Mentha piperita L. (M_EO) and Rosmarinus officinalis L. (R_EO) essential oils are well-known for their biological effects , but their potential as complementary treatment in colorectal cancer is underexplored. The aim of the present study was to investigate the M_EO and R_EO in terms of chemical composition, antioxidant, antimicrobial, and cytotoxic effects in a colorectal cancer cell line—HCT 116. The gas-chromatographic analysis revealed menthone and menthol, and eucalyptol, α-pinene and L-camphor as major compounds in M_EO and R_EO respectively. M_EO exhibited potent antimicrobial activity, moderate antioxidant activity and a low cytotoxic effect in HCT 116 cells. R_EO presented a significant cytotoxicity in colorectal cancer cells and a low antimicrobial effect. The cytotoxic effect on non-cancerous cell line HaCaT was not significant for both essential oils. These results may provide an experimental basis for further research concerning the potential use of M_EO and R_EO for anticancer treatment.Colorectal cancer is one of the most frequently diagnosed forms of cancer, and the therapeutic solutions are frequently aggressive requiring improvements. Essential oils (EOs) are secondary metabolites of aromatic plants with important pharmacological properties that proved to be beneficial in multiple pathologies including cancer.  Colorectal cancer is a heterogenous pathology characterized by a landscape of genomic and epigenomic alterations  ,2 and ocEOs are defined as secondary metabolites of aromatic plants with a complex composition consisting in a mixture of different chemical compounds (oxygenated compounds and hydrocarbons), that exert key roles in the protection of plants against different assailants , in the signal transduction pathways within the plant, and in human health due to the plurality of innate pharmacological properties ,8,13. ThMentha piperita L. (M. piperita) and Rosmarinus officinalis L. . Previous in vitro and in vivo studies reported the anticancer potential of EOs against different types of cancer, as: lung, breast, melanoma, mouth, liver, colon, brain, prostate, pancreatic cancer, and leukemia ,14. An aMentha piperita L. commonly named peppermint is a native genus of the Mediterranean region  and the weakest against Gram negative bacteria Pseudomonas aeruginosa  while the antifungal effect was significant on both fungi Candida albicans  and Candida parapsilosis . R_EO, used in concentration of 10 μg/mL, exhibited a more pronounced effect than the positive control against Pseudomonas aeruginosa . In almost all cases, MBC was equal to MIC, with the highest value for R_EO, at the highest concentration (10 μg/mL), against P. aeruginosa. The exceptions were observed in the case of R_EO_1 against S. pyogenes where MBC was ten time higher than MIC and in the case of M_EO_3 against C. albicans where MFC was double than MIC  for 24 h and the cell viability percentage was calculated using 3--2,5-diphenyltetrazolium bromide (MTT) assay. It was noticed a decrease in the cell viability percentages starting with the concentration of 150 μg/mL, and the highest decrease was calculated for the highest concentration tested—500 μg/mL 81.15%—. These rAnother aspect investigated in the present study was the impact of M_EO on HCT 116 cells’ morphology , an impoBased on data from cell viability test and cells’ morphology evaluation, it was verified if the cytotoxic signs induced by M_EO treatment were specific for apoptosis or necrosis by the means of Hoechst staining . The conIn order to verify the selective cytotoxicity of M_EO on cancer cells, human immortalized keratinocytes—HaCaT were incubated for 24 h with M_EO  and analyzed in terms of cell viability and celullar morphology. The viability assessment indicated that the concentrations tested had no toxic impact on the cells’ viability percentage, at the highest concentration—500 μg/mL being calculated a viability percentage higher than 95% . As regards the effect on HaCaT cells’ morphology, as it can be seen in A similar experimental protocol was applied for the evaluation of R_EO cytotoxic potential in HCT 116 cells as the one described above for M_EO, and it can be observed that R_EO induced a stronger reduction of cell viability percentage in cancer cells as compared to M_EO . R_EO inSince R_EO treatment triggered a dose-dependent reduction of HCT 116 cells’ viability, we also analyzed its impact on cells’ morphology . No signFor the Hoechst 33,342 staining assay of R_EO were selected the same concentrations (10 and 150 μg/mL) as for M_EO, the selection being done on the same consideration as in the case of M_EO. The lowest concentration of R_EO—10 μg/mL had no impact on HCT 116 cells nuclei, whereas the highest concentration verified—150 μg/mL was associated with the presence of apoptotic cells characterized by the presence of nuclear shrinkage, nuclear fragmentation, chromatin condensation , it was observed a slight decrease of cells’ viability (around 90%) and changes in cells’ confluency only at the highest concentration—500 μg/mL . The shape of the HaCaT cells was not modified following the R_EO treatment and no signs of cytotoxicity (roundish cells floating and cell debris) were detected .Colorectal cancer represents one of the leading causes of death worldwide and in the last years, the interest of researchers was oriented towards the potential role of EOs in the prevention and treatment of this type of cancer and other colonic-related pathologies . The benBased on the different bioactive constituents, EOs act as multi-target molecules becoming potential alternatives for chemoprevention and cancer complementary treatment . Mentha piperita L. and Rosmarinus officinalis L. essential oils was previously proved against different cancer types, as: human cervix carcinoma (HeLa cells), human lung carcinoma (A549 and SPC-A1 cells), human non-small cell lung cancer (H1299 cells), human breast adenocarcinoma (MCF-7 cells) [The anticancer potential of 7 cells) ,32,33,347 cells) , human h7 cells) ,36, and 7 cells) ,38, stilTherefore, we analyzed the effects of two EOs commercially available (M_EO and R_EO) in colorectal cancer cells—HCT 116 in terms of chemical composition, antioxidant potential, antimicrobial activity, and cytotoxicity.The results obtained in the present study from the GC-MS analysis of the chemical composition are in accordance with those presented in the literature. The major compounds of M_EO were menthone and menthol  and also compared to R_EO which did not show significant activity in these strains. The greatest inhibition induced by M_EO was observed against S. pyogenes, confirming the data from previous studies which mention the higher susceptibility of Gram-positive species to EOs [Biological activity is closely related to chemical composition, and the existence of certain functional groups significantly influences this activity . M_EO sas to EOs . Mentha x piperita L. aetheroleum, the peppermint essential oil’s medicinal use related to gastrointestinal illnesses is as symptomatic treatment for mild spasm of the gastrointestinal and bile tract, flatulence, and irritable bowel syndrome  [According to the latest EMA’s assessment report on pletion) . Moreovepletion) ,46. In rpletion) . Severalpletion) .To the best of our knowledge, the data regarding the cytotoxic effect of M_EO in colon cancer cells are lacking. In the view of these facts, the present study proposed the analysis of the M_EO potential cytotoxicity in HCT 116 colon cancer cells by assessing its impact on cells’ viability, cellular and nuclear morphology. 50 = 10.89 µg/mL; 16.16 µg/mL and 38.76 µg/mL, respectively) cells [50 = 165.24 ± 4.40 c/w) and lung (A549—IC50 = 183.00 ± 23.10 c/w) cancer cells [50 values determined for these compounds being 4.5, 4 and 1 mM) [Our results indicate that M_EO 10–500 µg/mL) treatment for 24 h exhibited a low cytotoxicity in HCT 116 cells characterized by a dose-dependent reduction of cells’ viability percentage , signifiy) cells , (ii) moer cells , rat glier cells . Differe0 µg/mL t50) of peppermint oil analyzed in fasted Wistar male rats was found to be 4441 ± 653 mg/kg after 24 h and 2426 mg/kg after 48 h [Another aspect verified was the potential toxicity of M_EO on a non-cancerous cell line—HaCaT . Even at the highest concentration tested—500 µg/mL, no significant changes were noticed in HaCaT cells’ shape and confluence, and the cells’ viability did not decrease under the percentage of 95% . The verter 48 h , and theter 48 h .Rosmarinus officinalis L. essential oil (R_EO). Multiple biological properties of Rosmarinus officinalis L. essential oil were shown in previous studies, including antioxidant and antimicrobial effects (being used as biopreservative in food industry), anti-inflammatory, antidepressant, antinociceptive, DNA-protective, anticancer, and based on the EMA’s report R_EO is recommended for the treatment of dyspepsia and mild spasmodic pathologies of the gastrointestinal tract [The other essential oil tested in the present study was the al tract ,51,52. A50 = 3.06 µg/mL), human breast cancer  [50 = 0.011 µL/mL; MCF-7, IC50 = 0.253 µL/mL, inhibition of proliferation and migration) [In terms of cytotoxicity, our results indicated that R_EO proved to be more active on HCT 116 colon cancer cells as compared to M_EO, the cells’ viability percentage at the highest concentration tested—500 µg/mL being around 50% . The dec8 µg/mL) ,53, huma8 µg/mL) , human cgration) , and ovagration) ,36. Studgration) .4-induced toxicity) [As regards the impact of R_EO on HaCaT non-cancerous cells, it was recorded a slight decrease in cells’ viability only at the 500 µg/mL concentration (around 90%) and a reduction of cells’ confluency . These roxicity) , human poxicity)  and HUVEoxicity) . Even thoxicity) .Different studies have shown the complementary effects obtained with the association between essential oils and 5-fluorouracil (5-FU). In mice transplanted with sarcoma 180 tumor cells and treated with peppermint essential oil/5-FU (50 and 100 mg/kg/day respectively 10 mg/kg/day) showed a greater inhibition of tumor growth and a less severe leukopenia compared to treatment with only 5-FU . The rosThe essential oils R_EO and M_EO analyzed in the present study were purchased from Fares Biovital Laboratories  and present the following identification items: M_EO—Ulei Menta R 20, 10 mL for internal use , and R_EO—Ulei Rozmarin A11, 10 mL for internal use batch number: 10/19, reference number: 6040/14.10.2011).® 30-2007™) and Dulbecco’s modified Eagle Medium (DMEM) high glucose—4.5 g/L (ATCC® 30-2002™) were purchased from ATCC . All reagents complied with the analytical standard purity and were used according to the manufacturers’ recommendations.The reagents applied to perform the experimental part were: N-hexane , 2,2-diphenyl-1-picrylhydrazyl , ascorbic acid , methanol 99% , Columbia blood agar, Sabouraud agar and Mueller-Hinton agar , gentamycin (GM) and fluconazole (FCZ) disks , phosphate saline buffer (PBS), fetal bovine serum (FBS), penicillin/streptomycin, trypsin-EDTA solution, dimethyl sulfoxide (DMSO) and 3--2,5-diphenyltetrazolium bromide (MTT) reagent , and Hoechst 33342 nuclear staining assay . The cell culture media, McCoy’s 5A Medium (ATCCv/v), solvent n-hexane. The NIST 02 and Wiley 275 libraries spectra databases were employed to compare and identify the volatile compounds. The linear retention indices (LRI) were determined according to Van den Dool and Kratz formula, related to a homologous series of n-alkanes (C8–C24) [The chemical composition of the EOs was identified by GC-MS analysis. GS/MS QP 2010 Plus  connected at AT WAX 30 m × 0.32 mm × 1 μm capillary column was utilized and helium with a flow rate of 1 mL/min was the carrier gas. Briefly, the following steps have been applied: (i) temperature program—initially 40 °C followed by an increase of 5 °C/min until reaching the final temperature of 210 °C (for 5 min), injector temperature 250 °C, ion source 220 °C; (ii) injection volume 1 μL at a split ratio of 1:50; dilution of samples 1:10 ((C8–C24) .The antioxidant capacity of the EOs was evaluated using the DPPH assay, according to the method described in the literature ,51 with Streptococcus mutans (ATCC 35668™), Streptococcus pyogenes (ATCC 19615™), Staphylococcus aureus (ATCC 25923™), Escherichia coli (ATCC 25922™), Pseudomonas aeruginosa (ATCC 27853™) and for antifungal activity against Candida albicans (ATCC 90029™) and Candida parapsilosis (ATCC 22019™)  using the Disk diffusion method for susceptibility testing, according to the Standard Rules for Antimicrobial Susceptibility Testing using Impregnated Disks. In brief, the following steps were realized: (i) the Mueller-Hinton agar, supplemented for streptococci with horse blood and ß-NAD, was inoculated with microbial standardized suspension (0.5 Mc Farland); (ii) 10 µL from each sample was added to a blank disk , placed on top of the agar; (iii) the plates were incubated at 37 °C for 24 h; (iv) the inhibition zone diameters were determined in millimeters. For all bacterial strains were performed triplicate disk-diffusion tests. The positive controls were gentamycin  for bacteria and fluconazole  for fungi, while negative control was the solvent (ethanol).The EOs antibacterial activity was tested against The dilution tests were conducted according to previous studies based on the recommendations of the Clinical Laboratory and Standard Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) ,64,65,66® CCL-247TM) and immortalized human keratinocytes—HaCaT. The HCT 116 cells were cultured in their specific culture medium—McCoy’s 5A medium, whereas the HaCaT cells [2 and a temperature of 37 °C in a humidified incubator. Two cell lines acquired from ATCC (American Type Cell Collection) and CLS Cell Lines Service GmbH , respectively, were used as in vitro models, namely: human colorectal carcinoma cells—HCT 116 (ATCCaT cells  required4 cells/200 µL complete medium/well) and when the confluence was reached were incubated with different increasing concentrations of EOs (10–500 µg/mL) for a 24 h period; (2) after the 24 h incubation period, the old medium was replaced by fresh culture medium (100 µL/well) and a volume of 10 µL/well of MTT solution (5 mg/mL) was added in each well followed by a 3 h incubation; (3) the formazan crystals formed were dissolved in 100 µL of solubilization buffer provided by the manufacturer during a 30 min repose at room temperature and dark; (4) the reduced MTT was spectrophotometrically analyzed at 570 and 630 nm, using the Cytation 5  microplate reader. All experiments were performed in triplicate. The results were expressed as percentage of viable cells (%). The stock solutions of M_EO and R_EO were prepared in DMSO, and the following dilutions were performed in culture medium.The impact of EOs on cell viability was assessed using the MTT assay. The experimental protocol was applied according to our previous studies  and adapTo identify the potential changes/alterations of cellular morphology following EOs treatment , the cells were observed using the Olympus IX73 inverted microscope  under bright field and photographed at the end of the 24 h incubation period. The images obtained were analyzed using the cellSens Dimensions v.1.8. Software .5 cells/1.5 mL complete medium/well) were cultured in 12-well plates and incubated for 24 h with two concentrations (10 and 150 µg/mL) of each essential oil: M_EO and R_EO; (2) after 24 h, the old medium was removed, the staining solution 0.5 mL/well (1:2000 in PBS) was added and the cells were incubated for 10 min at room temperature and dark; (3) the cells were washed with PBS (3x) and photographed using the Olympus IX73 inverted microscope . The nuclear staining images were analyzed using the cellSens Dimensions v.1.8. Software . As positive control for apoptosis was used a Staurosporine solution—5 µM (3 h at 37 °C) and for necrosis–Triton X-100 (30 min at 37 °C)—0.5%.To define the type of cytotoxic effect induced by essential oils treatment to colorectal cancer cells—HCT 116 it was used the Hoechst 33342 nuclear staining assay. The implemented protocol was detailed in our previous study  and was www.graphpad.com, accessed on 10 May 2022). The results were expressed as means ± SD. The differences between data were compared by performing the one-way ANOVA test and Dunnett’s multiple comparisons post-test. The statistically significant differences between data were labeled with * . The normal distribution was also verified by applying the Kolmogorov-Smirnov test (GraphPad Prism version 9.3.1) and the p value obtained was p < 0.0001.The software used for the statistical analysis was GraphPad Prism version 9.3.1 for Windows  exerted potent antimicrobial activity on S. pyogenes, poor cytotoxicity in colorectal cancer cells—HCT 166 and no toxic effects in non-cancerous cells—HaCaT. As regards the Rosmarinus officinalis L. commercial essential oil , showed low anti-microbial effects, and a significant cytotoxic effect in HCT 116 cells (reduced cell viability and apoptotic-like features) as compared to M_EO. Future research directions require the evaluation of biological effects in association with other classes of compounds  to evaluate synergic actions with a beneficial role in colon tumor diseases.Essential oils composed of complex mixtures of hydrophobic and volatile compounds present numerous pharmacological properties but the access to clinical studies is difficult based on their complexity but also on the significant variations between the compositions of the same oil having different geographical origins. Therefore, to obtain complete, reproducible and clinical results, an adequate physico-chemical and biological characterization is mandatory. The present study provides evidence that the


Therefore, we fused FGF21 to transferrin (Tf) via a furin cleavage site (F), to promote uptake from the intestine into the portal vein, yielding FGF21-F-Tf, and established its production in both seeds and leaves of commercial Nicotiana tabacum cultivars, compared their expression profile and tested the bioavailability and bioactivity in feeding studies. Since biopharmaceuticals need to be produced in a contained environment, e.g., greenhouses in case of plants, the seed production was increased in this setting from 239 to 380 g m–2 a–1 seed mass with costs of 1.64 € g–1 by side branch induction, whereas leaves yielded 8,193 g m–2 a–1 leave mass at 0.19 € g–1. FGF21-F-Tf expression in transgenic seeds and leaves yielded 6.7 and 5.6 mg kg–1 intact fusion protein, but also 4.5 and 2.3 mg kg–1 additional Tf degradation products. Removing the furin site and introducing the liver-targeting peptide PLUS doubled accumulation of intact FGF21-transferrin fusion protein when transiently expressed in Nicotiana benthamiana from 0.8 to 1.6 mg kg–1, whereas truncation of transferrin (nTf338) and reversing the order of FGF21 and nTf338 increased the accumulation to 2.1 mg kg–1 and decreased the degradation products to 7% for nTf338-FGF21-PLUS. Application of partially purified nTf338-FGF21-PLUS to FGF21–/– mice by oral gavage proved its transfer from the intestine into the blood circulation and acutely affected hepatic mRNA expression. Hence, the medication of NASH via oral delivery of nTf338-FGF21-PLUS containing plants seems possible.Non-alcoholic steatohepatitis (NASH) is a global disease with no effective medication. The fibroblast growth factor 21 (FGF21) can reverse this liver dysfunction, but requires targeted delivery to the liver, which can be achieved  To date, non-alcoholic steatohepatitis (NASH) is a widespread disease in developed countries with a total prevalence of 0.4 billion patients . NASH isvia oral delivery, rather than administered systemically, as proteins are absorbed in the intestine and directly transported to the liver via the portal vein before entering the blood circulation (A recently proposed drug candidate for the treatment of NASH is the fibroblast growth factor 21 (FGF21) , an autoculation .via the ganglioside GM1 receptor. However, CTB is a strong immune trigger, which limits the possibility of long-term application. Therefore, Ex-4 was fused to the non-immunogenic human iron-carrier protein transferrin (Tf), enabling the uptake via the enterocyte Tf-receptor. When applied orally, Ex-4-Tf reduced blood glucose levels by 31% compared to the 25 and 20% achieved with Ex-4-CTB and unmodified Ex-4, respectively , compared to 10−20 g kg–1 protein per leaf fresh mass (LFM) [corresponding to 83−167 g kg–1 leaf dry mass (LDM)] , and seeds have been used for several oral vaccination trails. In some of the corresponding feeding studies, tobacco seeds were used because the production of recombinant proteins is well established in this system, and they are edible due to the absence of alkaloids such as nicotine ] , and diss (LDM)] . For exao leaves    were used for targeting to the endoplasmic reticulum (ER). All constructs contained a C-terminal double His-tag for Immobilized Metal Ion Affinity Chromatography (IMAC) purification . BamHI/NruI, BsaI/BsaI, and NcoI/XbaI restriction sites were added to the 5′ and 3′ end of the final nucleotide sequence to allow directional cloning into the p9U , the MagnICON vectors pICH29921 and pICH31160 , ICF320 (MagnICON) and GV3101:pMP90RK (pTRAc).The amino acid sequences for the individual proteins  were assICH31160  and the ICH31160 , respectNicotiana tabacum cultivars VG and SL632 were used in this study. The seeds were surface sterilized in a saturated calcium hypochlorite solution with 0.1% (m v–1) Triton X-100 for 5 min. The seeds were rinsed with sterile distilled water several times and then germinated on Linsmaier and Skoog (LS) medium  supplemented with 30 g L–1 sucrose, 6.5 g L–1 plant agar and adjusted to pH 5.7. The plants were maintained at 24/22°C day/night temperature with a 16 h photoperiod. Subsequently, 4-week-old tobacco leaves were used for Agrobacterium-mediated transformation (–1 kanamycin and 0.5 g L–1 cefotaxim. Transgene integration was confirmed by PCR using FGF21- and Tf-specific primers (ormation . Regener primers .–1) Hakaphos Blue  or 0.1% (m v–1) Ferty 2 Mega .Transgenic individuals were transferred from tissue culture 4 weeks after the last subculture directly into 5-L pots containing peat soil . Plants were fertilized twice a week using 0.2%  and fertilized with 0.2% (m v–1) Hakaphos Blue or 0.1% (m v–1) Ferty 2 Mega, in both cases with additional illumination (140 μmol s–1 m–2) to ensure a 16 h photoperiod. A. tumefaciens was cultivated either in lysogeny broth (LB)  for 24−48 h at 28°C in an orbital shaker (150 rpm) before dilution 1:100 in the same medium. After another 24−48 h of incubation at 28°C, the bacteria were centrifuged  and resuspended in infiltration buffer  to a final ODnm600 of 0.1−0.5. For vacuum infiltration, N. benthamiana plants were submerged upside down into the bacterial suspension, a vacuum of 100 mbar applied for 2 min in a desiccator, and the plants then returned to normal growth conditions. N. benthamiana leaf material was harvested 10 days post-infection (dpi) for the MagnICON vectors and 5 dpi for the pTRAc vectors.The oth (LB)  or peptooth (LB)  containig, 20 min, 4°C) and the supernatant, containing the FGF21-transferrin fusion protein, collected.Each 100 mg seed or leaf material were homogenized in extraction buffer  at a biomass/buffer ratio of 1:10 and 1:3, respectively, using a FastPrep-24 5G  and 0.5 g Zirconia/Silica beads for 3× at 8 Hz for 40 s with 30 s breaks on ice in between. The homogenate was clarified by 2 × centrifugation  before final sterile filtration using Sartopore 2 Capsule (pore size of 0.45 and 0.22 μM) . The clarified extract , containing the FGF21-transferrin, was used for further purification.The extraction and purification for IMAC was carried at 4°C. Each 800 g leaf material was homogenized in extraction buffer  at a ratio of 1:3 biomass using an HR3655/00 Standmixer  3× for 30 s with 30 s breaks. In the standard protocol, the homogenate was initially clarified by a BP420 bag filter , then passed through a combination of K700  and KS50  depth filters  or applied to 2 × ultracentrifugation  extraction buffer, the FGF21-transferrin fusion protein was eluted with 3 CV elution buffer . A Vivaspin 15R centrifugal concentrator with a molecular weight cut off (MWCO) of 30 kDa  was used for buffer exchange into phosphate buffered saline (PBS) .His-tagged FGF21-transferrin was purified on an ÄTKA pure system  by applying 4 L filtrated extract to a XP26/20 column packed with 58 mL IMAC Chelating Sepharose Fast Flow resin charged with Nivia Pierce Coomassie Protein Assay-Kit .The total soluble protein (TSP) concentration in extracts and supernatants, including the FGF21-transferrin fusion protein and the host cell proteins (HCPs), was quantified –1) β-mercaptoethanol and 100 mM dithiothreitol (DTT). Samples that were extracted with the NuPAGE LDS sample buffer were directly applied to Western blot analysis. The samples were denatured at 70°C for 10 min, separated on Invitrogen Novex NuPAGE 4−12% (m v–1) Bis-Tris protein gels  at 200 V at 22−24°C for 35 min, and transferred to Amersham Protran nitrocellulose membranes  at 50 V at 22−24°C for 1 h using a Trans Blot Cell . The membrane was blocked with PBS containing 0.05% (m v–1) Tween-20 (PBST) and 5% (m v–1) skimmed milk powder for 1 h at 22−24°C. After washing 3× with PBST for 5 min, the membrane was probed at 4°C overnight with a polyclonal rabbit anti-His antibody  diluted 1:5,000 (0.1 mg L–1) or a polyclonal rabbit anti-FGF21 antibody  diluted 1:5,000 (0.2 mg L–1) in Signal Boost ImmunoReaction Enhancer solution I . Following another wash in PBST, the membrane was probed at 22−24°C for 1 h with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit diluted 1:5,000 (0.06 mg L–1) in Signal Boost ImmunoReaction Enhancer solution II. After another wash with PBST, the signal was detected using the NBT/BCIP alkaline phosphatase system . Densitometric analysis of Western blot signals were conducted using the AIDA Image Analyzer analysis software .For Western blot analysis, 100 μg or 10 μg TSP per sample were precipitated with trichloroacetic acid (TCA) and resuspended in NuPAGE LDS sample buffer, optionally supplemented with 0.5%  for both via anti-FGF21 Western blot analysis.Intact FGF21-transferrin fusion protein accumulation was obtained through multiplying the ELISA-derived total product concentration by the fraction of intact FGF21-transferrin determined –/– mice generated previously .Oral delivery experiments were performed in adult FGF21eviously  that wer studies , 1 mg kg–1) Triton X-100, pH 7.4] at a ratio of 1:3 tissue mass using a TissueLyser LT  for 3 min at 50 Hz, and subsequent freeze-thaw (−20°C overnight) and centrifugation .Plasma samples for the FGF21 ELISA were undiluted, and 30 mg ground tissue was homogenized in tissue extraction buffer [10 mM Tris-HCl and 0.02% . Gene expression was calculated as ddCT, using B2m as the normalizer gene . p-Values of 0.05 (* and letters), 0.01 (**) and 0.001 (***) were considered to indicate significant, strongly significant and highly significant differences as indicated in the individual experiments. There were no non-normal distributed data.Statistical analysis was performed in Origin 2020b . The Shapiro–Wilk- and N. tabacum VG  because this cultivar is nicotine-free and has been bred to a high seed yield of >9 tons SDM per hectare and year under open field conditions . However, SL632 has not yet been used for seed production in a greenhouse setting and thus required cultivation strategy adaptation to increase seed productivity.Biopharmaceutical safety is often ensured by the production in contained environments, e.g., greenhouses in case of plants . The corbacum VG , which i–1 (Under standard cultivation conditions (uncut), both tobacco cultivars grew up to a height of 1.98−2.20 m, and when harvested after 136 and 107 days, SL632 yielded ∼43 g SDM per plant compared to 18 g SDM per plant in case of VG . Notewor–1 .N. benthamiana used for transient expression (N. benthamiana with production costs of 0.19 € g–1 (SL632), 0.14 € g–1 (VG) and 0.13 € g–1 (N. benthamiana)  . Moreovehamiana) . In thishamiana) .via tobacco seeds, we used mature human FGF21 without O-glycosylation site to obtain a homogenous product as previously described , which limited the vegetative propagation to six passages. On the other hand, VG flowered only occasionally, was propagated for at least 19 passages and provided almost twice the number of positive transformants , 4. Note–1 SDM in seeds of the T1 descendants for both cultivars  and 1.54 (VG) mg m–2 a–1 in seeds , to identify potential steric hindrances prior to cloning, but did not observe any problems , FGF21 could be detected by ELISA in plasma obtained from the Vena cava in three and in liver homogenates in two out of four treated mice after 4 h , was unaffected by FGF21, while its downstream target, the fatty acid synthase (Fasn)  seemed also to be downregulated, while the expression of one of the targets of Ppara, Cpt1a , a key enzyme in β-oxidation,   , seemed e (Fasn) . Unexpecidation,  was unchidation, . However(PGC-1α) , even thWe showed that a plant-derived FGF21-transferrin fusion protein can be used for oral delivery of FGF21 as a potential therapeutic of NASH and provide an initial comparison of different production options and associated costs in transgenic seeds and leaves which both might be used for oral delivery.–2 a–1 in a greenhouse with manufacturing costs of 1.64 € g–1 SDM compared to 8,193 g m–2 a–1 and costs of 0.19 € g–1 LFM for leaves.We demonstrated for SL632 that the tobacco seed yield can be improved by pruning to 380 g m–1  than in transgenic leaves (41%) , indicator SL632 , even thor SL632 .in vivo trial using FGF21–/– mice .H-WH, HN, CB, SK, IB, and JB conceived the experiments. H-WH and CB conducted the experiments and analyzed the data. H-WH and HN wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


It is involved in general stress responses including heat, oxidative, surface, starvation stress, and macrophage infections. Despite having an extremely short half-life, SigB tends to operate downstream of at least three stress-responsive extra cytoplasmic function (ECF) sigma factors  and SigF involved in multiple signaling pathways. There is very little information available regarding the regulation of SigB sigma factor and its interacting protein partners. Hence, we cloned the SigB gene into pET28a vector and optimized its expression in three different strains of E. coli, viz., (BL21 (DE3), C41 (DE3), and CodonPlus (DE3)). We also optimized several other parameters for the expression of recombinant SigB including IPTG concentration, temperature, and time duration. We achieved the maximum expression of SigB at 25°C in the soluble fraction of the cell which was purified by affinity chromatography using Ni-NTA and further confirmed by Western blotting. Further, structural characterization demonstrates the instability of SigB in comparison to SigA that is carried out using homology modeling and structure function relationship. We have done protein-protein docking of RNA polymerase (RNAP) of Msmeg and SigB. This effort provides a platform for pulldown assay, structural, and other studies with the recombinant protein to deduce the SigB interacting proteins, which might pave the way to study its signaling networks along with its regulation.Sigma factor B (SigB), an alternative sigma factor (ASF), is very similar to primary sigma factor SigA ( Based on the structure and function, σ factors are classified into two major groups, viz., (i) sigma 70 (σ70), i.e., primary sigma and (ii) sigma 54 (σ54) which is involved in nitrogen fixation and found mostly in plant growth promoting rhizobacteria. Further, σ70 is classified into four major groups based on the domain organization, viz., groups 1, 2, 3, and 4. Group 1 (SigA) has an extended N-terminal region and four highly conserved regions, namely, regions 1, 2, 3, and 4. The group 2 (SigB) does not have the N-terminus extended region, but all the four conserved regions are present. The group 3 (SigF) has only three regions, viz., 2, 3, and 4 while the group 4  sigma factor) contains only regions 2 and 4. The regions 2 and 4 are extremely important in promoter recognition and binds at -10 and -35 regions of the promoter, respectively [Prokaryotic transcription is mediated by RNA polymerase (RNAP) which is made up of five core subunits αββ′ω thatσ70 being the principle σ factor contributes in the expression of all housekeeping genes under normal conditions [σ70 has broad range promoter recognition efficiency but ASF deal with very high promoter recognition stringency. By these mechanisms that regulate transcription initiation, the organism ensures its survival and adaptability [nditions , 4. Howenditions , 6. Due tability . SigB retability , 10, 11.tability –15.E. coli, it is only 2 minutes [In contrast to SigA, the half-life of Msmeg SigB is very short (less than 8 mins.) and in  minutes , 16–18.  minutes –22. This minutes , 23–26. E.coli DH5α  was used as cloning host, and BL21 (DE3), C41 (DE3) (Millipore Sigma (Novagen)), and CodonPlus (DE3) (Agilent Technologies) cells were used as expression host strains. The pET28a expression vector , Taq DNA polymerase , restriction enzymes , T4 DNA ligase , and molecular biology grade reagents were obtained from Sigma-Aldrich (USA). His-Tag monoclonal antibody and secondary anti-mouse IgG (H + L) (peroxidase/HRP conjugated) were from Puregene: Genetix Biotech Asia Pvt. Ltd., respectively.SigB (MSMEG_2752) gene of M. smegmatis mc2155. The primer pair used to amplify the SigB gene from the genomic DNA of M. smegmatis mc2155 was forward primer SigB_2752_EcoRI 5′-CGGAATTCATGGCAAATGCCACCACAAGCC-3′ and reverse primer SigB_2752_EcoRI 5′-CGGAATTCGGAGGCGTAGGAGCGGAGGCGG-3′ respectively, where underline sequences are showing the EcoRI restriction site. The condition for PCR amplification was initial denaturation at 95°C for 5 minutes, 35 cycles , and final elongation 72°C for 5 minutes. The amplified PCR product was ligated into the EcoRI digested and dephosphorylated pET28a expression vector. The ligation product was transformed into DH5α cells, and the recombinant positive clones (His-SigB) were confirmed by EcoRI restriction digestion and sequencing.The nondirectional cloning strategy was employed for the cloning of E.coli, viz., BL21 (DE3), C41 (DE3), and CodonPlus (DE3) [λDE3 lysogen that carries the gene T7 RNA polymerase under the control of lacUV5 promoter. So, IPTG  induction for the protein expression was performed to obtain maximum expression. The selected clone was inoculated from a single colony and cultured overnight in 5 ml Luria Bertani (LB) containing 50 μg/ml kanamycin at 37°C in a shaking incubator at 150 rpm. Next day, 1% (v/v) of secondary culture was incubated at 37°C until the OD600 of the culture reached ~0.4 O.D., and the culture was induced with 0.1 mM IPTG and grown for 3 hrs at 37°C at 150 rpm. For analysis of the His-SigB protein expression, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by [600 equivalent cell lysate prepared in 4× sample loading buffer , 200 mM DTT, and 0.05% bromophenol blue) after heat denaturation at 100°C was loaded onto the gel. For visualization, the gel was stained by Coomassie brilliant blue (R-250) followed by destaining.The clones containing His-SigB in positive orientation were transformed into three expression host strains of us (DE3) . These eribed by . For SDSE. coli strains and the optimum protein expression, we selected best expressing host strain for a range of IPTG concentrations, viz., 0.05, 0.1, 0.25, and 0.5 mM and induced at approximately <0.4 OD600 at 37°C 150 rpm for 3 hrs. To determine the solubility of protein at various IPTG concentrations, the pellet/supernatant fractionation of the lysate was performed. The harvested cell cultures were washed and resuspended in 1× phosphate buffer saline  and thereafter sonicated at 15% amplitude (ultrasonic homogenizer) for 2-3 min. The lysate was clarified by centrifugation at 13,000 rpm for 20 min at 4°C containing soluble His-SigB in the supernatant fraction while the pellet fraction was resuspended in equal volume of lysis buffer (1× PBS). The protein bands were visualized by electrophoresis in denaturing SDS-PAGE after staining with Coomassie R-250 as mentioned above. Semiquantitative analysis of the gels was performed with Quantity One software (Bio-Rad).Commonly, IPTG was used to achieve high level of expression for recombinant proteins. To optimize the concentration of IPTG on To optimize suitable temperature for protein expression, the best expressing host strain cultured at different temperatures, viz., 37°C, 25°C, and 16°C. The cells were induced with 0.1 mM IPTG at 37°C and 25°C for 3 hours, while at 16°C for 12 hours with shaking at 150 rpm. The protein bands were visualized by SDS-PAGE, and solubility fractionation analysis was done as above.To check the best time point for the expression of His-SigB in the best expressing host strain, it was induced with 0.1 mM IPTG at 25°C/150 rpm for different time intervals, viz., 0, 15, 30, 60, 120, and 180 min. The expression profile was visualized in 10% SDS PAGE.The recombinant His-SigB was purified by affinity using Ni-NTA beads as per manufacturer's instructions. Briefly, the cells were lysed by sonication at 15% amplitude in buffer A (Tris.Cl 50 mM (pH 8.0), NaCl 300 mM, imidazole 10 mM, Triton X-100 1% (v/v), PMSF 2 mM, lysozyme 1 mg/ml), and the soluble lysate was clarified by centrifugation at 13,000 rpm  for 15 min. at 4°C. The preequilibrated Ni-NTA beads (Qiagen) in buffer A were incubated with the lysate at 4°C for 30 mins., washed thrice with buffer B (Tris.Cl 50 mM (pH 8.0), NaCl 300 mM, Imidazole 50 mM, Triton X-100 0.5% (v/v)), and eluted thrice with buffer C (Tris.Cl 50 mM (pH 8.0), NaCl 300 mM, imidazole 400 mM, Triton X-100 0.5% (v/v)).μg/μl, 1 : 1000 in 1% PBST; Puregene) for 1 hr., washed with PBST, and further incubated with secondary antibody anti-Mouse IgG  in PBST. Finally, the blot was washed thrice by PBST, developed in ClarityTm ECL Bio-Rad, and analyzed by the ChemiDoc Imaging System (Bio-Rad).The purified His-SigB was resolved by SDS-PAGE and transferred to the PVDF membrane followed by blocking in 5% (w/v) skimmed milk powder (HiMedia laboratories) in 1× PBST . The blot was washed thrice by 1× PBST and incubated with anti-His antibody  database [2 server [2 server utilizes multiple templates to produce the accurate model of the protein. In case of M. smegmatis SigA, three templates were used, namely, 5TW1 (RNA polymerase sigma factor) [M. tuberculosis) [E. coli) [M. smegmatis SigB model was build using two templates 5TW1 (RNA polymerase sigma factor) [M. tuberculosis) [The protein sequences of database . To cons2 server . Phyre2  factor) , 6C05 (cculosis) , and 4YGE. coli)  with per factor)  and 6C05culosis)  with perculosis) .https://www.chemcomp.com/Products.htm), workbench. Structure issues such as missing atoms and missing chains were automatically corrected using quick prep module. Optimal hydrogen positions and charges were optimized using protonated 3D module using the MOE software recommended settings. We have used here core enzyme of 6YED [The protein-protein docking utilizes a multistage method for generating poses followed by their rankings. Here, we have used MOE platform  for the  of 6YED  PDB with of 6YED  was conslac operator for the IPTG inducible expression of downstream recombinant genes. However, these genes can only be expressed when T7 RNA polymerase is made available in expression hosts containing a DE3 lysogen under the control of IPTG inducible lacUV5 promoter in their genomic DNA [E. coli containing the DE3 lysogen, viz., BL21 (DE3), C41 (DE3), and CodonPlus (DE3) for the optimization of the expression of recombinant His-SigB. Empty pET28a vector and uninduced recombinant His-SigB clones were used as negative control for the expression host strains BL21 (DE3), C41 (DE3), and CodonPlus (DE3) in The pET28a vector belongs to the pET series of vectors that contain a T7 promoter with omic DNA . Hence, CodonPlus (DE3) depicted the maximum expression of the recombinant His-SigB protein; hence, it was decided to optimize the maximum protein expression at different IPTG concentrations (0.05-0.5 mM). The expression of His-SigB at IPTG concentrations: 0.05, 0.1, and 0.25 mM was comparable which was approximately 30% higher than the expression at 0.5 mM concentration , β (chain C), β′ (chain D), and ω (chain E) . The ith SigB . Arg846 E. coli and occur commonly in GC-rich genomes like Mycobacterium [Although, SigB is dispensable for growth in both Msmeg and Mtb , 37, nevacterium , 40. SigDuring the optimization with different IPTG concentrations, it was clearly observed that the total protein expression was higher at 0.1 mM IPTG concentration Figures . The IPTM. smegmatis RNA polymerases SigA and Sig B showed 60% sequence identity. Sigma factor A differs from sigma factor B in possessing N-terminal domain, which may provide stability. Our docking studies demonstrated that SigB binds to RNAP where chain C and chain D of RNAP play a significant role in molecular interactions. In silico comparative binding studies of SigA and SigB demonstrated that they both bind to similar regions of RNAP. In future, our aim will be to cocrystallize SigB-RNAP together to better understand the mechanism of their molecular interactions.However, the highly unstable nature of this protein can be further improved by using various stabilizers and (or) another cofactor so that future experiments like protein-protein interactions and crystallization of SigB can be carried out smoothly. Mycobacterium in coordination with ECF and other sigma factors. Higher quantities of soluble and purified recombinant SigB are essential for the study of its structure, function, and regulation but there is dearth of literature that depicts it. In this report, we demonstrate an efficient method for the production of soluble and purified recombinant His-SigB protein that can be used as a tool to unravel the structure, function, and regulation of its signaling cascade.SigB has significant similarities with house-keeping sigma factor SigA with regards to the domain organization; however, it has drastically short half-life as compared to SigA. Despite its short half-life, it is also involved in multiple stress response regulatory pathways of


Aggressive invasiveness is a common feature of malignant gliomas, despite their high level of tumor heterogeneity and possible diverse cell origins. Therefore, it is important to explore new therapeutic methods. In this study, we evaluated and compared the effects of graphene (GN) and reduced graphene oxides (rGOs) on a highly invasive and neoplastic cell line, U87. The surface functional groups of the GN and rGO flakes were characterized by X-ray photoelectron spectroscopy. The antitumor activity of these flakes was obtained by using the neutral red assay and their anti-migratory activity was determined using the wound healing assay. Further, we investigated the mRNA and protein expression levels of important cell adhesion molecules involved in migration and invasiveness. The rGO flakes, particularly rGO/ATS and rGO/TUD, were found highly toxic. The migration potential of both U87 and Hs5 cells decreased, especially after rGO/TUD treatment. A post-treatment decrease in mobility and FAK expression was observed in U87 cells treated with rGO/ATS and rGO/TUD flakes. The rGO/TUD treatment also reduced β-catenin expression in U87 cells. Our results suggest that rGO flakes reduce the migration and invasiveness of U87 tumor cells and can, thus, be used as potential antitumor agents. Gliomas are the most common primary malignancy of the central nervous system . Stage IMoreover, the common feature of gliomas is high invasiveness, despite strong biological heterogeneity . Thus thMoreover, fibronectin, the dominant component of the extracellular matrix (ECM), is involved in the development of GBM intrusion. GBM shows increased invasion activity in the presence of fibronectin . HoweverTherefore, when it comes to rGO flakes, a crucial discovery may be the reduction in the content of oxygen-containing functional groups that may disrupt signaling pathways in the cell membrane or directly interact with cellular structures such as integrin α5, integrin β1, β-catenin, N-cadherin, PAN-cadherin, and FAK in comparison to GO. Fiorillo et al. demonstrated that the presence of multiple oxygen-containing functional groups in GO favors an adhesion to U87 tumor cells . We prevThe Łukasiewicz Research Network—Institute of Microelectronics and Photonics, Warsaw prepared the GN and rGO. To obtain the GN/ExF graphene flakes, direct graphite exfoliation using Capstone (a fluorinated surfactant) was adopted. The graphite oxidation and exfoliation were used for obtaining the GO, according to a modified Marcano method. Marcano et.al. found that excluding NaNO3 increases the amount of KMnO4 and performs the reaction in a 9:1 mixture of H2SO4/H3PO4, which improves the efficiency of the oxidation process. This improved method provides a greater amount of hydrophilic oxidized graphene material. Moreover, the GO produced by Marcano et al.’s method is more oxidized than that prepared by Humme et al.’s method and does not generate toxic gas . Then th–8 mbar.The multi-chamber ultra-high vacuum (UHV) system PREVAC  was used for X-ray photoelectron spectroscopy (XPS). The spectra were obtained by using a hemispherical Scienta R4000 electron analyzer . Complementary equipment such as the Scienta SAX-100 X-ray source  equipped with the XM 650 X-ray Monochromator  (0.2 eV band) was used. The pass energy of the analyzer was set to 200 eV for survey spectra (with 500 meV steps), and 50 eV for regions (high-resolution spectra): C1s, O1s, N1s, S2p, and F1s (with 50–100 meV step). The base pressure in the analysis chamber was 5·109 mbar. During the spectra collection, it was not higher than 2 × 10® HTB-14™) and bone marrow stromal Hs5 (ATCC® CRL-11882™) cell lines were used from the American Type Culture Collection . Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum  and 1% antibiotic-antimycotic mixture containing penicillin and streptomycin  at 37 °C under 5% CO2 and 95% humidity in an INCOMED153 .For cell culturing , the hum4 cells were cultured in 96-well plates for 24 h. Then the culture medium was replaced by another containing 10% solutions of GN and rGO flakes at the concentrations of 10, 25, 50, and 100 μg/mL. As controls, the cells incubated without flakes were used. After 24 h, 10 μL of 0.33% neutral red solution in DPBS  was added for 4 h at 37 °C. Subsequently, the culture medium with neutral red was removed, and the cells were washed with 200 µL of fixative solution  for 3 min. The wells were then supplemented with 100 µL of solubilization solution (1% acetic acid in 50% ethanol), incubated for 10 min at room temperature, and mixed gently by pipetting. Measurements were performed at a wavelength of 540 nm using a spectrophotometer  and a reference 690 nm. Cell viability was expressed as a percentage compared with the control.For the cell viability studies, the neutral red assay was used. A total of 2 × 104 cells/well cells were seeded and cultivated in two-well culture-inserts and grown until a monolayer was obtained. The growth area in the culture-insert was 0.22 cm2 per well. The cell-free gap width was 500  ±  100 µm. After a few days of incubation, inserts were removed and the cell layer was washed with PBS . The plate was filled with 1.8 mL of the medium in each well. Two hundred microliters of aqueous solutions of GN and rGO flakes at a concentration of 250 µg/mL were added to the appropriate wells. Cells were cultured for 48 h and then fixed after 0, 12, 24, and 48 h with 4% paraformaldehyde and stained with May-Grünwald Giemsa stain. Photographs were taken at the same area at 2.5× magnification of the wound using an inverted light microscope. Images were analyzed using the ImageJ software .Cell invasion was assessed using two-well culture-inserts  in 6-well plates. A total of 2 × 105 cells were used for labelling. Tracking dye green was diluted 1:1000 in assay buffer and 500 μL was added to 500 μL Hs5 cell suspension. Tracking dye orange was diluted 1:50 in assay buffer and 100 μL was added to 900 μL U87 cell suspension. Cells were incubated for 30 min at 37 °C. After staining, the cells were washed thrice with 20 mM HEPES buffer (HBBS). Cell pellets were suspended in 1 mL of growth medium DMEM and applied to the IBIDI dish 35 mm (Cat. No:81156) in a ratio of 1:1. A co-culture was incubated for 24 h (after the culture medium was replaced with that containing 10% solutions of the flakes of GN and rGOs at the concentrations of 25 μg/mL) and visualized on a FV10-ASW 4.2  in 200 repetitions every 5 min—Multi Area Time Lapse. The images were analyzed using two fluorescence channels for Ex/Em = 490/520, Ex/Em = 540/570, and differential interference contrast (DIC). After visualization, a film was created using the software FV3000 , and the selectivity of the studied cell flakes in the co-cultures was analyzed. For more detailed analysis, films in the oib format were analyzed using the (Fiji Is Just) ImageJ program with the TrackMate overlay . Then the Trans-Blot Turbo Transfer System  was used for protein transfer to polyvinylidene difluoride (PVDF) membranes and blocked with I-BLOCKTM reagent (0.2%)  in PBS with Tween-20 (0.1%) for 60 min. The primary antibodies such as integrin α5 monoclonal antibody , integrin β1 monoclonal antibody , N-cadherin monoclonal antibody , β-catenin polyclonal antibody , and FAK monoclonal antibody  were incubated with the membranes overnight at 4 °C. Then they were washed in PBS with Tween-20 (0.1%) and incubated with the diluted secondary antibodies such as goat anti-mouse IgG H&L  and goat anti-rabbit IgG H&L  for 1 h. The Novex™ AP chemiluminescent substrate (CDP-Star™)  was used for protein detection and then visualized using Azure c400 . For protein normalization, the glyceraldehyde-3-phosphate dehydrogenase  was used as the loading control. The background corrections were performed using ImageJ® 1.48v  [Immunoblotting was used to evaluate integrin α5, integrin β1, N-cadherin, β-catenin, and focal adhesion kinase (FAK) expression. The cells were cultured as described in the section “Cell cultures”. Ice-cold lysis buffer TX100  supplemented with protease  at a ratio of 100:1 (TX100: protease) was used for whole-cell protein extracts isolation. Cells were centrifuged for 30 min at 12,000× MD, USA) .p < 0.05.GraphPad Prism 8.4.3  was used for the data analysis. Differences between the groups were tested using Bonferroni’s tests and Dunnett’s multiple comparison tests. All mean values are presented as standard deviations or standard errors. Differences were considered statistically significant at The XPS survey spectra of the samples are shown in p < 0.0001) reduction in viability was observed in the groups treated with rGO/ATS flakes at concentrations of 50 μg/mL (29.16% ± 2.54) and 100 μg/mL (7.98% ± 1.25) and in the groups treated with rGO/TUD flakes at a concentration of 100 μg/mL (58.31% ± 2.06). In healthy Hs5 cells, the most significant reduction in viability (p < 0.0001) was observed in the groups treated with rGO/ATS at 25 μg/mL (29.95% ± 5.27), 50 μg/mL , and 100 μg/mL (13.55% ± 4.13). Interestingly, in the groups treated with 100 μg/mL rGO/TUD, a greater reduction in viability was observed in healthy Hs5 cells(31.8% ± 5.6), compared to the U87 tumor cells. Based on the results of the neutral red test, the optimal concentration of 25 µg/mL GN and rGO flakes was selected for further experiments.First, we assessed the effect of GN and rGO flakes on the viability and cytotoxicity of glioblastoma multiforme (U87) and normal (Hs5) cell lines using a neutral red assay . In the A wound healing assay  was perfp < 0.0001) and 30.86% (p < 0.01) upon treatment with rGO/ATS and rGO/TUD flakes, respectively. In the case of healthy Hs5 cells, mobility decreased significantly by 13.41% (p < 0.05), 36.06% (p < 0.0001), and 28.36% (p = 0.0001) after treatment with GN/ExF, rGO/ATS, and rGO/TUD, respectively, compared to control cells.Next, we analyzed the motility , Table 3p < 0.01) after treatment with GN/ExF (log2RQ = 0.39 ± 0.09812), rGO/ATS (log2RQ = 0.4 ± 0.09812), and rGO/TUD (log2RQ = 0.41 ± 0.09812), whereas in healthy Hs5 cells (p < 0.0001), it was observed only after rGO/ATS treatment (log2RQ = 0.64 ± 0.09322). Expression analysis of FAK showed a statistically significant (p < 0.01) reduction in its expression in U87 glioma cells after treatment with rGO/ATS (log2RQ = −0.36 ± 0.09078) and rGO/TUD (log2RQ = −0.34 ± 0.09078), whereas in healthy Hs5 cells, it was observed after treatment with all tested flakes: GN/ExF (log2RQ = −0.31 ± 0.1084), rGO/ATS (log2RQ = −0.49 ± 0.1084), and rGO/TUD (log2RQ = −0.65 ± 0.1084).The next stage of this study was to evaluate the influence of the studied forms of GN and rGO on the expression of the selected adhesion and cell migration markers in U87 glioma cells and healthy Hs5 cells . A statiChanges in expression at the protein level were confirmed using Western blotting. Expression levels of proteins involved in cell adhesion and migration  were analyzed in U87 glioma cells and compared with the corresponding protein levels in healthy Hs5 cells treated with different graphene flakes (GN and rGO) . No signIn the present investigation, we hypothesized that the cytotoxicity of rGOs may result mainly from the direct contact of their surface functional groups with the GBM cell membrane and may lead to the disruption (activation/inhibition) of signaling pathways in the plasma membrane and inside the cell, thus modifying the mRNA and protein expression of molecules related to migration and adhesion. The study of the interaction between the GN materials and the expression of selected proteins was thought to reveal the mechanisms underlying the cytotoxicity of these materials in the cell membrane and their innovative application potential in therapy. Therefore, the present study focused on elucidating the migration and adhesion mechanisms of U87 GMB cells treated with GN and rGO flakes in comparison to normal Hs5 cells.p < 0.01) for GO and rGO; GO had an IC50 of 26.27 μg/mL, which turned out to be more toxic to U87 cells than rGO, with an IC50 of 132.40 μg/mL). They also showed that rGO showed no toxicity against normal cell lines, with cell viability above 90%, even at a concentration of 50 µg/mL [During a first stage of this study, the cytotoxicity of GN and rGO flakes was analyzed using the neutral red test . Having 50 µg/mL .Next, we performed a wound healing assay to assess the migration of U87 and Hs5 cells after treatment with GN and rGO flakes . We obseThe factors that regulate cell mobility are cell adhesion molecules and ECM receptors . These iβ-catenin is a key component of the canonical Wnt/β-catenin pathway. It accumulates in the nucleus, where it interacts with transcriptional co-regulators, including Tcf-4 and Lef-1, to form the B-catenin/Lef/Tcf complex, which regulates the transcription of several genes involved in proliferation, differentiation, survival, and apoptosis . It appeβ-catenin, a component of the cell adhesion complex, binds to cadherin and thus regulates cell–cell adhesion. Changing the binding of β-catenin to cadherin reduces cell adhesion while promoting cell migration and epithelial-mesenchymal transition . In the Focal adhesion kinase (FAK) is a multifunctional regulator of cell signaling in the tumor microenvironment . During Analyzing the results in terms of the differences between the tested materials, it is evident that rGO/ATS causes a strong decrease in viability, mobility, and mRNA expression of β-catenin and FAK, as well as an increase in the expression of N-cadherin mRNA in U87 cells. At the protein level, after treatment with rGO/ATS flakes, an increase in the expression of integrin βl and a decrease in the expression of FAK in U87 cells was observed. In contrast, rGO/TUD caused a decrease in viability in U87 glioblastoma cells, but lower than in the case of the aforementioned flakes, as well as inhibition of migration, reduced mobility, an increase in mRNA expression of integrin α5 and N-cadherin, and a decrease in the extrusion pressure of β-catenin and FAK. After treatment with rGO/TUD flakes, an increase in the expression of integrin βl and a decrease in the expression of β-catenin and FAK were observed at the protein level. For comparison, GN/ExF flakes in U87 glioma cells caused a lower cytotoxic effect, did not inhibit migration, decreased cell mobility to a lesser extent, and, at the mRNA level, increased integrin α5 and N-cadherin expression, and decreased β-catenin expression. At the protein level, there was a slight reduction in the expression of PAN-cadherin and FAK. These differences probably result from the structure of these flakes . It seems that flakes with more oxygen-containing functional groups have relatively high potential and hydrophilicity, which is directly related to the size of the GN flakes that can more easily adhere to the cell wall.This study expands our knowledge about the functional mechanism of carbon flakes. We showed that they can affect cell mobility by interacting with cell membrane receptors, such as integrins α5 and β1, β-catenin, cadherins, and FAK, through their surface functional groups. However, they are not selective for tumor cells and interact similarly with normal cells, although the strength of this interaction is weaker, or have an opposite effect on normal cells compared to the tumor cells. To the best of our knowledge, we observed for the first time a significant decrease in FAK expression at both mRNA and protein levels in U87 glioma cells treated with rGO/ATS and rGO/TUD flakes. Moreover, although the final effect is similar in normal and cancerous cells , it must follow different pathways because the expression of key proteins related to mobility  is not consistent in these cells. Further research should be carried out on a more advanced research model, which would verify whether changes in the expression of the studied molecules responsible for migration and invasiveness increase/decrease the virulence of these tumors.


VSG 3′ untranslated region (UTR). To identify positive VSG regulators in bloodstream-form Trypanosoma brucei, we used genome-scale screening data to prioritize mRNA binding protein (mRBP) knockdowns that phenocopy VSG mRNA knockdown, displaying loss of fitness and precytokinesis accumulation. The top three candidates were CFB2 (cyclin F-box protein 2) (Tb927.1.4650), MKT1 (Tb927.6.4770), and PBP1 (polyadenylate binding protein 1) (Tb927.8.4540). Notably, CFB2 was recently found to regulate VSG transcript stability, and all three proteins were found to associate. We used data-independent acquisition for accurate label-free quantification and deep proteome coverage to quantify the expression profiles following the depletion of each mRBP. Only CFB2 knockdown significantly reduced VSG expression and the expression of a reporter under the control of the VSG 3′ UTR. CFB2 knockdown also triggered the depletion of cytoplasmic ribosomal proteins, consistent with translation arrest observed when VSG synthesis is blocked. In contrast, PBP1 knockdown triggered the depletion of CFB2, MKT1, and other components of the PBP1 complex. Finally, all three knockdowns triggered the depletion of cytokinesis initiation factors, consistent with a cytokinesis defect, which was confirmed here for all three knockdowns. Thus, genome-scale knockdown data sets facilitate the triage and prioritization of candidate regulators. Quantitative proteomic analysis confirms the 3′-UTR-dependent positive control of VSG expression by CFB2 and interactions with additional mRBPs. Our results also reveal new insights into the connections between VSG expression control by CFB2, ribosomal protein expression, and cytokinesis.Variant surface glycoproteins (VSGs) coat parasitic African trypanosomes and underpin antigenic variation and immune evasion. These VSGs are superabundant virulence factors that are subject to posttranscriptional gene expression controls mediated via the IMPORTANCE VSG expression represents a key parasite virulence mechanism and an example of extreme biology. Posttranscriptional gene expression controls in trypanosomatids also continue to be the subject of substantial research interest. We have identified three candidate VSG regulators and used knockdown and quantitative proteomics, in combination with other approaches, to assess their function. CFB2 is found to control VSG expression via the VSG 3′ untranslated region, while other data support the view that MKT1 and PBP1 also form part of a CFB2 mRNA binding complex. Remarkably, we also find the depletion of cytoplasmic ribosomal proteins upon CFB2 knockdown, consistent with translation arrest observed when VSG synthesis is blocked. Proteomic profiles following knockdown further yield insights into cytokinesis defects. Taken together, our findings confirm and elaborate the role of CFB2 in controlling VSG expression and reveal new insights into connectivity with translation and cytokinesis controls. Trypanosoma brucei is transmitted by tsetse flies and causes lethal and debilitating diseases in both humans and livestock. Antigenic variation involves the expression of a dense coat of superabundant variant surface glycoprotein (VSG) derived from a single VSG gene at a time  that, in most cases, bind the mRNA 3′ untranslated regions (UTRs). The VSGs are among only a few genes with a known regulatory motif within the mRNA 3′ UTR. In this case, a “16-mer” is required for high-level expression in the bloodstream form  (n = 178) were assessed for these phenotypes, and the top three candidates were selected for further analysis  cells following knockdown and as assessed by flow cytometry; ratios of 4C to 2C (G1-phase) plus S-phase cells were increased 3-, 1.3-, and 3.3-fold following CFB2, MKT1, and PBP1 knockdown, respectively (We constructed tetracycline (TET)-inducible RNAi strains, targeting either CFB2, MKT1, or PBP1 for knockdown. Each strain was grown in tetracycline, and growth was monitored for 48 h. Consistent with the loss of fitness reported previously in the genome-scale knockdown screen , we obsen screen , we obseectively . Each knectively . Indeed,ectively .VSG 3′ untranslated region (UTR) (TGATATATTTTAACAC) that is thought to bind a specific positive regulator (VSG 3′-UTR-associated reporter was targeted to the region immediately downstream of the VSG expression site promoter and comprised green fluorescent protein (GFP) fused to an antibiotic-selectable marker, the blasticidin resistance gene (BSD)  . This seegulator . The VSGne (BSD) . The expne (BSD) . Thus, t10.1128/msphere.00069-22.2DATA SET S1Data Set S1, XLSX file, 2.5 MB.Mass spectrometry data and oligonucleotides used in this study. Download Copyright © 2022 Bravo Ruiz et al.2022Bravo Ruiz et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the T. brucei 927 genome reference strain and the VSG expression sites from the T. brucei Lister 427 strain. Samples were separated into two batches, containing the MKT1 and PBP1 knockdowns in the first batch and the CFB2 knockdown in the second. In both cases, the reporter strain was added as a reference. To assess the depth of proteome coverage, we compared the proteomic data sets from the parental strain with our previously reported transcriptomic data set  were detected in one or the other from two independent proteomics experiments using the reference strain, while 62% were detected in both experiments  (an implementation of a library-free data-independent acquisition [DIA] method) mass spectrometry (MS), which provides accurate label-free quantification (LFQ) and deep proteome coverage . The datdata set , includieriments , represehttps://gustavo-e64.pages.dev (MKT1), https://gustavo-e84.pages.dev (PBP1), and https://gustavo-e146.pages.dev (CFB2).Next, we used cluster analysis to compare the full data set derived from all 24 mass spectrometry runs. Cluster analysis of differentially expressed proteins revealed excellent correspondence and clustering of triplicate runs from the reference strain and the knockdowns . Importa10.1128/msphere.00069-22.1FIG S12) of the GFP reporter strain replicates in the first (GFPA.1 to .3) and second (GFPB.1 to .3) batches was performed. The top diagonal visualizes the protein intensity value (log2) distribution with histogram counts. Download FIG S1, PDF file, 0.3 MB.Assessment of reproducibility in the proteomics analysis. A pairwise comparison of the protein intensity values , as indicated by plotting the protein abundance against the fold change (FC) . Each prnockdown . These rt manner .VSG mRNA knockdown  binding protein PABP2 in association with VSG mRNA   was undepression , 26, whipression  and D. Tpression  and centpression , are alspression .Endoreduplication and continued rounds of mitosis specifically following CFB2 knockdown may also be linked to differential kinetoplastid kinetochore protein (KKT) expression . These cVSG 3′ UTR, specifically following CFB2 knockdown. All three proteins interact in association with the VSG transcript, however  medium at 37°C with 5% CO2  as the trigger for RNAi (https://dag.compbio.dundee.ac.uk/RNAit/). Before transfection, knockdown constructs were linearized using AscI (New England BioLabs).For gene-specific knockdown RNAi constructs, target gene fragments of 400 to 600 bp were amplified and cloned into the pRPafor RNAi . The necfor RNAi  were desfor RNAi  . For GFP:BSD detection, a mouse anti-GFP  (Roche) primary antibody was used. Mouse anti-EF1α  (Millipore) primary antibody was used for a loading control. As a secondary antibody, goat anti-mouse coupled to horseradish peroxidase  (Bio-Rad) was used. Blots were developed using an enhanced chemiluminescence kit (Amersham) according to the manufacturer’s instructions. Densitometry was performed using a ChemiDoc XRS+ system (Bio-Rad).Cell extracts from ∼5 × 107 TET-induced (24 h) and uninduced cells from each RNAi strain were harvested and fixed by adding 1% formaldehyde in supplemented phosphate-buffered saline (PBS)  dropwise and with regular shaking. The cells were incubated for 10 min at room temperature, washed in 1 mL of supplemented PBS, and then resuspended in 250 μL of supplemented PBS. Samples were stored at 4°C in the dark until further processing. Before flow cytometry analysis, cells were centrifuged for 10 min at 1,000 × g and resuspended in 1 mL of supplemented PBS containing 0.01% Triton X-100 (Sigma-Aldrich). The cells were incubated for 30 min at room temperature, centrifuged for 10 min at 700 × g, and washed once in 1 mL of supplemented PBS. Cells were then resuspended in 400 μL supplemented PBS with 10 μg/mL propidium iodide (Sigma-Aldrich) and 100 μg/mL RNase A (Sigma-Aldrich) and then incubated at 37°C in the dark for 45 min. Samples were then run on a BD FACSCanto flow cytometer for cell cycle analysis. FlowJo v10 was used for data analysis and visualization.Approximately 1 × 106 TET-induced and uninduced cells were fixed in 1% formaldehyde in 1 mL of culture medium at 37°C for 5 min and then at room temperature for 10 min. Cells were rinsed twice in PBS for 10 min, with spins at 1,000 × g, and then resuspended in 600 μL of ice-cold 1% bovine serum albumin (BSA) in water. Cells were dried on microscopy slides overnight at room temperature, rehydrated in PBS for 5 min, and then mounted in Vectashield antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector) under a coverslip. Cells were visualized at a ×63 magnification with oil immersion under a Zeiss Axiovert 200M microscope with Zen pro software (Zeiss) in the DAPI and phase channels. The cell cycle stages of at least 100 cells were identified and counted manually.For microscopy, approximately 1 × 107 cells after growth for 24 h under standard conditions with or without TET. Cell extracts were resuspended in 100 μL of a solution containing 5% SDS and 100 mM triethylammonium bicarbonate and submitted to the Fingerprints Proteomics Facility at the University of Dundee to be analyzed by directDIA using Spectronaut software (Biognosys). Triplicate samples of each RNAi strain were submitted for proteomics analysis in two batches: the first including the MKT1 and PBP1 RNAi strains and the second including the CFB2 RNAi strain. The reporter strain was included in triplicate in both batches as a further control. Samples were processed using trypsin  using 200 μg from each sample, and the final peptide quantification yielded between 72 and 120 μg. For liquid chromatography-mass spectrometry (LC-MS) analysis, 1.5 μg of each sample was injected onto a nanoscale C18 reverse-phase chromatography system  and then electrosprayed into a Q Exactive HF-X mass spectrometer (Thermo Scientific). For liquid chromatography, buffers were as follows: buffer A was 0.1% (vol/vol) formic acid in MilliQ water, and buffer B was 80% (vol/vol) acetonitrile and 0.1% (vol/vol) formic acid in MilliQ water. Samples were loaded at 10 μL/min onto a trap column  equilibrated in 0.1% trifluoroacetic acid (TFA). The trap column was washed for 3 min at the same flow rate with 0.1% TFA and then switched inline with a Thermo Scientific resolving C18 column . The peptides were eluted from the column at a constant flow rate of 300 nL/min with a linear gradient from 3% buffer B to 6% buffer B in 5 min and then from 6% buffer B to 35% buffer B in 115 min and, finally, to 80% buffer B within 7 min. The column was then washed with 80% buffer B for 4 min and reequilibrated in 35% buffer B for 5 min. Two blanks were run between each sample to reduce carryover. The column was kept at a constant temperature of 40°C.Proteins for each RNAi strain were extracted from ∼5 × 10m/z range from 350 to 1,650), with the RF lens at 40%, the automatic gain control (AGC) target set to custom, the normalized AGC target set at 300, the maximum injection time mode set to custom, the maximum injection time at 20 ms, and source fragmentation disabled. The MS survey scan was followed by tandem MS (MS/MS) DIA scan events using the following parameters: multiplex ions set to false; collision energy mode set to stepped; collision energy type set to normalized; high-energy collisional dissociation (HCD) collision energies set to 25.5, 27, and 30; orbitrap resolution at 30,000; first mass at 200; radio frequency lens at 40; AGC target set to custom; normalized AGC target at 3,000; and maximum injection time of 55 ms. Data for both MS and MS/MS scans were acquired in profile mode. Mass accuracy was checked before the start of sample analysis.The data were acquired using an easy-spray source operated in positive mode with spray voltage at 2,500 kV and the ion transfer tube temperature at 250°C. The MS system was operated in DIA mode. A scan cycle comprised a full MS scan . Trypsin was set as the enzyme with a maximum of two missed cleavages. Fixed modification was set for carbamidomethyl, and variable modifications were set for protein N-terminal acetylation, oxidation of methionine, dioxidation of methionine and tryptophan, glutamine to pyroglutamate, and deamidation of asparagine and glutamine. The identifications were filtered at an FDR of 1% at both the peptide and protein levels. The protein LFQ method was set to Quant 2.0, and data filtering was set to T. brucei Lister 427 strain, for protein identifications, we used the protein database of the T. brucei 927 reference strain, obtained from TriTrypDB (https://tritrypdb.org/tritrypdb/app/), combined with predicted proteins from the VSG expression sites from the Lister 427 strain .We extracted the protein abundance values from the first batch, MKT1, PBP1, and the control reporter strain (“GFPA”), and the second batch, CFB2 and the control reporter strain (“GFPB”), using the CellRanger pivot tables. We then used the removeBatchEffect function in limma  or the Chttps://www.ebi.ac.uk/pride/archive/projects/PXD031351). The code to reproduce the analysis pipeline was deposited in GitHub (https://github.com/mtinti/Gustavo_DIA_RBP) and archived in zenodo (https://doi.org/10.5281/zenodo.5761826).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner


Size is the most important quality attribute of giant pumpkin fruit. Different concentrations and application frequencies of α-naphthaleneacetic acid (NAA) and 24-epibrassinolide (EBR) were sprayed on the leaves and fruits of giant pumpkin at different growth stages to determine their effects and the mechanism responsible for fruit size increase. NAA+EBR application improved source strength, and further analysis indicated that NAA+EBR markedly boosted net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and the expression level and activity of galactitol synthetase (GolS), raffinose synthetase (RS), and stachyose synthetase (STS), resulting in an increase in the synthesis of photoassimilate, especially stachyose. Concomitantly, NAA+EBR spray increased stachyose and sucrose contents throughout pumpkin fruit growth and the concentrations of glucose and fructose at 0 and 20 days post-anthesis (DPA) in peduncle phloem sap, implying that such treatment improved the efficiency of assimilate transport from the peduncle to the fruit. Furthermore, it improved the expression and activity of alkaline α-galactosidase (AGA), facilitating assimilate unloading, providing carbon skeletons and energy for fruit growth, and increasing fruit weight by more than 44.1%. Therefore, exogenous NAA and EBR increased source capacity, transportation efficiency, and sink strength, overall promoting the synthesis and distribution of photoassimilate, ultimately increasing fruit size. Cucurbita maxima, Cucurbitaceae) are a special type of pumpkin with a very large single fruit, often weighing hundreds of kilograms [Giant pumpkins  are two important plant growth regulators that play multiple physiological roles in promoting cell elongation and division, seed germination, fruit development, and plant stress responses ,9,10,11.−1 EBR) and T4 (10 mg∙L−1 NAA) had no significant (p < 0.05) difference relative to the control, while the other 13 treatments showed marked differences. In particular, T10 (20 mg∙L−1 NAA + 1.0 mg∙L−1 EBR) led to the highest fruit weight, i.e., 112.4 ± 3.8 kg; approximately 1.28-fold higher than the control fruit weight. This was followed by T14 (30 mg∙L−1 NAA + 1.0 mg∙L−1 EBR) and T11 (20 mg∙L−1 NAA + 1.5 mg∙L−1 EBR), with values of approximately 107.1 and 106.3 kg, respectively. Thus, spraying pumpkin leaves and fruits with NAA+EBR promoted increased fruit weight, with T10 having the most significant effect (Great pumpkin single-fruit sprayed with different concentrations of NAA and EBR at 20 days post-anthesis (DPA) showed a higher fruit weight than that of the control (87.6 kg). The single fruit weight in T1 (0.5 mg∙Lt effect .−1 NAA + 1.0 mg∙L−1 EBR were significantly higher than that of the control and the one-time application of growth regulator at 20 days after flowering (T10). The fruit weight increased most obviously when NAA+EBR was applied 4 times at 20 d after planting, 0, 10, and 20 DPA, respectively (T18). The single-fruit weight obtained under T18 was approximately 127.2 ± 3.6 kg, indicating an increase of 44.1% and 15.3% compared with that of the control and T10 at 20 DPA, respectively. The effect of NAA and EBR on the increased single-fruit weight was not directly proportional to spraying time. The single-fruit weight obtained under T19 (Applied 20 mg∙L−1 NAA + 1.0 mg∙L−1 EBR for 5 times) was lower than that of T18, although not significantly (p < 0.05)  of 20 mg∙L < 0.05) .2 concentration (Ci); however, the effects on Pn, Gs, and Tr were inconsistent. The diurnal variation in the Pn of pumpkin was similar across treatments, which began to increase after 8:00 a.m. and gradually decreased after reaching a peak. The Pn value observed under T18 was notably higher than that recorded for the control. However, at 0 DPA, the maximum value of Pn in the T18 and control groups appeared at 10:00, whereas, at 20 DPA, the maximum value was recorded at 12:00. From 8:00 a.m. to 16:00 p.m., Gs showed a downward trend; however, at 20 DPA, it was higher than that at 0 DPA. Spraying NAA and EBR markedly increased Gs compared with that in the controls before 12:00 at 0 and 20 DPA; however, there was no significant difference in Gs between 14:00 and 16:00 p.m. Tr first increased and then decreased. The peak value of Tr occurred at 14:00 p.m.. Furthermore, at 20 DPA, Tr was substantially higher than at 0 DPA, and T18 led to higher Tr than those recorded for the controls. Pn, Gs, and Tr at 20 DPA were higher than those at 0 DPA, indicating that more assimilates must be produced and more water must be supplied during the rapid fruit-expansion period, and the T18 treatment obviously increased the photosynthesis rate.The effects of T18 on the photosynthetic characteristics of giant pumpkin at 0 and 20 DPA were assessed . General(CmGolS1), raffinose synthetase (CmRS), and stachyose synthetase (CmSTS) were consistent with changes in enzyme activity during the growth and development of giant pumpkin leaves. The expression of CmGolS1 was higher in the early stages of fruit development and lower after 50 d. In turn, CmRS expression was maintained at a relatively stable level during fruit development, while CmSTS expression increased after the fruit entered the rapid growth period and gradually decreased after 50 d . Changes in sucrose and fructose contents during fruit development were consistent with those of stachyose. Meanwhile, glucose content reached a maximum value at 10 DPA and then gradually decreased, aligning with raffinose. Lastly, fruit sucrose, glucose, and fructose contents after NAA+EBR treatment were higher than those in the control treatment . Thus, sEpipremum aureum [CmGolS1, CmRS, and CmSTS, respectively [CmAGA1 and CmAGA2 are important for stachyose catabolism. In this study, we found that AGA activity and the expression of CmAGA1 and CmAGA2 were markedly increased in NAA-treated leaves, indicating an enhancement of stachyose metabolism in fruit and an increase in the substances needed for fruit growth.To reach maturity, fruit growth depends on photoassimilate from source organs to enhance sink strength by supporting cell division and growth, ultimately promoting the transfer of photoassimilate to the growing fruits . NAA is m aureum . In thism aureum ,21. Aftem aureum ,23. Singm aureum . Pumpkinectively ,24,25. Iectively ,27. In t−1. This result is similar to others recently reported [2 assimilation level [BRs mediate many physiological processes in plants, including increasing leaf area, regulating photosynthesis, promoting carbohydrate metabolism, increasing plant biomass, and increasing grain size and yield ,28,29,30reported ,18. BRs reported . Applyinon level ,33. The on level ,35. In ton level . BRs repon level  and grapon level ,25. In t−1) of exogenous NAA, which led to a higher fruit weight than that of the control, although notably lower than that observed when 20 mg.L−1 exogenous NAA was applied. Pumpkin fruit weight with five exogenous applications of NAA+EBR was significantly lower than that observed with four applications of NAA+EBR. This result may be due to insufficient concentrations of exogenous NAA and EBR to inhibit pumpkin growth. Nonetheless, the effects of high concentrations of NAA and EBR on pumpkin fruit weight decreased. Because of the high temperature in the pumpkin cultivation area during the summer, cultivation in this season is not convenient, as heat markedly restricts fruit growth. This may be an important reason for the evident difference in fruit weight relative to that reported in the world records. Fruit weight significantly increased by applying NAA and EBR; however, this increase was limited. Further research is required to determine whether the fruit weight of giant pumpkins can be adjusted by increasing the nutrient area using growth regulators or improving horticultural cultivation facilities or practices to reduce summer temperature and extend the fruit growth period in this cultivation region. Although this study did not analyze the residues of NAA and EBR in pumpkins, a large number of previous studies showed that NAA dissipated rapidly in plants, and the residues of NAA were lower than the safety limit of pesticides in agricultural products and food , and EBR residues could not be detected in the plants in which EBR content was widely used [Auxin and BRs have cooperative effects and many similar physiological functions, such as synergistic involvement in the formation of lateral roots. In addition, it was also reported that BRs regulated auxin polar transport . NAA or ely used ,42,43,44−1 NAA + 1.0 mg∙L−1 EBR, and the optimal periods of application were spraying on the 20 days after planting, 0, 10, and 20 DPA.Taken together, exogenous NAA+EBR increased pumpkin fruit weight via an all-around coordinated action involving the source (leaf), transport (peduncle phloem sap), and the sink (fruit). Application of NAA+EBR can improve the photosynthetic capacity of leaves (sources), promote the synthesis of assimilates and water supply, enhance the transport efficiency of assimilates from the peduncle phloem sap to the growing fruit, and also improve sink strength, which was conducive to the unloading of assimilates, thus providing carbon skeletons and energy for fruit growth, ultimately promoting the increase in pumpkin fruit weight. According to the results, the recommended combination of growth regulators for pumpkin production was 20 mg∙L2O5: K2O = 15:15:15), 10 kg of CaSO4 fertilizer, 10 kg of rape seed leaves, and 20 kg of cow dung were applied to each planting hole. The row spacing was 5 m × 2 m. The main vine of the plants retained 40 knots, and the secondary vine retained 3–4 knots. Each plant retained only one fruit .The Atlantic giant pumpkin, which belongs to the Indian variety, was purchased from the Sustainable Seed Company . Pumpkin seeds were sown on 1 February 2021, and, at the three-leaf stage, seedlings were transplanted into the plastic greenhouse of Lehou Manor Agricultural Ecological Park, in Shatou Town, Yangzhou City, on 10 March. Pumpkin seedlings were planted in 50 cm-deep holes approximately 1–1.5 m in diameter; 10 kg of compound fertilizer (N: PDuring the rapid fruit expansion period (20 DPA), giant pumpkin leaves (both sides) and fruits were sprayed with different concentrations of NAA and EBR until dripping, and distilled water (Tween-20 with a volume fraction of 0.1%) was used as the control. Each treatment was repeated 3 times, and each repetition included 20 plants. Specific concentrations of NAA and EBR are listed in To further elucidate the beneficial effect of NAA and EBR on giant pumpkin fruit-weight, the combination treatment producing the largest single-fruit weight in After treatment, samples were collected at 10-day intervals, and the leaves of the fruit-setting node and fruits were collected; this was repeated three times for every five leaves or fruits. The phloem sap of the pumpkin peduncle was collected as previously described ,6,45. PePumpkin fruits were harvested when growth stopped, and single-fruit weight was measured using an electronic scale. 60 pumpkin fruits were measured for each treatment and the average value was displayed.2 concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr) of the leaves located on the fruiting-node were measured using a portable photosynthesis measurement system Li-6400XT  from 8:00 to 16:00 h at 0 and 20 DPA. The measurements were made five times at 2 h intervals. Five pumpkin plants from each cultivar were measured at each measuring time.Net photosynthetic rate (Pn), mesophyll CO® One-Step gDNA Removal, and cDNA Synthesis Super Mix  was performed for cDNA synthesis according to the manufacturer’s instructions. iTaq Universal SYBR® Green Supermix  was used to determine the expression of related genes, according to the manufacturer’s instructions. The reaction system comprised reaction mix 12.5 μL, cDNA 1.0 μL, 0.5 μL for the forward primer, 0.5 μL for the reverse primer, and 5.5 μL double distilled water (ddH2O). Polymerase chain reaction (PCR) amplification was performed using a CFX96 Touch Real-Time PCR Detection System . The cycling program was as follows: 94 °C for 2 min; 35 cycles of 94 °C for 10 s, 60 °C for 5 s; and a slow temperature increase from 65 to 95 °C at a rate of 0.5 °C ∙min−1. The expression level of each gene was normalized to that of the pumpkin internal reference gene, EF-1a, and calculated using the 2−ΔΔCT method. Primers used in this study are listed in Total RNA was extracted from pumpkin leaves and fruits using an RNAiso Plus kit , according to the manufacturer’s instructions. Reverse transcription was performed using EasyScript−1 HEPES-NaOH buffer  and centrifuged . Supernatants were retained and dialyzed overnight with a buffer containing 25 mmol∙L−1 HEPES-NaOH buffer and 1 mmol∙L−1 DTT, and the enzyme solution was collected.To detect GolS, RS, and STS, 0.5 g of the frozen ground sample was homogenized with 2 mL 50 mmol∙L−1 HEPES-NaOH buffer (pH 7.0), 20 mmol∙L−1 β-mercaptoethanol, 5 mmol∙L−1 MgCl2, and 4 mmol∙L−1 DTT; and different enzymes were added to different reaction substrates. Specifically, the reaction system for determining GolS activity also contained 40 mmol∙L−1 sucrose, 20 mmol∙L−1 inositol, and 5 mmol∙L−1 UDP galactose; in turn, the system for determining RS activity contained 20 mmol∙L−1 inositol galactoside; the system for determining STS activity contained 40 mmol∙L−1 raffinose and 20 mmol∙L−1 galactoside. Subsequently, 50 μL of the enzyme solution was added to a water bath at 30 °C for 3 h and boiled for 5 min. After cooling, high-performance liquid chromatography (HPLC)-Agilent Technologies 1260 Infinity  was used for the detection of enzymatic activities, which were expressed as the amounts of galactoside, raffinose, and stachyose produced for GolS, RS, and STS, respectively.Activities of GolS, RS, and STS were measured according to the methods of Li et al., 2011 , and Wan−1 HEPES-NaOH buffer with 2 mmol ∙L−1 MgCl2, 1 mmol∙L−1 EDTA, and 1 mmol ∙L−1 DDT, pH7.4) for 15 min at 25 °C and centrifuged at 18,000× g∙min−1 for 30 min. Then, 5% (w/v) polyethylene glycol (PEG6000) was added to the supernatant, prior to centrifugation for 30 min. This step was repeated with 50% (w/v) PEG6000. The precipitate was redissolved in 25 mmol∙L−1 HEPES NaOH buffer with 1 mmol∙L−1 DTT and dialyzed overnight. The assay was performed using a 100 μL reaction system containing 100 mmol∙L−1 HEPES NaOH buffer (pH 7.4) and 16 mmol∙L−1 stachyose; 30 μL of the extracted enzyme was also added. After 15 min, 100 μL of 5% (w/v) Na2CO3 was added, and the mixture was boiled for 5 min to terminate the reaction. The amount of stachyose consumed was measured using HPLC to determine enzyme activity.AGA was extracted as previously described by Miao et al., 2007 , and Wang∙min−1 for 15 min. The supernatant was then placed in a rotary evaporator, evaporated under reduced pressure at 40 °C, dissolved in ddH2O and chloroform (2.5 mL), shaken, and centrifuged to remove the organic phase. The pH of the aqueous phase was adjusted to 7.0 using NaOH (0.1 mol∙L−1 NaOH) and evaporated to dryness under reduced pressure. Finally, the solution was dissolved in 1 mL ddH2O and passed through a 0.45 μm needle filter. Using the same chromatographic conditions as the standard, the stachyose, raffinose, sucrose, glucose, and fructose contents were calculated using the external standard method. For HPLC, an Agilent Hi-Plex Ca (Duo)  column (300 mm × 6.5 mm) was used as the chromatographic column; ultrapure water was used as the mobile phase, and the column temperature was 80 °C; the flow rate was 0.5 mL∙min−1; and the injection volume was 5 μL. Retention time and peak area were used to qualitatively and quantitatively analyze soluble sugar contents.Soluble sugars were extracted and determined in pumpkin tissues according to Miao et al., 2007 , and ZhaData were sorted using Microsoft Excel 2016 . Meanwhile, SPSS Statistics 24.0  was used to determine significant differences among treatments. GraphPad Prism v9.0  was used to visualize and generate plots.