Coronary artery disease (CAD) is a multi-factor complex trait and is heritable, especially in early-onset families. However, the genetic factors affecting the susceptibility of early-onset CAD are not fully characterized.CYP17A1 gene from a Chinese Han family with CAD. To validate the effect of this variation on atherosclerosis and early-onset coronary artery disease, we conducted studies on population, cells, and mice.In the present study, we identified a rare nonsense variant in the CYP17A1-deficient mice present the phenotype of metabolic syndrome with hypertension, increased serum glucose concentration, and presentation of central obesity and fatty liver. Furthermore, CYP17A1 knockout mice or CYP17A1 + ApoE double knockout mice developed more atherosclerotic lesions than wild type (WT) with high fat diary. In cell models, CYP17A1 was found to be involved in glucose metabolism by increasing glucose intake and utilization, through activating IGF1/mTOR/HIF1-α signaling way, which was consistent in CYP17A1 knockout mice with impaired glucose tolerance and insulin resistance.The mutation precisely congregated with the clinical syndrome in all the affected family members and was absent in unaffected family members and unrelated controls. Similar to the human phenotype, the CYP17A1 as a key protein participating in the pathophysiology of the atherosclerotic process and the possible mechanism of CYP17A1 C987X mutation induced atherosclerosis and early-onset CAD involving glucose homeostasis regulation was revealed.Through our study of cells, mice and humans, we identified Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01061-z. Coronary artery disease (CAD) has a familial component. Moreover, the genetic contribution to CAD is more pronounced in premature forms of CAD , 2. A reRecently, whole genome scans have been increasingly used and performed on a large number of patients with complex diseases such as CAD. By affected sibling-pair analyses, genome regions that were over shared between siblings were screened subsequently for specific sequence variations associated with the disease . A speciPlease see Additional file CYP17A1 gene  on chromosome 10  were significantly higher than those of wild type (WT) individuals , high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), urea nitrogen, creatinine and uric acid, were similar in both CYP17A1-C987X and WT individuals  was identified in one family but not in 150 sporadic CAD subjects. The CYP17A1 c. -14G > A mutation congregated with the CAD phenotype within this family  with inherited CAD  form of CYP17A1, the C987X mutant produced a new truncation protein (about 40KD)  which caused a 20% truncation of the CYP17A1 protein and exhibited lower protein levels  or CYP17A1(C987X), the mRNA abundance of CYP17A1were increased, regardless of the amounts or time transfected, CYP17A1(WT) is more expressive than CYP17A1(C987X)  was more stable with a half-life of ≈ 8 h  and CYP17A1 (C987X), we performed immunofluorescence experiments by co-staining transfected CYP17A1(WT) or CYP17A1(C987X) together with the endogenous ER marker KDEL or mitochondria marker COXIV. CYP17A1-FLAG was largely colocalized with KDEL  or CYP17A1 (C987X) plasmid. It showed that over-expression CYP17A1, promoted the expression of the glucose transporter enzymes in both HEK239T and Huh7 cells , the expression of PDHA1and COXIV were detected. Our data showed that CYP17A1 up-regulated PDHA1 and COXIV expression in Huh7 cell, not in HEK293T , glucose depleted medium (G−/Gln+) or glutamine depleted medium (G+/Gln−). Our results demonstrate that transient in HEK293T cells expression of CYP17A1 WT or C987X can’t increase cell proliferation  and the anti-IgG IP were similarly performed, and the identified proteins served as nonspecific binders. The CYP17A1-binding candidates specifically identified from anti-CYP17A1 IP of WT samples are listed  , dehydroepiandrosterone sulfate (DHEAS), estrogens (E2), 17 hydroxyprogesterone (17-OHP), which were in consistent with CYP17A1−/− mice HFD feeding at 6 or 9 months  and postprandial blood glucose (PBG) than WT  . In 6 or 9 or 12 months, a significant difference in plaque size was found, larger size of atherosclerotic plaques in As shown in Fig. CYP17A1 as a disease mutation leads to familial early-onset CAD. Our data supply further expansion of genes that may predispose to early-onset CAD and also support further research into genetic determinants of cardiovascular risk. Then we conducted a detailed functional study of CYP17A1 and its mutation in human, cells and animals. CYP17A1 was found to be involved in glucose metabolism by increasing glucose intake and utilization, though activating IGF1/mTOR/HIF1-α signaling way. Due to the unstable of C987X mutation and its impaired function, it caused the loss of original function. In consistent with cellular results, CYP17A1 knockout mice, apparent impaired glucose tolerance, insulin resistance, corresponding changes in the levels of hormones, accelerated vascular endothelial injury and promoted formation of atherosclerosis. Taken these together, our results suggest that CYP17A1 may also be involved in glucose metabolism, in addition to the classical steroid regulation. When CYP17A1 is mutated or functional impaired, it may be involved in the occurrence and development of coronary atherosclerosis through a variety of ways.For the first time, we identified a rare nonsense variant in It has been shown that IGF1 has insulin-like metabolic effects as an essential regulator of cell growth, differentiation and apoptosis, which are regulated by a variety of factors including hormones, nutritional status and genetics. It plays a crucial role in the pathophysiological processes of cardiovascular diseases, endocrine and metabolic diseases, and tumors . Recent CYP17A1, cytochrome P450 family 17 subfamily A member 1, is also named as CPT7, CYP17, P450C17, and S17AH. It is a cytochrome P450 monooxygenase encoding 17α-hydroxylase, 17, 20-lyase, involved in drug metabolism, synthesis of cholesterol, steroids, and other lipids and participating in the process of carbon–carbon bond scission [CYP17A1 is a key microsomal enzyme that converts pregnenolone and progesterone to their major downstream products: DHEAS, cortisol, testosterone, and estradiol [CYP17A1 may related to atherosclerosis by participating in the regulation of steroid. HMGCR and LSS over express with the transfection of CYP17A1 plasmid in cells, strongly evidenced that CYP17A1 may related to cholesterol metabolism. Aherrahrou et al. also found the increased atherosclerosis in CYP17A1 XY knockout mice lacking testosterone associated with altered lipid profiles [CYP17A1 with glucose metabolism has not been clarified clearly to date. In this context, we generated CYP17A1-deficient mice and cells models to discuss its impact on glucose metabolism. Lu et al. found CYP17A1/17-hydroxyprogesterone/ glucocorticoid receptor dependent pathway in the liver that mediates obesity-induced hyperglycemia [CYP17A1 gene in Huh7 cell could regulating glucose metabolism by increasing glucose intake, promoting glucose utilization, and improving insulin resistance by IGF1 signaling way, and we confirmed the expected disorder of glucose metabolism in CYP17A1-deficient mice with impaired glucose tolerance and insulin resistance. As for the difference of conclusion, we think it may be caused by the difference of background mice. Relevant research results are obtained by building 2 common models of severe obesity (leptin receptor deficient db/db mice and leptin deficient ob/ob mice), whereas, our background mice were C57BL/6. At the same time, CYP17A1 is mainly produced in adrenal glands and sexual tissues in mice, while Lu et al. interfered with the expression of CYP17A1 gene in the liver. What we performed was the complete knockout mice of CYP17A1 gene. Therefore, we believe that CYP17A1 at the systemic level has another way action of blood glucose regulation. As reported, the atherosclerosis was enhanced in castrated female [CYP17A1 is mutated or functional impaired, it may cause long-term fluctuation of blood glucose and increase the level of free lipids, which can aggravate lipid toxicity and glucose toxicity, further increase oxidative stress, cause endothelial dysfunction.Early-onset CAD (before 55 years in men and 60 in women) is more likely to reflect a genetic predisposition. Because of shared lifestyle risk factors and genetic predisposition, compared with the general population, siblings have at least double the risk, offspring and partners are also at increased risk , 42. As scission . Publishscission , 48, whiscission –52. As Cstradiol . It’s maprofiles . The relglycemia . Howeverd female , the athCYP17A1 involvement in atherosclerotic and it also gives a new sight to the possible function of CYP17A1∆ECD protein.Then the corticosterone release was stimulated by inflammatory cytokines and nutritional overflow, which in turn accelerates the development of atherosclerotic changes. The result of co-IP suggests the possible interacting proteins and provides a new direction for our further study to reveal the possible mechanism of CYP17A1 is also associated with essential hypertension and involved in corticoid and androgen biosynthesis, we failed to measure the relative endogenous hormone levels of Family 1 members.A limited number of patients with early-onset CAD were collected, and none C987X mutations were detected. It may helpful to understand the physiological function of CYP17A1 gene, by examining the expression of genes or proteins related to glucose metabolism in the CYP17A1 knockout mice. However, our animal model, which was a whole-body knockout of CYP17A1 gene, could not well simulate the mechanism of human CYP17A1 and its variants,its another limitation of our study. In vivo regulatory pathways may be more complex. It remains unclear whether alternative pathways are involved in the regulation of blood glucose and targeted knockout animal models are needed to better mimic the real situation in vivo and reveal its more precise regulatory mechanisms.Given that Given differences between mice and humans, functional studies through more clinical samples may be of more clinical value.These findings indicate a role for CYP17A1 in glucose homeostasis and associate its altered function with an inherited form of the early-onset CAD.Additional file 1: Figure S1. Schematic of whole-exome sequencing data processing and variant identification. Figure S2. The c.-14G>A variant in CYP17A1. (A) Schematic of identification CYP17A1 as a candidate gene for CAD susceptibility. (B) Pedigrees of the family 2 with c.-14G>A mutation. CYP17A1 genotype  are shown below each square or circle. Figure S3. CYP17A1 over-expression involved in glycometabolism. (A) Relative mRNA levels of genes related to glycolytic pathway (screened by RNA-seq) in stably expressing C987X mutation or in HEK293T cells with over-expressed CYP17A1 WT or C987X.Vector as control. (B) CYP17A1 promotes the glucose uptake in HEK293T cells. HEK293T cells transfected with plasmids were labeled with intracellular level of 2-NBDG and subjected to measurement of flow cytometry. HEK293T cells cultured in a 6-well culture plate were transiently transfected with 3μg/well of plasmids encoding CYP17A1(WT) or CYP17A1(C987X). After 48h transfection, supernatants were harvested and subjected to the detection of glucose(C) or lactate(D) using assay kits. (E) After 48h transfection, cell lysates were prepared and subjected to western blot analysis. β-actin expression was used as a loading control. The effect of CYP17A1 and C987X mutation on key enzymes of cholesterol synthase in HEK293T and Huh7 cells were detected. (F) The influence of glucose and glutamine on CYP17A1-mediated cell proliferation. HEK293T cells with transient CYP17A1 WT or C987X expression were cultured in three different media: the complete medium with both glucose (Glu+) and glutamine (Gln+); the culture medium with glucose (Glu+) but without glutamine (Gln-); the culture medium without glucose (Glu-) but with glutamine (Gln+). After 24h incubation, cell proliferation in each culture condition was evaluated by CCK8 assay. (G)ATP level was measured by an assay kit. Data are presented as mean ± SD from at least three independent experiments. (H) The expression of CYP17A1 protein in different media, detection by WB. n=3 biological repetition. *, p < 0.0.5; **, p < 0.01; ***, p < 0.005, ****, p < 0.001.n.s., no significance. Figure S4. Identification of CYP17A1-binding proteins. (A)Schematic of the work flow of immunoprecipitation (COIP) followed by tandem mass spectrometry (MS/MS). (B) The top twenty candidate proteins that specifically identified in anti-CYP17A1 IP from HEK293T cell transfected with WT plasmids sample shown in (A). Figure S5. Identification of CYP17A1 functional paths. (A) Schematic of the work flow of combine analysis of CO-IP and RNA-seq. (B) Parts of candidate gene that specifically related to functional paths shown in (A). (C-F) Functional path clusters  and interaction network  in differences unique to wild-type (C and D) or to C987X truncation mutant (E and F). (G)Silver staining of IP. Specific protein (about 35 kD) between CYP17A1 and CYP17A1ΔECD were pointed with an arrow. Figure S6. Diagram of CYP17A1-/-mice. Upper part: (A) Representative photos of of appearance(A) in chow fed CYP17A1-/- heterozygote, homozygous and WT mice at 12 mo of age and relative diagram of the anatomy (B), 9 month of HFD appearance(C) of WT, CYP17A1-/- and CYP17A1-/-ApoE-/- mice. Lower part: Abdominal, epicardial and liver necropsy photos of representative 12-mo-old CYP17A1-/- heterozygote, homozygous and WT mice, showing increased adiposity in CYP17A1-/- vs. WT mice. Livers of CYP17A1-/- mice tend to display increased fat accumulation displaying yellow coloration compared with WT (F vs. C). Figure S7. Characterization of CYP17A1 whole-body knockout mice. (A)WT, CYP17A1-/- mice (n=8) were fed on chow diet or HCD (0.25% cholesterol). Weights were measured at 3,6,9,12 month. Data is expressed as mean ± SD. Statistical analyses, two-way ANOVA. (B)Tissues taken from 12-mo-old WT and CYP17A1-/- mice were immediately weighted by micro balance. Organ coefficient was calculated. Biochemical detector to detect serum AST(C) and ALT(D). (E)Blood pressures were measured indirectly by tail arteries in awake mice. Data are expressed as median with SD. Nonparametric test. #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001, compared with WT mice fed with HCD diet. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with WT mice fed with chow diet. ns, not statistically significant compared with WT mice fed with chow diet. NS, not statistically significant compared with WT mice fed with HCD. Figure S8. Adipose and liver histology in CYP17A1-/- mice. Upper part: Hematoxylin and eosin (H&E) staining of gonadal adipose sections from WT (A), CYP17A1-/-(B) mice at 12 mo of age. Lower part: H&E staining of liver sections. H&E staining of liver sections from chow fed female CYP17A1-/- mice at 3, 6,9 and 12 mo-old (A-D). WT female mice at 3, 6, 9and 12 mo(E-H), respectively. All histology images are X40 magnification; scale bar in bottom right =50μm. Figure S9. Metabolic characteristics of CYP17A1 whole-body knockout mice. WT and CYP17A1-/- mice at 6 or 12mo-old  with chow diet, WT and CYP17A1-/- mice a t6 or 9 mo-old (n=6, each month/each genotype with HCD. Plasma DHEA, DHEAS, plasma Estrogen, plasma aldosterone, plasma cortisol, plasma insulin, plasma resistin and plasma 17-hydroxyprogesterone were measured. Data are expressed as mean ± SD. Statistical analyses, two-way ANOVA. #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001, compared with WT mice fed with HCD diet. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with WT mice fed with chow diet.


The research elucidates the stress degradation behavior of Ertugliflozin, which is used for the treatment of type-2 diabetics. The degradation was conducted as per ICH guidelines and Ertugliflozin is relatively stable in thermal, photolytic, neutral, and alkaline hydrolysis conditions; however, considerable degradation was detected in acid hydrolysis and oxidative hydrolysis. Degradation products were identified by ultra-high-performance liquid chromatography-mass spectrometry, isolated by semi-preparative high-performance liquid chromatography, and structural characterization using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Total four degradation products were identified and isolated in acid degradation, which are degradation products 1, 2, 3, and 4. Whereas in oxidative conditions, degradation product 5 was identified. All the five degradation products formed are novel, which was not reported earlier. This is the first time documented complete structural characterization of all five degradation products by using a hyphenated analytical technique. High-resolution mass, and nuclear magnetic resonance spectroscopy were used in the present study to get concrete confirmation of degradation products structures. The current method is also used to identify degradation products with shorter runtime in the future. It’s available as a solitary medicine and combined with sitagliptin and metformin HCl. The available literature in the market reveals some analytical and bioanalytical technique development validation, and stability suggesting investigations are accessible with the drug (Ertugliflozin) and the mixture of sitagliptin and metformin. The medication Ertugliflozin (brand name Steglatro) is used to treat type 2 diabetes. The Food and Drug Administration authorized it for use as a monotherapy and in a fixed dosage combination with either sitagliptin or metformin in the United States4. It was approved for use as a monotherapy or combination treatment in Europe in March 2018. Ertugliflozin belongs to the family of medicines known as gliflozins and is SGLT2 inhibitor. Segluromet is sold in conjunction with metformin, and Steglujan is offered in combination with sitagliptin.In type 2 diabetes medication, Ertugliflozin is used as an inhibitor22H25ClO7 (Mol. Wt.: 436.13) and the chemical name is 5-(4-chloro-3-(4-ethoxybenzyl)phenyl)-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol. Ertugliflozin is in pyroglutamic acid salt form and appears as a white non-hygroscopic crystalline powder. It is soluble in acetone and ethanol, slightly soluble in acetonitrile and ethyl acetate, and only in water sparingly soluble. Figure 11, with sitagliptin18, in combination with both metformin and sitagliptin19. Several bioanalytical strategies utilizing LC–MS/MS approaches have been reported to detect sitagliptin in plasma and Ertugliflozin21. However, until now, there was no literature available to explain the Ertugliflozin degradation products and their characterization. None of the literature provided the NMR, high-resolution mass spectrometry, and IR studies of the degradation products. The present study explains the detailed structural characterization for all 5 degradation products of Ertugliflozin. Hence, the current research was endeavored to provide concrete evidence for the degradation products structures, which involved the chromatographic method development of UHPLC-MS method for ERG and its 5 degradation products, well resolved in 4 min runtime along with HRMS/MS, IR, and NMR  experiments.Ertugliflozin is marketed under the brand name Steglatro. Its molecular formula is CErtugliflozin active pharmaceutical gift sample was obtained from leading pharma organizations in Hyderabad. Reagents and chemicals used for the research were sodium hydroxide, hydrochloric acid, 30% hydrogen peroxide  formic acid (LCMS grade), acetonitrile (HPLC grade), procured from Honeywell Research Chemicals, India. The water used for the analysis was from a milli-Q instrument from Millipore, Amsterdam, Netherlands. Dimethylsulfoxide-d6 (NMR grade) from Cambridge Isotope Laboratories, Inc. D, 99.9% + 0.03% v/v.The analytical instruments and supported software engaged for the present work are UHPLC-MS instrument from Acquity UHPLC frontend with waters single quadrupole detector with and maslynx 4.2 software. HRMS instrument from Thermo with Dionex ultimate 3000 LC frontend q-exactive orbitrap MS with ESI ion source and x-calibur software. Purification instrument from Waters binary module 2545, detector-2489 and autosampler 2707 with chrom scope-2.1 software. NMR instrument from Bruker Avance neo 400 MHz with topspin 4.09 software. Analytical balance from Sartorius-SQP-F, and Infrared spectroscopy instrument from Shimadzu ir-afinity-1S and lab solution software.−1 gas was set for the flow of desolvation and that of cone gas was set at 60 L h−1. The liquid chromatography-mass spectrometer instrument was operated using MassLynx 4.1 application manager. Samples were maintained at 10 °C temperature and were run with shorter chromatographic runtime of 4.0 min and an injection volume of 0.5 µL.Liquid chromatography resolution was achieved on Waters make Single Quadrupole mass Detector (SQD2) attached with Acquity UHPLC & Photo diode array detector (PDA) front-end. Dual polarity- negative and positive with ESI (electrospray ionization) source with waters single quadrupole mass spectrometer was utilized for mass analysis MS optimization was done by using scan mode from 100 to 1200 Daltons (Da). The desolvation temperature was kept at 350 °C. 700 L h−1 and column temperature of 35 °C. A photo diode array (PDA) detector was used to monitor the eluents. The diluent used in the ratio of 1:1 (v/v) mixture of water and acetonitrile. All 6 peaks were separated with good peak shape and resolution in this condition. The final method development trace is shown in Fig. In pursuit of an appropriate, reliable, and reproducible method, different buffers using different columns were screened to optimize the resolution. Initially, the method development was done using trifluoroacetic acid, formic acid, ammonium bicarbonate buffers, and different columns like acuity phenyl, C18, and CSH C18. During copious trials, 0.1% formic acid buffer with acquity BEH C18 showed positive and encouraging results and the remaining trials got either poor resolution or resolution. Further, extensive trials were made on acquity BEH column with various flow rate and gradient conditions. The optimum separation of all degradation products and API was achieved on the Waters Acquity UPLC BEH C18 column  column. The mobile phase included mobile phase-A and B having 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Separation was attained using gradient program Time (min)/B conc (%): 0/3, 0.5/3, 2.5/98, 3.5/98, 3.6/03, 4/03 at a flow rate of 0.4 mL minSamples were analysed using an ESI source equipped with Thermo Q Exactive orbitrap MS; UHPLC Dionex Ultimate 3000 with PDA detector as front-end. Utilized instrument source parameters were Spray Voltage: 3.5 kV; Aux gas flow rate: 14; Capillary Temperature: 270 °C; Sweep gas flow rate: 3; Aux gas heater Temperature: 440 °C. Sheath gas flow rate: 53. Mass data were acquired using xcalibur software. Reserpine (monoisotopic mass: 608.2734 Da) was used as a standard to check the mass analyser accuracy. Chromatographic conditions were like UHPLC-MS. The HRMS data for all degradation products are shown in Fig. Waters semi-preparative HPLC 2489 dual UV detector, 2545 pump module, and 2707 sample manager with auto fraction collector-III. The total instrument was controlled with ChromScope-2.1 software, and in-house packed Luna C18 150 × 25 mm, 5 µm was used to isolate the degradation products. All isolated pure fractions were lyophilized using Lyofreeze lyophilizer.4)2CO3 was utilized, and the resulting solution was solidified using lyophilization. After diluting the peroxide solution, evaporated to get a free solid. For preparative HPLC purification, the identical sample was dissolved in a small amount of the mobile phase.Degradation was seen in acidic and peroxide environments. To neutralize the acid hydrolyzed sample, a saturated solution of  with shim system of Z-gradient with sensitivities of 225:1 and 480:1. TMS (tetramethyl silane) signal at zero ppm was used as a reference for 1H and 39.5 ppm septet signal of DMSO-d6 for 13C.Shimadzu IR-Afinity-1S model with lab solutions software was used to recognize the functional groups like alcohol, ketone and chloro ketone present in the compounds. KBr was used as a dispersion medium to prepare the sample pellets.26, various Forced degradation parameters were employed, i.e., thermal, photolytic oxidation, neutral, acid, and base hydrolytic conditions. Forced degradation study of ERG was performed as mentioned in the ICH guidelines for stability study. The ERG standard solution (10 mg mL−1), One milliliter of the solution was transferred into a 10 mL volumetric flask and diluted up to the mark with 30% hydrogen peroxide solution. The solution was stirred at room temperature. One milliliter of the solution was taken and make up to 10 mL with ACN water (1:1 v/v). To obtain a final concentration of 100 µg mL−1, it was taken for further studies to get the degradation behavior. For photodegradation, 25 mg of ERG sample was exposed to 254 nm UV light for 48 h in UV chamber. For thermal degradation, 25 mg of ERG sample was kept at 100 °C for 48 h and it was used for further studies to observe the degradation behaviour.As per ICH stability guidelines−1) was prepared in ACN and water (1:1 v/v). 1 mL of ERG standard stock solution was transferred to a 10 mL volumetric flask. Then make up to the mark using water for neutral degradation, 1 N NaOH for alkaline degradation and 1 N HCl for acidic degradation . The solution was kept stirring at 60 °C temperature. One milliliter of the solution was taken, neutralized, and transferred to 10 mL volumetric flask. Then the volume was makeup to mark with water to obtain a final concentration of 100 µg mL−1. It was taken for further studies to get the degradation behavior.For neutral, acid and base hydrolysis study, a standard stock solution of ERG  acid  and conditions. Detailed degradation conditions and results are displayed in Table ERG compound is highly stable in neutral, basic hydrolysis, photolytic and thermal, conditions and did not show any degradation, which confirms the stability of ERG under the above conditions. The drug was found to be labile to acid hydrolysis and peroxide conditions. As a result, ~ 15–20% of degradation was observed in both peroxide  promotes the formation of 3. This in turn react with acetonitrile (used as solvent for the solubility purpose) to form DP-1 through Ritter chemistry.The formation of degradation impurities was as follows, for the Formation of DP1, Initial acid mediated electrophilic substitution of protonated formaldehyde to the ortho position as in compound 1 followed by Ritter chemistry with acetonitrile afforded DP1. As per the literature of Journal of Analytical and Applied Pyrolysis, 113 (2015) 621–62928 under Bronsted acidic condition.Formation of DP2, the proposed degradation pathway for the formation of DP2 is based on the literature 30. The formed acetic acid involved in the esterification of the DP2 under the existing hot acidic condition. In a similar way, the free hydroxymethyl group undergoes chlorination via SN2 mechanism. Accordingly, the acid protonates the hydroxyl group and is successively displaced by the chloride to form the corresponding DP4.Formation of DP3/DP4, as per the literature of Journal of Analytical and Applied Pyrolysis, 113 (2015) 621–629, sugar moiety in the target molecule undergoes gradual decomposition to form formaldehyde/acetaldehyde and acetic acid in different proportionsFormation of DP5, Th proposed mechanism for the formation of DP5 is through the oxidative cleavage of aryl ethoxy group to form the corresponding phenol i.e., DP5.Using mass spectrometry (UHPLC-MS), individual samples were analysed to determine the outcomes of all the stress studies. All degradants formed over a time, and from each constraint, study conditions are outlined in the experimental section. Five substantial degradation products were identified, isolated, and characterized by UHPLC-MS, Prep-HPLC, HRMS, 2D-NMR and FTIR techniques. Most of these degradation products are formed by the rearrangement of six-member carbohydrate ring of API into five-member furan ring.The degradation products formed as considerable percentage of > 5% as a result of degradation was observed under peroxide and acid stress conditions. Luna C18 column  and acetonitrile, 0.1% formic acid in aqueous were used as a mobile phase for purification. Consecutive injections of crude sample solutions were injected, and the fractions collection was done based on UV response and later mass confirmed by LC–MS. Collected separately fractions of various degradation products and lyophilized to get a free solid.+ and ammonia adduct [M + H + NH3]+ ions were detected as 437.1350 and 454.1617, respectively, which confirms the molecular formula for Ertugliflozin is C22H25ClO7. The confirmed data of Ertugliflozin is mentioned at HRMS, HRMS/MS spectrum  in HRMS for the molecular formula C25H30ClN5O8 shown in Fig. 1H NMR and D2O exchange experiments. On comparison of 1H and D2O data confirm the presence of five exchangeable protons in the compound and in aromatic region missing of 1,4 disubstituted ring pattern and presence the pattern of 1,2,4 trisubstituted ring. The key difference from API compound is the presence of secondary amide (H32), methylene protons (H31) and acetyl group (H34). In 1H NMR, a total of 30 protons were present, the secondary amide is at 8.08 ppm, and aromatic protons were shown between 6.0 and 8.0 ppm. Primary and secondary alcoholic protons are at 4.5–5.5 ppm, and aliphatic protons resonance between 1.0 and 4.5 ppm. N-methylene protons are showing at 4.15 ppm and acetyl protons are at 1.85 ppm. O-methylene is at 4.00 ppm, and aromatic methylene protons are at 3.97 ppm. In 13C NMR a total of 25 carbons were showing; most downfield carbon is acetamide carbonyl showing at 169.26 ppm, followed by O-ethyl group attached quaternary carbon is at 154.36 ppm. The remaining aromatic carbons were between 100 and 140 ppm, aliphatic carbons were showing at 10–90 ppm. In HSQC experiment confirmed as H31 methylene protons attached carbons is showing at 37.16 ppm, and N-acetyl protons attached carbon is showing at 22.47 ppm. In the gDQ-COSY experiment, H31 protons showed correlation with the H32 proton. In 1H-15N HSQC experiment H32 proton was confirmed as an amidic NH proton, and its nitrogen value is showing at 115 ppm. In 1H-13C HMBC experiment, H31, H32, and H34 protons showed connectivity to C33 carbon, and H31 protons showed connectivity to C9, C10 and C11 carbons. All the key information of 1D and 2D NMR data confirms that 1,4 disubstituted ring converted to benzylacetamide. Finally, it formed N--6,8-dioxabicyclo[3.2.1]octan-5-yl)benzyl)-2-ethoxybenzyl)acetamide and the 2D NMR spectrum was depicted in Figs. The Ertugliflozin treatment with acid resulted in degradation product-1 (DP-1). This degradation product was isolated, and its mass was confirmed as [M + H]−1 in FT-IR spectra specifies the presence of NH and OH groups, and 1645 cm−1 stretching frequency directs the presence of amide keto groups. The stretching frequency at 811 cm−1 leads to the existence of the C–Cl group. A significant FT-IR spectrum established the existence of keto, amide, alcohol, and chloride functional groups, as presented in Table Further, stretching frequency at 3415 cm+ was achieved as a protonated molecule with − 2.0463 ppm error for the calculated molecular formula C21H19ClO4 shown in Fig. 1H NMR data of DP-2 showed extreme differences from API compound. Here we could not see the bicyclic ring protons and noticed the primary alcohol proton at 5.56 ppm as a triplet, it was exchanged in D2O exchange. The methylene protons show as a doublet at 4.51 ppm, furan ring protons showing at 6.59 and 7.28 ppm with J value 3.2Hz. In aromatic region 1,4 disubstituted ring, 1,2,4 trisubstituted pattern protons were present like parent compound at 6–8 ppm, and in aliphatic region O-ethoxy protons, methylene protons were shown between 1.0 and 4.5 ppm region. In 13C NMR, a total of 19 carbons were shown due to symmetry C9–C13 and C10–C12 carbons showing each as one signal. Most downfield carbon is a ketone, and it is resonating at 180.11 ppm. Quaternary carbons of furan ring are showing at 150.15, 161.7 ppm, and O-ethoxy attached aryl carbon is at 157.05 ppm; remaining aryl carbons are showing at 110–140 ppm. In the aliphatic region 4 carbons were showing, key methylene carbon is at 55.94 ppm, by HSQC experiment confirmed the same. Other key information from HSQC experiment is identifying the furan ring protonated carbons. From COSY experiment, primary alcohol proton H26 showed a correlation with H25 methylene protons, and furan ring protons H21, H22 are shown correlation with each other. From g-HMBC experiment confirms H25 protons showing connectivity to C22 and C23 carbons and H1, H3 protons show connectivity to C18 carbon. Based on NMR data concluded that bicyclic ring has been rearranged and formed 2,5 substituted ring furan rings as structure depicted in Figs. The DP-2 compound was formed in acid hydrolysis of the ERG compound. To acquire structural information, DP-2 HRMS analysis was performed and 371.1037 [M + H]−1 in FTIR spectra confirms the presence of OH groups, and 1637 cm−1 stretching frequency specifies the presence of keto groups. The stretching frequency at 756 cm−1 directs the presence of the C–Cl group. FT-IR significant frequencies established the existence of alcohol, keto, and chloro functional groups, as depicted in Table Further, stretching frequency at 3481 cm+ as 413.1139 with − 2.8246 ppm error, which confirms the calculated molecular formula C23H21ClO5 shown in Fig. 1H NMR, a total 21 number of protons are present, with those 9 protons from the aromatic region and furan ring protons shown at 6.79 and 7.32 ppm with J = 3.6Hz. Aliphatic region, a total 12 number of protons were present, in that 3 methylene groups are showing between 3.5 and 5.5 ppm, and acetyl group protons were showing at 2.07 ppm. In 13C NMR number of carbon count is 21, in those 16 carbons from aromatic and 5 carbons from aliphatic region. The most downfield carbon is ketone at 180.22 ppm, followed by O-acetyl is at 169.86 ppm, and O-ethoxy attached aryl carbon at 157.05 ppm. The furan ring quaternary carbons are at 150.92 and 154.92 ppm, protonated carbons are at 122.12 and 112.85 ppm, remaining carbons were shown between 110 and 140 ppm, aliphatic carbons were shown at 10–65 ppm. To identify protonated carbons from total number of carbons, conducted HSQC experiment and it confirms that O-methylene carbons C25 is 57.47 ppm and C15 is 62.86 ppm and furan ring protonated carbons were at 122.12 and 112.85 ppm. In COSY experiment H21, H22 protons show correlations. To check 2J and 3J correlations DP-3 compound HMBC experiment helped a lot, here the key correlations were H25, H28 protons are showing connectivity to C27 carbon, H25 showed connectivity to C22, C23 carbons also and H1, H3 protons showing connectivity to C18 carbon along with these remaining connectivity’s are fitting to the structure. The structure of degradation product-3 was (5-(4-chloro-3-(4-ethoxybenzyl)benzoyl)furan-2-yl)methyl acetate, and the 2D NMR spectrum was depicted in Figs. Acid hydrolysis of ERG compound resulted in the DP-3 compound. To acquire structural information_ of DP-3, HRMS analysis was executed and observed protonated molecule [M + H]−1 stretching frequency indicates the presence of keto groups, and signals at 1349, 1039 cm−1 confirm the presence of C–O groups. No signal in the region of 3400–3000 cm−1 confirms that OH and NH protons are not present in the structure. The stretching frequency at 756 cm−1 directs the presence of the C–Cl group. Significant FT-IR spectrum confirmed the presence of keto, and chloro functional groups, as tabulated in Table Further confirmation done with FT-IR and 1744 and 1645 cm+ 389.0694 with − 2.9529 ppm error for the molecular formula C21H18Cl2O3 shown in Fig. 1H NMR data shows 18 protons, and a minor difference was observed between 1H NMR data of DP-2 and DP-4 i.e., missing of the alcoholic proton, in DP-4, H25 methylene is showing as a singlet at 4.93 ppm. The furan ring protons were shown at 6.82 and 7.31 ppm with J = 3.6Hz. Two strong doublets H9, H13, and H10, H12 are showing at 7.15, 6.86 ppm, H1, H2, and H3 protons are between 7.5 and 7.9 ppm, and aliphatic protons are showing between 1 and 5 ppm. In 13C NMR number of carbon count is 19, due to symmetric nature in 1,4-disubstituted ring instead of 6 observed 4 signals. Most downfield carbon is ketone is resonating at 180.21 ppm, rest of the carbons showing at 110–160 ppm. In aliphatic region four carbons were showing, the key methylene carbon at C25 is at 36.88 ppm, O-ethoxy carbons are at 14.63 and 62.86 ppm, methylene carbon C7 is at 37.37 ppm. By HSQC experiment validated the protonated carbons, furan ring protonated carbon shifts showing at 122.26 and 112.62 ppm. In HMBC experiments observed, two important connectivity’s in that H25 proton showing 2J correlation with C23 carbon and 3J correlation with C22 carbon. The second important connectivity is H1, H3 protons showing connectivity C18 carbon. All important information of NMR data confirms that bicyclic ring has been rearranged and 1,5 substituted furan ring has been formed. As a result, the structure forming (4-chloro-3-(4-ethoxybenzyl)phenyl)(5-(chloromethyl)furan-2-yl)methanone and the 2D NMR spectrum was depicted in Figs. DP-4 resulted in treating ERG with acid. After isolation, HRMS was recorded to know its mass. For structural information of DP-4, HRMS analysis was performed and obtained the protonated molecule [M + H]−1 indicates the presence of the keto group, and signals at 1305 and 1246 cm−1 indicate the presence of C–O groups. The stretching frequency at 841,703 cm−1 directs the presence of the C–Cl group. No single in the region of 3400–3000 cm−1 confirms that OH and NH protons are not present in the structure. FT-IR signals in spectrum conforms the structure includes of Chloro, and keto functional groups, as shown in Table FTIR spectrum indicates the presence of signal 1649 cm+ of 409.1037 with − 2.9086 ppm error for molecular formula C20H21ClO7 shown in Fig. 1H and D2O saying that, in 1H-NMR data showing 21 protons, in D2O exchange experiment 5 protons were exchanged and showing 16 protons. Hence confirmed the presence of five labile protons in the compound. The major difference from DP-5 compound to the parent compound is missing of O-ethoxy group and the presence of phenolic OH proton at 9.24 ppm; remaining 4 labile protons are showing between 4.5 and 5.5 ppm . In aromatic region 1,4 disubstituted ring protons are at 6.66 and 6.97 ppm, chloro substituted ring protons are at 7.1–7.5 ppm, aliphatic methine and methylene protons are showing between 3 and 4 ppm. In 13C NMR 7 carbons were showing in aliphatic region and 11 signals were showing in aromatic region. More de-shielding carbon is phenolic OH located quaternary carbon, remain aromatic signals were shown between 100 and 140 ppm. In aliphatic region O- attached methylene and methine carbon were showing between 55 and 90 ppm, aromatic methylene carbons are at 37.70 ppm. in HSQC experiment confirmed the shifts of methylene and methine carbons. In COSY experiment H24 proton showed connectivity to H19, H23 shows with H20, the H22 proton is with H21 proton, and H28 shows correlation with H25 protons. These connectivity’s are helped to identify the methine and methylene protons. In HMBC experiment, few important 3J correlation are observed H1, H3 and H27 protons are showing connectivity to C16 carbon. Also, H7 protons showing 3J connectivity to C3, C5 and C9, C13; 2J connectivity to C4, C8 carbons. NMR and mass data confirmed that ethoxy group leaved from the parent and formed phenolic OH on 1,4 disubstituted ring. The structure of the DP-5 has been elucidated and the NMR spectrum portrayed in Figs. −1 stretching frequency. Stretching frequency at 755 cm−1 directs the presence of the C–Cl group. No signal in the region of 1750–1600 cm−1 confirms the absence of the keto group in the structure. Central frequencies established the presence of alcohol, Chloride functional groups, and data is tabulated in Table Degradation product-5 was formed by treating the API compound with Hydrogen peroxide. For DP-5 structural information, HRMS analysis was performed and observed the protonated molecule [M + H]The degradation study of Ertugliflozin under stressed conditions was examined following ICH guidelines. The API was subjected to oxidative, acidic, alkaline, neutral, photolytic, and thermolytic degradation conditions. The drug was stable in basic, neutral, thermal, and photolytic conditions, and no degradation products were observed. However, five degradation products were formed in acid and oxidative stress hydrolysis conditions. These degradants are developed because of rearrangement of the ring, which is labile to acid hydrolysis , and DP-5 was formed by removing the ethyl group in the presence of peroxide conditions. All degradation products were separated and characterized entirely using various analytical techniques like NMR (1D and 2D experiments) and HRMS/MS experiments. FT-IR data gave an additional add-on to confirm the structures. All five DPs are novel products and are not reported in any literature. The current study provides the complete structural interpretation of Ertugliflozin and all 5 degradation products using HRMS, FTIR, and 2D-NMR studies. It also reports well developed UHPLC-MS method to separate all the degradation products with good resolution.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.Supplementary Figure S7.Supplementary Legends.


SOST is regulated by mechanical stimuli, cytokines, and all-trans retinoic acid (ATRA) and mediates antianabolic effects on bone formation as an inhibitor of the canonical Wnt/β-catenin pathway. Interestingly, skeletal muscle has recently been identified as another source of sclerostin, suggesting that the musculature may play an important role in maintaining bone mass. However, regulators of muscular SOST expression are virtually unknown. This study investigates the influence of ATRA and the provitamin A derivative beta-carotene (β-C) on sclerostin synthesis in muscle cells. The impact of ATRA, its synthetic analog TTNPB, and β-C on Sost transcription was analyzed by qRT-PCR in C2C12 myotubes and the secreted sclerostin protein by ELISA. ATRA strongly increases the sclerostin synthesis in C2C12 myotubes in a dose-dependent manner. The stimulating effect of ATRA and TTNPB on Sost is largely reduced in the presence of the retinoic acid receptor inhibitor AGN193109. β-C also increases the Sost expression, but this effect vanishes when β-C is coincubated with beta-carotene 15,15′-monooxygenase 1 (BCMO1)-specific siRNA. Thus, ATRA is a potent stimulator of sclerostin release in muscle cells. β-C can also increase Sost mRNA abundance, but this effect depends on the conversion to a retinoid.Sclerostin is a protein secreted by osteocytes whose encoding gene  Skeletal muscles and bones are anatomically and biochemically linked and play critical roles in locomotion and metabolism in humans . The intSOST gene and is mainly synthesized and secreted in bone tissues by osteocytes and osteoblasts benzoic acid; 1–104 nM)  or β-C  for 24 h. In some experiments, C2C12 myotubes were pre-stimulated with or without the pan-RAR antagonist AGN193109 -2-naphthalenyl]ethynyl]benzoic acid; 1 µM; Tocris, Bristol, UK) 3 h before being treated for an additional 24 h with ATRA, TTNPB (both 100 nM), or β-C (400 nM).Murine C2C12 muscle myoblasts  were cultured under proliferative conditions at 37 °C and 5% COBcmo1, 1 × 105 C2C12 myoblasts were seeded per well for 48 h in growth medium. After 48 h of proliferation, the growth medium was removed, and the cells were washed with PBS and then transfected in antibiotic-free differentiation medium using 100 nM ON-TARGETplus Mouse SMARTpool Bcmo1 siRNA (L-057708-01-0010) or 100 nM ON-TARGETplus nontargeting control siRNA (D-001810-10-20) and 7.5 µL of DharmaFECT 1 transfection reagent . After 72 h of Bcmo1 silencing, the siRNA-containing antibiotic-free differentiation medium was renewed. After an additional 24 h of silencing, the C2C12 myotubes were incubated with 400 nM β-C or with vehicle alone in the presence or absence of Bcmo1-specific or nontargeting siRNA for another additional 24 h. In the C2C12 myotubes treated with Bcmo1-specific siRNA, the relative Bcmo1 expression was 1.72 × 10−6 ± 4.5 × 10−7 arbitrary units and 1.70 × 10−5 ± 2.29 × 10−6 in the control cells treated with nontargeting siRNA (p < 0.001). For the silencing of TM Reverse Transcription System . For cDNA amplification, an RT–PCR was performed using a Rotor-Gene Q Cycler  with 2 µL of the synthesized cDNA and the following program: 95 °C for 3 min and 40 cycles of 95 °C for 10 s, 59 °C for 30 s, and 72 °C for 30 s. Amplified RT–PCR products were loaded on a 1.5% agarose gel and visualized by Midori Green . The following primers (5′ → 3′ orientation) were used:Untreated murine C2C12 myotubes were used for the total RNA extraction with TriFast reagent . The cDNA was synthesized at 25 °C for 5 min, 42 °C for 1 h, and 70 °C for 15 min using 1.2 µg of total RNA, random primers, and the GoScriptBcmo1: Mouse F: CCCTCGGATAAATTATGCTTACR: GGACATCATCTTCATCCTTCTM Reverse Transcription System and random primers . For the determination of relative Sost and Gapdh expression, a quantitative reverse-transcription real-time PCR (qRT–PCR) was performed using a Rotor-Gene Q Cycler  and GoTaq qPCR Master Mix  under the following conditions: Sost: 95 °C for 3 min and 40 cycles of 95 °C for 10 s, 59 °C for 30 s, and 72 °C for 30 s; Gapdh: 95 °C for 3 min and 20 cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s. The calculated relative mRNA transcript level of Sost was normalized to the expression level of Gapdh in the same cDNA sample. The quantification of Sost gene expression is presented as 2−ΔCT (ΔCT = CT [target gene] − CT [reference gene]) transformed data [The total RNA was extracted from the murine C2C12 myotubes using a TriFast reagent  according to the manufacturer’s protocol. Next, 1.2 µg of total RNA was used for cDNA synthesis  with the GoScriptmed data . The folSost:Mouse F: TCAGGAATGATGCCACAGR: GTACTCGGACACATCTTTGGapdh:Mouse F: GGTGAAGGTCGGTGTGAACGR: CTCGCTCCTGGAAGATGGTGC2C12 myoblasts were cultured and differentiated into myotubes as described above and treated at day 3 of differentiation with 10 nM ATRA, 100 nM ATRA or vehicle alone for another 24 h. Next, the cell culture supernatant was collected and stored at −80 °C. For the quantification, cell culture supernatants were concentrated using Vivaspin 6 centrifugal concentrators , and the sclerostin protein was subsequently determined using an ELISA kit  according to the manufacturer’s protocol. n represents the number of independent experiments. Data were tested for normal distribution using the Shapiro-Wilk normality test. Comparisons of more than two treatments were tested for significant differences with one-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison  or the Kruskal–Wallis test followed by Dunn’s multiple comparison test for non-normally distributed data. Differences were considered significant at p < 0.05. The data are shown as arithmetic means ± SEM; Sost expression in C2C12 myotubes, differentiated cells were treated with increasing doses of ATRA for 24 h, and the mRNA abundance of Sost was measured by qRT–PCR. As illustrated in Sost mRNA abundance in a dose-dependent manner. To investigate whether Sost gene expression induced by ATRA is associated with increased sclerostin secretion, we analyzed the sclerostin protein concentrations by ELISA in the cell culture supernatant. Strikingly, the sclerostin protein secretion of C2C12 myotubes into the cell culture medium was also increased in a dose-dependent manner after 24 h of treatment with increasing concentrations of ATRA  [Sost gene expression in bone [Sost gene expression following ATRA treatment. Our experiments also investigated the molecular mechanisms through which ATRA exerts its stimulatory effect on sclerostin. Because RAR is the classic transcription factor to which ATRA binds to mediate its genomic effects , it was β, or -γ . The RARed RAR-γ . The reme (AMPK) ,45, whic in bone . TherefoBcmo1, which is consistent with data from muscular cells of nonmurine origin [trans retinal, which is confirmed by the observation that β-C, similar to ATRA, could stimulate Sost expression in C2C12 myotubes via the activation of RAR. Interestingly, β-C does not have an ATRA-independent effect because in the presence of siRNA-mediated Bcmo1 knockdown, the stimulatory effect of β-C on the relative Sost mRNA abundance vanished. This result shows that β-C must first be metabolized to all-trans retinal by Bcmo1 and then metabolized to ATRA to induce stimulation of Sost gene expression. Additionally, our data show that C2C12 myotubes can express e origin . This fiBCMO1 expression, thus preventing excess retinoid and ATRA production via BCMO1 [The vitamin A metabolite ATRA affects bone and muscle tissue homeostasis ,29,30,31ia BCMO1 . Howeveria BCMO1 , it can ia BCMO1 , which iia BCMO1 . Based oSOST expression is higher in patients with multiple myeloma and sarcopenia than in control subjects [Sost.Moreover, whether ATRA-mediated sclerostin formation mediates possible paracrine effects on the surrounding musculature is an open question. Because muscular subjects  and circsubjects , it is tsubjects . Howeversubjects , which cSince it has already been demonstrated that dietary vitamin A intake is related to the concentrations of other circulating myokines, such as myostatin , our datSost gene expression depends on the conversion of β-C to all-trans retinal by Bcmo1. These results provide evidence for the regulatory importance of vitamin A derivatives for muscle–bone crosstalk via the release of sclerostin with antianabolic effects on bone and provide further insight into the complex myokine-mediated homeostasis between muscle and bone.Taken together, this study is the first to show a stimulatory effect of ATRA and β-C on sclerostin synthesis and release in C2C12 myotubes by activating RAR. However, the β-C effect on


Botrytis cinerea causes gray mold disease in leading crop plants. The disease develops only at cool temperatures, but the fungus remains viable in warm climates and can survive periods of extreme heat. We discovered a strong heat priming effect in which the exposure of B. cinerea to moderately high temperatures greatly improves its ability to cope with subsequent, potentially lethal temperature conditions. We showed that priming promotes protein solubility during heat stress and discovered a group of priming-induced serine-type peptidases. Several lines of evidence, including transcriptomics, proteomics, pharmacology, and mutagenesis data, link these peptidases to the B. cinerea priming response, highlighting their important roles in regulating priming-mediated heat adaptation. By imposing a series of sub-lethal temperature pulses that subverted the priming effect, we managed to eliminate the fungus and prevent disease development, demonstrating the potential for developing temperature-based plant protection methods by targeting the fungal heat priming response.Priming is a general and important stress adaptation mechanism. Our work highlights the importance of priming in fungal heat adaptation, reveals novel regulators and aspects of heat adaptation mechanisms, and demonstrates the potential of affecting microorganisms, including pathogens through manipulations of the heat adaptation response. Botrytis cinerea is a notorious plant pathogen that causes gray mold disease, leading to massive crop losses worldwide. Its optimum growth temperature is 18–22°C, and gray mold disease is widespread in relatively cool environments. At temperatures just a few degrees higher than the optimum, the disease completely disappears; however, the fungus remains viable in warm climate and resumes growth and infection when temperatures drop to the optimal range. How B. cinerea copes with high temperatures and remains viable in warm environments, including occasional extreme heat waves, is unclear.Temperature shifts alter the expression of thousands of genes. In budding yeast, the most significantly enriched gene ontology (GO) categories that are upregulated under heat stress include heat shock proteins (HSPs), oligosaccharide metabolism, protein folding, and protein catabolic and recycling processes , 2. ProtExcess heat causes protein misfolding and aggregation, rapidly endangering cell viability. To cope with proteotoxic damage, organisms have evolved a quality control (QC) system that removes misfolded proteins by promoting their refolding using chaperones or by proteolytic degradation through the ubiquitin–proteasome and autophagy systems , 12. WheAlthough the protein QC system is evolutionarily conserved, fungi exhibit fundamental differences in their responses to moderate and severe heat stress. The upregulation of molecular chaperones and certain metabolic processes represents the predominant response of yeast to moderately high temperatures , whereas under severely high temperatures SHTs; 42–46°C), protein aggregates form and translation shuts down –46°C, pr. SimilarB. cinerea, namely, it greatly improved the ability of the fungus to cope with a subsequent potentially lethal heat stress. Using transcription profiling and proteomics analysis of soluble and aggregated proteins, we were able to differentiate the priming from a general heat stress effect and showed that priming improves protein solubility during severe heat stress. Among priming-associated proteins, we identified one serine-type peptidase (STP) to be essential for the priming response.Here, we show that MHT had a strong priming effect on 2O2) and cell wall (SDS) stress generated similar response curves (We characterized spore germination and the growth of germ tubes (GTs) and mycelia at selected temperatures between 22°C and 37°C. Both spore germination and mycelial growth decreased with increasing temperatures above 22°C . Spores e curves , suggestWe produced GTs at 22°C or 29°C, transferred them to 37°C, and monitored cell death by propidium iodide (PI) staining. Transfer from 22°C to 37°C resulted in massive cell death, with ~80% of GTs showing positive PI staining after 6 h and 100% after 12 h at 37°C . When GTPhaseolus vulgaris) plants with spore suspension, incubated them at 22°C or 29°C for 16 h, transferred them to 37°C for 4 h, and then incubated them at 22°C for 3 d. When inoculated plants were incubated at 29°C followed by 37°C, they developed less severe symptoms than plants incubated at 22°C throughout the experiment (control) but more severe symptoms than those incubated at 22°C + 37°C, with average lesion diameters of 2.0 cm, 2.75 cm, and 1.6 cm, respectively  was applied, and 1,329 DGEs were determined, 144 under MHT (29°C), 878 under SHT (22°C + 37°C), and 965 under SHT-P . HeatmapB. cinerea serine-type peptidase genes by heatmap analysis. All 21 genes were upregulated under MHT or SHT-P, whereas most were expressed at low levels under OT and SHT  under the three heat treatments compared to the control (2 (SHT-P/OT)} – {log2 (SHT/OT)} ≥ 1 for proteins more abundant at SHT-P than at SHT and (ii) log2(SHT-P/OT) > 1 and log2(SHT/OT) < 1 for proteins more abundant at SHT-P but not at SHT compared to OT. This analysis yielded 355 proteins  (R = 0.594) (R = 0.477) (2(treatment/OT) < 0 and all proteins with log2(treatment/OT) ≥ 1 and calculated their proportions. Under SHT-P, 11.56% of all gene–protein pairs had negative correlations   and SHT = 0.594)  and some= 0.477) . We alsoelations , a much R = 0.04) for the 355 priming-specific candidate proteins. In 54.6% . To evaluate priming responses, we produced GTs at 29°C, transferred them to 37°C, and stained them with PI. Compared to the wild type, strain ∆ll death . Similar to 37°C .bcin_06 g00620, bcin_06 g00330, bcin_15 g04670, and bcin_15 g03150). To determine whether the lack of a priming phenotype in a single-deletion strain of these genes resulted from functional redundancy, we generated the strain Δ4stp, with deletions of all four S53 genes. Similar to the single-deletion strain, the Δ4stp strain did not show developmental defects and had a normal priming response (not shown). Hence, the role of these four S53 genes in priming remains unverified.Four of the STPs belong to peptidase group S53 . To further examine the physiological relevance of the priming defects, we examined the effect of priming on biomass production of the ∆bcprm1 strain. We produced uniform wild-type and ∆bcprm1 cultures in malt medium at 22°C, transferred them to 29°C for 12 h to induce priming, and measured the biomass. We then incubated cultures for 12 h at 37°C, followed by 22°C for 24 h, and measured their biomass once again  or suspended in PDB at 22°C under continuous fluorescent light supplemented with near UV light. Additional media that were used for specific experiments included malt medium  and GB5-Glc medium .5 /mL. A 20-µL droplet of spore suspension was placed on a coverslip and incubated under continuous light at the specified temperature and time. After incubation, the slides were visualized under a light microscope and germination rates were scored. Each experiment was repeated at least four times, with >200 randomly selected spores each time.Fungi were cultured on PDA for 7 d. Spores were collected by washing with PDB and filtered through two layers of Miracloth, and the spore density was adjusted to 5 × 10Cultures were initiated from 4 mm plugs that were cut from the edge of a 2-d-old colony. The plugs were placed in the center of a Petri dish containing PDA and incubated at the indicated temperatures under continuous light. Colony diameter was recorded after 3 d, the diameter of the initial inoculation plug (4 mm) was subtracted, and radial growth was calculated. Each experiment was performed with three replications (three plates per treatment) and was repeated three times.4 /mL) was placed on a coverslip and incubated under the indicated conditions. After incubation, the slides were visualized under a light microscope and GT length was recorded. Each experiment was repeated three times, with more than 120 randomly selected GTs each time.A 20-µL droplet of spore suspension (5 × 102) PDA cube and incubated at 22°C for 72 h. The number of germinated and un-germinated spores was scored, and germination rate was calculated. Each experiment was performed at least three times with >200 spores per treatment.Fungi were cultured on PDA for 7 d to allow mycelia and spores to develop. The plates were transferred to 37°C or 42°C for the indicated time. After heat treatment, spores were collected into PDB, and spore density was adjusted to 200 spores/mL. To determine germination rates, a 5-µL spore suspension was placed on a small (1 cm5 /mL PBD) were placed on a coverslip, incubated at 22°C or 29°C for 12 h under continuous light, and transferred to 37°C. Following incubation at 37°C, the GTs were stained with 10 µg/mL Propidium iodide  for 15 min. Samples were visualized under a Zeiss Axio imager M1 fluorescence microscope  using a rhodamine filter. The number of dead cells (PI-positive) was counted, and the proportion of dead cells was calculated. Each experiment was repeated at least three times with more than 120 randomly selected GTs each time.Spores (2 × 10GTs were suspended in PBS containing 100 µM DiBAC4(5) , incubated for 15 min at room temperature, and visualized under a Zeiss Axio imager M1 fluorescence microscope  using a rhodamine filter. The fluorescence intensity of each germ tube was quantified using ImageJ, and the mean signal intensity of each treatment was calculated. The experiment was repeated four times with at least 50 randomly selected GTs each time.5 /mL) in the center of a Petri dish containing PDA. The plates were incubated under continuous light at 22 or 29°C for 12 h and then at 37°C for 6 or 9 h. After heat treatment, the cultures were incubated at 22°C for 3 d, and the colony diameter was measured. Each experiment was repeated six times with five replications per treatment.Colonies were initiated by placing a 5-µL droplet of spore suspension (5 × 105 /mL) were incubated in liquid GB5-Glc on a coverslip at 22°C for 6 h, transferred to 42°C for 2 h (heat treatment), and incubated at 22°C for 22 h (recovery). The cycle was repeated three times, and the levels of cell death were determined after each cycle by PI staining. The experiments were repeated at least three times with >300 randomly selected GTs each time.Spores (2 × 10Phaseolus vulgaris L. genotype N9059) plants as described previously  per treatment.To evaluate the effects of high-/low-temperature cycles on pathogenicity, inoculated plants were first incubated at 22°C for 6 h to allow spores to germinate and produce GTs and then transferred to 42°C for 2 h, followed by 22°C for 22 h. This cycle was repeated three times. The plants were then incubated at 22°C for three more days. The experiments were repeated five times with three plants (two leaves per plant) per treatment.N-Amp Tissue PCR Kits . For cDNA synthesis, total RNA was treated with DNase I  and first-strand cDNA was synthesized from 1 µg of DNA-free RNA using a RevertAid First Strand cDNA Synthesis Kit . qRT-PCR was performed with SYBR Premix Ex Taq II  using a StepOne Real-Time PCR instrument . Relative fold changes of mRNA levels determined by RNA-seq were normalized to the expression of the ribosomal protein gene bcrsm18, which has relatively stable mRNA expression.Genomic DNA extraction was performed using Extract-B. cinerea cultures were produced by spreading 500 µL of spore suspension (107 /mL) on a cellophane-covered PDA plate. Treatments . RNA and cDNA quality and quantity were evaluated by performing a Qubit assay with the TapeStation 4200 system . Sequencing libraries were constructed with barcodes using NEBNext multiplex oligos for Illumina . Pooled libraries of 16 samples were sequenced at the Faculty of Life Sciences, Tel Aviv University, Israel, in one lane of the Illumina NextSeq 500 platform  using the 75 bp single-end RNA-seq protocol.B. cinerea reference genome  using STAR-2.7.3a. Over 97% of the reads in each sample were mapped to the B. cinerea genome  , and 3′-end low-quality bases (<Phred 20) were trimmed. An average of more than 24 million reads per sample was obtained, with high quality (Phred score > 34). The number of reads after QC and trimming ranged from 19,604,233 to 29,043,828 . The clea genome , and 11,reatment . RT-PCR reatment . Quantifidb/app) .g for 6 min at 4°C using a tabletop centrifuge, and each supernatant was transferred to a new 1.5-mL Eppendorf tube. The samples were centrifuged at 5,000 × g for 5 min at 4°C to precipitate remnants of cellular debris, and the supernatant was transferred to a new tube. A sample of the protein extract was measured in a spectrophotometer, and the amount of total protein in each sample was calculated using a 1-mg/mL standard sample of bovine serum albumin . To separate the soluble and aggregated proteins, extracted proteins were centrifuged at 16,000 × g for 20 min at 4°C, and the supernatant was carefully removed, transferred to a new tube, and stored at –80°C as the soluble protein fraction. The pellet was washed twice with 300-µL sodium phosphate buffer  with centrifugation at 16,000 × g for 20 min between washes. The supernatant was removed, and the clean pellet was stored at –80°C as the protein aggregate fraction.To isolate soluble and aggregated proteins, fungal material was produced as described for the RNA-seq experiment, except that the initial incubation at 22°C or 29°C was performed for 14 h instead 8 h to allow the accumulation of sufficient biomass. Soluble and aggregated proteins were extracted from the samples according to Koplin and Brennan , 48 withFor soluble proteins, samples were mixed with protein loading buffer and boiled for 5 min. For protein aggregates, the protein pellet was dissolved in 20 µL of 8-M urea, mixed with protein loading buffer, and boiled for 5 min. The amounts of proteins were normalized to represent an equal amount of total proteins in all samples. The protein samples were separated by SDS-PAGE and stained with Coomassie brilliant blue.18 tips , dried, and resuspended in 0.1% formic acid. The samples were analyzed by LC-MS/MS using a Q Exactive Plus mass spectrometer  fitted with a capillary high-performance liquid chromatograph . The peptides were loaded onto a homemade capillary column  packed with Reprosil C18 Aqua  in solvent A (0.1% formic acid in water). The peptide mixture was resolved with a linear gradient (5%–28%) of solvent B (95% acetonitrile with 0.1% formic acid) for 105 min, followed by a 15 min gradient of 28%–95% and 15 min at 95% acetonitrile with 0.1% formic acid in water at a flow rate of 0.15 µL/min. Mass spectrometry was performed in positive mode  using a repetitively full MS scan, followed by collision-induced dissociation  of the 10 most dominant ions (>1 charges) selected from the first MS scan. The AGC settings were 3 × 106 for the full MS scan and 1 × 105 for the MS/MS scans. The intensity threshold for triggering MS/MS analysis was 1 × 104. A dynamic exclusion list was enabled with an exclusion duration of 20 s.Proteomic analysis was performed at the Smoler Proteomics Center, the Technion, Israel, as previously described . SamplesThe mass spectrometry data from all three replications were analyzed using MaxQuant software v.1.5.2.8 . for pea5 /mL) was placed on a coverslip and incubated at 29°C for 8 h. The suspension medium was carefully removed and replaced with fresh PDB supplemented with 2 × protease inhibitor , 0.5 mM or 2 mM PMSF , or an equal volume of PDB (control treatment). The samples were incubated at 29°C or 37°C for 4 h, the GTs were stained with PI, and the death rate was calculated. Each experiment was repeated at least three times with more than 150 randomly selected GTs per sample.A 20-µL droplet of spore suspension (10http://grna.ctegd.uga.edu/). To assemble a single sgRNA duplex (33 µM), 0.2 nm each of Alt-R CRISPR-Cas9 crRNA (2 µL) and Alt-R CRISPR-Cas9 tracrRNA (2 µL) were mixed with Nuclease-Free Duplex Buffer (2 µL). The mixture was incubated for 5 min at 95°C and allowed to cool at room temperature. To assemble a Cas9/sgRNA ribonucleoprotein (RNP) complex, 3 µg Cas9 (3 µL), 33 µM sgRNA duplex (2 µL), and 5 µL of Cas9 working buffer were mixed and incubated at 37°C for 30 min.Deletion strains were generated using a marker-free CRISPR-Cas9 genome-editing method  with twoB. cinerea was performed according to Leisen et al. . In all graphs, results represent the mean values of at least three independent experiments, each with at least three replications per treatment. Details of statistical analyses are presented in the figure legends, and figures were generated using Prism software (GraphPad 8.0).The statistical significance between means of treatments was evaluated by Student’s


HMOX1, CAT, and GPX1. All PS-MPs decreased the intracellular H2O2 levels, which can be attributed to mitochondrial stress responses, such as increased mitochondrial DNA content, footprint, and morphology. Altogether, we demonstrated uptakes and changes in redox and mitochondrial parameters for all PS-MPs, with the 200 nm spheres showing the most profound effects. This suggests that the induction of defensive responses in Caco-2 cells mainly correlates with the number of particles taken up.Currently, we lack crucial knowledge on how the physicochemical properties of particles affect cellular health, resulting in an important gap in our understanding of the human toxicity of microplastics (MPs). Our aim was to evaluate the impact of the size and the shape of MPs on uptake and the intracellular effects in a human epithelial colorectal adenocarcinoma (Caco-2) cell line. Spherical (200 nm and 2 µm) and fibre-/fragment-shaped (8.9 ± 10.1 µm by 1.14 ± 0.97 µm) polystyrene microplastics (PS-MPs) were used to study their uptake and the potential to induce redox and mitochondrial stress responses after 24 h of exposure. We demonstrated the cellular uptake of both spherical and fibre-/fragment-shaped MPs in a size-dependent manner. In response to 2 µm spheres, we observed differential expressions of redox-related genes, including  Plastics are ubiquitous due to their ease of use, mechanical properties, and low cost . The annMicroplastics (MPs) are commonly defined as plastic particles smaller than 5 mm originating from a variety of sources including cosmetics, clothing, and industrial processes. MPs are classified as either primary MPs, which are intentionally manufactured for industrial or household applications including facial scrubs and toothpaste , or secoWhen MPs are deposited in the environment, they can be ingested by marine organisms from different trophic levels, entering our food web. In addition to seafood and drinking water, MPs have also been found in food products and beverages, including honey and beer ,12. IngeAlthough recent studies have discovered MPs in human blood  and in pThe variability of MPs’ physicochemical properties and the associated complexity in the interaction with the human biological systems require an in-depth investigation at the fundamental level into carefully chosen model systems in order to elucidate MPs’ fate . A humanw/v for the fluorescent particles and 10% w/v for the non-fluorescent particles. The particle information of the manufacturer is provided in Commercially available red fluorescent and non-fluorescent polystyrene beads, functionalized with carboxylic surface groups (-COOH), were purchased from Magsphere Inc. . In total, 2 particle diameters were used in this study: 200 nm and 2 µm. The material was supplied by the manufacturer in an aqueous suspension of 2.5% g at room temperature (22 °C) and a relative humidity of 40%. The fibres were then captured between collector poles 12 cm away from the nozzle tips, collected, and stored in a sealed glass container at room temperature.For the synthesis of PS microfibres and fragments (PSMFs), polystyrene granules (Mw = 190 kg/mol) and tetrahydrofuran  were purchased from VWR Chemicals  and used without further purification. The polymer solutions were prepared by slowly adding the polymer to the solvent while continuously magnetically stirring for 24 h in hermetically sealed vials to ensure a homogeneous mixture. A stock suspension of 12.5 wt% was prepared. The polystyrene fibres were then synthesised using a home-built centrifugal spinning setup  . BrieflyThereafter, the fibres were ground twice in a small amount of liquid nitrogen using a mortar and pestle, collected in MilliQ with 0.01% Tween 80  and passed through a nylon mesh filter  using vacuum filtration. The supernatant was then filtered through a polycarbonate filter (10 µm pore size) . After drying the PSMF at 65 °C, a final mass of 5 mg was obtained. Then, half of the PSMF was stained with iDye Poly Pink , while the rest was suspended in phosphate-buffered saline . For the staining, dry PSMFs (2.5 mg of PSMF per mL) were added to 100 mg/mL of iDye Poly Pink solution. This solution was heated at 70 °C for 2 h in the dark. Next, the stained PSMF were poured through a polycarbonate filter (10 µm pore size) using vacuum filtration and rinsed with PBS. Thereafter, they were dried at 65 °C in the dark, weighed, and suspended in PBS. After 20 min of gamma-irradiation at 30 Gy, all stock suspensions of PSMF were stored at 4 °C. To avoid external contamination, all procedures were performed in a fume hood without the use of plastic consumables. Based on the limited mass of PSMFs available, a selection of cellular and molecular parameters was measured.For the commercially available microbeads, transmission electron microscopy (TEM) samples were prepared by placing the pioloform-coated 150 mesh copper grid on a drop of the particle suspension (100 µg/mL) for 10 min. The grids were pre-treated with alcian blue to render a positive charge. After incubation, the TEM grid was removed from the droplet and dried with filter paper from below to avoid a loss of the particulate material. The particle size distribution was measured by quantitative TEM analysis . BrieflyThe PSMFs were characterised using a Nikon Eclipse Ts2 stereomicroscope . A set of ten micrographs were randomly recorded in brightfield mode, equipped with a Nikon DS-Fi3 camera operating at 40× magnification. Analyses were performed for at least 350 particles using ImageJ 2.9.0/1.53t software to determine the particle diameter and length.® HTB-37TM, LGC Standards, Wesel, Germany) were cultured and maintained in 75 cm2 cell culture flasks  containing Dulbecco’s Modified Eagle Medium , supplemented with 10% heat inactivated foetal bovine serum , 1% penicillin/streptomycin , and 1% non-essential amino acids . The cells were cultivated at 37 °C and 5% CO2, and the medium was refreshed twice a week. For the experiments, cells with passage numbers between 30 and 40 were used to establish a confluent Caco-2 monolayer . The exposure conditions selected for the experiments  were based on preliminary concentration range-finding experiments (0 to 500 µg/mL of PS-MPs) and on the ability to compare the findings with literature references.Adherent human epithelial colorectal adenocarcinoma cells  in DMEM without FBS. After detaching with 0.05% trypsin , the cells were washed twice in FACS buffer [PBS  + 2% FBS]. The cells were collected by centrifugation at 300× g for 5 min between each step. Afterwards, the cells were fixed in 4% paraformaldehyde  for 10 min at RT, and washed again in FACS buffer. A total of 10,000 gated cells were measured with FACS. The percentage of cells associated with fluorescent PS-MPs was determined by plotting the fluorescence (FL2-H) versus the cell number. The percentages were corrected for auto-fluorescence by subtracting the control values.The association (=attachment and/or uptake) of MPs was quantitatively assessed with fluorescence-activated cell sorting . The cells were seeded in a 24-well plate at a density of 3 × 104 cells/24-well on ethanol-sterilized coverslips and cultured for 5 days to generate a confluent Caco-2 monolayer. After exposure to 100 µg/mL PS200RF, PS02RF, and PSMF (stained with Idye Pink) for 24 h in DMEM without FBS, the cells were washed twice with the PBS   and stained with 25 µM CellMask Green Plasma Membrane Stain  in phenol-free DMEM medium  for 40 min at 37 °C and 5% CO2. Next, the cells were washed twice in PBS  and fixed in 4% formaldehyde  for 10 min at room temperature (RT). After washing 3 times with PBS , the cells were counterstained with 1 µM Hoechst 33342  for 10 min at RT. Finally, after three washings in PBS , the coverslip with cells was mounted onto a glass microslide using Shandon Immu-mount  and allowed to dry at 4 °C for 24 h. To distinguish between PS-MPs attached to the cell surface and PS-MPs taken up by cells, the XYZ acquisition mode was used . For each condition, 3 z-stack images were scanned manually at 40× magnification for the presence of PS-MPs inside the cells (Ex/Em: 505–545/560–630). A semi-quantitative analysis was carried out by manually selecting images that included the entire thickness of the cell layer but did not include the particles attached to the cell boundary. Then, the number of PS-MPs within the entire cell layer, as well as the total number of cells in the scanned image, were counted. The results were presented as the number of particles per ten cells. The surface covered by PS-MPs inside the cells was calculated by the following formula: [(number of PS-MPs inside cells of the scanned image × 4πr2)/(number of cells within scanned image)]. The results were presented as the surface (nm2) PS-MPs per 10 cells.Immunofluorescence stains were used to determine the qualitative uptake of the fluorescent MPs using confocal laser scanning microscopy , equipped with high power diode lasers at 405, 488, 514, and 633 nm, and mounted on an inverted laser-scanning microscope Axio Observer Z1/7, Zeiss, Zaventem, Belgium). Briefly, the cells were seeded at a density of 3 × 10® 30–1010 K, LGC Standards, Wesel, Germany. Briefly, 6 × 104 Caco-2 cells were seeded in a 96-well clear culture plate and grown in DMEM medium with FBS to a confluent monolayer. Next, the monolayer was exposed to 0, 10, and 100 µg/mL of PS200RF, PS02RF, and PSMF for 24 h in DMEM medium without FBS at 37 °C and 5% CO2. After exposure, 10 µL of MTT reagent was added to the suspension and incubated at 37 °C and 5% CO2 for 2–4 h. The incubation time was dependent on the presence of an intracellular punctate purple precipitate, which was visualized periodically using a Nikon Eclipse Ts2 stereomicroscope . Then, 100 µL of detergent reagent was added and the plate was left at room temperature overnight. Thereafter, the absorbance at 570 nm was measured using a microplate reader .Several assays were used to assess the effects of PS-MPs on the cell viability. First, the metabolic activity was measured using the 3--2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT) cell proliferation assay . Briefly, Caco-2 cells were seeded at a density of 6 × 104 cells in a 24-well culture plate and cultured for 5 days to obtain a confluent monolayer. The cells were exposed to 0, 10, and 100 µg/mL PS200RF, PS02RF, and PSMF for 24 h in DMEM medium without FBS. Next, 45 min before the end of exposure, 100 µL of 10× CyQUANTTM lysis buffer or MilliQ was added to the control cells to serve as a control of the maximum lactate dehydrogenase (LDH) activity and a control of the spontaneous LDH activity, respectively. After exposure, 50 µL of the cell medium was transferred to a clear 96-well plate. After adding a reaction mixture for 30 min at RT in the dark, the stop solution was added to stop the reaction and the absorbance of LDH release was measured using a microplate reader  at 490 nm and 680 nm absorbance. The following formula was used to calculate the percentage cytotoxicity: [(Particle-treated LDH activity – Spontaneous LDH activity)/(Maximum LDH activity – Spontaneous LDH activity)] × 100. Second, the membrane integrity was assessed using the CyQUANT3 cells/96-well culture plate in DMEM medium with FBS and monitored until a confluent monolayer was reached. After 24 h of exposure in DMEM with FBS, the cells were washed with PBS and incubated with 0.1% 5-CFDA-AM for 30 min . Finally, the fluorescence intensity (Ex: 485/20 nm/Em: 528/20 nm) was measured using a microplate reader . Third, the 5-carboxyfluorescein diacetate—acetoxymethyl ester (5-CFDA-AM) fluorometric assay  was used to measure a combination of the esterase enzymatic activity and the membrane integrity in a Caco-2 confluent monolayer after exposure to 0, 10, and 100 µg/mL PS200NF and PS02NF. Briefly, Caco-2 cells were seeded at 5 × 10For all cell viability assays, the possible optical interference was checked by assessing the absorbance/fluorescence of PS200RF, PS200NF, PS02RF, PS02NF, and PSMF in the same experimental setups without cells. If optical interference was observed, the values obtained were used as blanks.4 cells/24-well and grown to a confluent monolayer. After exposure to 0 and 100 µg/mL PS200RF, PS02RF, and PSMF for 24 h in DMEM medium without FBS, the cells were washed in PBS, trypsinised, resuspended in DMEM medium with FBS and pelleted by centrifugation (300× g). After removing the supernatant, the cells were immediately frozen in liquid nitrogen and stored at −80 °C for further processing. The total RNA was isolated from the cells by phenol-chloroform extraction. The purity  and the concentration of the sample (absorbance at 260 nm) were determined using Nanodrop spectrophotometer . After TurboDNase treatment , RNA was converted to cDNA using the Superscript III First-Strand Synthesis Supermix  according to the manufacturer’s instructions. The gene expression was measured using the 384-well format of the QuantStudio 5 RT-qPCR system . A mastermix (7.5 µL) containing Fast SYBR Green PCR Master Mix , 0.3 mM forward and 0.3 mM reverse primer, and RNAse free water, together with 2.5 µL of 10 ng/µL cDNA, were added to each well. The following program was initiated on the Quantstudio 5 RT-qPCR system : 95 °C for 20 s, 40 cycles of 95 °C for 1 s, and 60 °C for 20 s. The 2–∆∆Ct method, normalized for three reference genes  levels by several assays, i.e., the Peroxy-orange 1 (PO-1) fluorescent probe  and the OxiselectTM Hydrogen Peroxide/Peroxidase Assay Kit .Because the change in the redox balance is a well-known phenomenon associated with particle exposure, we determined the intracellular hydrogen peroxide , grown in DMEM medium with FBS for 4–5 days at 5% CO2 and 37 °C until they reached 90–100% confluence. After 24 h of exposure to 0, 10, and 100 µg/mL of PS200NF and PS02NF, the cells were washed twice with PBS  and incubated for 40 min with 5 µM PO-1 staining solution at 37 °C and 5% CO2. Next, 20 min before the end, 300 µM H2O2  was added to a well to serve as a positive control. After incubation, the cells were rinsed twice with prewarmed PBS and immediately imaged with CLSM  at 10× magnification and Ex/Em: 543/545–750. Two tile scans (1.79 mm × 1.79 mm) were recorded for each condition with the ZEN blue 3.1 software . The mean fluorescence intensity (MFI) ± standard error to the mean (SEM) was calculated by the random selection of 5 circles (circle diameter: 504.2 µm) on each image. In addition, the cells were also visualised using the same experimental setup without staining solution in order to detect possible autofluorescence , the cells were exposed to 0, 10, and 100 µg/mL of PS200NF, PS02NF, and PSMF for 24 h in DMEM medium without FBS at 37 °C and 5% CO2. After exposure, the cells were washed twice with PBS , trypsinised, resuspended in DMEM medium with FBS, and centrifuged at 300× g for 5 min. The pellet was resuspended in 70 µL of 1× Assay buffer and 3–5 glass beads were added to disrupt it for 1 min at 30 Hz . Subsequently, 50 µL of each sample and standard were transferred to a black 96-well microplate , followed by the addition of 50 µL of the OxiselectTM acetyl-3, 7-dihydroxyphenoxazine/horseradish peroxidase (ADHP/HRP) working solution to each well. The fluorescence intensity was measured using a microplate reader (Ex/Em: 544 nm/590 nm) . To normalise the data, the total protein content was determined using the Bio-Rad protein assay  according to the manufacturer’s instructions.The Oxiselect4 cells/24-well and grown to a confluent monolayer (5 days). After exposure to 0, 10, and 100 µg/mL PS200RF, PS02RF, and PSMF for 24 h in DMEM medium without FBS, the cells were washed in PBS, detached with 0.05% trypsin, and pelleted by centrifugation . After removing the supernatant, the cells were immediately frozen in liquid nitrogen and stored at −80 °C. DNA was isolated from the cells using phenol-chloroform extraction. The purity and concentration of the sample were determined using Nanodrop spectrophotometer . To ensure a consistent DNA input of 5 ng for each RT-qPCR reaction, the samples were diluted and checked using the Quant-iT™ PicoGreen® dsDNA Assay Kit . The relative mtDNAc was measured in triplicate using a previously described quantitative real-time PCR assay with minor modifications [–∆∆Ct method. MtDNAc was expressed as the ratio of the mtDNA copy number to single-copy gene number (M/S) relative to the control.As the mitochondrial DNA fluctuates in response to the cellular environment, we determined the mitochondrial DNA content (mtDNAc) using RT-qPCR. Caco-2 cells were seeded at 6 × 10ications . All rea2+, +Mg2+, 37 °C), 300 µL of MitoSOX Red stain (2.5 µM diluted in prewarmed PBS) was applied for 10 min . After rinsing twice with PBS, live imaging was performed using CLSM  at 40× magnification and Ex/Em: 510/580. For each condition, 2 tile scans (300 µm × 600 µm) were recorded using the ZEN blue 3.1 software . The MFI ± SEM was calculated by randomly selecting 5 circles (circle diameter = 100 µm) for each image. The MFI shows mitochondrial superoxide production because the dye selectively targets the mitochondria in living cells, and it is rapidly oxidized by superoxide and not by other reactive oxygen or nitrogen species. In addition, both non-fluorescent particles and cells were also visualised using the same experimental setup without the staining solution to detect possible autofluorescence  was loaded onto a confluent Caco-2 monolayer grown in an 8-well IBIDI chamber that was treated with 0, 10m and 100 µg/mL PS200NF for 24 h. After removing the culture medium and washing twice with PBS . Then, 300 µL of MitoTracker Red CMXRos , diluted in prewarmed PBS, was incubated on the cells in the incubator  for 45 min, followed by 2 washes in prewarmed PBS. Live imaging was immediately performed using CLSM  at 40× magnification and Ex/Em: 579/599. For each condition, a tile scan (300 µm × 600 µm) was recorded using the ZEN blue 3.1 software . Based on the binary morphological skeleton obtained in ImageJ using the MiNA tool as described by Valente et al. (2017) [Because exposure to environmental stressors might affect mitochondrial functioning, we assessed the mitochondrial network morphology. After 24 h of exposure of the Caco-2 monolayer to 0, 10, and 100 µg/mL PS200NF in 8-well IBIDI chambers, the culture medium was removed and washed twice with prewarmed PBS (+Ca. (2017) , the mit® Pro 16  software. The normality and homoscedasticity were tested with the Shapiro-Wilk and Bartlett’s tests, respectively. Normally distributed data were statistically tested with a one-way analysis of the variance (ANOVA) with Tukey’s or Dunnett’s post hoc tests to assess significant differences between the control and treatment groups. If the assumptions of normality and homoscedasticity were not satisfied after log or square root transformations, a non-parametric multiple comparison procedure using the Dunn method was performed. A p-value < 0.05 was considered statistically significant. For the gene expression data, agglomerative hierarchical clustering  was performed with the free PAST software  using the UPGMA method computed with Euclidean distances. The cophenetic correlation coefficient was calculated to assess the significance of each dendrogram developed.All cellular assays were performed in triplicate and repeated twice in independent experiments unless stated otherwise. The results are presented as the mean ± standard deviation (SD) or the standard error of the mean (SEM) relative to the control. Gene expression was carried out in 3 independent experiments with 6 biological replicates and the results were presented as the mean ± SEM relative to the control. All statistical analyses were performed using the JMPImage analysis of the TEM images showed a minimum Feret diameter ± SD of 0.180 µm ± 0.017 µm for the PS200RF and 2.073 µm ± 0.132 µm for the PS02RF, indicating a spherical shape A–D. TheiSmall changes in metabolic activity were observed after 24 h of exposure to all PS-MPs A–C. BaseThe membrane integrity of the Caco-2 monolayer was affected by two micron PS spheres. Based on the LDH assay, PS02RF (10 µg/mL) caused 3.27 ± 0.88% more membrane damage compared to the control but optical particle interference was observed that attenuated the effect B. For 5-n = 11) diameter of 2.52 ± 1.34 µm and length of 15.66 ± 9.96 µm were (partly) taken up (2.81 ± 1.21 particles/10 cells) (6 ± 15.6 × 106 nm2/10 cells versus 140.3 × 106 ± 31.4 × 106 nm2/10 cells)  C. A semi0 cells) D. It sho0 cells) D.HMOX1 (Fold Change (FC): 2.09 ± 0.32, p = 0.0042), CAT , and GPX1 . Following PS200RF exposure, only NRF2 was differentially expressed , while PSMF exposure did not alter the gene expression significantly  and 44 ± 4% (p < 0.0001) lower H2O2 levels (based on PO-1 staining) were observed for the 10 and 100 µg/mL PS200NF, respectively, as well as for the 10 µg/mL PS02RF   and PSMF exposure  compared to the control  compared to the unexposed cells A. Signifficantly A. Hierarficantly , while fol group . We furted cells B–D, with = 0.02) B,C. For  control D. p = 0.005) for 10 µg/mL exposure   and the mean number of mitochondrial network branches  were more present after exposure to PS200NF compared to the controls (p = 0.02). Finally, the mitochondrial superoxide levels were also significantly increased after exposure to PS200NF  (MtDNAc was higher compared to the control after exposure to PS200RF with a significant fold change difference of 2.89 ± 0.51 (exposure A. No cha = 0.07) B. In addcontrols C. For PS 0.0001) A, but th 0.0001) B.Microplastics (MPs) are a major concern for both the (marine) environment and human health. MPs were found in human stool , blood , and pla10 particles/mL) compared to the PS 2 µm spheres (100 µg/mL: 2.26 × 107 particles/mL). Another explanation for the differences in particle uptake is the different uptake mechanisms that are involved in the uptake of sub-micron and micron-sized PS spheres. A recent review by Manzanares and Cena (2020) emphasised that, although other pathways can be involved, receptor-mediated endocytosis is the most prominent uptake mechanism of sub-micron particles with an upper size limit of 150–200 nm [Our study found that spherical PS-MPs of 200 nm and 2 µm, as well as a combination of fibre- and fragment-shaped PS-MPs, were taken up by Caco-2 cells. Most previous studies investigated the uptake of mainly spherical micro- and nanoplastics, as shown in a recent review . For a w0–200 nm . The upt0–200 nm . Apart f0–200 nm , while S0–200 nm . These s0–200 nm ,38.7 particles/mL) [Although higher uptake was observed for the PS 200 nm spheres compared to the PS 2 µm spheres and PSMFs, subcytotoxic effects in metabolic activity and membrane integrity were mainly found for PS 2 µm spheres and PSMFs. Based on the MTT assay, we did not detect changes in the metabolic activity of PS 200 nm and 2 µm spheres in Caco-2 cells after 24 h of exposure in medium without FBS (cell viability > 90% compared to controls), but mild effects were observed for PSMF, especially at exposure concentrations higher than 100 µg/mL (500 µg/mL: ± 70% compared to control condition). Based on the LDH and 5-CFDA AM assays, the membrane integrity was mainly affected by the PS 2 µm spheres; however, the cell viability was still >70% compared to the unexposed cells. Contradictory findings were reported regarding the cytotoxicity of MPs ,41,42,43cles/mL) , and Chocles/mL) . We shoucles/mL) ,48,49. THMOX1, CAT, and GPX1 was observed after exposure to PS 2 µm spheres, suggesting an important role of the heme oxygenase 1 (HO-1) system. Dong et al. (2020) [2) compared to low-dose concentrations (10 µg/cm2). The authors postulated that high exposure resulted in massive ROS generation that overcame the cellular anti-oxidative enzyme capacity, leading to oxidative stress, while low exposure resulted in a compensatory increase in HO-1 as an attempt to maintain the redox balance [2O2 to H2O in the mitochondria serve as the first line of antioxidant defence against ROS [2O2 levels were found after exposure to the lowest concentration of PS 2 µm spheres, but this observation was assay dependent. Overall, the altered intracellular redox balance could explain the subcytotoxic changes observed at the cellular level following PS 2 µm sphere exposure.We further investigated the underlying redox-related signatures as reactive oxygen species (ROS) are often proposed as a mediator of particle-induced responses. We also looked at mitochondrial responses as mitochondria are a major source of ROS and actively participate in the regulation of cellular metabolisms . At the . (2020)  found an balance . The latinst ROS . In this2O2 levels and more mitochondria were detected as evidenced by a higher mtDNAc. It is well understood that mtDNAc fluctuates in response to the physiological environment surrounding the cell and might, therefore, reflect an altered cellular redox balance [Although no significant gene expression changes were observed after exposure to both PS 200 nm spheres and PSMF, lower intracellular H balance . Exposur balance ,54. Chan balance . Cortes  balance ,44, resp balance . We hypo balance , supportThis study acknowledges both its strengths and limitations. First, particle interference with existing assays complicates experiments and result interpretation. In our research, we discovered that some absorbance-based assays are susceptible to interference caused by particles to the optical read-out. Furthermore, non-fluorescent PS 2 µm spheres became fluorescent in the standard assay, indicating interaction of the dyes on the surface of the PS spheres. This is plausible considering the surface charge density of the particles . WhetherOur study revealed that, in addition to spherical PS-MPs, fibre- and fragment-shaped PS-MPs are also taken up by Caco-2 cells. Uptake clearly depends on the particle size. Although subcytotoxic changes were observed mainly for the micron-sized spherical and fibre-and fragment-shaped PS-MPs, a redox response was found to be induced for all the particles. This response varied in time and location, depending on the particle type, size, and number, with the PS 200 nm spheres having the most profound effects. Nonetheless, our results showed that the interaction between mitochondria and ROS, including the HO-1 system, are key regulators of the microplastic-induced defensive response in Caco-2 cells. Since the existence of other additional key factors cannot be ruled out, further research is needed to delve deeper into intracellular redox dynamics and to fully understand the underlying mechanisms of varying sizes, shapes, surface groups, and charges of MPs. In addition, it should be noted that particle interference in the assay results can lead to misinterpretation, underpinning the importance of measuring a combination of parameters for hazard profiling of MPs.