed with 2.5% glutaraldehyde. Then, cells were embedded in epoxy resin and imaged by JEM-1400 Plus TEM  following standard TEM procedures.The DLBCL cells were seeded (2 × 106 cells/mouse/100 μL) were subcutaneously injected in the right flank of the mice. The tumor-bearing mice were randomly divided into three groups (n = 6 per group) when tumor volume reached 80–100 mm3. ZY0511 was suspended in 6% PEG4000, 2.4% Tween-20, and 91.6% ultrapure water, followed by nanocrystalline suspension preparation by high-pressure homogenization. ZY0511 (50 mg/kg and 100 mg/kg) or solvent were administrated once daily by intraperitoneal injection for 21 days. Tumor volumes and body weights were monitored every three days. Calculation formula: V (mm3) = (a × b2)/2 . At the end of the experiment on day 21, the tumor tissues were stripped and fixed in 4% paraformaldehyde for further experiments.NOD/SCID mice aged 5- to 6-week were purchased from the GemPharmatech Co., Ltd  and fed under specific pathogen-free (SPF) barriers. The SU-DHL-6 Cells  to analyze the expression of KDM1A mRNA in human DLBCL tissues and normal lymph nodes. The results showed that high expression of KDM1A was observed in human DLBCL tissues compared with that in normal lymph nodes  had a higher survival rate than that of the KDM1A high expression group (KDM1A-high) based on the data from Genomicscape website (http://www.genomicscape.com/), suggesting that high KDM1A expression in DLBCL tissues was associated with poor prognosis of patients  for gene set enrichment analysis (GSEA), it was found that ZY0511 treatment induced the change of a subset of LSD1 downstream transcriptional programs in SU-DHL-4 , and Z-VAD-FMK cotreatment reduced apoptotic SU-DHL-4 cells by about 70% (from 53.13 to 15.72%). ZY0511 increased the number of apoptotic SU-DHL-6 cells by 12.4 folds (from 6.62 to 81.78%), and Z-VAD-FMK cotreatment reduced apoptotic SU-DHL-6 cells by about 49% (from 81.78 to 41.8%)  compared with ZY0511 group. In SU-DHL-6 cells, Z-VAD-FMK cotreatment increased the number of live cells by about 53% (from 1.13 × 105 to 1.73 × 105 cells/mL) compared with ZY0511 group ,and ribosomal protein S6 (S6) in a concentration-dependent manner . The difference of chemical structure might be the one of reason to explain above difference. Our previous study found that ZY0511 formed hydrogen bond with Q358 of LSD1, and a π–π reaction between ZY0511 and FAD which is different from TCP derivatives. As FAD is a very important co-factor in cancer progression, inhibition of FAD by ZY0511 might be responsible for, and enhanced the anticancer effect of ZY0511 compared with TCP derivatives. However, the underlying mechanism needs to be further revealed. Moreover, the clinical trials of GSK2879552 against AML and SCLC were terminated because of adverse events and serious adverse events, suggesting that TCP derivatives may has toxicity. Thus, the further structure–activity relationship studies of ZY0511 may lead to develop safer and more potent LSD1 inhibitors which inhibits proliferation of DLBCL, and expand the clinical use of LSD1 inhibitors.ZY0511 is a potent LSD1 inhibitor developed by our group . We founSKP2 and CDC25A) and thus facilitates the expression of S-phase genes and G1-S phase transition [SKP2 expression by 46% and CDC25A expression by 40%. Thus, we speculate that ZY0511 increases the level of H3K9me2 and thereby represses expression of the targeted genes of SKP2 and CDC25A. However, whether ZY0511 down-regulate the expression of CDK4 and Cyclin D1 by directly up-regulating H3K9me2 at these genes promoter needs further study. Collectively, the cell cycle progression was blocked by ZY0511, further leading to inhibition of cell proliferation in vitro.Cancer is characterized by aberrant cell cycle. Cyclin-dependent kinases (CDKs) interact with cyclin to regulate cell cycle progression and proliferation . Cyclin ansition . We founIt is well known that cells with cell cycle arrest eventually progress to apoptosis. Apoptosis plays a crucial role in maintaining cellular homeostasis and controlling the cell proliferation . There aSQSTM1 (gene encodes p62 protein) and affecting autophagy level through its H3K4 demethylase activity. Inhibition of LSD1 activates autophagy by stabilizing SQSTM1/p62, thus reduces malignant cell growth [SQSTM1 expression by 1.9 folds and elevated the protein level of p62. As LSD1 inhibits the transcription of SQSTM1 by removing the lysine residues of histone H3K4 [SQSTM1 by up-regulating H3K4me and thereby induces autophagy. It is also reported that LSD1 inhibits autophagy in neuroblastoma by SESN2 (Sestrin2)-dependent pathway [SESN2 expression, and obviously suppressed mTOR/P70S6K axis by inhibiting the level of phosphorylation of mTOR, p70S6K, and S6, which is consistent with above studies. Moreover, these data are consistent with our previous findings that ZY0511 predominately elevates the level of DDIT4 [Together with apoptosis, autophagy involves in controlling cell fate. Autophagy is originally identified as a cell survival mechanism, sequestering, degrading, and recycling cellular material. Accumulating studies suggest that autophagy facilitate DLBCL cell death by providing a scaffold for the cell death machinery . Xu’s stl growth . Our resone H3K4 , thus ZY pathway . SESN2 i pathway , 47. Ourof DDIT4 , a supprof DDIT4 , 49.Several studies have suggested that inhibition of autophagy could reduce anti-tumor T cell responses , becauseIn summary, we have demonstrated that ZY0511 showed anti-tumor effect against human DLBCL cells by blocking G0/G1 cell cycle progress and triggering apoptosis. Additionally, ZY0511 induced autophagy through mTOR/P70S6K axis. Moreover, ZY0511 inhibited tumor growth in vivo without adverse events. Therefore, our findings suggest that ZY0511 might be a promising treatment strategy for treating DLBCL.Supplementary file1 (DOCX 16 kb)Supplementary file2 (DOCX 28 kb)Below is the link to the electronic supplementary material.


UCA1 is frequently upregulated in a variety of cancers, including CRC, and it can play an oncogenic role by various mechanisms. However, how UCA1 is regulated in cancer is largely unknown. In this study, we aimed to determine whether RNA methylation at N6-methyladenosine (m6A) can impact UCA1 expression in colorectal cancer (CRC).http://gepia.cancer-pku.cn).qRT-PCR was performed to detect the level of UCA1 and IGF2BP2 in CRC samples. CRISPR/Cas9 was employed to knockout (KO) UCA1, METTL3 and WTAP in DLD-1 and HCT-116 cells, while rescue experiments were carried out to re-express METTL3 and WTAP in KO cells. Immunoprecipitation using m6A antibody was performed to determine the m6A modification of UCA1. In vivo pulldown assays using S1m tagging combined with site-direct mutagenesis was carried out to confirm the recognition of m6A-modified UCA1 by IGF2BP2. Cell viability was measured by MTT and colony formation assays. The expression of UCA1 and IGF2BP2 in TCGA CRC database was obtained from GEPIA (Our results revealed that IGF2BP2 serves as a reader for m6A modified UCA1 and that adenosine at 1038 of UCA1 is critical to the recognition by IGF2BP2. Importantly, we showed that m6A writers, METTL3 and WTAP positively regulate UCA1 expression. Mechanically, IGF2BP2 increases the stability of m6A-modified UCA1. Clinically, IGF2BP2 is upregulated in CRC tissues compared with normal tissues.These results suggest that m6A modification is an important factor contributing to upregulation of UCA1 in CRC tissues.The online version contains supplementary material available at 10.1186/s12935-021-02288-x. Colorectal cancer (CRC) is the third most commonly diagnosed cancer, and is the second leading cause of cancer-related death worldwide . In ChinLong non-coding RNA (lncRNA) is a transcript with a length > 200 nucleotides, lacking the coding capacity. Evidence accumulated over the past decade indicates that the lncRNAs are frequently dysregulated in cancer and they can play key roles in tumorigenesis. In particular, CRC represents one of the tumors showing the most relevant dysregulation of lncRNA functions , 7. NumeAlthough the crucial role of UCA1 in CRC development has been well characterized, the mechanism underlying the upregulation of UCA1 in CRC is still largely unknown. In this study, we demonstrated that m6A modification of UCA1 enhances its RNA stability, resulting in the high level of UCA1 in CRC. We further showed that knockout of m6A writer METTL3 or WTAP decreases UCA1 RNA level in CRC cells, whereas rescue experiment  restores the expression level of UCA1. In vivo RNA pulldown assay indicated that UCA1 is recognized by IGF2BP2 on the m6A site in CRC cells. Silencing of IGF2BP2 impairs the RNA stability of UCA1, highlighting the importance of m6A modification in regulation of UCA1 expression in CRC.WTAP (56501) and METTL3 (96391) were purchased from Cell Signaling Technology , YTHDF1 (17479-1-AP), YTHDF2 (24744-1-AP), YTHDF3 (25537-1-AP), IGF2BP1 (22803-1-A), IGF2BP2 (11601-1-AP), IGF2BP3 (14642-1-AP) were from Proteintech . Secondary antibodies conjugated with IRDye 680 were purchased from LICOR Biosciences . Anti-N6-methyladenosine antibody (17-3-4-1) were purchased from MilliporeSigma . PCR primers were purchased from IDT .We collected 46 patients with CRC diagnosed in the First People’s Hospital of Chenzhou City between 2014 and 2016. This study was approved by the Ethics Committee of the First People’s Hospital of Chenzhou. All procedures were implemented according to the guidelines of the Declaration of Helsinki. All patients provided written informed consent. Fresh colorectal neoplasms and matching normal tissues (located > 2 cm away from the tumor boundary) were stored in liquid nitrogen until RNA isolation.HCT116, DLD-1, 293 T were purchased from ATCC . HCT116 and DLD-1 were cultured with RPMI 1640 from Sigma supplemented with 10% fetal bovine serum . 293 T cells were grown in DMEM with 10% FBS. All media were supplemented with 2mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin . Cells were incubated at 37 ℃ and supplemented with 5% CO2 in the cell incubator.Cells were transfected with plasmid or siRNAs using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer’s instruction. The siRNA sequences targeting IGF2BP2 were designed and synthesized by Genepharma Company , and they were listed in Additional file Lentivirus was packaged in 293 T cells using the third generation of packaging system. Virus was collected 48 h after transfection. Lentivirus infection was carried out 6-well plates by mixing 500 μl virus supernatant and 500 μl medium containing 8 μg polybrene.Total RNA was isolated from cells using Trizol reagent (Invitrogen), and then cDNA was synthesized using GoScript™ RT System . Quantitative RT-PCR (qRT-PCR) was performed using a standard SYBR Green method . Actin was used as an internal control. The primers were listed in Additional file 3 /well HCT-116 or DLD-1 cells were seeded into a 96-well flat-bottomed plate. Cell numbers were evaluated for next 3 days. Ten microliters of CCK-8 reagent was added to each well, after which the plate was incubated at 37 °C for 2 h. Subsequently, the absorbance at 450 nm was measured in each well by using a spectrophotometer . All assays were performed in triplicate.Cell proliferation was performed using Cell Counting Kit-8 (CCK-8) Kit . According to the manufacturer’s instruction, as previously described . Briefly2 for 10–15 days. Then the cells were fixed with 4% paraformaldehyde and stained with crystal violet dye. The number of macroscopic colonies was counted, and images were obtained of the representative colonies.For the colony formation assay, 500 cells/well in the growth phase were seeded in six-well plates. The cells were cultured in cell incubator at 37 °C in 5% COCells were harvested and proteins were extracted and quantified as previously described . ProteinWTAP (#53741) and METTL3 (#53739) expression vectors for rescue experiments were obtained from Addgene . S1m expCRISPR/Cas9 system  was usedWe used m6A antibody to pull down m6A modified RNA, and IGF2BP2 antibody to pull down RNA. Total RNA was extracted from a 10 cm dish and dissolved in 50 μl RNase-free water. We used 5 μl as RNA input and the remaining RNA was added to 500 μl pulldown lysis buffer containing RNase inhibitor. In the case m6A pulldown, the total RNA was first precleaned with 1 µl mouse IgG and then the IgG was removed by protein A/G beads, followed by incubation with control IgG or primary antibody overnight at 4 °C. Finally, precipitated RNA was isolated with Trizol and the RNA level of UCA1 was determined by qRT-PCR.The procedure of in vivo pulldown of UCA1 using S1m-tagged system was similar to what had been previously described . The HCT6 stable HCT-116 UCA1-KD cells or control cells in 0.2 mL PBS with 50% matrigel as described previously [BALB/c nude mice (4–6 weeks old) were purchased from Hunan SLAC Jingda Experimental Animal Company . All animal experiments were approved by the Ethical Committee of the First People’s Hospital of Chenzhou City, University of South China. Six mice in each group were subcutaneously injected with 5.0 × 10eviously . Five weAll statistical analyses were performed using the GraphPad Prism program. The continuous variables are summarized as mean and standard deviation (SD) unless stated. One-way analysis of variance (ANOVA), paired t test and unpaired t test were used for statistical comparison. All P values were two-sided and P values < 0.05 were considered as significant.To determine the expression of UCA1 in CRC, we first analyzed CRC data from TCGA. We found that the level of UCA1 was significantly higher higher in CRC (n = 599) than in normal tissues . For example, both UCA1 KD1 and UCA1 KD2 suppressed UCA1 expression by ~ 50%  with CRISPR/Cas9 in colorectal cell lines HCT116 and DLD-1. The knockout (KO) clones were identified by Western blot. Shown in Fig. To further confirm the METTL3-mediated UCA1 expression, we performed rescue experiments, i.e., re-expression of METTL3 in the KO HCT116 and DLD-1 cells Fig. A & C. AsSince we have shown that METTL3 and WTAP positively regulate UCA1 expression, we set up to explore the biological function of METTL3 and WTAP in CRC. METTL3 KO inhibited cell proliferation abilities in HCT116 cells Fig. A. A simiThe m6A readers recognize m6A and affect the fate of the methylated RNA. There were two major m6A “readers” families, YTH family and IGF2BP family. To determine which m6A reader is involved in UCA1 m6A, we performed in vivo RNA pulldown assay to identify UCA1 m6A readers using the procedure outlined in Fig. To determine the consequences of interaction between UCA1 and IGF2BP2, we suppressed expression of IGF2BP2 by IGF2BP2 siRNAs. As shown in Fig. We then reasoned that as a UCA1 regulator, IGF2BP2 may also play a role in CRC. Thus, we analyzed IGF2BP2 expression in TCGA CRC data, and the expression of IGF2BP2 was significantly increased in CRC as compared to normal tissues Fig. D. AnalysAlthough UCA1 was initially identified in human bladder cancer cell line, subsequent studies revealed that it is overexpressed in a wide range of human cancers, including CRC , 29. Ext6-methyladenosine (m6A) modification, the most frequent chemical modification in eukaryotic RNA, is involved in many biological processes including cancer progression [Ngression , 37. Emegression , 39.M6A modification is a dynamic process and three key players, i.e., writers, erasers and readers, fine tune the dynamic balance of m6A level in cells . WritersIn this regard, we show that UCA1 expression can be regulated by m6A modification in CRC. Three lines of evidence support the regulatory role of the m6A system in UCA1 expression. (1) KO of METTL3 and WTAP causes a reduction of UCA1 level; (2) re-expression of METTL3 or WTAP in the corresponding KO cells increases UCA1 expression; (3) similarly, IGF2BP2 can also positively regulate UCA1 level. In addition, there is a positive correlation between UCA1 and METTL3, UCA1 and WTAP in the CRC clinical specimens. Given that UCA1 plays an oncogenic role in a variety of cancer, these findings support the importance of m6A-mediated UCA1 expression.As critical components of the m6A system, both METTL3 and WTAP have been shown to play an important role in various types of cancers. For example, METTL3 has been reported as an important oncogene in many tumor types, such as liver cancer, breast cancer and CRC –43. It hIGF2BP2 is an important RNA binding protein, which regulates RNA location, stability and translation. Moreover, accumulating evidence supports the role of IGF2BP2 in cancer. For example, the up-regulated IGF2BP2 promotes pancreatic cancer cell proliferation by activating the PI3K/Akt signaling pathway . Our recIn summary, we demonstrate that m6A plays an important role in regulation of UCA1. In this m6A system, m6A-modified UCA1 is recognized by IGF2BP2, which subsequently stabilizes UCA1. Clinically, like UCA1, METTL3, WTAP and IGF2BP2 are also upregulated in CRC tissues. Therefore, further dissection of this regulatory system will provide new insight into UCA1-mediated tumorigenesis in CRC.Additional file 1. Table 1: The list of primers used in this study.