Abnormality of darto tissue has been suggested as the pathophysiology of chordees. Collagen is one of the most abundant fibrous proteins within the extracellular matrix. In this study, we determined the expression of COL1A1 and COL6A1 in darto tissue was determined by reverse-transcriptase polymerase chain reaction (qPCR). The penile curvature severity was classified as mild (< 30 degrees), moderate (30–60 degrees), and severe (> 60 degrees).We included 60 children < 18 years old, consisting of 20 distal hypospadias, 20 proximal hypospadias patients, and 20 controls in our institution from 2017 – 2020. The expression of COL1A1 and COL6A1 expression was significantly downregulated in the distal (0.88 (0.38–2.53) and 0.54 (0.16–4.35), respectively) and proximal 0.76 (0.33–2.57) and 0.57 (0.18–1.38), respectively) hypospadias groups compared to controls (1.85 (0.24–4.61) and 0.93 (0.17–4.06), respectively) with p-values of 0.024 and 0.018, respectively. Furthermore, there was a moderate correlation between COL1A1 and COL6A1 expression . Interestingly, COL1A1 and COL6A1 were also significantly downregulated in the moderate and severe chordee groups compared to the mild chordee groups, with p-values of 0.003 and 0.037, respectively.qPCR showed that COL1A1 and COL6A1 expression might affect abnormalities in darto tissue and penile curvature severity in hypospadias patients.Aberrant  Hypospadias is the most frequent congenital penile anomaly that affects male external genitalia, with an incidence of approximately 1 in 250 male newborns. It is caused by tissue underdevelopment on the ventral aspect of the penis, resulting in an abnormal location of the urethral opening on the penile underside [Hypospadias is associated with curvature of the ventral penis, called the chordee. It is caused by the insufficiency or disorganization of the complex growth  of the ventral penis . ResectiThe extracellular matrix (ECM) is a noncellular macromolecular network that supports cellular processes, such as proliferation, migration, differentiation, and homeostasis . One of collagen 1 (COL1A1) and COL6A1 in patients with hypospadias and associated them with the severity of penile curvature.Fibrillar collagens are widely distributed in tissues providing tensile strength. Most fibrillar collagen is type 1 collagen, which can be found throughout the tissue beneath the urethral plate , 7. TypeWe included 60 children < 18 years old, consisting of 20 distal hypospadias, 20 proximal hypospadias patients, and 20 controls in our institution from 2017 – 2020. Detailed history and thorough examination of patients with hypospadias were performed by one urologist. We harvested periurethral darto tissue during urethroplasty in hypospadias patients and circumcision in normal penis samples.Fragments of the darto tissue were kept in RNAlater  solution before extraction. The tissue was extracted using Genezol RNA solution . cDNA was synthesized using an Excel RT Reverse Transcriptase Kit  with PCR conditions of 25 °C for 10 min (denaturation), 42 °C for 50 min , and 85 °C for 5 min (extension).COL1A1 (F: 5′-TACAGCGTCACTGTCGATGGC-3′ and R: 5′-TCAATCACTGTCTTGCCCCAG-3′), COL6A1 (F: 5′-GACCTCGGACCTGTTGGGTAC-3′ and R: 5′-TACCCCATCTCCCCCTTCAC-3′) [GAPDH (F: 5′-GCACCGTCAAGGCTGAGAAC-3′ and R: 5′-TGGTGAAGACGCCAGTGGA-3′) [COL6A1, COL1A1, and GAPDH, respectively) for 30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. PCR products were then separated by 2% agarose gel along with a 100-bp DNA ladder   with the following primer genes: TCAC-3′) , and GAPTGGA-3′) . qPCR cop < 0.05 was considered statistically significant.Data were analyzed for their normality distribution using the Kolmogorov–Smirnov test. Kruskal–Wallis and Mann–Whitney U tests were used for the data that were not normally distributed, while the normally distributed data were analyzed with one-way ANOVA tests. COL1A1 and COL6A1 expression was significantly downregulated in the distal (0.88 (0.38–2.53) and 0.54 (0.16–4.35), respectively) and proximal (0.76 (0.33–2.57) and 0.57 (0.18–1.38), respectively) hypospadias groups compared to controls (1.85 (0.24–4.61) and 0.93 (0.17–4.06), respectively) with p-values of 0.024 and 0.018, respectively .Moreover, there was no significant difference in COL1A1 and COL6A1 expression between proximal and distal hypospadias or severe and moderate penile curvature Table . FurtherCOL1A1 and COL6A1 expression in hypospadias patients with moderate and severe penile curvature. These results are consistent with previous research, which found that the mean number of total collagen fibers in dartos tissue in hypospadias was lower but had thicker fibers compared to normal patients [. proposed no difference in collagen intensity between normal areas and under the urethral plate of patients with hypospadias [Here, we are able to show downregulated patients . They alpatients . Anotherpatients . Eros etospadias .Type 1 collagen is the most abundant collagen. It is the predominant component of interstitial membranes. It is also associated with fibrosis and fibrogenesis . HayashiCOL6A1 as the most studied type of collagen in the beaded filament-forming collagen group. It plays a central role in cell attachments and connections between tissues and the surrounding matrix [This paper also investigated the gene expression of g matrix . Type 6 g matrix . In lungg matrix . Howeverg matrix . We founThe etiology of chordee in hypospadias remains unclear. Resection of dartos tissue usually can straighten the penis in patients with chordee and buried penis, suggesting that the pathophysiology of these anomalies is related to dartos tissue . The comCollagen is a part of the tissue backbone. During tissue turnover, it is formed and degraded to maintain tissue health and homeostasis. Imbalance of that process leads to fibrosis. Fibrosis is known as excessive formation of connective tissue, which damages the structure and function of its tissue . ChordeeCOL1A1 and COL6A1 expression among groups based on penile curvature in the hypospadias group. Mostly, severe forms of hypospadias are related to a significant chordee and a urethral meatus located proximal to the midshaft of the penis [We also investigated the significant difference in he penis . Anotherhe penis . Many fahe penis . The Darhe penis . Finallyhe penis .COL1A1 and COL6A1 expression might affect abnormalities in darto tissue and penile curvature severity in hypospadias patients.Aberrant


The loading capacity of Fe3O4-PVA@SH on Aspirin and the drug release kinetics of loaded drugs were studied. The obtained Fe3O4-PVA@SH exhibits excellent drug release properties in simulating the human body fluid environment (pH 7.2). Since magnetically targeting drug carriers are readily available and have excellent biocompatibility and the characteristics of drug release. This work’s development, preparing amphiphilic magnetically targeting drug carriers in drug delivery and other fields, has great significance.Currently, magnetic applications have great potential for development in the field of drug carriers. In this paper, Fe The polymer’s purpose is to make Fe3O4 nanoparticles as a magnetic core to be better and more uniformly distributed in the drug carrier. When Fe3O4 nanoparticles are coated with high molecular polymers, they can be used in drug carriers. Superparamagnetic iron oxide was used extensively in the detection of atherosclerotic plaque. And expanded-pore nanoparticles functionalized with N-isopropyl acrylamide and poly(ethylene glycol) were applied for temperature control release of bovine hemoglobin. Compared with small molecule drugs with passive targeting, polymer-drug carriers generally exhibit better pharmacokinetics. [Nanotechnology helps develop new pharmaceutical agents, drug delivery, and the synthesis of drug carriers . Playinginetics. Table 1.T3O4 is widely used in human treatment because of its stable quality, superparamagnetic properties, and easy realization. Fe3O4 nanoparticles are widely used as biomaterials and exhibit superparamagnetic properties in magnetic resonance imaging (MRI), targeted hyperthermia, drug delivery, and immobilized proteins [In the drug carrier’s actual application, the targeted drug carrier should have good biocompatibility and accurately target the body fluid environment’s desired location ,28.As a proteins .However,proteins .Among vaproteins .It has gproteins .Pharmacoproteins .Suppose the magnetical drug carriers want to show excellent biocompatibility. In that case, it needs to improve the solubility of the water of their carriers and improve their solubility of lipids. The feature of this study is to improve the lipid solubility of drug carriers through thiohydrazide-iminopropyltriethoxysilane (TIPTS). TIPTS is not only playing a coupling role in the polymerization reaction but also improve the lipid solubility. Good biocompatibility requires good amphiphilic ,51. The 9H8O4, is a widely used medicine. Aspirin is a cyclooxygenase (COX) inhibitor. It mainly reduces the synthesis of thromboxane (TXA2) by inhibiting COX activity, thus preventing platelet aggregation and blood clotting. [Aspirin , with a chemical formula Clotting.  Aspirin 3O4 is used as a magnetically targeted magnetic carrier. They were improving the water solubility of Fe3O4 by the reaction of Fe3O4 and PVA to get the Fe3O4-PVA. At the same time, the swelling characteristics of PVA makes the entire drug carrier exhibit slow-release characteristics. The Fe3O4-PVA reacts with thiohydrazide-aminopropyl-riethoxysilane (TIPTS) to synthesize Fe3O4-PVA @ SH improving the fat solubility of the drug carrier. On the other hand, the hydroxyl group on Aspirin can form a hydrogen bond(-OH) with the hydroxyl group(-OH) and thiol group(-SH) on Fe3O4-PVA @ SH and is loaded on the Fe3O4-PVA @ SH carrier, which is transported to the lesion site by the carrier and exerts efficacy. The mechanism is shown in In this project, Fe2.2.1.2 · 4H2O), iron(III) chloride hexahydrate(FeCl3 · 6H2O), [2SO4) was purchased from Acros Organics . thiohydrazide-iminopropyltrithoxy-silane (TIPTS) [Poly (PVA)  87.0–89.0 mol%, viscosity 80.0–110.0 mpa.s), Aspirin , Iron(II) chloride tetrahydrate (FeCl· 6H2O),  ammonia  (TIPTS) , homemad2.2.3 · 6H2O (4 g) and FeCl2 · 4H2O (2 g) were dissolved in 100 mL of distilled water. The solution ultrasound for an hour. Then, 30 mL of ammonia (NH3) was added to the solution and stirred for five hours at 70°C. The entire reaction system was carried out under nitrogen protection. Finally, the product was rinsed repeatedly, rinsed with water, and freeze-dried.FeCl2.3.3O4 (3 g) was ultrasound for an hour and add it in the PVA solution; the ammonia solution was added to adjust the pH appropriately. The entire reaction system was reacted for 5 h under the protection of nitrogen. The black product was washed with distilled water. Until the solution as the whole system reached a neutral pH, and the samples were freeze-dried.PVA (3 g) was dissolved in 100 mL of distilled water and stirred using a mechanical stirrer to dissolve. Fe2.4.3O4-PVA(1 g) were dispersed into 100 mL of DMSO. The solution ultrasound for 2 h and add H2SO4 until the solution system’s pH is 1 to 2.Then the entire reaction system was stirred at 35°C for 2 h. Also, the product was centrifuged, rinsed with water, and freeze-drying. At this point, we get Fe3O4-PVA@SH. The experimental process is shown in The thiol coupling agent(4 g) and Fe2.5.−1 in nitrogen flow.X-ray powder diffraction (XRD) spectra were taken on a Holland PANalytical X− Pert PRO X-ray diffractometer with Cu−Kα radiation. Fourier transforms infrared (FTIR) spectra were performed on the IRAffinity-1 spectrometer. Infrared spectrum analysis. Scanning electron microscopy (SEM)and Energy Dispersive Spectrometer (EDS) images were recorded using the JSM-6380 LV microscope. Contact angle measuring instrument . Differential scanning calorimetry (DSC) was carried on a NETZSCH STA 449 C analyzer with a heating rate of 20 °C min2.5.1.3O4-PVA@SH were determined. The swelling ratio was calculated as follows:The swelling properties of the FeWd and Ws are the weight of dried Fe3O4-PVA@SH before and after immersing in aqueous solution for 48 h, respectively.2.5.2.3O4-PVA@SH for Aspirin, we typically left 45 mg of Fe3O4-PVA@SH to soak in 10 mL of an aqueous solution of Aspirin(0.085 mmol/L) at 37°C temperature. After predetermined intervals time, the supernatant solution was collected for analysis by UV spectrophotometer. The amount of Aspirin loaded by Fe3O4-PVA@SH the was calculated from the following mass balance equation:To investigate the loading kinetics of the FeQt (mmol/g) is the amount adsorbed per gram of Fe3O4-PVA@SH at time t, Co is the initial concentration of Aspirin in the solution (mmol/L), Ct is the concentration of Aspirin at time t (mmol/L), V is the volume of the solution (L), and m is the mass of the Fe3O4-PVA@SH used(g).2.5.3.3O4-PVA@SH were kept immersed in 3 mL water of pH = 7.2 at 37°Cand placed on a shaking machine a certain shaking frequency to simulate the process of drug release in the human body. At particular intervals, the supernatant solution was collected for analysis by a UV spectrophotometer. Each experiment was carried out in triplicate.For the drug release experiment, the release of Aspirin was determined with a UV−vis spectrophotometer at λmax = 287 nm at a function of time. The typical procedure used as follows: the above aspirin-loaded Fe3.3.1.3O4-PVA, TIPTS and Fe3O4-PVA@SH nanocomposite. Compared with standard cards. We can be seen that the peaks at 2θ = 30.1°, 35.5°, 43.3°, 57.3°, and 62.7° were assigned to the characteristic peaks of Fe3O4 (JCPDS card No. 72–2303), demonstrated that Fe3O4 particles were successfully formed in the PVA matrix. The peaks at 2θ = 26.6°, 43.4°, 54.8°, 56.6°, and 63.6° are given to the typical carbon peak of TIPTS(JCPDS card No.26–1097) and confirmed the PVA’s semicrystalline properties. The XRD patterns of Fe3O4 -PVA shows that the synthesis of Fe3O4-PVA was successful [3O4-PVA@SH is shown in 3O4-PVA, TIPTS, and Fe3O4-PVA @ SH in the figure. Some characteristic peaks of TIPTS have partially deviated. This phenomenon is because that PVA and TIPTS are coated outside Fe3O4, which makes the particle size of Fe3O4 change massive, the crystal form changes slightly. All shows that PVA and TIPTS are added to the core structure, so this analysis’s composite construction is quite evident.According to the XRD pattern, ccessful . The XRD3.2.3O4-PVA are shown in −1 is attributable to the -OH stretching vibration of PVA. The peak at 2902 cm−1 in the infrared spectrum of Fe3O4-PVA is due to the stretching vibration of -CH. The peak at 1416 cm−1 is due to the stretching vibration of -C-C-, the rise at 1096 cm−1 is the stretching vibration peak of Fe-O-C, and the rise at 569 cm−1 is due to the stretching vibration of Fe-O- vibration peak. The existence of characteristic peaks indicates that we successfully synthesized Fe3O4-PVA. 3O4-PVA@SH. The mountain seen in TIPTS at 3435 cm−1 is due to the stretching vibration of -OH. The peak at 2583 cm−1 in Fe3O4-PVA @ SH is -SH, the height at 1432 cm−1 is caused by the hydrocarbon bending vibration of -CH3, and the stretching vibration of -CH causes the peak at 2928 cm−1. The height at 1373 cm−1 is the typical stretching vibration of -C-C-. The height at 1083 cm−1 is the stretching vibration peak of Fe-O-C. The height at 1628 cm−1 is the stretching vibration peak -NH, the peak at 798 cm−1 is attributed to the stretching vibration peak of Si-O-CH3. There is no stretching vibration of Fe-O, indicating that Fe3O4 is encapsulated to form a magnetic core drug carrier system Fe3O4 with thiols and hydroxyl groups. The synthesis of Fe3O4-PVA@SH was successful based on the synthesis of Fe-O-C, Si-O-CH3, and the presence of -SH and -NH, which confirms the formation of Fe3O4-PVA@SH.Infrared spectroscopy was performed to analyze the chemical changes between the incorporated components. The FT-IR spectra of PVA and Fe3.3.3O4-PVA synthesized. Accordingly, the synthesis of uniformly distributed spherical structures with a particle diameter of about 60 nm can be seen. 3O4-PVA @SH with a particle diameter of about 100 nm. Among them, the spherical irregularities are a mixture of PVA and TIPTS. Fe3O4 nanoparticles in which the polymeric that cover the core are well visible.3O4-PVA. The samples contain C, O, and Fe elements. The elemental analysis obtained from this spectrum indicates that C: O: Fe atomic ratio is 1:1.7:1.1. 3O4-PVA@SH the samples contain N, C, Fe, Si, O, and S(contain N) element3.4.3O4, Fe3O4-PVA and Fe3O4-PVA@SH are 91.84 degrees, 54.38 degrees, and 59.32 degrees. 3O4, Fe3O4-PVA, and Fe3O4-PVA@SH are 65.17 nm, 74.62 nm, and 88.52 nm, respectively. It has been well documented that the size of less than 100 nm is favorable for passive targeting. Fe3O4 is coated with polyvinyl alcohol, making Fe3O4-PVA have an increased particle size and increased hydrophilicity compared to Fe3O4. And Fe3O4-PVA@SH is grafted with TIPTS outside, so the particle size of Fe3O4-PVA@SH is also slightly increased compared to Fe3O4-PVA. And improve the lipid solubility, so the corresponding hydrophilicity has been reduced. Nanoparticle size change again proves the formation of magnetically targeted drug carrier Fe3O4-PVA@SH. Respectively, Fe3O4 has some hydroxyl groups on the surface, but the number is too small to meet the drug carrier’s hydrophilicity requirements. PVA is a hydrophilic polymer. PVA has strong hydrophilicity. PVA has many hydroxyl groups and is coated on Fe3O4 to improve the carrier’s hydrophilicity. The mercapto group (-SH) is less water-soluble than the hydroxyl group (-OH), so the presence of the mercapto group (-SH) makes the carrier’s water solubility slightly lower. At the same time, a suitable drug carrier also needs to have excellent lipophilicity. The thiol group on TIPTS has strong nucleophilicity. The introduction of the thiol group improves the lipid solubility. Improving amphiphilicity allows magnetic targeting drug carriers to have excellent hydrophilicity and lipophilicity, and biological activity is much improved. Moreover, The magnetic targeting drug carrier can form a hydrogen-bonded co-loading drug with the drug, pass through the layers of cells, and target the drug to be transported under the action of an external magnetic field to exert the drug effect and improve the bioavailability.3.5.3O4, Fe3O4-PVA, and Fe3O4-PVA@SH were measured by VSM at 300 K  evaporation. At around 100°C to 200 °C, there is a loss regarding side -OH elimination and TIPTS defunctionalization reactions decomposition and 200°C to 330 °C C-C cleavage chain rupture and is decomposed. At a temperature higher than 330°C, residual carbon and Fe3O4 has remained.Various ingredients of the nanoparticles were also characterized by differential Scanning Calorimeter (DSC) in 3.7.3O4-PVA@SH synthesized at 20°C, 25°C, 30°C, and 35°C was studied, as shown in 3O4-PVA @ SH synthesized at 20°C, 25°C, 30°C, and 35°C were 148%, 134%, 129%, and 118%, respectively. Also, the stability of the Fe3O4-PVA@SH was studied. First, the Fe3O4-PVA@SH were immersed in an aqueous solution for 48 h, and then the Fe3O4-PVA@SH were separated, and the aqueous residue solution was evaporated and weighed. It is found that all the Fe3O4-PVA@SH showed tiny (below 5 wt %) weight loss.Also, the swelling ratio of the FeIt is well known that the molecular chain structure of the PVA determines the swelling rate, and the reaction temperature can affect the void structure between the molecular chains, thereby affecting the swelling ratio. It can be seen from 3.8.3O4-PVA@SH. The dry Fe3O4-PVA@SH was immersed in the aspirin solution for 12 hours to carry out the process of loading aspirin. As shown in 3O4-PVA@SH is mainly because the PVA chain can adsorb aspirin molecules through strong interactions . There are a lot of sulfhydryls (-SH) groups on TIPTS. Disulfide bonds will be formed between the sulfhydryl polar groups at the end of TIPTS and dispersed between the pores of Fe3O4-PVA@SH. For better detection, the drug loading process and drug loading kinetics of Fe3O4-PVA @ SH were studied. The effect of contact time on Fe3O4-PVA@SH loaded aspirin is shown in 3O4-PVA@SH was 2.11 mg/g at 4 h and 3.21 mg/g at 10 h . The release rate of Fe3O4-PVA was gentle at 4 h to 6 h, which was due to the slow release of Aspirin adsorbed by the internal pores of PVA swelling. After 6 h, the release rate of Fe3O4-PVA is almost zero, and the release behavior stops. It can be seen that the drug release rate of Fe3O4-PVA @ SH has a slow-release process after 6 h and continues to 8 h. We think that the disulfide bond is formed between the thiol groups of TIPTS, and the disulfide bond break requires more energy than the hydrogen group, which slows the release of Aspirin and makes Fe3O4-PVA @SH reach a 3 h slower than Fe3O4-PVA release time.Because the Fe3O4-PVA @SH showed high drug loading levels during the experiment. At the same time, drug release experiments showed that the drug release rate and quantity of Fe3O4-PVA@SH reached the release requirements of the 2019 US Pharmacopoeia Aspirin sustained-release tablets. We prepared a targeted magnetically drug carrier with a high potential for drug delivery and prevented the whole body’s excess distribution and eliminated its side effects. Our magnetic systems can easily reach the target point by applying an external magnetic field, while the superparamagnetic requires a smaller area than previously published works. When these nanoparticles begin to decompose inside the body, soluble iron is harmful and can be used in the patient with iron-deficiency anemia.PVA acts as a matrix, and -OH on the PVA chain can form strong interactions with drugs . At the same time, magnetic iron oxide nanoparticles provide targeting for the carrier. TIPTS is used as a biomaterial to improve the lipid solubility of magnetic polyvinyl alcohol. The thiol group on TIPTS can form a disulfide bond internally. It can also include a disulfide bond with the cysteine-rich(Cys) sulfhydryl group in the Cys subdomain of the cell surface mucosal glycoprotein. Well, the auxiliary PVA shows a better sustained-release effect. Fe4.In conclusion, we successfully proved that the coupling agent (TIPTS) could be used in biology as a material for improving amphiphilicity and improving the liposolubility of magnetic drugs. Aspirin can be administered orally by loading on a magnetically targeting nanocarrier. The magnetic targeting drug carrier prepared experimentally has an excellent drug loading rate and a stable release for 8 hours. The present work is of interest for opening up enormous opportunities to make full use of magnetic carrier material in drug delivery and other applications, because of their easy availability, cost-effective productivity, and profitable drug release performance.


NAT2*6) but lack the wild-type allele in their tumors due to loss of heterozygosity (LOH) at 8p22. These tumors were sensitive to treatment with a cytotoxic substrate of NAT2 (6-(4-aminophenyl)-N-pyrazin-2-amine, APA), and pointed to NAT2 loss being a therapeutically exploitable vulnerability of CRC tumors. To better estimate the total number of treatable CRC patients, we here determined whether tumor cells retaining also other NAT2 low activity variants after LOH respond to APA treatment. The prevalent low activity alleles NAT2*5 and NAT2*14, but not NAT2*7, were found to be low metabolizers with high sensitivity to APA. By analysis of two different CRC patient cohorts, we detected heterozygosity for NAT2 alleles targetable by APA, along with allelic imbalances pointing to LOH, in ~ 24% of tumors. Finally, to haplotype the NAT2 locus in tumor and patient-matched normal samples in a clinical setting, we develop and demonstrate a long-read sequencing based assay. In total, > 79.000 CRC patients per year fulfil genetic criteria for high sensitivity to a NAT2 LOH therapy and their eligibility can be assessed by clinical sequencing.Therapies targeting somatic bystander genetic events represent a new avenue for cancer treatment. We recently identified a subset of colorectal cancer (CRC) patients who are heterozygous for a wild-type and a low activity allele ( Surgery followed by conventional chemotherapy remains the preferred treatment of CRC. Although several targeted therapies are used in the clinical management of unresectable CRCs3, the development of novel therapeutic avenues is warranted because of resistance development. Several recent strategies seek to target somatic loss of bystander genes located near tumor suppressor genes for cancer therapy10. We recently reported that LOH at the NAT2 locus on 8p22 can be exploited for therapy using APA, a cytotoxic low molecular weight compound11. The NAT2 gene encodes N-acetyltransferase 2, a cytosolic enzyme involved in phase II metabolism of xenobiotics, which is highly polymorphic and has > 100 known single nucleotide variants (SNVs) in the protein coding sequence. The majority of these variants give rise to protein products with lower acetylator activity than the wild-type allele12. The expression of NAT2 is largely confined to epithelial cells of the gastrointestinal tract and liver, but NAT2 protein has also been detected in lung, bladder and other tissues exposed to exogenous compounds14. Due to chromosome arm LOH at 8p22 during the early development of CRC, a patient heterozygous for a rapid and a slow NAT2 allele may lose the rapid variant in the tumor cells, effectively rendering them deficient in NAT2 activity and sensitive to a cytotoxic substrate of NAT211. As the normal epithelial cells of gut and liver express also the wild-type NAT2 allele, they can evade APA toxicity11. The number of patients standing to benefit from treatment with APA will depend on (1) the fraction of the CRC patient population that has a targetable NAT2 intermediate acetylator phenotype , and (2) the fraction of CRCs that lose their rapid NAT2 allele. It is therefore essential to understand which specific slow NAT2 alleles confer sensitivity to APA and to determine their prevalence in CRC patient populations in order to advance to clinical implementation.Colorectal cancer (CRC) is the third most common form of cancer in the world with more than 1.4 million newly diagnosed cases and almost 700.000 deaths in 2012NAT2 alleles differs in populations from geographically separated regions, but combinations of alleles encoding intermediate or low NAT2 enzymatic function are present in all15. For example, the wild type allele NAT2*4 and the alleles NAT2*11, NAT2*12, NAT2*13 encode proteins with a rapid acetylator phenotype, whereas the allele groups NAT2*5, NAT2*6, NAT2*7 and NAT2*14 confer the slow acetylator phenotype17. We focused our previous studies11 on tumor cells expressing the wild-type NAT2*4 (rapid) or the NAT2*6 (slow) acetylator allele, and it is currently unknown which, if any, alleles arising from the compendium of NAT2 slow acetylator variants that can be exploited in LOH-based therapy using APA. Once these variants are identified, accurate haplotype resolution at the NAT2 locus of normal and tumor samples is required for diagnostic purposes. Sanger sequencing and single nucleotide polymorphism (SNP) arrays are limited with respect to haplotype resolution as they rely on statistical inference to resolve complex genotype data18. More recently, haplotype phasing of high-throughput short-read next-generation sequencing (NGS) data has been employed, but this method heavily relies on high density of SNPs for accurate inference19. As sequencing methods based on single-molecule real-time (SMRT) sequencing can provide more reliable resolution of alleles and haplotypes at a comparable cost per sample21, we hypothesize that the implementation of these long-read based assays will improve the resolution of NAT2 haplotypes.The distribution of NAT2 alleles belonging to the groups NAT2*5, NAT2*7 and NAT2*14. Next, we genotype NAT2 in two independent CRC patient cohorts to determine frequencies of alleles and LOH events. For diagnostic purposes, we finally develop a low-cost long-read sequencing protocol for the multiplexed NAT2 haplotyping of patient-matched normal and tumor samples.In the present study, we seek to determine the number of patients who can benefit from a cytotoxic compound (APA) targeting LOH of NAT2 Fig. . First, NAT2 allele group frequencies from the phased haplotypes of 2054 individuals sequenced by the 1000 Genomes Project22 and observed that the slow acetylator alleles from the groups NAT2*5, NAT2*6, NAT2*7 and NAT2*14 represented 29.3%, 26.5%, 7.7% and 2.8% of the total alleles, respectively. The fraction of heterozygous individuals with the slow acetylator variants *5 (rs1801280), *6 (rs1799931), *7 (rs72554617) and *14 (rs1801279) were 35.7%, 36.7%, 13.0% and 5.0%, respectively. However, only heterozygous individuals with one slow and one rapid acetylator allele (NAT2*4 or NAT2*12) are considered for allele-selective therapy. Heterozygosity was present in 15.1% (NAT2*5/Rapid), 13.4% (NAT2*6/Rapid), 5.7% (NAT2*7/Rapid) and 1.6% (NAT2*14/Rapid) of samples. We detected only one allele from the group NAT2*10 and no other slow acetylator alleles in the dataset. The data suggest that ~ 35.8% of the global population has an intermediate NAT2 acetylator phenotype through heterozygosity of prevalent rapid and slow NAT2 alleles. Given that ~ 21% of colorectal tumors undergo LOH at 8p2223, we estimate that ~ 79,000 individuals heterozygous for a NAT2 slow allele could potentially be eligible for therapy every year   as the most frequent followed by *4 (26.5%), *6 (23.5%), *7 (4.1%) and *12 (< 0.1%)  and *6 (31.3%), followed by NAT2*4 (19.6%) and NAT2*7 (4.5%)  had *4/5A, *4/5C or *4/7B genotypes. Again, LOH events were detected in 19.6% of the total cohort and particularly in 40% of heterozygous patients with the NAT2*4/*5B haplotype. Analyses of these two cohorts revealed five different NAT2 genotypes in heterozygous patients losing wild-type NAT2 in their tumors , rs1799931 (NAT2*7) and rs1801279 (NAT2*14) was confirmed by PCR-coupled Sanger sequencing  and NAT2_14 variants (Vmax/Km = 0.03 and 0.38 µl/min/106 cells), leading to > 20 and > 100-fold less efficient NAPA formation as compared to the wild-type NAT2 clone (Vmax/Km = 15.5 µl/min/106 cells)  showed much higher acetylation efficiency, comparable to the wild-type enzyme and one clone also displaying substrate inhibition kinetics  and NAT2_14 (IC50 = 0.15 µM) slow acetylator variants showed a > fourfold decrease in viability when compared to wild-type NAT2  comparable to wild-type cells , compared to 4540 ± 749 for Pool B. While the amplicon mean coverage was comparable, a larger standard deviation was observed in Pool B. While RKO, DLD-1 and VaCo-703 cells were homozygous in the sequenced region and carried the NAT2*6A allele as suggested by Sanger sequencing, HepG2 cells were polymorphic in 7 sites and the haplotype calling from long-read sequencing revealed the NAT2 slow-acetylator alleles NAT2*6A and NAT2*5B , barcode de-multiplexing and read alignment we observed that mean NAT2 coverage per sample was 1547 × in the normal tissues and 2229 × for the tumor samples, which was deemed sufficient to call the haplotypes. We called the haplotypes from the reads covering all 7 allele-classifying polymorphic sites   the increase of coverage to 2235 × did not reveal a second allele. In sample N_ID:148 with similarly low coverage (238 ×), two alleles were resolved (NAT2*5B/*6A). The allele groups NAT2*4, *5, *6 and *7 were detected in 22.3%, 50.7%, 20.9% and 6.1% of patients, respectively. The data from the complete NAT2 haplotypes implied that 25 individuals (33.8%) were intermediate acetylators, consistent with the genotype and imputed haplotype data from the SNP arrays and NGS sequenced cohorts. We detected LOH of NAT2 in 19 tumor samples (25.7%) of which 13 were from individuals with an intermediate acetylator phenotype. The LOH events in the intermediate acetylators indicated that the wild-type allele was lost in 3 cases . The long-read sequencing approach additionally identified a novel wild-type NAT2*4 allele with a rare synonymous SNP (rs144176822). We conclude that SMRT sequencing enables NAT2 haplotype resolution for multiple samples by analyzing sequencing reads spanning the NAT2 gene.After confirming that NAT2 haplotype resolution is feasible by SMRT sequencing, we aimed to resolve the genotype of 74 CRC tumor and patient-matched normal tissues, which included the Swedish cohort. We sequenced the barcoded 26. Cancer vulnerabilities emerging from these genomic losses have been proposed as promising putative targets for the selective treatment of solid tumors. Examples include the loss of bystander metabolic enzymes lost in conjunction with tumor suppressor genes, enabling inhibition by collateral lethality7. Similarly, loss of essential genes in the spliceosome or proteasome can render cancer cells sensitive to further suppression of these genes10. In a previous study, we have focused on LOH events reducing the genetic complexity of tumors and have demonstrated that NAT2 could serve as a target in colorectal cancer11. We focused on NAT2*6 heterozygous individuals which lose the functional NAT2 allele in their tumors  and demonstrated that cell lines and tumors expressing the slow acetylator NAT2*6 allele are more sensitive to APA treatment than those expressing a rapid acetylator NAT2 allele. Based on structural similarity with kinase inhibitors, we additionally performed a kinome screen to profile binding of APA and its acetylated product NAPA with respect to > 300 kinases, and identified differential binding to Aurora Kinase A (AURKA) which is a known drug target in cancer. We were able to determine binding of APA to AURKA, activation of phospho-AURKA in slow but not rapid acetylator tumor cells, and mitotic arrest along with formation of multipolar mitotic spindles in CRC cells expressing the slow acetylator variant. In this study, we hypothesized that individuals carrying other slow variant alleles may also be eligible for APA treatment. Tumor cells encoding the slow acetylator variants NAT2*5 and *14 indeed responded to APA treatment and had similar kinetic profiles when compared to cells expressing the *6 allele group. This is in concordance with previous analyses of sulfamethazine N-acetylation in human hepatocytes, where NAT2-mediated substrate processing occurs at similar rates for all slow acetylator variants27. In contrast, cells encoding the NAT2*7 variant showed higher efficiency of APA acetylation and responses to APA treatment that are comparable to cells expressing wild-type NAT2 activity. We hypothesize that this effect is particular for APA.The vast majority of human cancers exhibit genomic instability at the chromosomal level. Such cancers acquire somatic changes in chromosomal number and structure throughout tumor progressionNAT2 in two different colorectal cancer cohorts revealed several combinations of alleles that encode intermediate acetylator phenotypes. For patients heterozygous for NAT2, we observed an almost equal probability of losing either allele after the tumor undergoes LOH. The LOH frequency for the NAT2 locus was > 23% in both cohorts, which is considerably higher than previously reported28. This discrepancy could be a result of population specific factors, a difference in the number of patients and most importantly of the amount of SNP variation probed. Besides detecting a loss of the wild type NAT2*4 allele in tumors from NAT2*4/6A individuals (17%), we observed that the same allele can be lost in the tumors of NAT2*4/*5B (15%) and NAT2*4/*7B (27%) individuals. The largest number of intermediate acetylators in both patient cohorts carried a NAT2*4/*5B genotype and, despite the lower LOH frequency observed in the NAT2*4/*5B genetic background compared to the LOH frequency in NAT2*4/6A or NAT2*4/*7B backgrounds, the NAT2*4/*5B intermediate acetylators contributed with the most cases of potentially eligible patients . All intermediate acetylators in our cohorts which lost a wild-type allele in the tumor, account for ~ 6% of cases. These results significantly expand the population of patients eligible for LOH therapy and motivate the development of practical diagnostic tools to identify this clinically relevant subset. No carriers of the NAT2*14 slow acetylator allele were identified, presumably because this allele is not present in the studied populations29. As NAT2*14A or NAT2*14B haplotypes are found in Spanish (0.39%), German (0.13%) and US Caucasian (0.12%) populations, eligible patients could be found in these cohorts15. Despite their low frequency, our functional data suggests that tumors encoding the NAT2*14 variant are equally sensitive to APA treatment and should be considered for LOH-base therapy.Haplotyping of 18. Alternatively, haplotype resolution can be performed from short reads obtained from next-generation sequencing, but the output is highly dependent on the frequency of the polymorphism that needs to be inferred. We developed SMRT sequencing of NAT2 as a practical diagnostic technology, assigning the genetic variation found in the NAT2 locus to each DNA strand of the individual and potentially allowing the identification of LOH events in the patient’s tumor. By using SMRT sequencing, we were able to resolve the different NAT2 alleles present in several cell lines and developed a pipeline for multiplexed analysis. We resolved both NAT2 alleles  variable tumor purity, (2) tumor heterogeneity and clonality of LOH events, and (3) potential allele biases in the library preparation, sequencing and analysis of the samples. From the three implemented methods, the long-read SMRT sequencing can be considered the best haplotyping strategy since the evidence for each allele is direct and based on independently sequenced single molecules covering both directions of the DNA strands. This is clearly demonstrated by the detection of the rare g.18400582G > T polymorphism and its unambiguous assignment to a novel 14. Cancers of the liver, lung and kidney are also prone to LOH in the NAT2 locus on chromosome 8p2223, in similar frequency to that of colorectal cancer  and the Ethics Committee of the Hospital District of Helsinki and Uusimaa following participant informed consent. Signed informed consent or authorization from the National Supervisory Authority for Welfare and Health has been obtained for all Finnish sample materials used. All analyses were performed in accordance with relevant guidelines and regulations.NAT2*5, NAT2*7 and NAT2*14 slow acetylator variants within the Lv125C backbone sequence were acquired from LabOmics. The day before transduction, 50,000 cells were plated in a 24-well plate. Viruses were diluted in 250 µL of normal growth medium containing 7.5 mg/mL sequa-brene (Sigma) and added to each well. After 24 h of incubation at 37 °C, the virus-containing medium was replaced with fresh normal growth medium. After 48 h, transduced cells were selected with 1 µg/mL puromycin (Thermo Fisher Scientific) for 10 days. Next, 3 cell pools of each NAT2 slow acetylator variant were subjected to single clone selection and screening. To confirm the genotype of each clone, genomic DNA was isolated following a standard protocol for cultured cells (Macherey–Nagel) and amplification of the lentiviral NAT2 insert was performed by PCR with the forward primer 5′-GCGGTAGGCGTGTACGGT-3′ and reverse primer 5′-ATTGTGGATGAATACTGCC-3′. Each reaction contained 1 × Phusion HF Buffer (Thermo Scientific), 0.2 mM dNTPs, 0.2 µM forward and reverse primers, 0.02 U/µL Phusion DNA Polymerase (Thermo Scientific) and 6 ng of genomic DNA. The PCR was performed in a 2720 Thermal Cycler (Applied Biosystems) and consisted of 1 cycle of 98 °C for 30 s and 30 cycles of 98 °C for 10 s, 61 °C for 15 s and 72 °C for 20 s followed by 1 cycle of 72 °C for 10 min. Next, reactions containing 20 ng of PCR product and 4 pmol of the corresponding primer were prepared for Sanger sequencing on an ABI 360xl instrument (Applied Biosystems). Sequence traces were analyzed in SnapGene Viewer.The human colorectal cancer cell line RKO (ATCC) was maintained in McCoy’s 5A medium supplemented with 10% Fetal Bovine Serum and 1% Pen Strep (Life Technologies). Lentiviral particles encoding NAT2*4 acetylator variant was generated in a previous study11 by transfection of a pCMV6-Entry-NAT2 mammalian expression vector containing a Myc-tagged NAT2 open reading frame. To obtain a rapid acetylator phenotype, site directed mutagenesis of rs1799930 (A590G) was performed using the QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies) in 50 μL reactions containing 1 × reaction buffer, 125 ng of each primer, 0.2 mM dNTPs and 20 ng of dsDNA template. Primers used for site-directed mutagenesis included forward 5′-GAAGAGGTTGAAGAAGTGCTGAGAAATATATTTAAGATTTCCTTGG-3′ and reverse 5′-CCAAGGAAATCTTAAATATATTTCTCAGCACTTCTTCAACCTCTTC-3′, and were designed using the QuikChange algorithm (Agilent Technologies). Transfection of RKO cells was performed using Lipofectamine 2000 DNA Transfection Reagent (Invitrogen). The resulting stable clones were expanded under G418 selection (1 mg/mL) and confirmed to encode the wild-type NAT2 sequence by Sanger sequencing using primers forward 5´-GATCCGGTACCGAGGAGAT-3´ and reverse 5´-TTGCTGCCAGATCCTCTTCT-3´.The rapid T Kit (Thermo Fisher Scientific). Overexpression of NAT2*5, NAT2*7 and NAT2*14 transcripts was detected by RT-qPCR using TaqMan probes against NAT2 (Hs04194721_s1) and the reference gene ACTB (Hs01060665_g1). Clones with similar levels of NAT2 expression were chosen for downstream analyses.Direct cDNA extractions were performed from cell lysates using the TaqMan Gene Expression Cells-to-C6 cells were lysed by addition of RIPA buffer (Thermo Scientific). Protein concentration was determined by the Novex BCA assay (Life Technologies) and 50 µg of sample were loaded and separated in a 4–12% SDS-PAGE gel (Life Technologies) at 180 V for 1 h. Sample transfer was performed at 20 V for 8 min using nitrocellulose iBlot Transfer Stacks (Thermo Scientific), which was later blocked with 5% BSA and probed overnight at 4 °C with rabbit Myc-tag  and mouse β-actin  primary antibodies. After incubation with rabbit and mouse secondary antibodies , proteins were visualized by addition of SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) in an ImageQuant LAS 4000 imaging system . Immunoblots were quantified in ImageJ and protein expression was quantified relative to β-actin loading control. Unprocessed immunoblot scans are provided in Supplementary Fig. Pellets from 2 × 10Cells were plated in six replicate wells of a 96-well plate at a density of 3000 cells per well. After 24 h, all wells were imaged in real time using the IncuCyte ZOOM live cell analysis system (Essen BioScience) and cell confluence was determined every 12 h for a period of 7 days.m/z transitions 353 > 323 and 395 > 143 were monitored for APA and NAPA respectively.Cells were plated at a density of 18,000 cells per well in a 96-well plate. The next day, different concentrations of the substrate 6-(4-aminophenyl)-N-pyrazin-2-amine (APA) were added to the cells and the reaction was quenched after 30 min by addition of 99.8% MeOH. N-acetyl APA (NAPA) formation was detected by LC–MS/MS on a XEVO TQ (Waters) instrument couples to an Acquity UPLC (Waters) using a HSS T3 column . The mobile phases consisted of (A) 0.05% heptafluorobutyric acid and propionic acid, and (B) 0.05% heptafluorobutyric acid and propionic acid in acetonitrile. The 2 1420 microplate reader .Cells were seeded at a density of 8000 cells per well in a 96-well plate. The next day, medium containing different concentrations of APA was added. After 72 h, cell viability was determined in an MTT assay by addition of resazurin (Sigma-Aldrich) followed by fluorescence readouts at 590 nm in a VictorNAT2 was amplified by PCR with the forward primer 5′-CACACGAGG AAATCAAATGCTA-3′ and the reverse primer 5′-CACTGAGGCCAGATCTCCTATC-3′. The reaction was performed with the following concentrations of the reagents: 1 × Phusion HF Buffer (Thermo Fisher Scientific), 200 µM dNTPs, 0.5 μM forward and reverse primers, 0.02 U/μL Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and 20 ng of genomic DNA. The PCR was performed in a 2720 Thermal Cycler (Applied Biosystems) following the touch-down protocol with 1 min of initial denaturation at 98 °C, 3 cycles of 98 °C for 30 s, 70 °C for 30 s, 72 °C for 2 min, 3 cycles of 98 °C for 30 s, 68 °C for 30 s, 72 °C for 2 min, 3 cycles of 98 °C for 30 s, 65 °C for 30 s, 72 °C for 2 min, 25 cycles of 98 °C for 30 s, 64 °C for 30 s, 72 °C for 2 min and 10 min of final elongation at 72 °C. Unprocessed agarose gel images are provided in Supplementary Fig. NAT2 region, Sanger sequencing reactions were prepared containing 20 ng of purified PCR product and 0.1 μM of the corresponding primer. Samples were analyzed in an ABI3730XL DNA Analyzer (ThermoFisher). To perform multiplex SMRT sequencing, the same primer pairs were tagged with an unique barcode sequence of 15 bp  according to the manufacturer’s protocol. The exon 2 of ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/]. The genomic variation in the coding part of NAT2 was extracted in a form of phased alleles for each individual. The NAT2 allele definitions were taken from12 and matched to the variation at the rs200893121, rs72466456, rs45477599, rs149283608, rs1801279, rs1041983, rs1801280, rs45532639, rs146405047, rs12720065, rs139351995, rs1799929, rs72554617, rs79050330, rs1799930, rs45618543, rs56387565, rs138707146, rs45518335, rs55700793, rs1208, rs56393504 and rs1799931 positions in the alleles from the 1000 Genomes Project to identify the allele group and the specific NAT2 alleles present in each individual.The full chromosome 8 dataset for 2054 individuals from 1000 Genomes Project Phase 3  was downloaded from [NAT2 alleles were imputed from the genotype data. The LOH was estimated as allelic imbalance in one or more informative positions, where there was an observed ≥ 10% shift of the number of sequencing reads supporting each allele.Tumor and normal tissue samples were initially sectioned from a cohort of 173 stage I-III colorectal cancer patients. During this process, two patients were removed from the study cohort due to mismatches found between their tumor and corresponding normal samples. DNA was extracted from each sample in a Freedom EVO robot (Tecan) using magnetic nanoparticles. To discard tumors with microsatellite instability (MSI), PCR-based MSI detection was performed with the MSI Analysis System Version 1.2 (Promega), where the insertion or deletion of the repeating units NR-21, BAT-26, BAT-25, NR-24, MONO-27, Penta D and Penta C was evaluated. Fragments were sequenced in a 96-capillary 3730xl DNA Analyzer (Thermo Fisher Scientific) and MSI panels were analyzed using the GeneMapper software (Thermo Fisher Scientific). We discarded 24 MSI colorectal tumors, and proceeded with the analysis of tumor cell content in the remaining 147 samples. We identified 72 patients with tumors containing > 40% of tumor cell content and discarded 8 patients who had previously received chemotherapy or radiotherapy. We additionally removed samples from 7 patients for which not enough DNA was available for downstream analyses. These selection criteria identified 56 patients with tumor and matched normal samples suitable for whole genome sequencing. Sequencing libraries were prepared in duplicates from 100 ng of DNA by using the TruSeq Nano DNA sample preparation kit (Illumina), targeting an insert size of 350 bp. Sequencing was performed in a HiSeqX System (Illumina) with a paired-end 150 bp read length. The library preparation and sequencing were performed at the SNP&SEQ Technology Platform . Tumor and normal samples were sequenced with a 90 × read depth. The variant bases in positions corresponding to the SNPs rs1801279, rs1041983, rs1801280, rs1799929, rs144176822, rs1799930, rs1208, rs1799931, rs2552, rs4646247, rs971473, rs45547533, rs721398 and rs721399 were called separately for the normal and tumor samples. The 32. The tumor and respective normal DNA samples were genotyped with an Infinium Omni2.5–8 (Illumina Inc.) array at the Estonian Genome Center. The B-Allele Frequencies and Log-R ratios for the polymorphisms rs45529434, rs7832071, rs1801279, rs1041983, rs1801280, rs1799929, rs144176822, rs1799930, rs45607939, rs149460636, rs1208, rs1799931, rs2552, rs4646247, rs971473, rs45547533, rs721398 and rs721399 were extracted with Illumina Genome Studio software. The NAT2 haplotypes of 1511 patients with informative calls at the allele-classifying positions rs1801279, rs1041983, rs1801280, rs1799929, rs72554617, rs79050330, rs1208 and rs1799931 were imputed from the SNP genotypes. The variation in all genomic positions was used estimate LOH by comparing the data from the normal and tumor sample. We considered the allelic imbalances for LOH if the observed shift in B-allele frequency of the tumor compared to normal was ≥ 0.10 .We derived 1611 cases with colorectal adenocarcinoma from the ongoing Finnish CRC collection 33. The second NAT2 exon of each sample was amplified by PCR following the aforementioned protocol, however the primers contained additional sample-unique barcode sequence to allow amplicon identification. The barcode sequences were those recommended by Pacific Biosciences platform and the HPLC-purified primers were obtained from Sigma-Aldrich (Supplementary Table DNA isolated from 74 fresh frozen tumor and patient matched normal tissues was obtained from CRC patients with stage II-IV disease. Extraction was performed on a liquid handling workstation Tecan Evo 150 MCA LiHa RoMa as previously describedSupplementary Information 1.Supplementary Information 2.


P < 0.05). Corresponding CpG sites in the promoter regions these markers were hypomethylated in the acute phase of KD and significantly increased after IVIG therapy. In conclusion, both M1 and M2 markers showed increased mRNA expression in the acute phase of KD. CpG site methylation may be one of the mechanisms governing macrophage polarization in KD.Kawasaki disease (KD) is a common systemic vasculitides in children younger than 5 years of age. Activated macrophages are key drivers of vascular inflammation in KD. The aim of this study was to examine differences in M1 and M2 macrophage marker expression in patients with KD. Blood samples were obtained from 18 healthy controls and 18 patients with KD at 24 h prior and 21 days after to intravenous immunoglobulin therapy. GeneChip Human Transcriptome Array 2.0 and Illumina HumanMethylation450 BeadChip were used to examined the mRNA expression and corresponding CpG site methylation ratios of 10 M1 surface markers and 15 M2 surface markers. Of the markers examined 2 M1 markers  and 8 M2 markers  showed increased mRNA expression in the acute phase of KD which decreased after IVIG therapy ( Kawasaki disease (KD) is one of the most common systemic vasculitides in children younger than 5 years of age. First described by Dr. Tomisaki Kawasaki in 1967 . If inadAlthough the precise immunopathogenesis of KD is unclear, histopathology of the coronary arteries has revealed clues to the immunologic sequence of events that lead to coronary artery dilatation and destruction in patients with KD. Edema of the tunica media layer of the coronary arteries begins around the 6th to 8th day after disease onset. This is then followed by increased infiltration of lymphocytes, monocytes, and neutrophils by the 10th day of disease, which then infiltrates all layers of the coronary artery. This panarteritis is characterized by the formation of proliferative granulomatous inflammation consisting mostly of monocytes and macrophages , 5. MacrMacrophages are considered an important component of innate immunity and may initiate inflammation and subsequently adaptive immune responses. Recently it has been found that activated macrophages can also be divided into M1 and M2 subsets, which mirror the Th1 and Th2 polarization seen in T-helper cells . PolarizTo date, there have been no studies that specifically examine the role of macrophage M1/M2 polarization in patients with KD. Results from the limited number of existing studies regarding macrophage polarization in other forms of autoimmune vasculitis reveal that both M1 and M2 activation may be involved . As an eInitially, a total of 18 patients with KD and 18 healthy controls were enrolled in this study for Human Transcriptome array analysis, and another 12 healthy controls and 12 patients with KD were enrolled for Human Methylation Beadchip analysis. We then enrolled an additional 30 healthy controls and 30 patients with KD for confirmation of initial screening results with RT-PCR, 10 of which were analyzed by pyrosequencing.Patients were diagnosed with Kawasaki disease if they fulfilled the criteria proposed by the American Heart Association, namely fever which persists for more than 5 days and at least four of the following five clinical criteria: oral mucosal involvement , bilateral non-exudative conjunctival injection, cervical lymph node enlargement of over 1.5 cm in diameter, changes in the peripheral extremities , and a polymorphous rash . PatientBlood samples from the KD group were obtained at two time points: the first blood draw was performed within 24 h prior to the initial dose of IVIG therapy during the acute phase of KD (KD1), and the second blood draw was performed at least 21 days after initial IVIG therapy during the convalescent phase (KD2). All blood samples were centrifuged and the buffy coat containing white blood cells was then removed for further experiments. Total RNA was then extracted from white blood cells according to manufacturer's instructions . Total RNA samples were checked for quality and quantity and all RNA samples were confirmed to have a RIN  of ≥7 as previously described . Total gFor unbiased results, 18 mRNA samples each from the healthy control group (HC group), KD1 group (KD patients 24 h prior to IVIG therapy) and the KD2 group (KD patients 21 days after IVIG therapy) were combined into three pooled RNA libraries each containing six RNA samples. We then used GeneChip® Human Transcriptome Array 2.0  to determine gene expression profiles of macrophage surface markers which are commonly expressed on M1 and M2 macrophages after literature review –16. PrioDNA samples taken from the 12 healthy controls, and the 12 Kawasaki disease patients at two time points, 24 h prior to IVIG therapy (KD1) and 21 days after IVIG therapy (KD2) were then used to examine DNA methylation patterns of M1 and M2 macrophage surface markers. Two hundred nanograms of bisulfite-converted genomic DNA were then applied to the Illumina HumanMethylation450 (M450K) BeadChip assay as described in previously , 17. Cyt−, also known as 2−ΔΔCT.We then selected the following markers for RT-PCR confirmation of mRNA expression levels: IL1R1, TLR2, TLR4, IL1T2, ARG1, and TLR5. All mRNA samples were first transformed into cDNA according to manufacturer instructions . Samples were then prepared by adding 2.5 ng/μL of cDNA with 0.2 μL each of forward and reverse primers (10 μM) and 5 μL of SYBR Green PCR Master Mix  before performing quantitative RT-PCR on the LightCycler R 480 Real-Time PCR System . 18S was used as an internal control. IL1R1—Forward (F) AAAGATGACAGCAAGACACCTG, Reverse (R):GTTTGCAATCCTTACCACGCAA; TLR2—(F): GAGTTCTCCCAGTGTTTGGTGT (R):CACACCATCAGAACCCTGTC; IL1R2—(F):ATGACACCCACATAGAGAGCG (R):GAAGAGCGAAACCCACAGAGT; ARG1—(F):ACTTAAAGAACAAGAGTGTGATGTG (R) CGCTTGCTTTTCCCACAGAC; TLR5—(F):TGCTACTGACAACGTGGCTT (R):CCAGGAAAGCTGGGCAACTA; TLR4—(F):ATGCCAGGATGATGTCTGCC (R):TGGATTTCACACCTCCACGC; 18S—(F):GTAACCCGTTGAACCCCATT (R):CCATCCAATCGGTAGTAGCG. We then calculated the relative quantification of mRNA expression levels by using the comparative threshold cycle (CT) method  by using the following equation: 2To confirm the percentage of methylation of selected CpG, further pyrosequencing was performed in a separate cohort. First 0.5 μg of genomic DNA underwent bisulfite modification according to manufacturer instructions  and with eluted in Tris buffer (10 mM) for a final volume of 20 μl. Polymerase chain reaction (PCR) with a reaction mixture containing bisulfite-converted DNA, PyroMark PCR Master Mix (Qiagen), and biotinylated forward and reverse primers of the selected CpG sites. Primers used are as follows: IL1R1 (cg05886087) F:TTTTTGGAAATAGATTGTTTAAGAATGAGA, R:ATAATAATAACTTCCTTCCTTCTAATACAC; TLR2 cg06618866) F: AGGTTGGGTAGAAGAAGAGAGT, R: AACACTTAACTTTCCCTATAATTACCA; IL1R2 (cg20340242) F: ATTGTAGTTTGGGGGTGT, R: TTCACCTCCAACAAAAACTCA; ARG1 (cg01699630) F: TTAGGGTTGGAAGGGATGTGATA, R:AACCAAAAAATAACAATCAACAATCTTAC; TLR5 (cg05858079) F:AGGGTTTTAGAGTTTTTTTAGGGTAGTA, Reverse:AATAACTTATCCCCAATCACTTTC; TLR4 (cg13730105) F:GAGGGAAAGTTTAGAGGAGTT, R:ACCACCCATTCACAAAACCACTAC. We then calculated the percentage of methylation at for all CpG sites by comparing relative peak height differences at each site . Data from HTA 2.0 microarray was compared using one-way ANOVA and Bonferroni Correction was used to account for multiple testing. Beta-values from the M450K Array were first transformed into M-values by using the following equation: M-value = log2 . M-valuep = 0.023; 1 ± 0.013 vs. 1.185 ± 0.034, p = 0.011, presented as mean ± standard error, respectively). IVIG therapy resulted in a significant decrease in expression of three genes including TLR2, TLR4, and SOCS3 .In this study we examined the mRNA expression of 10 M1 surface markers  in three groups, healthy control (HC), acute KD (KD1) and in KD patients 21 days after IVIG therapy (KD2) . We founp < 0.029; 1 ± 0.069 vs. 2.491 ± 0.171, p = 0.001; 1 ± 0.134 vs. 2.364 ± 0.423, p = 0.013; 1 ± 0.126 vs. 2.390 ± 0.360, p = 0.011; 1 ± 0.120 vs. 3.178 ± 0.459, p < 0.002; 1 ± 0.099 vs. 1.799 ± 0.143, p = 0.011; 1 ± 0.029 vs. 1.616 ± 0.197, p = 0.045; 1 ± 0.070 vs. 2.261 ± 0.257, p = 0.003 presented as mean ± standard error, respectively), which were then significantly decreased after IVIG therapy . One marker, CXCR2 showed a statistically significant decrease in mRNA expression after IVIG therapy . Two markers, CXCR4 and FCER2, had decreased mRNA expression in the acute phase of KD  which increased after IVIG therapy .We also examined the mRNA expression of the 15 M2 cell surface markers  in both KD patients and healthy controls . Of the p = 0.771, 1 ± 0.128 vs. 1.452 ± 0.504 p = 0.388 presented as mean ± standard error, respectively), which increased after IVIG therapy , although these changes were not statistically significant. IL1R1 on the other hand, was lower in the acute phase of KD (KD1) , and increased after IVIG therapy (KD1 vs. KD2 0.655 ± 0.190 vs. 0.789 ± 0.344 p = 0.739 presented as mean ± standard error), but these changes were again not statistically significant.We then chose three M1 markers  and three M2 markers  with the highest fold change between acute KD (KD1) and healthy controls (HC), for RT-PCR confirmation of mRNA expression levels in a separate cohort of 30 KD patients and 30 healthy controls . For thep = 0.129, 1 ± 0.207 vs. 1.098 ± 0.235, p = 0.754 presented as mean ± standard error, respectively), which decreased after IVIG therapy , although these changes were not statistically significant. TLR5 showed lower expression in the acute phase of KD , which increased slightly after IVIG therapy , but was also not statistically significant.For the three M2 markers selected, IL1R2 and ARG1 showed higher average expression in acute KD (KD1)  to examine the CpG site methylation trends of the 10 M1 and 15 M2 surface markers included in this study (full results in p < 0.001) within the IL1R1 gene, cg06618866  within the TLR2 gene, cg 13730105  within the TLR4 gene. Likewise, for the M2 markers three corresponding CpG sites if the highest negative beta-value fold change were selected including cg20340242  within the IL1R2 gene, cg01699630  within the ARG1 gene, cg05858079 .Of the three M1 markers with the highest fold change in mRNA expression on HTA 2.0, we identified three corresponding CpG sites the highest negative beta-value fold change between KD1 and HC groups, indicating that these sites were likely to be hypomethylated. Selected CpG sites included cg05886087 , and became hypermethylated after IVIG therapy  The CpG site selected for the M1 marker TLR2 (cg06618866) showed significant hypermethylation after IVIG therapy . There were no significant changes in methylation of the Cpg site selected for the M2 marker TLR5, cg05858079 , TLR4 (cg13730105), and the M2 markers IL1R2 (cg20340242) and ARG1 (cg01699630) were significantly hypomethylated in the acute KD group  and three M2 markers  with the highest mRNA expression fold change on HTA 2.0 were selected for confirmation. Pyrosequencing of the six CpG sites selected was performed in a separate cohort of 10 KD patients and 10 healthy controls. Four of the CpG sites selected corresponding to the M1 markers IL1R1 (cg05886087), TLR4 (cg13730105), and the M2 markers IL1R2 (cg20340242) and ARG1 (cg01699630) were significantly hypomethylated in the acute KD group and were then hypermethylated after IVIG therapy, which mirrored the increased mRNA expression of these genes on HTA 2.0 microarray. This suggests that CpG site methylation may be one of the many mechanisms which regulate M1 and M2 macrophage polarization in KD.In our previous research, we have found that both Th-1 and Th2 subset activation are involved in the acute phase of KD. Th1 associated cytokines including IL-2 IL-6, TNF-α, and IFN-γ were significantly increased in the acute phase of KD and decrease after IVIG therapy –23. In aMonocyte derived macrophages can be to activated into either M1 or M2 macrophages according to the tissue microenvironment. As mentioned earlier, stimuli such as IFN-γ, TNF-α or LPS promotes macrophage polarization toward the M1 phenotype, and IL-13 and IL-4 promotes macrophage polarization into the M2 phenotype. In addition, there appears to be plasticity among the M1/M2 phenotypes; once a macrophage is activated into the M1 phenotype, it can still be repolarized into the M2 phenotype according to dynamic changes in the tissue microenvironment, and vice versa . In thisBoth M1 and M2 marker mRNA expression decreased after IVIG therapy. Although there are no other studies to date that specifically examine M1 and M2 macrophage activation in KD, one study of 28 patients with KD showed that CD14+ CD16+ monocytes were elevated in the acute phase of KD . CD14+ aOf the 10 M1 macrophage markers and 15 M2 macrophage markers examined in this study, we found that the M1 markers TLR2, TLR4, and IL1R1 and the M2 markers TLR5, IL1R2, and ARG1 had the highest levels of mRNA expression in the acute phase of KD. Previous research has found that toll-like receptors, particularly toll-like receptor 2 (TLR2) is associated with the development of KD. In human patients with KD, increased expression of TLR2 positive CD14 monocytes occurs in the acute phase of KD , and is Both IL1R1 and IL1R2 belong to the interleukin-1 receptor family. IL1R1 which is expressed on M1 macrophages, acts as a receptor of the cytokines IL-1a, IL-1b, and IL-1 receptor antagonist (IL-1RA). Conversely, IL1R2 which is expressed on M2 macrophages, acts as a decoy receptor for IL-1a, IL-1b, and IL-1RA thereby inhibiting the further signal transduction of these cytokines . The IL-Furthermore, we found that the M2 marker ARG1 showed significant increase of mRNA expression in the acute phase of KD. ARG1 diverts arginine away from the inflammatory iNOS (inducible nitric oxide synthase) pathway, thereby modulating inflammatory responses . Althougde novo methylation). There is evidence that increased expression of DNMT1 promotes polarization toward the M1 macrophage phenotype possibly through regulation of the PPAR-γ pathway , TLR4 (cg13730105), and the M2 markers IL1R2 cg20340242) and ARG1 (cg01699630) were significantly hypomethylated in the acute KD group and were then hypermethylated after IVIG therapy. Previous studies have found that epigenetic regulation of gene expression particularly through DNA methyltransferases (DNMT) may be one of the ways which regulate M1 and M2 macrophage polarization. DNMTs are enzymes that bind methyl groups to DNA strands, and include DNMT1, which maintains DNA methylation by adding methyl groups to hemimethylated strands of DNA during replication; and DNMT3a and DNMT3b, which add methyl groups to previously unmethylated DNA , the heterogeneity of cells used is one of the limitations of our study. In our study we found that acute Kawasaki disease (KD1) was associated with a higher percentage of neutrophils (KD1 vs. HC 55.28 ± 2.79% vs. 31.05 ± 1.95%), and a lower percentage of lymphocytes (KD1 vs. HC 33.62 ± 2.56% vs. 59.75 ± 2.14%) when compared to the healthy controls. The ratios of neutrophils and lymphocytes normalized and were comparable to healthy controls 21 days after IVIG therapy (KD2), and the percentage of monocytes was similar in all three groups . In our study we found that a much higher percentage of M2 related markers showed increased mRNA expression in the acute phase of KD, but decreased after IVIG therapy, particularly IL1R2, TLR5, and ARG1. While TLR5 is also expressed on neutrophils, it is more highly expressed in monocytes , 48.In conclusion, this is the first study to specifically examine the role of M1 and M2 macrophage polarization in patients with KD. We found that both M1 and M2 markers showed increased expression in the acute phase of KD which decreased after IVIG therapy, with the M1 markers TLR2, TLR4, and IL1R1 and the M2 markers TLR5, IL1R2, and ARG1 showing the highest levels of mRNA expression. We also found that the corresponding promoter CpG sites of these markers were hypomethylated in the acute phase of KD, and hypermethylated after IVIG therapy, which suggest that CpG site methylation may be one of the mechanisms governing macrophage polarization in KD. Further functional studies are needed to confirm the role of macrophage polarization in the development of KD, which may prove to be a novel therapeutic target in the future.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109430 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109351.The data analyzed in this study can be found in The studies involving human participants were reviewed and approved by Chang Gung Medical Foundation Institutional Review Board. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.MG participated in the study design, analyzed data, and wrote the main manuscript. L-SC, Y-HH, and F-SW participated in data collection and analysis. H-CK was responsible for study designs and edited the main manuscript. All authors have reviewed the manuscript prior to publication.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.


T389 and p70S6KT421/S424. Furthermore, the levels of mTORS2448, p70S6KT389, p70S6KT421/S424 and RPS6S235/S236 were decreased after DHA treatment in KYSE30 and KYSE150 cells. We then explored the proteins targeted by DHA to inhibit the mTOR-p70S6K-RPS6 pathway. Results of the in vitro kinase assay revealed that DHA significantly inhibited phosphorylation of mTORS2448 by binding to AKT1 and p70S6K kinases. In vivo, DHA inhibited the tumor growth of ESCC patient-derived xenografts and weakened p-mTOR, p-p70S6K, and p-RPS6 expression in tumor tissues. Altogether, our results indicate that DHA has antiproliferative effects in ESCC cells and can downregulate mTOR cascade pathway partially by binding to AKT1 and p70S6K. Thus, DHA has considerable potential for the prevention or treatment of ESCC.Dihydroartemisinin (DHA), a sesquiterpene lactone with endoperoxide bridge, is one of the derivatives of artemisinin. In addition to having good antimalarial properties, DHA exhibits anticancer effects including against malignant solid tumors. However, the mechanism by which DHA inhibits the progression of esophageal cancer, especially esophageal squamous cell carcinoma (ESCC), is unclear. In this study, DHA was found to inhibit the proliferation of ESCC, and the underlying molecular mechanisms were explored. DHA inhibited ESCC cells proliferation and anchorage-independent growth. Flow cytometry analysis revealed that DHA significantly blocked cell cycle in the G1 phase. The results of human phospho-kinase array revealed that DHA downregulated the levels of p70S6K Esophageal cancer, a malignant disease, ranked the seventh in morbidity and the sixth in overall mortality in the world. Its two common pathological types are esophageal squamous cell carcinoma (ESCC) and adenocarcinoma . ESCC isArtemisia annua, a plant belonging to Asteraceae family, is a source of artemisinin  and GAPDH . DHA was purchased from Tokyo Chemical Industry . MK-2206  was purchased from Selleckchem. LY2584702  was purchased from Med Chem Express.The primary antibodies and their dilution ratio in western blot analysis were as follows: p-mTOR-S2448 ; mTOR ; p-p70S6K-T389 ; p-p70S6K-T421/S424 ; p70S6K ; p-S6 Ribosomal Protein -S235/S236 ; RPS6 ; 2 incubator.KYSE30 cell line was provided by the Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University. KYSE150 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences . The cells were cytogenetically tested using STR technology for authentication. Cells were cultured in Roswell Park Memorial Institute 1,640 (RPMI1640) medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO3 cells per well. After approximately 14 h, media were replaced with growth medium containing DHA at different doses . The cells treated with DHA were cultured for 0, 24, or 48 h and then fixed in 4% paraformaldehyde. The cells were counted using 4′,6-diamidino-2-phenylindole (DAPI) staining and an In Cell 6,000 Analyzer .Cells were seeded in 96-well plates at 8 × 103 cells per well. After approximately 14 h, media were replaced with growth medium containing DHA at the different doses . The cells treated with DHA were cultured for 0, 24, 48, 72, or 96 h and were fixed in 4% paraformaldehyde. The cells were counted using DAPI staining and an In Cell 6,000 Analyzer.Cells were seeded in 96-well plates at 2 × 103 per well) were suspended in the top layer containing 10% FBS, 0.33% agar, 45% sterile water, 1% glutamine, 0.1% gentamicin and different concentrations of DHA . The cells were cultured at 37°C in a 5% CO2 incubator for 8 days, and colony numbers were counted using an In Cell 6,000 Analyzer.Basal Medium Eagle  supplemented with 10% FBS, 0.5% agarose, 9% sterile water, 1% glutamine, 0.1% gentamicin and different concentrations of DHA was spread in a 6-well plate at a volume of 3 ml per well. After approximately 2 h of solidification of the medium, cells  and identified using a BCA protein assay kit . Proteins (30–50 μg) were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with horseradish peroxidase (HRP)-IgG secondary antibody  for 2 h at room temperature and protein bands were detected using chemiluminescence reagent by Fluor Chem E . Densitometry analyses were performed for all western blots by ImageJ .After incubation in serum-free medium for 24 h in 60 mm dishes, media were replaced with complete medium (10% FBS) containing different concentrations of DHA  and incubated for 24 h. The cells were fixed in ice-cold 70% ethanol overnight, incubated with RNase (50 μg/ml) for 1 h, and stained with propidium iodide (50 μg/ml) for 30 min. Cell cycle distribution was measured and analyzed using FACScan flow cytometry (BD FACSCanto) and FlowJo7.6.1 software, respectively.KYSE30 and KYSE150 cells were treated with DHA (10 μM) for 12 h. Phosphorylated proteins were analyzed using the Human Phospho-Kinase Array Kit  according to the manufacturer’s instructions. The array membranes were blocked with 1.0 ml of Array Buffer one and incubated with 500 μg cell lysates. The detection antibody cocktail was added, followed by incubation with streptavidin-HRP. Signals were detected using the Chemi Reagent Mix, and array images were analyzed using ImageJ software. The negative control signal was subtracted from each spot, and values were normalized to the positive control present in each membrane. Then, the ratio of signal intensity (treated/untreated) was calculated.KYSE30 and KYSE150 cell lysates (500 μg) were incubated with DHA-Sepharose 4B (or Sepharose 4B only as a control) beads  in a reaction buffer . After incubation with gentle rocking overnight at 4°C, the beads were washed five times with the reaction buffer  and binding was visualized using western blot analysis.in vitro kinase assay with 100 ng of active AKT1  or p70S6K . Reactions were conducted in 1X kinase buffer  containing 200 μM ATP at 30°C for 30 min. Reactions were stopped by adding 6X protein loading buffer, and proteins were detected using western blot analysis.The purified mTOR protein  was used as the substrate for an 3) = (length × width × width/2).Severe combined immunodeficient (SCID), 6–8 weeks old female mice  were used as the PDX mouse model following the guidelines of the Ethics Committee of Zhengzhou University . The two cases of ESCC in PDX animal experiments were named LEG20 and LEG24. The tumors were divided into 0.1–0.2 g fragments and inoculated into the back of SCID mice. Mice were randomly assigned to the following groups: control group, 25 mg/kg DHA-treated group, and 50 mg/kg DHA-treated group. Each mouse was administered either DHA or DMSO three times per week by intraperitoneal injection. The tumor volume and weight of each mouse were measured every 3 days. Tumor volume was calculated based on the dimensions of the individual tumor and according to the following formula: Tumor volume , p-p70S6K (1:50), and p-RPS6 (1:200) overnight at 4°C. After incubation with HRP-conjugated secondary antibodies at 37°C for 15 min, the tissues were detected with 2,4-diaminobenzidine substrate (DAB) and counterstained with hematoxylin. All slides were scanned by using TissueFAXS and analyzed by using HistoQuest 4.0 software .Mice in the control group were monitored until tumors reached about 1.0 cmp < 0.05 was considered statistically significant.All quantitative results are expressed as mean ± SD. Statistically significant differences were obtained by using Student’s t-test or one-way ANOVA. in vitro.To explore the effect of DHA on ESCC cells, KYSE30 and KYSE150 were treated with various doses of DHA. Through DHA cytotoxicity assay, we chose 0, 1, 5, 10, or 15 μM for subsequent experiments. As the concentration increased, DHA significantly inhibited the growth of KYSE30  and KYSEG0/G1 phase. To further confirm the effect of DHA on the cycle, we examined the protein levels of cyclin-dependent kinase 2 (CDK2) and p21 using western blot analysis. DHA attenuated the expression of CDK2 in KYSE30  were suppressed. Moreover, we explored the molecular pathways by which DHA affected mTOR signaling. We found that DHA weakened mTORC1 signaling by reducing the levels of phosphorylated mTOR at Ser2448 (p-mTORS2448), p70S6K at Thr389 (p-p70S6KT389), p70S6K at Thr421/Ser424 (p-p70S6KT421/S424), and RPS6 at Ser235/Ser236 (p-RPS6S235/S236). Treatment with DHA at 0, 5, 10, or 15 μM for 24 h downregulated the levels of p-mTORS2448, p-p70S6KT389, p-p70S6KT421/S424 and p-RPS6S235/S236 in KYSE30 cells  for 12 h. The results indicated that DHA inhibited the phosphorylation of p38α, p70S6K, eNOS, Fgr, STAT5b, PLC-and PYK2 . Among t30 cells . Further30 cells . Similar30 cells .in vitro pull-down assays  as PDX models for further studies. The PDX tumor tissues were implanted into the back of the neck of SCID mice; either control or DHA (25 or 50 mg/kg body weight) was administered through intraperitoneal injection every 2 days. The results showed that DHA significantly inhibited tumor growth compared with the control group  analysis of PDX LEG20 and LEG24 tumor samples. The phosphorylation of mTOR, p70S6K, RPS6 were strongly inhibited in the DHA-treated group . We investigated the effect of DHA on p-mTORed group , which aed group . These rin vitro and in vivo via the AKT1-mTOR-p70S6K axis. Esophageal cancer is one of the most common malignant tumors in the world. Despite aggressive surgical resection and chemoradiation, its prognosis is still poor. There is an urgent need to identify new drugs capable of inhibiting ESCC progression. Chinese herbal medicine is an important resource for discovering new medicines, and natural products derived from herbal medicines have significant anticancer activity  and p70S6K (T389) phosphorylated S2448 site of mTOR in an in vitro kinase assay. Moreover, DHA inhibited phosphorylation of mTOR by binding to AKT1 and p70S6K , the Science Foundation of Henan Education Department (Grant no. 20A310023 and 19A310026) and the Supporting Plan of Scientific and Technological Innovation Team in Universities of Henan Province (No. 20IRTSTHN029).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.


Systemic sclerosis (SSc) is a multi-system connective tissue disease characterized by the increased deposition of extracellular matrix proteins such as collagen and fibronectin. Although the pathogenesis is not completely understood, a number of studies suggest that free radicals could be the major contributors to the disease. Indeed, different studies demonstrated how oxidative stress could contribute to the fibrotic process activation at the level of the skin and visceral organs. Emerging evidences highlight the beneficial effects of sildenafil, a phosphodiesterase type 5 inhibitor (PDE5i), which protects different cell lines from the cell damage induced by reactive oxygen species (ROS). These data make sildenafil a good candidate for therapeutic treatment aimed to protect biological macromolecules against oxidative damage, thus preserving cell viability. The purpose of this study was to evaluate the sensitivity of SSc dermal fibroblasts to an oxidative insult and the ability for sildenafil to prevent/reduce the DNA damage due to ROS action. Additionally, we evaluated the capacity for sildenafil to influence redox homeostasis and cytotoxicity, as well as cell proliferation and cell cycle progression. We demonstrated that SSc fibroblasts have an increased sensitivity to a pro-oxidant environment in comparison to healthy controls. The sildenafil treatment reduced ROS-induced DNA damage, counteracted the negative effects of ROS on cell viability and proliferation, and promoted the activity of specific enzymes involved in redox homeostasis maintenance. To our knowledge, in this report, we demonstrate, for the first time, that sildenafil administration prevents ROS-induced instability in human dermal fibroblasts isolated by SSc patients. These results expand the use of PDE5i as therapeutic agents in SSc by indicating a protective role in tissue damage induced by oxidative insult. Systemic sclerosis (SSc) is a multi-system connective tissue disorder characterized by the increased deposition of extracellular matrix proteins, where increased oxidative stress plays a role in the activation of the fibrotic process at the level of the skin and visceral organs ,2,3.In fact, it is known that excessive oxidative stress contributes to vascular damage, jeopardizes the function of the endothelial system, leading to immune system involvement, and it participates in the establishment and maintenance of fibroblast activation ,5.Several authors have shown that SSc patients have a reduced antioxidant capacity; however, the exact stage of the disease at which the increase in reactive oxygen species (ROS) occurs is still uncertain ,7. The pControversial results exist about the possible beneficial effects of the treatment with different antioxidants in patients with SSc. The favorable outcome depends on the nature of the antioxidant substance, stage of the disease, and the duration of treatment ,16,17,18Emerging evidences suggest specific protective effects of phosphodiesterase type 5 (PDE5) inhibition with sildenafil in protecting them from genotoxic damage induced by ROS in different cell lines ,20,21. PThe high potential antioxidant capability of sildenafil ,27 makesThe objective of this study was to evaluate the sensitivity of SSc dermal fibroblasts to oxidative stress and the ability for sildenafil to prevent/reduce the DNA damage induced by ROS. Additionally, we evaluated the capacity of sildenafil to influence redox homeostasis and cytotoxicity, as well as cell proliferation and cell cycle progression. To our knowledge, this is the first study on the redox characterization of SSc fibroblasts and their response to ROS-induced DNA damage, where it also highlights the possible capacity of sildenafil to determine an increased protection against oxidative stress-related alterations2+/Mg2+-free, bovine serum albumin (BSA) fraction V, glutamine, antibiotics, collagenase type IV, NaOH, Bradford reagent, 4′,6-Diamidino-2-phenylindole (DAPI), hydrogen peroxide, sildenafil citrate salts (98%), propidium iodide, ribonuclease enzyme, a redox-sensitive probe, and all reagents for Western blotting were from Sigma Aldrich . Fetal calf serum was from Gibco® . 2-mercaptoethanol was from Life Technologies, Inc. Laboratories . Cy3-labeled secondary antibodies (Abs) were from Jackson Laboratory ; peroxidase secondary Abs and all reagents for Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) were from Millipore . Plastic ware for cell cultures and disposable filtration units for growth media preparation were purchased from Corning . Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium (1:1) (with and without phenol red), phosphate-buffered saline (PBS) CaHuman dermal fibroblasts (Hfbs) were isolated at the SSc clinic within the Leeds Institute of Rheumatic and Musculoskeletal Medicine  and processed as previously described . Cells w2O2 (50 and 100 µM) in the presence or absence of sildenafil. Cells were then lysed in Radioimmunoprecipitation assay (RIPA) buffer , 1% nonyl phenoxypolyethoxylethanol (NP40), 0.25% sodium deoxycholate, and 0.1% SDS, water to volume) supplemented with protease and phosphatase inhibitor cocktails .Healthy and SSc fibroblasts were treated for 24 h with H® software version 4.6.8 . Western blot analysis was performed for three/four independent experiments. The list of primary antibodies utilized for the Western blot is reported in For the immunoblot analysis, an equal amount of proteins 20–30 μg) was resolved in SDS-polyacrylamide  gels and transferred on polyvinylidene fluoride (PVDF) or nitrocellulose membranes, as previously described  μg was r. Thereaf4 cells were seeded onto glass coverslips in the growth medium. For γH2aX and RAD51 localization, healthy and SSc fibroblasts were treated with sildenafil and then stimulated for 0.5 h or 1 h with 50 µM or 100 µM H2O2. After stimulation, cells were washed out from the drugs and maintained in a drug-free medium for 4 h. Next, cells were washed and fixed with 95% ethanol and 5% acetic acid for 5 min and incubated with a blocking buffer containing 3% BSA/TBS for 30 min at room temperature. Primary Abs were incubated 1 h in blocking buffer, followed by the Cy3-conjugated secondary Ab (1.500). For method specificity, slides lacking the primary Abs were processed. Details on the primary antibodies are reported in A total of 1 × 103 cells/well) were seeded in 96-well plates and incubated 1 h (T0) or 24 h (T24) with 50 µM or 100 µM H2O2 in the presence or absence of sildenafil. As previously described [Healthy and SSc fibroblasts  or cultured for 7 (7 d) and 14 days (14 d) in drug-free media. Cell viability was assessed each day by trypan blue (0.05% v/v solution in PBS) exclusion mixed in a ratio of 1:1. Cell number counting was assayed by a hemocytometer, and only cells excluding trypan blue dye were analyzed. The number of viable cells at each time point was derived, averaging at least five different fields for each well. Each experimental point was repeated in duplicate or triplicate. Results are expressed as the number of cells. Experiments were performed four times with different cell preparations.For the cell viability assay, healthy and SSc fibroblasts were seeded in 6-well plates and incubated for 1 h with 50 µM or 100 µM H4 healthy and SSc fibroblasts were seeded, with or without sildenafil, and then stimulated for 24 h with 50 µM or 100 µM H2O2. Propidium iodide (PI) assay was performed as previously described [v/v) alcohol at −20 °C overnight. After centrifugation, cells were suspended with 50 µM PI solution and ribonuclease A. DNA content was analyzed with FACScan CytoFLEX . Experiments were performed in triplicate with different cell preparations.To evaluate the cell cycle progression, 25 × 10escribed . Briefly3/mL in 6-well plates and incubated for 1 h with H2O2 50 µM or 100 µM in the presence or absence of sildenafil. At the end of treatment, cells were loaded with 200 µL of 5 µM dichloro-dihydro-fluorescein diacetate (DCFH-DA) dissolved in dimethylsulfoxide (DMSO) in PBS solution and maintained at 37 °C in the dark. The loading buffer was removed after 40 min, and the cells were incubated at 37 °C for an additional 30 min in a prewarmed growth medium. Then, the cell layer was washed with serum-free DMEM, and the level of ROS was determined by a Spark® fluorescence plate reader  at 488/525 nm. Results obtained were expressed as absorbance arbitrary units/mg of proteins tested.For intracellular ROS levels analysis, healthy and SSc fibroblasts were seeded at 5 × 102O2, were lysed in RIPA buffer. Cell lysate (150 μL) was added to 25 μL 0.2% butylated hydroxytoluene (BHT) and 600 μL of 15% aqueous trichloroacetic acid (TCA). Then, the mixture was centrifuged at 4000× g for 15 min at 4 °C. The deproteinized supernatant was transferred in a cryotube and added with 600 μL of thiobarbituric acid . Samples were then heated at 100 °C for 15 min in boiling water. The absorbance was determined at 535 nm by a spectrophotometric method and compared to standard MDA  solution. The levels of TBARS were expressed as nmol/mg of proteins.TBARS levels were assayed by spectrophotometric analysis, as previously described . The met7 healthy and SSc fibroblasts, treated with sildenafil and then stimulated for 1 h with 50 µM or 100 µM H2O2, were collected and suspended in 1:1 (v/v) 5% sulfosalicylic acid (SSA). Cells were lysed by freezing and thawing and, then, were centrifuged at 10,000× g for 5 min at 4 °C. The deproteinized supernatant was analyzed for the total glutathione content. Oxidized glutathione (GSSG) was measured in samples where reduced GSH was masked by pretreatment with 2-vinylpyridine (2%). Ten microliters of the sample were added to the reaction buffer (700 μL nicotinamide adenine dinucleotide phosphate (NADPH) (0.3 mM), 100 μL DTNB (6 mM), and 190 μL H2O). The reaction was started by adding 2.66 U/mL glutathione reductase and followed at 412 nm by the TNB stoichiometric formation. Samples Δ optical density (ΔOD)/min412 were compared to those obtained by using glutathione standards. Results were normalized for protein contents.Intracellular reduced (GSH) and oxidized (GSSG) glutathione amounts were evaluated by a 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB)-glutathione reductase recycling assay, as previously described . Briefly2O2. After treatment, cells were immediately collected. Intracellular superoxide dismutase (SOD) and catalase (CAT) activities were measured using commercial assay kits , as previously described [Healthy and SSc fibroblasts were treated with sildenafil and then stimulated for 1 h with 50 µM or 100 µM Hescribed . Experim4 cells/mL in 96-well tissue culture plates and incubated 1 h with 50 µM or 100 µM H2O2 in the presence or absence of sildenafil. Cell culture supernatants were assayed for the macrophage migration inhibitor factor (MIF) by the magnetic bead-based multiplex assay according to the manufacturer’s protocol. As previously described [Healthy and SSc fibroblasts were plated at 2 × 10escribed , data act-test. In all cases, p-value ≤ 0.05 was considered significant. All data were expressed as the mean ± SE.The statistical analysis was conducted using GraphPad Prism software 8.0 . To test the normality of the quantitative variables, the Kolmogorov-Smirnov or Shapiro-Wilk tests were applied. Normally distributed, continuous variables were analyzed using one- or two-way ANOVA and a Student’s 2O2 for 0.5 h), SSc Hfbs showed higher sensitivity to oxidative stress compared to healthy ones, with a percentage of γH2aX-positive cells of 46.4 ± 17.9% after 30 min (p < 0.05 vs. control (ctr)), reaching 93.0 ± 5.7% after 1 h  (2O2), SSc Hfbs showed increased γH2aX by 62.9 ± 12.7% after 30 min  and 82.7 ± 9.1% after 1 h (p < 0.01 vs. ctr) (2O2 100 µM (p < 0.01 vs. ctr)  A. At thevs. ctr) A. Differvs. ctr) A.2O2 100 µM for 1 h in the presence or not of sildenafil. As shown in 2O2: 8.8% ± 5.2% vs. 65.5 ± 12.2%, p < 0.05) and SSc fibroblasts ; however, the presence of sildenafil significantly decreased the percentage of γH2AX-positive cells by 32.8 ± 4.9% in healthy  and 56.1 ± 0.9% in SSc fibroblasts , respectively. When the same analysis was extended to RAD51, a protein involved in DNA repair mechanisms [p < 0.01)  or in SSc fibroblasts , although the number of RAD51 foci was significantly higher in the SSc (p < 0.01). Of interest, the highest dose of H2O2 (100 µM) induced major recruitment of RAD51 only in healthy Hfbs  (2O2 (50 µM) only in SSc Hfbs , whereas no differences were observed in healthy Hfbs, where the level of RAD51 remained quite similar to that without sildenafil (p > 0.05) (2O2 (100 µM), the presence of sildenafil increased RAD51 recruitment exclusively in SSc Hfbs . No significant changes were observed in healthy Hfbs exposed to the pro-oxidant condition between the presence and absence of sildenafil (p > 0.05). Finally, the analysis of serine/threonine kinase (CHK2) modulation, a key component of the DNA damage response, highlighted that, in a pro-oxidant environment (50 µM and 100 µM H2O2 for 24 h), there was an increase of its phosphorylated form in SSc Hfbs , independently of the presence of sildenafil (p > 0.05)  A. Exposu < 0.01) A. The pr > 0.05) A. At hig > 0.05) B. No sig2O2 administration (50 µM and 100 µM H2O2 for 24 h), a flow cytometric analysis was performed. As shown in p > 0.05). However, in SSc, the highest dose of pro-oxidant induced an increase of cells blocked in the G1 sub-phase . Furthermore, the pretreatment with sildenafil reduced the proportion of cells in the G1 subphase by 77% after H2O2 50 µM  and by 83% after H2O2 100 µM   B.p < 0.05). No significant effects were observed for CASPASE 3 cleavage, Bcl-2, or Bax (p > 0.05). The presence of sildenafil reduced the activation of LC3 II, the level of p62, and ERK phosphorylation (p < 0.05).To better characterize this phenomenon, specific protein markers of the apoptotic and autophagic processes were analyzed. As shown in 2O2 exposure (p < 0.05 and p < 0.01) independently from the presence of sildenafil, although at 24 h, the presence of sildenafil showed a trend towards improving the cell proliferation (p = 0.0523) (2O2 exposure at 7 d (H2O2 vs. H2O2 + S: (2.9 ± 0.5) × 104 vs. (4.1 ± 0.7) × 104, p < 0.05) and 14 d (H2O2 vs. H2O2 + S: (3.2 ± 0.7) × 104 vs. (5.9 ± 0.1) × 104, p < 0.05), whereas, in SSc Hfbs, it was observed with both H2O2 doses at 7 d (50 µM H2O2 vs. 50 µM H2O2 + S: (1.7 ± 0.5) × 104 vs. (3.0 ± 0.4) × 104, p < 0.05 and 100 µM H2O2 vs. 100 µM H2O2 + S: (1.0 ± 0.2) × 104 vs. (2.2 ± 0.3) × 104, p < 0.01) and 14 d (50 µM H2O2 vs. 50 µM H2O2 + S: (2.3 ± 0.6) × 104 vs. (4.0 ± 0.6) × 104, p < 0.05 and 100 µM H2O2 vs. 100 µM H2O2 + S: (1.3 ± 0.3) × 104 vs. (2.8 ± 0.8) × 104, p < 0.05)  A,B. Long 0.0523) C. In hea < 0.05) C.Previous experiments have been performed to demonstrate that sildenafil per se did not retain scavenger activity on oxygen-derived free radicals when added to the culture medium (data not shown).p < 0.05). Hydrogen peroxide administration induced a significant dose-dependent increase in ROS levels, particularly in SSc fibroblasts . Interestingly, the combined treatment with sildenafil reduced H2O2-induced ROS levels in healthy fibroblasts at both doses tested , whereas, in SSc cells, there was a significant reduction of ROS only at the highest dose , respectively. No significant differences were found with sildenafil administration alone (p > 0.05) (As shown in  > 0.05) A.p < 0.01) . Differently, healthy Hfbs showed a significant reduction of the GSH/GSSG ratio only after exposure to the highest dose of H2O2   (p > 0.05). At the level of the antioxidant enzyme activities, we found substantial differences between two types of fibroblasts depending on the enzyme examined (p = 0.0938), and its level was not influenced by the presence of H2O2 and/or sildenafil (p > 0.05), the analysis of superoxide dismutase (SOD) activity highlighted distinct and significant differences between the two groups. Indeed, SOD activity was ≈ 37.7% lower in SSc compared with the counterpart healthy . In the presence of ROS, both cell lines were responsive, with a significant increase of SOD activity only at the higher dose . Moreover, while in healthy ones, the combined treatment with sildenafil induced a major increase in SOD activity in both H2O2 doses tested , in SSc, this increase was present only at the low dose  .The evaluation of the glutathione homeostasis revealed significant differences in the GSH/GSSG ratio between the experimental groups. In particular, in SSc, the total glutathione levels were already found lower compared with the counterpart healthy ones at the basal level  , also sh < 0.05) B. Pretre < 0.05) B. Both texamined C. In par < 0.05) C. The prp > 0.05), whereas SOD2 was significantly upregulated only in SSc cells exposed to H2O2 (p < 0.05), independently from the presence of sildenafil (p > 0.05)  D.p < 0.05)  and a massive increase in SSc ones    , the ana < 0.05) E. The pr < 0.01) E. Intere < 0.01) E.p > 0.05). Under pro-oxidant conditions, TBAR levels increased in both cell types tested, showing the greatest sensitivity of SSc. Indeed, already at a low dose of H2O2, there was a significant increase in TBAR in SSc cells . Only at the highest concentration of hydrogen peroxide, there was a significant increase in TBAR in both cell types . The concomitant presence of sildenafil induced no significant variation in TBAR levels after the low dose of H2O2 (p > 0.05), whereas, at the highest dose, sildenafil reported TBARS levels similar to the control in both experimental groups (p < 0.01). No significant differences were found with sildenafil administration alone (p > 0.05).As shown in The present study demonstrates for the first time that the PDE5 inhibitor sildenafil reduces the sensitivity to DNA damage of human SSc fibroblasts exposed to a pro-oxidant environment, improving their genomic stability. In addition, sildenafil counteracts the negative effects of ROS on cell viability and proliferation, inhibiting the activation of the autophagic pathway and modulating the activity of specific enzymes involved in redox homeostasis. Taken together, these results suggest that sildenafil may be a possible candidate for therapeutic treatment aimed at counteracting oxidative stress adverse effects.There is considerable evidence implicating sildenafil as a key molecule capable of preventing ROS-induced DNA damage in several different types of cells, a condition present during the progression of many diseases—as, for example, in atherosclerosis, cardiovascular diseases, and systemic sclerosis ,20,21,392O2, indicating the proceeding of HR repair; however, and more markedly in SSc cells, sildenafil modified ROS-induced effects. In particular, in the presence of low levels of DNA damage, the PDE5i determined a reduced redistribution of RAD51. Presumably, the extent of DNA damage did not exceed the intrinsic capacity of the cells to repair themselves. Differently, in the presence of massive DNA damage, sildenafil improved the cellular response by increasing the efficiency of cell repair systems.In the present study, we utilized a cell culture of human dermal fibroblasts from healthy and SSc patients already validated as a useful model to investigate the mechanisms involved in SSc fibrosis ,40,41. WCHK2 is a checkpoint kinase and a key component of the DNA damage response regulating the binding of RAD51 to DNA ,46. Geno2O2 [There are evidences of antioxidative effects of sildenafil on different cell lines and animal models ,54,55,562O2 . Here, f2O2 , and thi2O2 .The molecular mechanism behind the pharmacologic actions of this PDE5i relates to its potential to increase intracellular cyclic guanosine monophosphate (cGMP) . The regAlthough our in vitro system represents a simplified model reproducing the alteration of the redox homeostasis determined by the pathological state ,66,67, wParticularly, our findings offer compelling evidences indicating that the administration of sildenafil in SSc fibroblasts exposed to a pro-oxidant: (i) protects against ROS-induced DNA damage, (ii) preserves the cell cycle kinetics, and (iii) improves redox homeostasis .Therefore, the aforementioned effects suggest a possible positive impact of sildenafil not only on the treatment of the first signs of the disease, such as endovascular damage and decreased blood flow in the digital vein (Raynaud’s phenomenon), but, also, on the last stages when skin fibrosis occurs.However, further studies are needed to deepen the molecular mechanisms by which sildenafil acts as an antigenotoxic drug and antioxidant, as well as to establish the feasibility and efficacy of this PDE5i in the clinical setting of patients at risk of developing SSc and in those where the pathology is already in progress.