Razumab™ (world’s first biosimilar ranibizumab) is approved for several macular disorders including wet age-related macular degeneration (AMD). We evaluated the safety and efficacy of biosimilar ranibizumab in wet AMD.This prospective, multicentre, rAnibizumab bioSimilar Safety Efficacy postmarkeTing (ASSET) study enrolled patients aged ≥ 50 years with wet AMD having best-corrected visual acuity (BCVA) between 20/40 and 20/320. The patients received intravitreal biosimilar ranibizumab 0.5 mg every 4 weeks for 24 weeks. Safety endpoints included the incidence of adverse events (AEs), serious AEs (SAEs), and immunoreactivity after 6 months. The efficacy endpoints were the proportion of patients who lose fewer than 15 letters, increase in BCVA, change in central retinal thickness (CRT), and change in Visual Function Questionnaire-25 (VFQ-25) score, from baseline to 24 weeks.Of the 126 enrolled patients, majority (95.24%) of the patients received all 6 doses of biosimilar ranibizumab . Nineteen AEs were reported ; majority (78.9%) were mild. There were no serious AEs reported, except one AE of death which was unrelated to the study drug. None of the patients discontinued the study due to an AE. The most common ocular AE was increase in intraocular pressure (4 events) and non-ocular AE was pyrexia (5 events). A total of 7.9% (10/126) patients prior to dosing and 7.1% (9/126) patients post-treatment were positive for anti-ranibizumab antibodies. No AEs suggestive of immunogenicity were noted. At 24-weeks, 97.60% patients in the intent-to-treat (ITT) population (N = 125) and 97.41% patients in the per-protocol (PP) population (N = 116) lost < 15 letters from baseline visual acuity. In the ITT and PP populations, 31.20% and 32.76% patients, respectively, showed improved visual acuity by ≥ 15 letters. Significant improvements in BCVA  and VFQ-25  were seen; CRT reduced significantly .Razumab™ (world’s first biosimilar ranibizumab) was well-tolerated without new safety concerns and significantly improved visual acuity in wet AMD patients.Trial registration CTRI/2016/03/006739. Registered 18 March 2016—Prospectively registered, http://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=13141&EncHid=&userName=2016/03/006739The online version contains supplementary material available at 10.1186/s40942-021-00293-w. Age-related macular degeneration (AMD) is a progressive neurodegenerative disease and a leading cause of visual loss in elderly patients . Wet -A plays a vital role for angiogenesis and vascular permeability, and it is overexpressed in wet AMD , 9. Antith February 2015) by the highest Indian regulatory authority ‘Drugs Controller General of India (DCGI)’ for the treatment of neovascular (wet) AMD, diabetic macular edema (DME), macular edema following retinal vein occlusion (RVO) and for visual impairment due to choroidal neovascularization (CNV) secondary to pathologic myopia [Intas Pharmaceuticals Limited, India, has developed Razumab™, the world’s first biosimilar ranibizumab, which has been approved  was a phase 4, prospective, interventional, single-arm, non-comparative, multicentre, postmarketing study to evaluate the safety and efficacy of the world’s first biosimilar ranibizumab, Razumab™, in patients with wet AMD . The first patient was enrolled on 01 June 2016 and the last patient completed the study on 06 June 2018. The study was conducted at 16 sites across India. The study protocol was approved by an institutional ethics committee of the respective study center. The study was conducted in accordance with the Declaration of Helsinki, good clinical practices and relevant regulatory guidelines. All patients provided written informed consent before enrolment into the study.We enrolled patients of either gender, aged ≥ 50 years, having active primary or recurrent sub-foveal lesion with CNV secondary to AMD, involving the foveal center, and having a best corrected visual acuity (BCVA) of 20/40 to 20/320 using the Early Treatment Diabetic Retinopathy Study (ETDRS) chart  in the study eye. Only one eye was evaluated, and if both eyes were eligible for inclusion, the eye with the better visual acuity was selected unless the investigator chose the other eye for treatment on medical grounds.Patients were excluded if they had received prior treatment with any intravitreal drug, ranibizumab, verteporfin or photodynamic therapy within the past three months; or had received prior treatment with systemic or intravitreal bevacizumab within the last six months; or underwent extrafoveal laser photocoagulation within one month in the study eye; had subfoveal fibrosis or atrophy in the study eye, had CNV in either of the two-eyes due to causes other than AMD; had intraocular condition that could require any intervention during the six months study period; had vitreous haemorrhage or rhegmatogenous retinal detachment or macular hole (stage 3 or 4); had active intraocular inflammation or ongoing infection; had a recent history of stroke, or a patient with known hypersensitivity to ranibizumab.Patients were screened within 4 weeks before study enrolment. Eligible patients received intravitreal injections of biosimilar ranibizumab at a dose of 0.5 mg every 4 weeks for 24 weeks for a total of 6 doses. A total of 16 study visits were planned for each enrolled patient including the screening visit and the end of study visit  and serious AEs (SAEs) including any hypersensitivity reactions, any significant laboratory abnormalities, and the proportion of patients developing anti-ranibizumab antibody after 6 months of treatment.Fundus fluorescein angiography (FFA) was performed for all patients on the day of screening to assess the lesion severity. The spectral domain-optical coherence tomography (SD-OCT) examinations were performed on the day of screening, baseline visit and at 3- and 6-months post-treatment visits. Slit-lamp examination, indirect ophthalmoscopy, and intraocular pressure measurements were done at screening, each dose administration visits and at the end of the study. Ophthalmic examination was done for safety assessment on the subsequent day of dosing using the safety questionnaire  version 20.1.Immunogenicity was evaluated by assessing the presence of serum anti-ranibizumab antibodies in all patients. A total of 9 samples , each of 6 mL, were collected from each patient to detect anti-ranibizumab antibodies in the serum. Blood samples were collected through the needle from a venipuncture site with the arm in downward position, and collected in vacutainers placed upright in a rack kept in ice cold water bath until centrifugation. The serum samples were centrifuged at 3000 ± 100 rpm for 10 min at room temperature (18–25 °C) to separate the serum. The serum samples were transferred using pasture pipette (dropper) to polypropylene cryovial in two aliquots  and stored at a temperature of − 22 °C ± 5 °C or below for interim storage until shipment for analysis. Samples were at controlled temperature (− 22 °C ± 5 °C or below)/packed with dry ice for transport along with data logger. After receiving the samples at the Bioanalytical Laboratory of Lambda Therapeutic Research Limited, Ahmedabad, India, the samples were stored at − 65 ± 10 °C until completion of analysis. The serum samples were analysed using a standard, globally acceptable and validated bridging enzyme-linked immunosorbent assay (ELISA) method for anti-ranibizumab antibodies at Lambda Therapeutic Research Limited.The efficacy assessments were done at baseline, Week 12 and end of the study (Week 24) using BCVA, central retinal thickness (CRT), and Visual Function Questionnaire (VFQ)-25  parameteBased on the requirement of the Indian apex drug regulator the ‘DCGI’ to generate the efficacy and safety data on Razumab in at least 100 patients, a total 126 patients were planned to be recruited into the study considering a drop out/withdrawal rate of ~ 20%. The safety variables were assessed using descriptive statistics such as number of observations, mean, standard deviation, median, and range for continuous variables, and percentages and frequency for categorical variables. The safety analysis was done on the safety population which was defined as patients who received at least one dose of biosimilar ranibizumab. The percentage of patients developing anti-ranibizumab antibodies during the 6 months of treatment along with the 95% CI was calculated in immunogenicity population, defined as the patients who received at least one dose of biosimilar ranibizumab in the study and had at least one pre-dose and one post-dose immunogenicity sample analysed.t-test to compare changes from the baseline to 24 weeks. The proportion of patients who lost fewer than 15 letters was presented. The efficacy analysis was done on an intent-to-treat (ITT) and per protocol (PP) populations. The ITT population was defined as all patients who received at least one dose of study treatment and had at least one efficacy assessment data. The PP population was defined as patients who completed the study without major protocol deviations impacting efficacy evaluations. The last observation carried forward (LOCF) approach was used to compute the missing data. Statistical analyses were done using SAS® Version 9.4 .The efficacy variables including change in BCVA, CRT and VFQ-25 were assessed using descriptive statistics and the p-value was calculated using paired A total of 149 patients were screened from 16 centres in India. Of which, 126 were enrolled and 23 were excluded as they did not meet the selection criteria (n = 11) or were not ready to provide consent for study participation (n = 12). All the 126 enrolled patients received biosimilar ranibizumab and were included in the safety and immunogenicity populations. The ITT population included 125 patients since one patient did not have any post-dose efficacy assessment due to deviation from the protocol procedure. Of the 126 patients, 116 (92.06%) completed the study and were included in the PP population; 10 (7.94%) patients did not complete the study since they did not return for follow-up (n = 1), died (n = 1), missed the visit (n = 1), investigator’s decision (n = 1) or withdrew consent (n = 6)  were males  patients received all 6 doses of biosimilar ranibizumab , 4 (3.17%) patients received 4 doses  and 1 patient each received five doses  and one dose .th dose of biosimilar ranibizumab due to another MI event which was unrelated to the study drug. Except for this patient, none discontinued the study due to an AE. There were no other SAEs reported in the study.Of 126 patients, 16 (12.7%) patients reported a total 19 AEs during the study  decreased from baseline 14.5 ± 3.38 to 13.9 ± 2.74 for left eye, and from 14.2 ± 3.19 to 13.9 ± 2.97 for right eye at Week 24. Majority of the AEs were mild (n = 15), 3 AEs  were moderate, and one AE (death) was severe. Except death, all other AEs resolved during the study. Out of 19 AEs, 10 AEs were ocular and were reported by 9 (7.14%) patients. The most common ocular AE was increase in intraocular pressure (4 AEs in 3 patients), followed by eye pruritus (2 AEs in 2 patients). The most common non-ocular event was pyrexia (5 AEs), reported in 5 (3.97%) patients. No event of hypersensitivity reaction to biosimilar ranibizumab was reported in the study.A total of 10  patients were positive for anti-ranibizumab antibodies prior to dosing, and 9 , 5 , 6 , 5 , 8 , 9 , 9 , and 8  patients at Weeks 2, 4, 6, 8, 12, 16, 20 and 24, respectively, were positive for anti-ranibizumab antibodies post treatment with biosimilar ranibizumab.At the end of 24 weeks, visual acuity of treated eye improved in both ITT and PP populations as assessed by efficacy parameters such as the 15-letter loss for visual acuity, BCVA, CRT and VFQ-25 score.The proportion of patients who lost fewer than 15 letters from baseline visual acuity was 98.40%  patients at Week 12 and 97.60%  patients at Week 24 in the ITT population, and 98.28%  patients at Week 12 and 97.41%  patients at Week 24 in the PP population. The mean (SD) BCVA improved significantly from 44 (16.27) to 50.3 (17.37) letters at Week 12  and 53.7 (17.83) letters at Week 24  in the ITT population and from 44.4 (16.38) to 50.8 (17.17) letter at Week 12  and 53.5 (17.84) letters at Week 24  in the PP population  CRT significantly decreased from 384.8 (146.44) µm to 273.1 (101.34) µm at Week 12  and 258.5 (74.77) µm at Week 24  in the ITT population and from 379.7 (141.57) µm to 275.2 (102.59) µm at Week 12  and 259.7 (74.00) µm  in the PP population  patients, 10 were ocular AEs reported in 9 (7.14%) patients; increase in intraocular pressure was the most common AE. Of 4 AEs of increase in intraocular pressure, only one was considered related to the study drug. Increase in intraocular pressure have been noted both pre- and post-intravitreal injections . The meaIridocyclitis, corneal edema, ocular hyperaemia, and dry eye were observed in < 1% of patients after biosimilar ranibizumab treatment. Similar incidence rates for these AEs were observed in the innovator ranibizumab postmarketing surveillance studies , 39. TheIn the current study, hypertension and nasopharyngitis were reported in one patient each. Incidence rates of hypertension, nasopharyngitis and headache ranged from 3 to 9%, while incidence rates of thromboembolic events ranged from 0.8 to 5.6% with innovator ranibizumab as reported in literature , 40, 41.Of 126 patients, only one patient discontinued the study due to an AE, i.e. death due to MI. This AE was considered unrelated to the study drug. Important ocular SAEs such as endophthalmitis and vitreous haemorrhage, and non-ocular SAEs such as thromboembolic events, which were reported in some large clinical studies of innovator ranibizumab, were not reported in our study , 42, 43.Patients with wet AMD lose visual acuity by 2 to 3 lines per year if the patient remains untreated , 45. BioThe study limitations included the unavailability of details pertaining to the patterns, size and type of CNV (classic/occult/mixed). Also, the details of the patients who had received previous treatments before the timelines as mentioned in the exclusion criteria, were not captured, and hence, the data on ‘treatment naïve’ versus ‘previously treated patients’ could not be provided.Overall, this prospective, postmarketing ASSET study of the world’s first biosimilar ranibizumab, Razumab™, in patients with wet AMD over 6 months showed similar safety and efficacy profiles to the evidence reported with innovator ranibizumab. Biosimilar ranibizumab was well-tolerated without any new safety concerns. Biosimilar ranibizumab also showed similar improvement in patients with wet AMD for 15 letter loss assessed by visual acuity, BCVA and retinal thickness, as reported with innovator ranibizumab. Long-term studies with a larger patient population may elicit better results.Additional file 1: Appendix S1. Safety Assessment Questionnaire


This study aimed to investigate whether quercetin exerts anticancer effects on oral squamous cell carcinoma (OSCC) cell lines and to elucidate its mechanism of action. These anticancer effects in OSCC cells were assessed using an MTT assay, flow cytometry (to assess the cell cycle), wound-healing assay, invasion assay, Western blot analysis, gelatin zymography, and immunofluorescence. To investigate whether quercetin also inhibits transforming growth factor β1 (TGF-β1)-induced epithelial–mesenchymal transition (EMT) in human keratinocyte cells, HaCaT cells were treated with TGF-β1. Overall, our results strongly suggest that quercetin suppressed the viability of OSCC cells by inducing cell cycle arrest at the G2/M phase. However, quercetin did not affect cell viability of human keratinocytes such as HaCaT  and nHOK  cells. Additionally, quercetin suppresses cell migration through EMT and matrix metalloproteinase (MMP) in OSCC cells and decreases TGF-β1-induced EMT in HaCaT cells. In conclusion, this study is the first, to our knowledge, to demonstrate that quercetin can inhibit the survival and metastatic ability of OSCC cells via the EMT-mediated pathway, specifically Slug. Quercetin may thus provide a novel pharmacological approach for the treatment of OSCCs. Oral squamous cell carcinoma (OSCC) is the most common malignancy, accounting for about 90% of head and neck cancers, and develops at the lips, tongue, salivary glands, gums, bottom of the mouth, pharynx, and surfaces within the oral cavity ,3,4,5,6.Cancer metastasis involves a complex process that includes primary tumor formation, angiogenesis, invasion, and metastatic colonization . In the Actinobacillus actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg), which are the major pathogens in PD [Quercetin is a plant flavonol of the flavonoid polyphenol group found in many fruits, vegetables, leaves, and grains ,22,23. Ans in PD ,28. Morens in PD ,30,31,32p < 0.01), whereas treatment with low concentrations (10–40 μM) reduced cell viability slightly (approximately 5–30%) (p < 0.05) compared to the control . However, the cell proportion in the G0/G1 phase decreased . The results indicate that quercetin suppressed the viability of OSCC cells by inducing cell cycle arrest in the G2/M phase.To determine the effect of quercetin on cell viability, OSCC cell lines  were treated with various concentrations (0~160 µM) of quercetin for 24 h, and cell viability was determined using the MTT assay. The results showed that quercetin decreased cell viability in OSCC cell lines A. In par control A. To detWe performed a wound-healing assay to evaluate the inhibitory effects of quercetin on migration. To observe the effects of quercetin on cell migration, the cell proliferation of OSC20, SAS, and HN22 cells was inhibited by treatment with thymidine (1 mM) for 2 h prior to treatment with quercetin (40 μM). After 24 h, the wound area of the control was almost completely reduced compared to the initial area; however, the quercetin-treated cells did not show any decrease A,B. The The effects of quercetin treatment on EMT and the activity of ECM-degrading enzymes in OSCC cell lines were analyzed by Western blot or gelatin zymography. OSC20, SAS, and HN22 cells were treated with different concentrations of quercetin  for 24 h. As shown in EMT-activating transcription factors (EMT-TFs), such as Twist, Slug, and Snail 1, were observed by Western blot and immunofluorescence. Twist and Slug were significantly downregulated in quercetin-treated OSC20 and SAS cells compared to the untreated cells A,B. Slugp < 0.05)  and nHOK  cells were treated with various concentrations (0~160 µM) of quercetin for 24 h, and cell viability was determined by an MTT assay. Cell viability of HaCaT cells and nHOK cells was not changed by the low concentration (10–40 μM) of quercetin compared to the control group but was significantly reduced in 160 μM quercetin treatment ( < 0.05) A. The re < 0.05) B. The ex < 0.05) C–H. TGF-To investigate whether quercetin also inhibits TGF-β1-induced EMT in normal cells, HaCaT was treated with 10 ng of TGF-β1 for 24 h, followed by various concentrations of quercetin for 24 h without changing the media A. To obsSuppression of cancer metastasis is considered an effective strategy for preventing cancer progression. Many studies have reported that a variety of natural products and compounds exert antitumor effects by inducing apoptosis and cell cycle arrest in cancer cells and reducing their metastatic capacities ,34. ThusThe EMT process contributes to greater interstitial cell adhesion profiles, as unregulated cellular or cell matrix adhesion and increased migration and invasion are required for the development of metastatic cancer . The abiIt has been found that transforming growth factor-beta 1 (TGF-β1) induces epithelial mesenchymal transition (EMT) in various cancer cells, promoting motility and invasiveness ,47. AddiOverall, our results strongly suggest that quercetin suppressed the viability of OSCC cells inducing cell cycle arrest in the G2/M phase . SignifiQuercetin was purchased from Sigma-Aldrich  and dissolved in DMSO  to prepare stock solutions (100 mM). TGF-β1 was purchased from R&D Systems  and dissolved in 4 mM HCl containing 0.1% fatty acid-free bovine serum albumin to a concentration of 1.0 mg/mL to prepare stock solutions (20 μg/mL). 3--2,5-diphenyltetrazolium bromide (MTT) and thymidine were purchased from Sigma-Aldrich . Antibodies against E-cadherin, vimentin, and Slug were purchased from Cell Signaling Technology , whereas fibronectin was purchased from BD Bioscience ; α-smooth muscle actin was purchased from Abcam , and Twist and beta-actin were purchased from Santa Cruz Biotechnology .TM Gold Keratinocyte growth basal medium  containing KGMTM-2 SingleQuotsTM supplements  at 37 °C in 5% CO2 and 95% O2 in a humidified environment. Further, both media contained 10% fetal bovine serum  and 1% penicillin and streptomycin . In this study, the three OSCC cell lines were treated with various concentrations of quercetin for 24 h; the HaCaT cells were treated with 10 ng of TGF-β1 for 24 h and then treated with various concentrations of quercetin.Three OSCC cell lines  and human keratinocyte cells such as HaCaT  and nHOK  were used in this study. OSC20 and SAS cells were grown in Dulbecco’s modified Eagle’s medium F-12, 1:1 mixture ; HN22 and HaCaT cells were grown in Dulbecco’s modified Eagle’s medium with high glucose ; and nHOK  were cultured in KBM4 cells per well in a 96-well plate and incubated with 10% FBS-containing media. However, the exception was that nHOK cells were seeded with 3 × 104 cells per well with serum-free media. After stabilization, the media was replaced with a media containing 10% FBS for 24 h. Then, the cells were treated with various concentrations of quercetin  with media containing 10% FBS. After 24 h, the media were replaced with MTT (500 μg/mL) in a fresh serum-free media, and the cells were incubated for 3 h at 37 °C. Then, the supernatant was removed, and 200 μL of dimethylsulfoxide (DMSO) was added to dissolve the MTT formazan. The absorbance of the plate was read at 570 nm using a Synergy HT micro plate reader .An MTT -2,5-diphenyltetrazolium bromide) assay was conducted to evaluate the cytotoxicity of quercetin on cell viability. Each cell line was seeded at 1 × 105 cells in 60 mm dishes. After cell stabilization, the cells were treated with 40 μM of quercetin for 24 h. Then, the cells were detached using trypsin and washed with phosphate-buffered saline (PBS). The harvested cells were then fixed with 75% ethanol at 4 °C overnight; subsequently, they were centrifuged and incubated with 20 μL RNase A (10 mg/mL) in 1 mL PBS at 4 °C for 30 min. Lastly, cells were stained with 25 μg/mL PI at room temperature for 10 min in the dark. The DNA content was then analyzed using a BD FACSCanto II flow cytometer .A cell cycle analysis was conducted to observe the cell cycle’s progression. Cells were seeded at a density of 5 × 105 per well. At 80% confluence, the cells were treated with 1 mM thymidine for 2 h before reagent treatment. Then, a wound was made in the middle of the well using a sterile 200 μL pipette tip. Further, wells were washed three times with PBS. OSCC cell lines were incubated with media containing 1% FBS and 40 μM of quercetin for 24 h. HaCaT cells were incubated with media containing 1% FBS and 10 ng TGF-β1 for 24 h; then, 40 μM of quercetin was added continuously for 24 h without discarding the existing media. Images were captured using a microscope . The rate of reduction of the wound area was calculated as a percentage of the remaining wound area compared to the initial wound area.A wound-healing assay was conducted to evaluate cell migration ability. Cells were seeded in 6-well plates at 5 × 10After treatment, cells were washed three times with cold PBS and lysed with 1X RIPA buffer containing a protease inhibitor cocktail and phosphatase inhibitors. The lysed cells were centrifuged at 12,000 ×g for 24 h. Subsequently, the supernatant was collected. Using the method of Bradford, the protein concentration was quantified as 20 μg. Samples were separated on 10% SDS-polyacrylamide gels. Then, SDS-gels were transferred electrophoretically onto polyvinylidene fluoride membranes (Bio-Rad) using a wet transfer kit. The transferred membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. The membranes were incubated overnight at 4 °C with the primary antibodies against E-cadherin ; claudin-1 ; fibronectin ; vimentin ; α-SMA ; slug ; and Twist  and β-actin . Continuously, the membranes were washed three times with 1X TNE buffer, and HRP-conjugated secondary antibodies  were applied for 3 h at room temperature. The antigen–antibody complexes were detected using a SuperSignal West–Femto reagent .5 cells per well in a 6-well plate with 10% FBS-containing media. After cell stabilization, the media were transformed into serum-free media and treated with different concentrations of quercetin for 24 h at 37 °C. Subsequently, the supernatant was collected and mixed with a sample buffer (50% 0.5 M Tris-HCl (pH 6.8), 40% glycerol, 8% SDS, and 0.01% bromophenol blue). Further, samples were loaded onto a 7.5% SDS-polyacrylamide gel containing 0.2% gelatin (Sigma). The gels were washed twice with PBS containing 2.5% Triton X-100 for 1 h and incubated with an incubation buffer at 37 °C for 48 h. Then, the gels were stained with Coomassie Blue R-250  for 1 h. Progressively, the gels were destained with a destaining buffer (8% acetic acid and 4% methanol) until a clear zone was visible.Gelatin zymography was conducted to detect matrix metalloproteinase (MMP) activity using the conditioned media. Each cell line was seeded at 5 × 105 cells onto a coverslip in a 6-well plate. In the case of OSCC, the cells were stained for transcription factor expression. The treated cells were fixed in 4% paraformaldehyde at room temperature for 15 min and washed three times with PBS for 10 min. Then, the cells were permeabilized by 0.02% Triton X-100 in PBS at room temperature for 20 min and washed with PBS. Diluted 1:100 primary antibodies (Slug and Twist) were applied to the cells overnight at 4 °C. On the following day, cells were washed three times with PBS, treated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies for 1 h at 37 °C, and washed three times. Then, 4 μg/mL Hoechst was used to stain the nuclei for 30 min at 37 °C. The treated HaCaT cells were stained to assess changes in their morphology. HaCaT cells were incubated with a phalloidin-conjugate working solution (1:100) and 4 μg/mL Hoechst instead of antibodies. Subsequently, the cells were washed and mounted on a slide glass. An image was then captured via confocal microscopy .Cells were seeded at 2 × 104 cells with serum-free media containing 10 ng TGF-β1, co-treated with TGF-β1 and quercetin (10 ng TGF-β1 and 40 μM) or without treatment. Then, 600 μL of culture medium containing 10% FBS was placed onto the lower chamber. The cells were next incubated at 37 °C in a 5% CO2 atmosphere for 44 h. After incubation, the cells on the upper chamber were removed using a cotton swab. The cells passing through the Matrigel and membrane were fixed with cold methanol for 30 min and washed with PBS. The membrane was stained with Mayer’s hematoxylin  and 1% Eosin Y solution (Muto). Photographs were captured under microscopy .An 8 μM pore size Transwell  was coated with 40 μL of Matrigel , mixing the serum-free media at a ratio of 1:3 and air drying for 3 h at 37 °C. HaCaT cells were incubated for 24 h with or without 10 ng TGF-β1 and detached using trypsin. The cells were seeded in a Matrigel-coated Transwell culture chamber at 5 × 10p-values of < 0.05 were considered statistically significant. All data are the means ± standard error of the mean (SEM).Statistical analyses were performed by a one-way analysis of variance (ANOVA) followed by a Bonferroni post-test, whereby three or more experimental groups were compared.


Arabidopsis to high light. Our results reveal that acclimation takes place on the one hand in the cytosol by differential phosphorylation of preproteins and resulting from the altered expression of the responsible kinases, and on the other hand at the level of the translocation machineries in the outer (TOC) and inner (TIC) envelope membranes. Intriguingly, while phosphorylation is more pronounced under high light, import itself shows a lower efficiency, along with a reduced accumulation of the Toc receptor proteins Toc34 and Toc159.Acclimation is an essential process in plants on many levels, but especially in chloroplasts under changing light conditions. It is partially known how the photosynthetic machinery reacts upon exposure to high light intensities, including rearrangement of numerous protein complexes. Since the majority of proteins residing within chloroplasts needs to be posttranslationally imported into the organelles, we endeavored to study how this important process is regulated upon subjecting plants from pea and  Plants as sessile organisms strongly rely on their abilities to adapt to fluctuating environmental conditions. The chloroplast as an environmental sensor plays a crucial role in plant acclimation. To maintain and control its protein homeostasis, drastic changes in the chloroplast proteome composition are required. As a consequence of endosymbiosis, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol as preproteins. The mandatory protein targeting and import mechanisms represent sophisticated checkpoints to control and modulate chloroplast function . After tThe import process itself is regulated at several levels: Two receptor proteins at the chloroplast surface, Toc159 and Toc34, vary their oligomerization state and thereby affinity to incoming preproteins depending on GTP-binding and hydrolysis . The samIn this study, we have investigated the reaction of protein targeting and import efficiency in response to high light exposure. Our results indicate that high light influences both cytosolic targeting mechanisms by triggering phosphorylation, as well as the translocation process, which was found to be reduced in response to high light.E. coli. The recombinant proteins were purified via a C-terminal His-tag and subjected to an in vitro kinase assay with purified STY8. a/b binding protein 1 (pLhcb1), as well as high chlorophyll fluorescence (pHCF101) were previously shown to be phosphorylated within their presequences -ATP and 2.5 μm ATP in a total volume of 25 μL of kinase buffer . The reaction was carried out for 15 min at 23 °C and stopped by adding 5 μL of SDS sample buffer. The proteins were separated on a 12% SDS-PAGE followed by autoradiography.Recombinant kinase or stroma extract was incubated with recombinant substrate in the presence of 3 μCi of [γ-Arabidopsis leaves were homogenized in homogenization medium  using liquid nitrogen and an electronic micropestle. The powdered suspension was incubated for 10 min at RT in the dark and centrifuged at 13,000× g, 4 °C for 10 min, the supernatant containing the soluble proteins was collected, and protein concentration was determined using Bradford reagent . SDS-PAGE and immunoblotting was performed as described [For immunoblot analysis, either isolated chloroplasts or total soluble proteins were used. To isolate total soluble proteins, escribed ,27.Total RNA was isolated from several leaves using the Plant RNeasy extraction kit . cDNA was synthesized from 1 μg of RNA (DNase treated) using iScript™ cDNA Synthesis Kit . All reactions were done in triplicate on three biological replicates. The following oligonucleotides were used: STY8QRT-PCR for 5‘-CCACGGATGGAACTGATGAGT-3‘, STY8QRT-PCR rev 5′-TACACGATCAGGCTTGAGAAA-3′, STY17QRT-PCR for 5′-AAGGTTTAAAAGATGCATTGA-3′, STY17QRT-PCR rev 5′-CATCAGTTCCATCCGTAGGTA-3′, STY46QRT-PCR for 5′- AGGTGCCAGAACGCATGTTCC-3′, STY46QRT-PCR rev 5′-TTGATAGCAACTTCCTGGCTA-3′, RCE1_qpcr_fr 5′-CTGTTCACGGAACCCAATTC-3′, RCE1_qpcr_rev 5′-GGAAAAAGGTCTGACCGACA-3′. The relative abundance of all transcripts amplified was normalized to RCE1 (At4g36800). For quantitative RT-PCR, the SYBR Green real-time PCR master mix  was used, and the reaction was performed in a Bio-Rad CFX96 real-time PCR detection system. Forty-five cycles were performed as follows: 1 s at 95 °C, 7 s at 49 °C, 19 s at 72 °C, and 5 s at 79 °C.35S Met/Cys . Translated preproteins were aliquoted, snap frozen, and stored at –80 °C until further use.All genes of interest were transcribed by means of SP6 polymerase  according to the manufacturer’s instructions. RNA was snap frozen in liquid nitrogen and stored at −80 °C. Translation war performed with the Flexi Retic Kit  for 90 min in the presence of 3, 0.2% (w/v) BSA, plus 2 µL translation product for 5 min at 25 °C. The reaction was stopped by addition of one volume of ice-cold washing buffer. Chloroplast were pelleted by centrifugation for 1 min at 1500× g, washed once, and finally resuspended in Laemmli buffer.Pea chloroplasts were isolated as described previously . Chlorop2, 5 mM EDTA, 20 mM HEPES-KOH, 10 mM NaHCO3 pH 8.0). The homogenate was filtered through one layer of cheese cloth and the procedure repeated three times with the same leaf material. The pooled filtrate was centrifuged for 4 min at 1200× g. The supernatant was discarded and pellets were carefully resuspended in residual isolation medium. Resuspended pellets were loaded onto a step Percoll gradient  and centrifuged at 1500× g for 6 min. The lower band containing intact chloroplasts was removed and washed once with at least three volumes of 50 mM HEPES, 0.3 M sorbitol, 3 mM MgSO4 pH 8.0 (wash buffer). The final pellets were resuspended in wash buffer depending on their size and chlorophyll concentration was determined according to Arnon [3, 0.2% (w/v) BSA, plus 2 µl translation product for 5 min at 25 °C. Afterwards, chloroplasts were pelleted at 1500× g, washed once in 200 µL wash buffer, and then resuspended in SDS loading buffer. Proteins were separated on SDS gels, which were Coomassie stained , STY17 (At4g35780), STY46 (At4g38470), pOE23 (At1g06680), pHCF101 (At3g24430), pNdhM (At4g37925) and pFNRL1 (At5g66190), pSSU (AAA34116), pLhcb1 (AT1G29930), CysP (AT5G06290), FNR (P10933), LHC (P27490), FBP (At3g54050)


Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death worldwide. Poor drug delivery to tumours is thought to limit chemotherapeutic treatment efficacy. Sonoporation combines ultrasound (US) and microbubbles to increase the permeability of cell membranes. We assessed gemcitabine uptake combined with sonoporation in vitro in three PDAC cell lines . Cells were cultured in hypoxic bioreactors, while gemcitabine incubation ± sonoporation was conducted in cells with operational or inhibited nucleoside membrane transporters. Intracellular active metabolite (dFdCTP), extracellular gemcitabine, and inactive metabolite (dFdU) concentrations were measured with liquid chromatography tandem mass spectrometry. Sonoporation with increasing US intensities resulted in decreasing extracellular gemcitabine concentrations in all three cell lines with inhibited membrane transporters. In cells with inhibited membrane transporters, without sonoporation, dFdCTP concentrations were reduced down to 10% of baseline. Sonoporation partially restored gemcitabine uptake in these cells, as indicated by a moderate increase in dFdCTP concentrations (up to 37% of baseline) in MIA PaCa-2 and PANC-1. In BxPC-3, gemcitabine was effectively inactivated to dFdU, which might represent a protective mechanism against dFdCTP accumulation in these cells. Intracellular dFdCTP concentrations did not change significantly following sonoporation in any of the cell lines with operational membrane transporters, indicating that the gemcitabine activation pathway may have been saturated with the drug. Sonoporation allowed a moderate increase in gemcitabine transmembrane uptake in all three cell lines, but pre-existing nucleoside transporters were the major determinants of gemcitabine uptake and retention. Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death worldwide ,2. Late Gemcitabine monotherapy is one of the three main chemotherapeutic drug regimens used in the palliative setting of PDAC patients worldwide . FollowiPaproski and co-workers demonstrated that in vitro inhibition of hENT1 by dilazep reduced average gemcitabine uptake 24-fold and sensitivity 13-fold in both PDAC and non-PDAC cell lines. Restoration of nucleoside membrane transport by transfection with an active nucleoside influx pump re-established gemcitabine uptake and sensitivity , suggestn = 10) were treated with gemcitabine followed by repeated intravenous boluses of SonoVue® microbubbles and US focused at their primary tumours. Sonoporation treated patients experienced tumour shrinkage, tolerated an increased number of treatment cycles, and survived longer than a historical control group, of comparable performance status, treated with gemcitabine alone [The combination of microbubbles and ultrasound (US) has been proposed to facilitate the formation of transient pores in biological membranes through a process commonly referred to as sonoporation , resultane alone . Similarne alone . It was ne alone ,21,22.® microbubbles and increasing US intensities, employing a frequency of 0.5 MHz and mechanical indices (MI) of 0.31–0.50–0.75, ISPPA 1.61–4.32–9.36 W/cm2 and ISPTA 0.052–0.14–0.30 W/cm2 [Mariglia and co-workers , however30 W/cm2 . The ult30 W/cm2 ,19,23. WWe hypothesised that the effect of sonoporation on cellular gemcitabine uptake could depend on the activities of hENTs or gemcitabine metabolizing enzymes.® G3 LOT  hypoxic cell culture bioreactors (hereafter entitled “Petakas”) from Tebu-Bio . Horse serum and sodium pyruvate were obtained from Thermo Fisher Scientific  and tetrahydrouridine (THU) from AH diagnostics . Reagents and equipment used for liquid chromatography tandem mass spectrometric methods (LC-MS/MS) are described elsewhere [Chemicals and reagents were purchased from Merck KGaA  unless otherwise stated, and were of analytical grade. Culture flasks and cryotubes were purchased from VWR , centrifuge tubes from Sarstedt  and Petakalsewhere ,25.2 at 37 °C. Media were complemented with 4 mM sodium pyruvate, 2 mM L-glutamine and 10% fetal bovine serum (FBS). Horse serum (2.5%) was added to the medium used for MIA PaCa-2. No antibiotics were used. Mycoplasma-tests performed on a regular basis were negative.The PDAC cell lines, BxPC-3, MIA PaCa-2 and PANC-1, were kindly provided by Prof. Anders Molven . Cell lines had been authenticated by DNA-fingerprinting  and was 6 cells per 25 mL medium. Petakas were kept in a horizontal position for 24 h to ensure even cell distribution over the surface, and then flipped to a vertical position with the air vent at the top, until the day of the experiments. At the day of experiments, cell confluency averaged 70–80%. A priori evaluation of cell growth had been performed for each cell line at four different seeding densities, and surface area coverage was quantified using MIPAR™ image analysis software [Two or three days before experiments with gemcitabine, cells were harvested using 0.05% trypsin-EDTA, counted and reseeded into Petakas A at a desoftware   60 min incubation with 10 µM gemcitabine, (2) 20 min pre-incubation with 100 µM dilazep followed by 60 min incubation with 10 µM gemcitabine and (3) 60 min co-incubation with 10 µM gemcitabine and 200 µM tetrahydrouridine (THU), an inhibitor of cytidine deaminase (CDA).® was prepared using the venting needle method. A total of 2 mL of saline  was slowly added to the vented vial and gently agitated for 30 s. Eighty µL Sonazoid® stock solution with 1.20 × 109 microbubbles per mL was added to 1 mL of the prepared gemcitabine solution, and injected into the Petakas. Air pockets were removed and the entire Petaka was exposed to US immediately thereafter. The Petakas  1.8%, ISPPA 3 W/cm2 and ISPTA 50 mW/cm2) and High , in addition to Control (no US). An ultrasound frequency of 2 MHz was chosen as this is commonly used in non-linear contrast imaging for Sonazoid microbubbles. In addition, this frequency is below the resonance frequency of Sonazoid, and would ensure the bubbles to resonate in phase with the ultrasound, maximising volumetric oscillations [In all experiments, we used one microbubble concentration and selected US intensities based on a priori optimisation, with the cell-impermeable dye calcein as “drug surrogate” . Sonazoi Petakas A were pl Petakas B was basllations . The medllations . The hig6 cells (abbreviated to pmol/106 throughout the manuscript).Quantification of gemcitabine and its metabolites was performed using an Agilent 1200 series HPLC-system  for chromatographic separation and an Agilent 6410 triple-quad mass spectrometer for mass detection. Concentrations of gemcitabine and dFdU in culture media samples were measured according to our previously published method , with opn = 3600 in total) were analysed using MIPAR™ image analysis software Version 3.0 [Cell viability following exposure to (1) 60 min 10 µM gemcitabine alone, (2) sonoporation (High) alone, (3) 60 min 10 µM gemcitabine combined with sonoporation (High), and (4) Control , was assessed by monitoring cell growth for up to ten days. BxPC-3 suspensions were diluted to 2500 cells/mL, MIA PaCa-2 and PANC-1 to 1000 cells/mL, and reseeded in triplicate into 24-well plates. Five daily snapshots from each well were captured using a Zeiss Vert.A1 microscope, Axiocam 105 colour camera and the Zeiss ZEN Pro 2012 blue edition software. Images . Two-sided independent student’s t-tests were used to compare means between experiments performed at two different ultrasound acoustic intensities (Medium or High) or no ultrasound (Control), within each cell line. Correction for multiple testing was not performed in this explorative in vitro study where the number of groups did not exceed 3 in any cell line or any experiment. A one-tailed Pearson’s correlation was used to describe linear relationships between US intensities and gemcitabine and—metabolite concentrations within each cell line. One-tailed was based on the assumption that increasing US intensities would have a one-directional effect on gemcitabine and—metabolite concentrations. Pearson’s was based on the assumption that the measures of US intensities, MI and ISPTA, represented continuous variables to be examined for a linear relationship to gemcitabine and metabolite concentrations. A p-value less than 0.05 was considered significant.Quantitative data were processed using Microsoft Office Excel (2016) and GraphPad Prism 8 . All experiments were performed with ® microbubbles, and treated with US at medium and high intensities, and without US (control). The contribution of membrane transporter activities in the cellular uptake of gemcitabine combined with sonoporation was elucidated by gemcitabine incubation ± the hENT-inhibitor dilazep. Data from cells with operational membrane transporters are displayed in All three cell lines were incubated with gemcitabine and Sonazoidp = 0.025) . A significant correlation was however observed between gemcitabine concentrations and MI , and dFdU concentrations and MI  in BxPC-3.In BxPC-3, after 60 min incubation with 10 µM gemcitabine and with application of the highest US intensity, mean extracellular gemcitabine concentrations were reduced from 9.0 ± 0.4 µM (Control) to 8.2 ± 0.4 µM (= 0.025) G. Extrac= 0.025) D and int= 0.025) A showed p = 0.005, r2 = 1.000) in MIA PaCa-2    Q and PAN = 1.00) R.p = 0.03) and further to 0.4 ± 0.09 µM at high intensity (p = 0.001) . No changes in dFdU concentrations were observed in MIA PaCa-2  to 0.2 ± 0.03 µM at medium US intensity (= 0.001) M. This tA PaCa-2 N or PANCA PaCa-2 O.6 at high US intensity (p = 0.005) in MIA PaCa-2 (6 (p = 0.036) in PANC-1 (p = 0.367) increase from 11.4 ± 0.9 (Control) to 12.8 ± 2.5 pmol/106 at high US intensity was noted , whereas in PANC-1 a correlation was seen between dFdCTP and ISPTA .Intracellular dFdCTP concentrations increased from 2.9 ± 0.2 (Control) to 4.8 ± 0.6 pmol/10A PaCa-2 K and fron PANC-1 L. In BxPas noted J. HoweveSixty minutes co-incubation with 10 µM gemcitabine and 200 µM THU resulted in dFdU concentrations below LLOQ (<0.1 µM) in all three cell lines D–F. Withp < 0.0001), but the growth rate (Hill slope) was the same for all groups (p = 0.942).Growth of the cell lines was followed for ten days after exposure to 10 µM gemcitabine, sonoporation (High), or both, and compared to untreated cells . In MIA To our knowledge, this is to date the most comprehensive in vitro study of gemcitabine cellular uptake combined with sonoporation, using diagnostic intensity US and microbubbles. The majority of in vitro sonoporation studies have used cell-impermeable fluorescent drug surrogates as indicators of membrane permeation. However, the extent of cellular chemotherapeutic drug uptake is rarely reported. This study fills into this knowledge-gap by quantitating extra- and intracellular concentrations of gemcitabine and metabolites. Moreover, our data demonstrate that gemcitabine uptake and metabolite accumulation following sonoporation depend on the activities of membrane transporters and metabolizing enzymes within the cells.In BxPC-3 with operational membrane transporters G, and inISPTA and dFdCTP concentrations in all three cell lines suggest that higher US intensities may be warranted [Our results indicated that sonoporation contributed only to a small proportion of cellular gemcitabine uptake compared to pre-existing nucleoside membrane transporters (hENT). When hENTs had been inhibited, dFdCTP concentrations were reduced to approximately 10–20% J–L of tharranted  in orderWhen CDA was inhibited , conversPrevious findings suggest that the sonoporation effect has a duration of up to and exceeding one hour ,35,36, s® microbubbles used by Mariglia and co-workers are smaller and stiffer than the Sonazoid® microbubbles used in our study, but they also used higher MIs that are known to induce inertial cavitation. Furthermore, Definity® has a much more neutral zeta potential compared to Sonazoid™ (−4.2 for Definity® vs. −82mV for Sonazoid™) [®, Definity® is a very unstable microbubble, in which the concentration and size distribution changes over time i.e., bubble dissolution rate varies depending on size, changing the concentration and bulk resonance frequency. These multiple differences in physicochemical characteristics make it difficult to directly determine a primary reason for the difference in response. In addition, the Definity® microbubbles were driven at 0.5 MHz which is more than 20 times lower than their fundamental resonance frequency [Growth curves over a 10-day period after exposure  indicatenazoid™) ,40 whichrequency . This suTo mimic useable clinical imaging frame rates (20–30 frames per second), the ultrasound treatment system was set to not receive and store any reflected ultrasound signal; hence we were unable to confirm if the ultrasound-induced inertial or stable cavitation. Based on literature values, at the medium ultrasound setting (MI = 0.2) we expect stable cavitation, whilst at the at the high ultrasound setting (MI = 0.378) we expect stable-inertial cavitation . It is iThe majority of in vitro research on US and microbubble assisted drug delivery has been performed using fluorescence labelled dyes that have no routes of spontaneous cellular entry ,21,22. AStudying sonoporation and gemcitabine-uptake in PDAC cells cultured in hypoxic Petakas is of particular interest. It has been demonstrated that cellular responses to sonoporation depend on the condition of the cells ,49, whicExperiments performed on plastic surfaces that do not mimic either mechanical or acoustic characteristics of tissue may increase acoustic aberration . FurtherSonoporation with diagnostic intensity US and Sonazoid microbubbles allowed a moderate increase in gemcitabine transmembrane uptake in all three cell lines, but pre-existing nucleoside transporters were the major determinants of gemcitabine uptake and retention. Cell growth after a single treatment with sonoporation combined with gemcitabine was well preserved, which may reflect a general treatment resistance in these cell lines. Moreover, the data underscore that specific PDAC cell lines may respond differently to sonoporation due to different intracellular gemcitabine metabolism.Future studies should include cells of multiple different origins, since a single response on a given cell line or drug does not represent a universally valid effect. Furthermore, sonoporation should be evaluated by using therapeutic drugs in more complex PDAC models that include multiple cell types, connective tissue components, and perfusion.


Wet-lab experiments are cost and time ineffective for the identification of disease-associated m7G sites. To date, tens of thousands of m7G sites have been identified by high-throughput sequencing approaches and the information is publicly available in bioinformatics databases, which can be leveraged to predict potential disease-associated m7G sites using a computational perspective. Thus, computational methods for m7G-disease association prediction are urgently needed, but none are currently available at present.Recent studies have confirmed that N7-methylguanosine  model. m7GDisAI predicts the potential disease-associated m7G sites by applying a matrix decomposition method on heterogeneous networks which integrate comprehensive similarity information of m7G sites and diseases. To evaluate the prediction performance, 10 runs of tenfold cross validation were first conducted, and m7GDisAI got the highest AUC of 0.740(± 0.0024). Then global and local leave-one-out cross validation (LOOCV) experiments were implemented to evaluate the model’s accuracy in global and local situations respectively. AUC of 0.769 was achieved in global LOOCV, while 0.635 in local LOOCV. A case study was finally conducted to identify the most promising ovarian cancer-related m7G sites for further functional analysis. Gene Ontology (GO) enrichment analysis was performed to explore the complex associations between host gene of m7G sites and GO terms. The results showed that m7GDisAI identified disease-associated m7G sites and their host genes are consistently related to the pathogenesis of ovarian cancer, which may provide some clues for pathogenesis of diseases.To fill this gap, we collected association information between m7GDisAI web server can be accessed at http://180.208.58.66/m7GDisAI/, which provides a user-friendly interface to query disease associated m7G. The list of top 20 m7G sites predicted to be associted with 177 diseases can be achieved. Furthermore, detailed information about specific m7G sites and diseases are also shown.The mThe online version contains supplementary material available at 10.1186/s12859-021-04007-9. The closer the association level is to 1, the stronger influence that variant exerts on the exact site. Initially, 812 m7G site-variant pairs with high confidence level were first extracted, then ranked according to the association level. Then 741 m7G site-disease pairs were further picked out with association levels higher than 0.8. Meanwhile, the sequence and genomic location information of m7G-variant pairs were collected correspondingly in this dataset. Specifically, it contains the genomic locations, host genes of m7G sites, site-centered 41 bp reference sequences as well as site-centered 41 bp alternative sequences.In the m7G-disease association dataset, 741 m7G sites were associated with 177 diseases via 741 variants in the m7G-variant dataset. Specifically, these variants are both m7G-associated and disease-associated. In other words, they cause the gain/loss of the m7G site and involve in various disease pathogenesis. Taking these variants as linkages, 177 diseases in ClinVar and GWAS were found to be associated with 741 variants, with implications for disease pathogenesis in m7G RNA methylation.In the m7G-disease association network reconstruction can be transformed into predicting the unknown entries in the m7G-disease association matrix, which can be solved by traditional matrix decomposition methods. However, the number of known associations is so small that matrix decomposition methods cannot achieve satisfactory performance in this case. Thus, we proposed a heterogeneous network-based m7G-disease association prediction method m7GDisAI which will be detailed in the next. The framework of m7GDisAI is shown in Fig. m7G-disease association dataset, the m7G-disease adjacency network was constructed to record their associations. To be more specific, let S = {s1, s2, …, sm} and D = {d1, d2, … dn} denote m m7G sites and n diseases respectively. Let 7G-disease pair 7G-disease association matrix ASD was provided in Additional file Based on the m7G similarity information plays a critical role in m7G-disease association prediction. To make full advantages of the information of m7G sites, a series of m7G similarity networks were constructed for further use in the heterogeneous network.As a kind of auxiliary information, m7G chemical similarity network (CSN) depicts the m7G similarities in terms of the chemical properties extracted from m7G site-centered sequences [i-th nucleotide in sequence N can be encoded by a vector mequences , 24. Spesi, denoted as CF (si), is the combination of these four vectors, in the form of a sequence consisting of {0,1}. Considering the binary numerical properties of the m7G chemical features, the Jaccard coefficient was applied to them. To be specific, for two sites si and sj, their pairwise chemical similarity is defined as , ,  and  respectively. Thus, the chemical feature of site ,1,1,  features were extracted for further similarity calculation. To be specific, CNF of the i-th nucleotide in a sequence is defined as the sum of all the instances of this nucleotide before the i + 1 position dividing i. Taking the sequence ‘TAAGTCCA’ as an example, the CNF for A is 0.5(1/2),0.667(2/3),0.375(3/8) at the 2nd, 3rd and 8th positions respectively. Thus, the CNF features of site si are denoted as CNF (si). Comparing with the m7G chemical features, CNF features pay more attention to the sequence context around the m7G site. Then the Cosine coefficient was adopted to calculate similarities of CNF since it reflects the similarity in trend rather than absolute values. For sites si and sj, the pairwise CNF similarity is defined as  was obtained with the weights between nodes si and sj, , and the adjacency matrix was indicated as ACNFSN , and its adjacency matrix was indicated as ASS. In addition, if α is 0, then ASS is ACSN, while if α is 1, then ASS is ACNFSN.The value of a taking  as weighADD, was also constructed by calculating pairwise disease semantic similarities. Generally speaking, functional similarity between molecules results in similar phenotypes, such as diseases. Based on this fact, many researchers [http://www.bio-annotation.cn:18080/DincRNAClient. By calculating all pairwise semantic similarities in D, a disease semantic similarity network was obtained and the adjacency matrix was indicated as ADD , indicated by adjacency matrix earchers , 25–27 uearchers , 29. Add7G-disease heterogeneous network and its adjacency matrix are shown in Fig. 7G-disease heterogeneous network was constructed by incorporating m7G-disease adjacency network, disease semantic similarity network DSSN, and m7G integrated similarity networks. It was represented by adjacency matrix A and mask matrix W, as  if it ranks above the threshold, false negative (FN) otherwise. Similarly, a candidate sample is considered as false position (FP) if it ranks above the threshold, true negative (TN) otherwise. By varying τ, true positive rate (TPR), false positive rate (FPR) can be calculated for Receiver Operating Characteristic (ROC) curve. It depicts the relative tradeoffs of prediction performance between TP and FP [Regardless of tenfold cross validation, global LOOCV and local LOOCV, for a given threshold P and FP . The areP and FP , 33.k, linear combination coefficient α, regularization parameters λ1 and λ2, that are required to be optimized to enhance the performance of m7GDisAI. To be specific, k is the number of independent factors that govern the pattern of the heterogeneous matrix A, and if k is too large, then the algorithm would be time-consuming. Then k is chosen from {70,90,110}. The linear combination coefficient α weights the contribution of m7G chemical similarity and m7G CNF similarity in m7G integrated similarity network, and it was taken from 0 to 1.0 with the step 0.1. In addition, regularization parameters λ1 and λ2 control the relative penalty extent of the factor matrices U and V respectively, and they were chosen from {2–2,2–1,20,21,22}. It is apparent that k, λ1 and λ2 directly influence the optimal solution of the two factor matrices U and V, while α only has an impact on the m7G similarity matrix ASS. Thus, α was first fixed to 0.5 or any other specific value between 0 to 1, and a grid search strategy was performed on k, λ1 and λ2. tenfold cross validation experiments were performed with all combination of k, λ1 and λ2 on the training set. m7GDisAI performed best when k is 90, λ1 is -2 and λ2 is -2 with AUC of 0. 728. For fairness, the impact of α on m7GDisAI was measured via tenfold cross validation experiments with fixed k, λ1 and λ2. To be specific, α is 0 means that ASS is ACHN, and m7GDisAI only utilizes m7G chemical similarities, while α is 1 indicates that ASS is ACNFHN, and m7GDisAI only utilizes m7G CNF similarities. Table α, and the highest AUC score is marked in bold.There are four parameters, rank α increases, AUC scores generally show an increased tendency except when α is 0.4, and reaches its maximum at 0.742 when α is 1. In other words, the more CNF similarities contribute, the higher the AUC scores achieved, and m7GDisAI has the best performance when only utilizes CNFHN. Table 7G site-centered sequence individually, while CNF features pay more attention to the context of site-centered sequence. Meanwhile, the Cosine coefficient reflects the similarity in trend instead of absolute value as the Jaccard coefficient calculates.In Table 7GDisAI, we conducted 10 runs of tenfold cross validation experiments by taking α as 1, which has the best performance in the Table 7GDisAI. Figure 7GDisAI. The AUC of global LOOCV was 0.769 while that of local LOOCV was 0.635. The ROC curves of LOOCV experiments are illustrated in the Fig. To further evaluate the robustness of mAs we can see from Fig. 7G sites were ranked in descending order according to their association scores with ovarian cancer, and the top 100 m7G sites were selected as potential ovarian cancer-associated sites. 98 host genes of these sites were further mapped out. To predict potential cellular processes and molecular functions that involve m7G methylation, we used the R package “clusterProfiler” to analyze and visualize the functional profiles of m7G host genes.Ovarian cancer is the most common cause of gynecological cancer-associated death . Over thp-value cutoff was set as 0.05, q-value cutoff 0.2, indicating statistical significance of associations between host genes and GO terms. Furthermore, “BH” method was used to adjust the p-value to control the false discovery rate, which was considered to be statistically significant. Considering the potentially biological complexities in which a gene may belong to multiple annotation categories, we utilized a gene-concept network to depict the linkages of gene and GO terms as a network. Figure GO terms include three subontologies, cellular component (CC), biological process (BP) and molecular function (MF), and they can be conducted via enrichGO function. In the parameter setting of the enrichGO function, we set the parameter “ont” to “ALL”, aiming at performing CC, BP and MF together. Additionally, the In Fig. To data, hereditary nonpolyposis colorectal cancer (HNPCC) is the third major cause of hereditary ovarian cancer, and HNPCC is caused by mutations in genes involved in DNA mismatch repair . MLH1 4 : Linux, Windows. Programming language: Python, R, JavaScript. Other requirements: Not specified. Python version 3.8.0 or higher, R version 4.0.3 or higher. License: GNU GPL. Any restrictions to use by non-academics: None.Project name: mAdditional file 1. Table S1: m7G-variant dataset. Additional file 2.  Table S2: The detailed information of diseases we collected.Additional file 3.  Table S3: m7G-disease association dataset.Additional file 4.  Table S4: m7G-disease association matrix ASD.Additional file 5.  Table S5: m7G chemical similarity matrix ACSN.Additional file 6.  Table S6: m7G CNF similarity matrix ACNFSN.Additional file 7.  Table S7: Disease semantic similarity network ADD. Additional file 8.  Table S8: Predicted ovarian cancer related m7G sites and their host genes.


Industrial wine yeasts owe their adaptability in constantly changing environments to a long evolutionary history that combines naturally occurring evolutionary events with human-enforced domestication. Among the many stressors associated with winemaking processes that have potentially detrimental impacts on yeast viability, growth, and fermentation performance are hyperosmolarity, high glucose concentrations at the beginning of fermentation, followed by the depletion of nutrients at the end of this process. Therefore, in this study, we subjected three widely used industrial wine yeasts to adaptive laboratory evolution under potassium chloride (KCl)-induced osmotic stress. At the end of the evolutionary experiment, we evaluated the tolerance to high osmotic stress of the evolved strains. All of the analyzed strains improved their fitness under high osmotic stress without worsening their economic characteristics, such as growth rate and viability. The evolved derivatives of two strains also gained the ability to accumulate glycogen, a readily mobilized storage form of glucose conferring enhanced viability and vitality of cells during prolonged nutrient deprivation. Moreover, laboratory-scale fermentation in grape juice showed that some of the KCl-evolved strains significantly enhanced glycerol synthesis and production of resveratrol-enriched wines, which in turn greatly improved the wine sensory profile. Altogether, these findings showed that long-term adaptations to osmotic stress can be an attractive approach to develop industrial yeasts. Saccharomyces wine yeasts includes spontaneous and naturally occurring events, such as heterozygosity, nucleotide and structural variations, introgressions, horizontal gene transfers, and hybridization, as well as human-enforced domestication effects [Saccharomyces cerevisiae lineages that are unique to several subpopulations . The history of the domesticated Saccharomyces breeds is mainly linked to fermentation processes; hence, there is a history of the evolution of wine yeast toward coping strategies that address fermentation-related challenges [Saccharomyces strains [An evolutionary history of  effects ,2,3,4. Iallenges . This inIndeed, wine yeast cells are subjected to a variety of stresses during winemaking processes, such as high osmolarity, high sulfite dosages, nutrient depletion, acid stress, anaerobiosis, as well as a constantly increasing concentration of ethanol. The latter in turn exerts high chaotropic activity , reducesS. cerevisiae exhibits high competitive abilities, manifesting in its dominance within microbial communities in both spontaneous and inoculated ferments [S. cerevisiae underlie in turn an adaptive evolutionary-based approach, which is used by many researchers to develop industrial microorganisms with novel characteristics. In general, adaptive laboratory evolution (ALE) [Because of its extremely phenotypic plasticity and intra-specific variability, ferments ,8. Colleon (ALE) ,10,11,12on (ALE) ,14.S. cerevisiae, serves as an antimicrobial chaotrope that prevents the growth and metabolism of other microbial species [The biotechnological usefulness of ALE strategy has been confirmed by several researchers to date, who employed the ALE approach to obtain thermotolerant , copper- species .Among other stress protectants that play a crucial role during winemaking processes is glycogen. It serves as the main carbon source and energy reserve as well as enhances cell viability under glucose deprivation conditions ,20.On the other hand, a high glucose concentration during fermentation affects the activity and stability of a wide range of yeast enzymes, for example, β-glucosidase ,2,21. ThBoth hyperosmolarity as well as a high glucose concentration or its deprivation are among the key parameters that are strictly linked to winemaking production, and they have a detrimental impact on yeast viability, growth, and fermentation performance ,2,23. ThIn this study, we subjected three industrial wine yeast strains to adaptive laboratory evolution under KCl-induced osmotic stress in order to develop wine yeast strains with increased fitness under high osmotic pressure as well as an increased ability for glycogen accumulation without the loss of their growth or fermentation abilities. Another aim of this study was to examine the levels of glycerol and resveratrol in wine products obtained with use of the KCl-evolved yeast strains.S. cerevisiae) [S. cerevisiae var. bayanus) [S. cerevisiae) [Three commercially available diploid wine yeast strains, previously described and characterized elsewhere ,25 were evisiae) , strain bayanus) , and strThe ancestral yeast strains, each from one single colony, were cultured in liquid Yeast Extract Peptone Dextrose (YPD) medium  at 28°C. The cells were grown for 6 generations before being diluted (1:100) into fresh YPD medium. Yeast cells were cultured for 250 generations under standard conditions (control) or under increasing concentrations of KCl, which was added to the YPD medium. The KCl concentration began at 0.5 M for 50 generations, followed by 1 M for next 50 generations, and reached a final concentration of: a) 1.25 M, or b) 1.5 M KCl for the last 150 generations. The concentration of 1.5 M KCl was established as non-cytotoxic through previous research for the yeast strains investigated. Both ancestral strains and the corresponding YPD-evolved derivatives served as controls for the corresponding KCl-evolved strains. The YPD-evolved derivatives of the ancestral strains 1, 2, and 3 are denoted in this study as 1_YPD, 2_YPD, and 3_YPD, respectively. The 1.25 M KCl-evolved derivatives of strains 1, 2, and 3 are abbreviated here as: 1_KCl_a, 2_KCl_a, and 3_YPD_a, respectively. In turn, the 1.5 M KCl-evolved strains 1, 2, and 3 are denoted as: 1_KCl_b, 2_KCl_b, and 3_KCl_b, respectively.At the end of the evolutionary experiment, samples of the ancestral and evolved (under standard conditions or in the presence of KCl) yeast strains were preserved as frozen (−80 °C) glycerol stocks. All analyses were performed with use of the yeast strains from glycerol stocks that were rescued in YPD agar plates  at 28 °C for 24 h and then grown under the same standard conditions  to enable the comparison and assessment of the features of the ancestral and evolved yeast cells.To determine growth kinetics, cells were washed, diluted (a) to a total volume of 150 μL with working solution of 5 × 106 cells/mL), and suspended in: a) YPD  medium (control), (b) YPD medium supplemented with 1.5 M KCl (osmotic stress), and (c) 30% YPGlu medium  (high glucose stress). Then, they were cultured at 28 °C, and their growth was monitored turbidimetrically at 600 nm in a microplate reader every 1 h during a 16-h period. All experiments were performed in triplicate.® Yeast Viability Kit  according to the manufacturer’s instructions. After staining with a mixture of FUN® 1 and Calcofluor® White M2R, cells were inspected under an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software. For each analysis, 200 cells were used.Cell viability was evaluated using a LIVE/DEADCell viability was evaluated for both ancestral strains and their evolved derivatives in: (a) YPD medium , (b) YPD medium supplemented with 1.5 M KCl, and (c) 30% YPGlu medium . Cell viability was assessed at the logarithmic phase of growth that was previously established for each growth condition. Cell viability was compared between the ancestral strains and their evolved derivatives separately for each growth condition variant.Preparation of agarose-embedded yeast DNA and PFGE separation of yeast DNA was performed according to Lewinska et al. .6, 1 × 105, and 1 × 104 cells mL-1 of a yeast exponential-phase culture (2 μL each) were spotted onto solid YPD medium , and, for stress tolerance analysis, onto solid YPD medium supplemented with 2 M KCl. Plates with yeast cells were incubated for 2 days at 28 °C, and then the growth of the evolved derivatives were compared to their corresponding ancestral strains. All experiments were performed in triplicate.To evaluate the effect of KCl-induced osmotic stress on the growth phenotype of the analyzed strains, spot test assays were performed Briefly, serial dilutions of 1 × 10Yeast colonies were evaluated for glycogen storage as described elsewhere . BrieflyLaboratory-scale fermentation in grape juice was performed as previously described  with smap < 0.05.The concentrations of glucose, fructose, glycerol, and resveratrol in the media were determined by HPLC at the end of the fermentation process. All experiments were performed in triplicate. Data are presented as the means ± SD. Statistical significance was assessed by one-way ANOVA and with the Tukey’s multiple comparison test. Significance was accepted at All experiments were performed on an HPLC Ultimate 3000 system  consisting of a charged aerosol detector ultra RS . Data processing was carried out with Chromeleon 6.8 software (Dionex). Chromatographic analysis was carried out at 35 °C. Samples were separated on a Shodex Ashipack NH2P-50 column packed with 5-μm shell particles (250 mm × 4.6 mm) and acetonitrile–water (75/25% v/v) mobile phase at a flow rate of 1.50 mL/min. The nitrogen (99.99%) gas flow rate was regulated automatically at 35 psi and monitored by the CAD device. The total time of analysis amounted to 20 min. HPLC-grade acetonitrile was obtained from J.T.Baker . The linearity of the detector response was determined by the square correlation coefficients of the calibration curves generated by injections of standard solutions. The range of linearity was established by injecting six different concentrations obtained by the dilution of a standard mixture of sugars and resveratrol in ultrapure water and methanol, respectively. Linearity was studied between 0.02 and 20 g/L and showed coefficients of determination (R2) ≥ 0.99 for all substances. The limit of quantification (LOQ) and limit of detection (LOD) for test substances were 0.02 and 0.01 g/L, respectively. The wine samples were prepared for analysis by centrifugation  and filtered through 0.45-um filter couplers PTFE .S. cerevisieae gene-specific primers: HOG1L: 5’-TTGACATCTCAGCCAGTTGC-3’ and HOG1P: 5’-TTTCCAAGGGTCTTGTTTGC-3’ for Hog1, GPD1L: 5’-TTTTGCCCCGTATCTGTAGC-3’ and GPD1P: 5’-CTCGCCTCTGAAATCCTTTG-3’ for Gpd1, GPD2L: 5’-TTTCCCAGAATCCAAAGTCG-3’ and GPD2P: 5’-CGGATTGACCGTTAAGCAAT-3’ for Gpd2, GPP1L: 5’-GCCAAGAAATGGTTCGACAT-3’ and GPP1P: 5’-CAACGATTTTACAGCCAGCA-3’ for Gpp1, GPP2L: 5’-GGTGCAGTTAAGCTGTGCAA-3’ and GPP2P: 5’-CCATTCCTGCCCTTCAGATA-3’ for Gpp2, as well as ActL: 5’-GCCTTCTACGTTTCCATCCA-3’ and ActP: 5’-GGCCAAATCGATTCTCAAAA-3’ for the actin gene. The PCR conditions were as follows: Denaturation at 95 °C for 5 min; 27–30 cycles of 95 °C for 30 s; 58 °C for 30 s for Hog1, Gpp1, and actin; 61 °C for 30 s for Gpp2 and Gpd2; or 62 °C for 30 s for Gpd1, followed by 72 °C for 30 s. The optimal amount of cDNA and the optimal number of cycles were determined for each primer pair to ensure the linearity of the PCR reaction. The expression of actin was used as a control. All experiments were performed in triplicate.The total RNA from the cells was isolated using a Universal RNA Purification Kit , treated with TURBO™ DNAse , and reverse transcribed with the use of an iScript™ Select cDNA Synthesis Kit  according to the manufacturer’s instructions. Semi-quantitative PCR were performed using OptiTaq PCR Master Mix  according to the manufacturer’s protocol with YRF1 genes) and copper and cadmium detoxification (CUP1 genes) [CUP1 are domestication-related traits in wine yeasts [Previously, we screened and characterized 17 industrial wine yeast strains for their response to generational and ethanol-mediated changes ,25, and 1 genes) . Such coe yeasts ,30. Simie yeasts ,31.Analysis of the strain 3 genome using next-generation sequencing revealed that this strain showed 47,575 high-quality variants, including single nucleotide variants and indels, compared to the reference S288C genome. Almost 60% of the affected genes were primarily involved in metabolic process, in particular protein metabolism and DNA/RNA metabolic processes . In addiIn this study, we subjected the abovementioned strains to KCl-induced osmotic stress for 250 generations. Although KCl is not present in grape must, it serves as an osmotic stressor that is widely used as a model for studies of osmotic stress in fungi ,19,32,33Because it is widely accepted that high osmotic pressure may affect the growth and viability of the yeast cells as well as the genome stability ,24,25, tChanges in the growth rate did not result from cell death as cell viability was not compromised (data not presented).Because genetic stability is one of the desirable characteristics of wine yeasts , we compAmongst other desirable features of wine yeast strains are high tolerance to osmotic stress as well as enhanced vitality. The latter term refers to the metabolic activity, fitness, or vigor of a starter culture and can be indirectly evaluated by measuring, i.e., storage molecules, including glycogen . TherefoAfter the end of the ALE experiment, we assessed an effect of 2M KCl-induced osmotic stress on the growth phenotype of the analyzed strains by using a spot test assay, which is a standard method to evaluate the growth phenotype ,36,37. WSacchaormyces accumulate storage carbohydrates very efficiently, as glycogen along with trehalose can reach 25% dry weight [GSY2, encoding glycogen synthase, exhibited enhanced viability under both laboratory growth and fermentation conditions in synthetic and natural musts. In particular, they showed increased resistance to glucose deprivation conditions [Similarly, adaptive evolution under osmotic stress conditions had an impact on strains 2 and 3 with respect to the ability for glycogen accumulation. This effect was observed for 2_KCl_a, 3_KCl_a, and 3_KCl_b strains throughout the 96 h of growth under control conditions and in the presence of 1.5 M KCl as was indicated by the reddish-brown coloration of yeast colonies after iodine staining B. Althouy weight . From thy weight  and are nditions . It shounditions .Because Tilloy et al.  used theTherefore, we performed a laboratory-scale fermentation in grape juice containing 18% or 30% sugars. At 21 days of fermentation, when sugars (glucose and fructose) in the media were exhausted, concentrations of glycerol in the media were determined. The highest increase in glycerol production was exhibited by one of the KCl-evolved derivatives of strain 1 (1_KCl_a strain), while KCl-evolved derivatives of strain 3 (3_KCl_a and 2_KCl_b) showed the lowest rises in glycerol synthesis, in comparison to the corresponding ancestral strains A.When 18% grape juice was used as fermentation media, concentrations of glycerol were 5.97 (± 0.62), 7.8 (± 0.82), 8.96 (± 1.32), and 6.86 (± 1.21) g/L for strains 1, 1_YPD, 1_KCl_a, and 1_KCl_b, respectively. In turn, for strains 3, 3_YPD, 3_KCl_a, and 3_KCl_b, concentrations of glycerol were 7.02 (± 0.78), 7.04(± 1.25), 8.89 (± 1.03), and 8.65 (± 0.93) g/L, respectively. In the case of strain 2, glycerol concentrations reached 5.68 (± 1.02), 7.24 (± 0.98), 8.24 (± 0.87), and 7.42 (± 1.24) g/L for strains 2, 2_YPD, 2_KCl_a, and 2_KCl_b, respectively A.Similarly, when fermentation was conducted in grape juice containing 30% sugars, the highest increase in glycerol production was observed for the strain 1_KCl_a, a derivative of strain 1, when compared to its ancestral strain. The lowest rises in glycerol content were observed when 30% grape juice was inoculated with KCl-evolved derivatives of strain 3. At the end of fermentation, the glycerol concentration reached 3.2 (±0.92), 5.43 (±0.99), 7.2 (±0.52), and 4.6 (±0.82) g/L for strains 1, 1_YPD, 1_KCl_a, and 1_KCl_b, respectively; 4.31 (±0.86), 5.2 (±0.99), 5.32 (±1.2), and 4.9 (±0.91) g/L for strains 2, 2_YPD, 2_KCl_a, and 2_KCl_b, respectively; and 4.76 (±0.89), 4.46 (±1.2), 5.26 (±0.99), and 5.81 (±1.2) g/L, respectively A.Our results showing adaptive evolution-mediated increases in glycerol production are consistent with data obtained by Tilloy et al. , who conMsn4 (multicopy suppressor of SNF1 mutation), encoding a stress-inducible transcriptional activator [It is widely known that intracellular glycerol is a key cellular osmoprotectant . Howeverctivator .Therefore, the next question that we asked was whether an enhanced tolerance of high osmotic stress as well as the increased levels of glycerol synthesis exhibited by the evolved strain might be linked to any changes in osmoresponsive gene expression. To answer this question, all investigated samples were collected after the end of the evolutionary experiment and were analyzed for their glycerol biosynthesis-related gene expressions.GPD1 and GPD2 [GPP1 and GPP2 [GPD1, GPP1, and GPP2 expression levels in the case of the KCl-evolved strain 3 (3_KCl_a strain), when compared to its ancestral strain , that is and GPD2 ,41,42, aand GPP2 ,44, whicl strain B.GPD1 and GPP2 [GPD2 [GPP1 [GPD2 during anaerobic fermentation was found to be of little significance [Nevertheless, it has been discussed whether all four genes played equally important roles in osmoadaptation. For example, Babazadeh et al. , who revand GPP2 . In suppP2 [GPD2  and GPP1D2 [GPP1 , respectD2 [GPP1 ,45. In aD2 [GPP1  showed tificance .GPD1 and GPP2 as well as GPP1 transcripts noticed in the 3_YPD_KCl_a , 0.60 (± 0.02), 0.99 (± 0.01), and 0.45 (± 0.02), respectively; and for strains 2_YPD, 2_KCl_a, and 2_KCl_b were 0.25 (± 0.02), 0.24 , 0.5 (± 0.01), and 0.24 (± 0.01) mg/L, respectively .In contrast, resveratrol concentrations were significantly decreased in the case of inoculations of grape juice with evolved derivatives of strain 3, when compared to its ancestral strain. The resveratrol concentration reached 0.93 (± 0.01) and 0.9 (± 0.01) mg/L for strain 3 growing in 18% and 30% grape juice, respectively. Decreases in its content were observed for strains 3_YPD (0.26 ± 0.01 mg/L), 3_KCl_a (0.52 ± 0.02 mg/L), and 3_KCl-b (0.46 ± 0.02) growing in 18% grape juice as well as for 3_YPD (0.24 ± 0.01 mg/L), 3_KCl_a (0.19 ± 0.02 mg/L), and 3_KCl-b (0.38 ± 0.02) growing in 30% grape juice .Saccharomyces yeasts [Sacchorymyces yeasts are often incorporated into mixed-culture fermentations; non-Sacchorymyces yeasts, in cooperation with Saccharomyces spp., greatly improve the chemical composition and sensory profile of wines [In general, many desirable enzymes, including β-glucosidases, which are responsible for modulating compounds of the grape must (by producing or releasing the preferred compounds from their precursors) and thereby affecting composition of wines, are found mainly in non-s yeasts ,2. Thereof wines ,2.Dekkera bruxellensis wine yeast [Polygonum cuspidatum into resveratrol [D. bruxellensis is generally considered the main spoilage yeast during winemaking due to the production of specific compounds like various pungent volatile phenols, associated with unpleasant aromas, which are collectively known as the phenolic taint or “brett” flavor [Vitis labrusca Bordô grapes were screened for β-glucosidase production; 33 out of the 308 analyzed strains were reported to be able to hydrolyze piceid into free resveratrol in synthetic media  [Hanseniaspora uvarum (n = 23), Hanseniaspora opuntiae (n = 4), Candida zemplinina (n = 3), S. cerevisiae (n = 1), Zygoascus meyerae (n = 1), and Zygosaccharomyces bailii (n = 1). With regard to S. cerevisiae, it also should be mentioned that only one strain out of 11 was able to hydrolyze piceid into resveratrol due to β-glucosidase activity [To date, several strategies have been developed to obtain resveratrol-enriched wines. Some of these approaches focus on developing genetically modified wine yeasts that will be able to produce resveratrol during fermentation. For example, Becker et al.  made an ne yeast . This enveratrol . Neverth” flavor ,2. Thereesculin) . Among tactivity .D. bruxellensis wine yeast, which previously had been subjected to the N-methyl-N0-nitro-N-nitrosoguanidine (NTG) mutagenesis, exhibited enhanced β-glucosidase activity towards piceid conversion into resveratrol [Here, we showed that adaptive evolution under osmotic stress can be a useful approach to develop yeast strains towards the production of wines with an increased content of resveratrol. Of course, there are some strain-dependent limitations. For example, the evolved derivatives of strain 3 failed to produce resveratrol-enriched wines. In fact, β-glucosidase activity, which is responsible for cleaving the glycosylated precursors of resveratrol, can be significantly decreased and/or inhibited under winemaking conditions, such as, i.e., low pH value, high initial glucose concentration, and high ethanol concentration . On the veratrol .Nevertheless, it also should be taken into consideration that many industrial relevant traits and performance parameters are polygenic and are not always recognized as being related to the particular trait. Besides, each biological system is the whole, which is greater than a sum of its parts. Due to these emergent properties, which result from numerous interaction effects between different organism elements, the final output of, i.e., metabolic engineering/reprogramming can be difficult or even impossible to predict . This isThe main aim of this study was to develop wine yeast strains with increased tolerance to high osmotic stress, enhanced ability to accumulate glycogen, as well as augmented capability to produce glycerol and resveratrol. All of these characteristics are desirable wine yeast properties that can greatly improve both the fermentation process as well as the sensory profile of wine. To achieve these goals, we employed adaptive evolution under osmotic stress and showed that (i) strains 2 and 3 greatly enhanced their tolerance to high osmotic stress, (ii) the evolved derivatives of strains 2 and 3 gained the ability to accumulate glycogen, (iii) strain 1 enhanced the synthesis of glycerol, while (iv) the evolved derivatives of strains 1 and 2 were able to produce resveratrol-enriched wines. Of course, it cannot be excluded that some of these adaptive-mediated changes might be stochastic and/or strongly depend on the genetic background of the individual strains. However, such an approach might be an attractive strategy to develop industrial wine yeast strains with desirable characteristics.