Glioma is highly lethal because of its high malignancy. Ubiquitination, a type of ubiquitin-dependent protein modification, has been reported to play an oncogenic or tumor-suppressive role in glioma development, depending on the targets. Ring finger protein 139 (RNF139) is a membrane-bound E3 ubiquitin ligase serving as a tumor suppressor by ubiquitylation-dependently suppressing cell growth. Herein, we firstly confirmed the abnormal downregulation of RNF139 in glioma tissues and cell lines. In glioma cells, ectopic RNF139 overexpression could inhibit, whereas RNF139 knockdown could aggravate the aggressive behaviors of glioma cells, including hyperproliferation, migration, and invasion. Moreover, in two glioma cell lines, RNF139 overexpression inhibited, whereas RNF139 knockdown enhanced the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and AKT serine/threonine kinase 1 (AKT). In a word, we demonstrate the aberration in RNF139 expression in glioma tissue samples and cell lines. RNF139 serves as a tumor-suppressor in glioma by inhibiting glioma cell proliferation, migration, and invasion and promoting glioma cell apoptosis through regulating PI3K/AKT signaling. The sampling procedure was conducted with the approval of the Ethic Committee of Hunan Brain Hospital. Immediately after specimen sampling, tissues were transferred to formalin or − 80 °C container until subsequent experiments.2.Normal human brain astroglia cell line SVG p12 (CRL-8621) was obtained from ATCC  and cultured in EMEM . Normal human astrocyte  was obtained from ScienCell  and cultured in Astrocyte medium . Glioma cell line U87 (HTB-14) was obtained from ATCC and cultured in EMEM (Gibco). Glioma cell line SHG-44 (3131C0001000700048) was obtained from Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences  and cultured in RPMI 1640 . Glioma cell line GOS-3 (ACC 408) was obtained from German Collection of Microorganisms and Cell Cultures  and cultured in Dulbecco’s MEM (Gibco). Glioma cell line TJ905 (3111C0001CCC000267) was obtained from Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences and cultured in DMEM-H: Dulbecco’s modified Eagle’s medium . All glioma cell lines were cultured in medium supplemented with 10% FBS . All cells were cultured at 37 °C in 5% COFor RNF139 overexpression, full-length RNF139 gene was combined in pLVX-puro (RNF139) and the empty vector was used as a negative control (vector-NC). For RNF139 knockdown, short-hairpin RNA against RNF139 gene, as well as their nontargeting sequences were reconstructed in pLVX-shRNA2-puro . The primer sequence for shRNA are listed in Table TRIzol reagent (Invitrogen) was used to extract total RNA from target cells or tissues, and then use the first strand cDNA synthesis kit (Promega) for reverse transcription. A SYBR Green PCR Master Mix (Qiagen) was used to detect the expression level according to the manufacturer’s instructions. With GAPDH expression as an internal reference, calculate the relative expression using the Ct method. The specific primers used are presented in Table Extract total protein from target cells, separate the total protein samples using SDS-PAGE, and transfer the protein samples to polyvinylidene fluoride membranes. For the blockage of nonspecific binding, the membranes were incubated with 5% skim milk dissolved in diphenyltris buffer saline for 2 h at room temperature. Incubate the membranes with the primary antibodies against RNF139 , PI3K , p-PI3K , Akt , or p-Akt  overnight at 4 °C. The incubation with the primary antibodies was followed by another incubation with the corresponding horseradish peroxidase coupled secondary antibody for 2 h at room temperature. Finally, the enhanced chemiluminescence visualization scan was performed.4 cells/well for a 24, 48, or 72 h of incubation. At the end of each incubation, 10 μl of 5 mg/ml MTT  was added to each well and the cells were subjected to another 4 h of incubation. Remove the supernatants, add 100 μl dimethyl sulfoxide  to each well, measure the absorbance value at 490 nm using a microplate reader.Target cells were transfected and seeded in 96-well plates at a density of 1 × 10Use 5-ethyl-2'-deoxyuridine (EdU) detection kit  for determining DNA synthesis. Cells were firstly transfected, incubated with EdU for 2 h, and collected. Then, fix the cells in 4% formaldehyde for 20 min and permeabilize the cells. Wash the cells with PBS, treat the cells with 200 μl Apollo ® reaction cocktail (Ribobio) for 10 min, and permeabilize the cells. Stain the cells with 100 μl Hoechst 33,342 . Observe the cells under a fluorescence microscope .Target cells were transfected and examined for cell apoptosis using a FITC-labeled AnnexinV/propidium iodide (PI) Apoptosis Detection kit . Immediately after staining, flow cytometry was performed . Early apoptotic cells were positive for Annexin V, and late apoptotic cells were positive for both Annexin V and PI.5 cells/well. Subsequently, the cells were cultured with serum-free medium for 12 h and then seeded in 100 μl serum-free media into each well of the Transwell at a density of 2 × 104 cells/well. In the lower chamber, add 700 μl complete medium containing 10% FBS. After 48 h, remove the cells stayed on the upper chambers, fix the cells on the surface of the lower chamber with 4% paraformaldehyde, and stain these cells with crystal violet. Count the number of cells on the surface of the lower chamber (invaded cells). Cell migration was evaluated similarly except for using Transwell without Matrigel.Target cells were transfected and evaluated for the invasion using Matrigel Transwell . The cells were seeded in a 6-well plate for 12 h at a density of 5 × 10t test was performed to analyze the statistical significance. The cell experiments were performed at least three times. A P value of < 0.05 was considered statistically significant.Each experiment was repeated for at least three times. Data were processed using the GraphPad software  and shown as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) and then Tukey’s multiple comparison test or Student’s N = 12). Consistently, the protein levels of RNF139 were significantly decreased in tumor tissues compared with those in adjacent non-cancerous tissues (N = 5)  or small interfering RNA targeting RNF139 (shRNA1-RNF139 and shRNA2-RNF139). The mRNA expression and protein levels of RNF139 were confirmed by qRT-PCR and Immunoblotting , phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphatase and tensin homolog (PTEN), tumor protein p53 (TP53), cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B), and so on, leading to dysfunction of the PI3K/Akt/mTOR, p53, RB transcriptional corepressor 1 (RB1), and other signaling pathways; however, the discovery of these aberrations also open up targeted therapies through targeting hyper-activated pathways [4]. The crucial role of the PI3K/Akt signaling pathway has been widely recognized in mediating cell proliferation, apoptosis, motility, as well as angiogenesis in glioblastoma (Brennan et al. Altogether, we demonstrate the aberration in RNF139 expression in glioma tissue samples and cell lines. RNF139 serves as a tumor-suppressor in glioma by inhibiting cancer cell proliferation, migration, and invasion and promoting cancer cell apoptosis through regulating PI3K/AKT signaling.


Muntinga calabura Linn.) is tropical tree that is known to produce edible fruit with high nutritional and antioxidant properties. However, its use as functional food is still limited. Previous studies suggest that fermentation with probiotic bacteria could enhance the functional properties of non-dairy products, such as juices. In this study, we analyze the metabolite composition and activity of Jamaican cherry juice following fermentation with Lactobacillus plantarum FNCC 0027 in various substrate compositions. The metabolite profile after fermentation was analyzed using UPLC-HRMS-MS and several bioactive compounds were detected in the substrate following fermentation, including gallic acid, dihydrokaempferol, and 5,7-dihydroxyflavone. We also found that total phenolic content, antioxidant activities, and inhibition of diabetic-related enzymes were enhanced after fermentation using L. plantarum. The significance of its elevation depends on the substrate composition. Overall, our findings suggest that fermentation with L. plantarum FNCC 0027 can improve the functional activities of Jamaican cherry juice.Jamaican cherry ( Muntingia calabura Linn.) is a plant species belonging to Elaeocarpaceae. This plant is indigenous to Southern Mexico, Northern South America, Central America, Trinidad, St. Vincent, and the Greater Antilles ). We identified the three putative metabolites of fermented Jamaican fruit juice, including gallic acid (A), dihydrokaempferol (B), and 5,7-dihydroxyflavone (C)  .L. plantarum strain CIR1 could produce gallic acid during fermentation. This bacteria could produce tannase, an enzyme that could facilitate bioconversion of tannic acid to gallic acid [L. plantarum strain FNCC 0027 used in this study might also produce tannase that can hydrolyze tannic acid presence in Jamaican fruits [Solanum retroflexum leaf using L. plantarum strain 75 could induce the production of various bioactive compounds, such as gallic acid, vanillic, coumaric, ellagic acid, quercetin, and catechin [L. plantarum FNCC 0027 also induces the production of bioactive compounds, including gallic acid, dihydrokaempferol, and 5,7-dihydroxyflavone. The production of dihydrokaempferol possibly due to the L. plantarum shikimic pathway that can convert flavonoid naringenin presents in Jamaican fruit into dihydrokaempferol [L. plantarum could produce intermediate compounds from dihydroflavonols class, such as dihydrokaempferol and dihydroxyflavone [A previous study reported that lic acid . L. plann fruits ,39. Rececatechin . Fermentempferol . Hydroxyyflavone ,42,43. Gyflavone ,45,46. T®, San Diego, MA, USA), starch , acarbose , Man Rogosa Sharpe/MRS broth , α-glucosidase from Saccharomyces cerevesiae , α-amylase from Aspergillus oryzae , amyloglucosidase from Aspergillus niger , 2,2-Diphenyl-1-picrylhydrazyl , 2,4,6-Tris(2-pyridyl)-s-triazine , 3,5-dinitrosalicylic acid , 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) , Folin–Ciocalteu’s phenol reagent , gallic acid, peroxidase. Other reagents were analytical grade or better.The chemicals and reagents used are p-nitrophenyl-α-D-glucopyranoside , the Indonesian Institute of Sciences (LIPI), Yogyakarta, Indonesia in March 2016. The fruits (250 g) were washed and kept at 4 °C before further use. Jamaican cherry juice was produced from the fruit using sterilized-commercial food juicer at room temperature.L. plantarum FNCC 0027 were propagated in MRS broth at 37 °C for 48 h under anaerobic conditions. The cells were harvested, pelletized, and re-suspended in sterilized phosphate buffer saline at 7.02 log CFU/mL for inoculation.First, Jamaican cherry juice was produced by homogenizing Jamaican cherry fruits with sterile water in a commercial blender at the concentration of 1 g/mL. The homogenized juices were filtered and pasteurized for 3 min at 85 °C . Fermentg [To obtain cell-free supernatant, the fermented juices were centrifuged for 15 min at 10,000× g . The res®, Basingstoke, England). The plate was incubated for 72 h at 30 °C ± 2 °C, and colony numbers was calculated using a colony counter. Plates with 15 to 300 colonies were considered for colony forming unit calculation. The viable colonies were converted into weighted mean colony forming units per milliliter (CFU/mL) using the following Equation (1):1 is the number of plates retained in the first dilution; n2 is the number of plates retained in the second dilution; and d is the first dilution factor. The viable colonies were then converted into log CFU/mL.Viable count was performed at different intervals over the course of 48 h fermentation . To perform viable count, 10 sterile 15mL test tubes were filled with 9 mL of sterilized phosphate buffer saline (PBS). Serial 10-fold dilutions in PBS were then prepared in the test tubes, using 1 mL of fermented juice as the starter. Viable count was next performed using pour plate method in duplicate, 1 mL of solution from each dilution series was mixed with 25 mL tempered (47 °C) Plate Count Agar . The pH measurement was performed at different intervals over the course of 48 h fermentation .® Go microplate spectrophotometer . Standard curve was produced using Gallic acid, the equation for the standard curve is y = 0.0104x − 0.0159 (R2 = 0.9988) where y is the absorbance at 735 nm and x is the concentration of Gallic acid in µg/mL. Total phenol was expressed as µg of Gallic acid equivalent (GAE) per mL of supernatant (µg GAE/mL).Total phenolic content was determined using methods developed by Zhou, Wang, Zhang, Yang, Sun, Zhang, and Yang , with mi® Go microplate spectrophotometer . Lower absorbance represents higher free radical-scavenging activity. The scavenging activity against DPPH was calculated using following Equation (2):0 was absorbance of control and Abs1 was the absorbance in the presence of supernatant.The DPPH scavenging assay was performed using 96-well plates following the method by Kosem, et al. , with mo® Go microplate spectrophotometer . The ABTS radical scavenging activity was calculated using Equation (2).The ABTS assay was performed according to Re et al. . In brie3.6H2O at 10:1:1 (v/v/v) ratio. Subsequently, 10 μL of supernatant was added to the 96-well plate containing 300 μL FRAP solution as an oxidizing reagent. After 5 min incubation in dark at 37 °C, the absorbance was measured at 593 nm using Tecan Infinite® 200 Pro microplate reader. The experiment was calibrated with FeSO4·7H2O and the results were expressed in terms of FeSO4·7H2O equivalents (μM).The FRAP assay was conducted according to Cecchini and Fazio , which w® 200 Pro microplate reader every minute for 30 min, ORAC values were expressed in term of Trolox equivalents (μM).ORAC were measured according to Zulueta, et al. . The 2,2® Go microplate spectrophotometer . Percent inhibition was calculated relative to the diabetes drug acarbose as the reference. Reaction system without supernatant was used as negative control while reaction system without α-glucosidase was used as a blank for correcting the background absorbance. The percentage of enzymatic inhibition activity was calculated using following Equation (3):A is the absorbance of the control and AbsB is the absorbance of the tested supernatant.The α-glucosidase inhibitory activity of the fermented supernatant was determined according to the chromogenic method, in a 96-well plate, according to our previous published method . First, ® Go microplate spectrophotometer . Acarbose was used as a positive reference standard. The percentage of α-amylase inhibition was calculated using Equation (2).The α-amylase inhibitory activities of the fermented supernatant were carried out according to procedure reported by Telagari and Hullatti . The ass® 200 Pro microplate reader.The amyloglucosidase  inhibitory activities of the fermented supernatant was carried out in 96-well plate according to Saul et al.  and Warr2O, B: Acetonitrile, both solvents containing 0.01% formic acid) for 30 min and held constant of 100% B for 10 min. The parameter was set in a capillary voltage of 4500 V, nebulizer nitrogen pressure of 2 bars, the dry gas flow of 9 L/min source temperature, ion source temperature 200 °C, and spectral rate of 3 Hz. The MS data subsequently analyzed using Bruker Compass Data Analysis 4.4 SR1(x64).High resolution MS was carried out using Bruker maXis 4G ESI time of flight mass-spectrometer  attached to an Ultimate 3000 HPLC . The UHPLC-method was performed using Reprosil 3 µm C18 100 Å, 10 × 3.3 mm  with linear gradient starts from 90% to 0% of A  was determined of each data. The analysis of variance (ANOVA) and student t-test was employed using SPSS 16 to determine the level of statistical differences between control and Jamaican juice formulation fermented with L. plantarum FNCC 0027 could significantly improve the functional activities of the substrates, including higher polyphenol content, antioxidant capacity , and inhibitory activity against diabetic-related enzymes . Various fermentation formulations with different ratios of Jamaican cherry juice and MRSB volume were studied in this study and we found that M1 formulation with 100% Jamaican cherry juice showed the highest functional activities compared to the other composition. This result indicates that the addition of MRSB is not required to facilitate effective fermentation and improvement of Jamaican cherry juice functional properties. In summary, the functional properties and beneficial activity of Jamaican cherry juice can be enhanced by fermentation with L. plantarum, one of “generally regarded as safe” (GRAS) strain. This work can be used as the basis for the development of new functional beverages using fermented Jamaican cherry juice as the main component.Our results demonstrated that fermentation of Jamaican cherry juice using


The antioxidant properties of CBD-enriched oil were investigated by Fe2+ chelating activity, Fe3+ reducing antioxidant power assay, O2●− scavenging activity, HO● scavenging ability and lipid peroxidation inhibitory assay, and its cytotoxicity, apoptosis- and oxidative stress-inducing effects on NHDF, MeWo, HeLa, HepG2 and HOS cells were determined. The CBD concentration in hemp oil was increased by CBDA soft decarboxylation optimized at 90 °C, for 1 h and the resulting oil was capable of reducing iron, scavenging free radicals and inhibiting lipid peroxidation in cell-free oxidative conditions. CBD-enriched oil promoted NHDF proliferation at up to 15 µg CBD/mL, while inducing apoptosis and ROS production and modulating antioxidant enzymes’ gene expression in cancer cells, being selective for osteosarcoma cells, and induced apoptosis by p53- and ROS-independent mechanisms. CBD-enriched hemp oil demonstrated antioxidant properties in oxidative conditions and promoted normal fibroblasts’ proliferation, while inducing apoptosis and ROS production in cancer cells.This study aimed to obtain and characterize extracted hemp oil enriched in cannabidiol (CBD) by decarboxylation of cannabidiolic acid (CBDA) and to give new insights into its antioxidant and anticancer effects. Optimization of CBDA decarboxylation in hemp oil was performed, and CBD and CBDA contents and purities were determined by flash chromatography,  Cannabis sativa L. or hemp was one of the first plants used to obtain fibers, oil and biomass and is cultivated specifically for its industrial uses. In addition, hemp-derived products are used as medicine, cosmetics, biofuel and animal feed, as well as for paper, varnishes, inks and phytoremediation [C. sativa L. and its derived products is regulated by European legislation, which states that hemp cultivation is permitted only if it contains less than 0.2% (w/w) Δ9-tetrahydrocannabinol (THC) due to its psychoactive effects (Regulation 1307/2013). Like in most European countries, hemp products are legal in Romania as long as their THC concentration is below 0.2%. In addition to THC, there are more than 100 other active compounds in hemp [ediation . The pro in hemp , and it  in hemp . However in hemp .On the other hand, the main targets of cancer treatments are the reduction in tumor sizes, the inhibition of angiogenesis and metastasis and, primarily, the induction of controlled cancer cell death, i.e., apoptosis. The principal issue of chemotherapeutic agents, however, is that they commonly have severe side effects, attacking normal, healthy cells as well as cancer cells. This issue could be mitigated by the use of natural compounds or mixtures, which have very few and mild side effects while exhibiting anticancer properties and/or alleviating chemotherapy fallouts.2●−), hydroxyl radical (HO●) or hydrogen peroxide (H2O2), are produced in living cells as secondary metabolic products and are required for normal cell function at physiological concentrations [2O2 activates various redox signaling pathways and exhibits either pro- or anti-apoptotic activities, depending on its concentration and cellular localization [The main proposed mechanism of action for cannabinoids in cancer cells is the induction of apoptosis by stimulating the production of reactive oxygen species (ROS) . It is wtrations . H2O2 aclization .It should be pointed out that also several pre-clinical in vitro and in vivo studies showed that among discovered cannabinoids, cannabidiol (CBD) alone or combined with standard chemotherapeutic drugs reduces cancer cells’ viability and tumor size, making it the most promising cannabinoid used to treat various diseases, including cancer . MoreoveBased on existing information, it can be said that hemp oil enriched in CBD could become one of the most powerful nutraceuticals recommended as adjuvants in numerous diseases, as well as for treatment for pain and in the reduction of chemotherapy side effects . In addi2●− scavenging activity, HO● scavenging ability and lipid peroxidation inhibitory assay), as well as its effects on normal dermal fibroblasts (NHDF), malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells, with emphasis on cytotoxicity, induction of apoptosis and oxidative stress. The results show that hemp oil enriched in CBD exhibits antioxidant properties in oxidative conditions and has significant anticancer effects on MeWo, HeLa, HepG2 and HOS cells, while being safe for normal fibroblasts, thus suggesting its potential application as an adjuvant in cancer therapy.This study aimed to obtain and characterize hemp oil enriched in CBD for use as a nutraceutical in cancer management and treatment. For this purpose, we determined the most efficient decarboxylation conditions of CBDA from extracted hemp oil. Furthermore, we investigated the cell-free antioxidant properties of CBD-enriched hemp oil . They were harvested when 50% of them were flowering and then air-dried in the dark.All extracts were done from a single strain of For this study, 500 g of the top third of the plant was used. The extraction procedure involved maceration of the finely ground plant in ethanol (ratio between dried plants and ethanol: 1:5) at room temperature for 30 min, under slow stirring. After filtration, the solid residue was washed again with ethanol at a ratio of 1:2 for 10 min and then filtered. The two supernatant solutions were combined and evaporated under vacuum, at 150 mbar, 55 °C, until the ethanol had been removed. The resulting hemp oil was kept at 4 °C until use.The hemp oil was processed by flash chromatography using a column of appropriate diameter and packed 1/3 with appropriate silica gel (230–400 mesh). A sample of 60 mg crude hemp oil was diluted with methanol and slowly introduced on the silica bed using the column wall as support. The elution of other compounds was performed with ethanol:acetonitrile 9:1, followed by methanol only for CBD elution.1H- and 13C-NMR, using a Bruker Avance NEO 400 MHz Spectrometer, equipped with a 5 mm inverse detection z-gradient probe, operating at 400.1 and 100.6 MHz for 1H and 13C nuclei. The sample was dissolved in dimethyl sulfoxide-d6 (DMSO-d6) and the spectra were recorded at room temperature. Chemical shifts were reported in ppm and referred to residual solvent signal (ref. 1H 2.512 ppm and 13C 39.47 ppm).The final fraction was collected and freeze-dried, obtaining 5 mg of solid white compound, representing purified CBD, from 60 mg crude hemp oil. The purity of the compound was verified by 2O with 0.1% HCOOH, pH 2, and B: methanol [The HPLC determination of CBDA and CBD from crude and decarboxylated hemp oil samples was performed using a Perkin Elmer HPLC system with a Flexar UV/VIS Detector at 220 nm and 30 °C. A 10 µL fixed injection loop was made in a Mediterranea Sea 18 column  with a 1 mL/min flow mobile phase in a gradient formed by A: Milli-Q Hmethanol  with 0.1By comparing two HPLC spectra of the same concentration of CBD and CBDA, respectively, the same peak areas were obtained. Due to this observation, CBDA concentrations were calculated using the same equation from the CBD calibration curve.In this study, the decarboxylation conditions were varied to screen for the best conditions to obtain maximum CBDA decarboxylation yield. Therefore, hemp oil was incubated at 500 mbar, 60 rpm, varying the temperature and time: (1) 70 °C, 1 h; (2) 70 °C, 2 h; (3) 70 °C, 3 h; (4) 70 °C, 4 h; (5) 80 °C, 1 h; (6) 80 °C, 2 h; (7) 80 °C, 3 h; (8) 80 °C, 4 h; (9) 90 °C, 1 h; (10) 90 °C, 2 h; and (11) 100 °C, 1 h.After decarboxylation, each sample was analyzed by HPLC using the method described in 2 × 4 H2O, 2 mM) was added. Methanol was used as a control. Then, 200 μL ferrozine (2 mM) was added and the total volume was adjusted to 2 mL with ethanol. The mixture was vigorously shaken and incubated at room temperature for 10 min. The Fe2+ chelating activity was estimated by measuring the absorbance of the Fe2+-ferrozine complex at 562 nm in a 96-well plate using a FLUOstar® Omega microplate reader . The inhibition percentage of Fe2+–ferrozine complex was calculated using the following formula: The chelating activities of CBD-enriched hemp oil, pure CBD and crude hemp oil compared to gallic acid (as a common antioxidant reference) were estimated using a previously described method , with sosorbance .2HPO4/KH2PO4, 0.2 M, pH 6.6) and 650 μL potassium ferricyanide (K3Fe(CN)6, 1%) and the mixture was incubated for 20 min at 50 °C. After 20 min, 650 μL trichloroacetic acid (10%) was added to the mixture and 910 μL of this solution was mixed with 910 μL distilled water and 180 μL ferric chloride . The absorbance was measured at 700 nm in a 96-well plate using a FLUOstar® Omega microplate reader . The reducing power (%) was expressed as the ratio between the absorbance for 15 μg CBD/mL and the absorbance for 45 μg CBD/mL.FRAP assay was performed using a protocol adapted from Li et al.  for CBD-2●− were determined with a method adapted after Xiong et al. [® Omega microplate reader . The scavenging percentage was calculated using the formula: The abilities of CBD-enriched hemp oil, pure CBD and crude hemp oil compared to gallic acid (as a common antioxidant reference) to scavenge Og et al. . In briesorbance .● scavenging abilities of CBD-enriched hemp oil, pure CBD and crude hemp oil compared to gallic acid (as a common antioxidant reference) were determined using a previously published method [4 × 7 H2O, 2.5 mM) and 400 μL of H2O2 were added and the mixture was incubated at 37 °C on a thermo-shaker for 1 h. After incubation, the absorbance was measured at 536 nm in a 96-well plate using a FLUOstar® Omega microplate reader . Methanol was used as a control. The scavenging percentage of HO● was calculated with the formula: 2O2 in reaction [HOd method , with soreaction .2 × 4 H2O solution (20 mM in 3.5% HCl). The resulting solution was mixed thoroughly and 50 μL potassium thiocyanate (KSCN) solution (30% in distilled water) was added. The absorbance of the resulting clear solution (50 μL) was recorded at 500 nm in a 96-well plate using a FLUOstar® Omega microplate reader . The inhibitory effect was calculated using the formula: Lipid peroxidation inhibitory activities of CBD-enriched hemp oil, pure CBD and crude hemp oil compared to gallic acid (as common antioxidant reference) were measured by a previously described method , with sosorbance .Normal dermal fibroblasts , malignant melanoma (MeWo), adenocarcinoma (HeLa), hepatocellular carcinoma (HepG2) and osteosarcoma (HOS) cells  were grown in alpha-MEM medium  supplemented with 10% fetal bovine serum  and 1% Penicillin-Streptomycin-Amphotericin B mixture .® AQueous One Solution Cell Proliferation Assay , according to the manufacturer instructions. Cells were seeded at a density of 0.5 × 105 (NHDF) or 1 × 105 cells/mL  into 96-well tissue culture-treated plates. After 24 h the medium in each well was replaced with 100 μL fresh complete medium  or various concentrations of CBD-enriched hemp oil . CBD-enriched hemp oil samples used for cell treatments were obtained by serial dilution of hemp oil with maximum yield of CBDA decarboxylation. Cells were treated for 48 h and 20 µL MTS reagent was added 1–3 h prior to absorbance readings at 490 nm on a FLUOstar® Omega microplate reader . Experiments were done in triplicate and treated cells’ viability was expressed as a percentage of control cells’ viability. The CBD-enriched hemp oil concentration that induced a 50% decrease in cell viability (IC50) was determined with GraphPad Prism 8 software from the dose–response curves for each cell line. The selectivity index of CBD-enriched hemp oil was calculated using the formula: SI = IC50 normal cells/IC50 cancer cells, as suggested by Badisa et al. [Cytotoxicity was measured using the CellTiter 96a et al. .5 (NHDF) or 1 × 105 cells/mL  into 24-well tissue culture-treated plates, allowed to adhere overnight, then incubated with fresh complete medium  or various concentrations of CBD-enriched hemp oil  for 48 h. CBD-enriched hemp oil samples used for cell treatments were obtained by serial dilution of hemp oil with maximum yield of CBDA decarboxylation. After incubation, the cells were visualized in bright field using a DMI 3000 B inverted microscope . Experiments were done in triplicate and images were acquired using the 10× objective for assessing morphological changes.Cells were seeded at a density of 0.5 × 105 (NHDF) or 1 × 105 cells/mL  into 24-well tissue culture-treated plates, allowed to adhere overnight, then incubated with fresh complete medium  or various concentrations of CBD-enriched hemp oil  for 48 h. CBD-enriched hemp oil samples used for cell treatments were obtained by serial dilution of hemp oil with maximum yield of CBDA decarboxylation. After incubation, the medium was removed and cells were stained with AO/EB solution (4 µg AO/EB/mL) for 1 min, then washed with PBS and visualized in complete medium, using a DMI 3000 B inverted microscope . Experiments were done in triplicate and images were acquired using the I3 filter and the 20× objective.A modified method of Ribble et al.  for AO/E2O2 production was measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), as previously described [5 (NHDF) or 1 × 105 cells/mL  into 12-well tissue culture-treated plates, allowed to adhere overnight, then incubated for 24 h with fresh complete medium (control) or specific concentrations of CBD-enriched hemp oil . After incubation, cells were loaded with 2 µM DCFH-DA for 20 min at 37 °C. After a gentle wash, the cells were scraped in a serum-free medium and the fluorescence was measured at 485/520 nm in 96-well black plates using a FLUOstar® Omega microplate reader . Experiments were done in triplicate, and H2O2 production was calculated as relative fluorescence units (RFU)/mg total cellular protein and expressed as fold change of control values. Total cellular protein was determined using the bicinchoninic acid assay.Intracellular Hescribed . Cells w5 (NHDF) or 1.25 × 105 cells/mL  into 6-well tissue culture-treated plates, allowed to adhere overnight, then incubated with fresh complete medium (control) or specific concentrations of CBD-enriched hemp oil  for 48h. After incubation, cells were rinsed once with PBS and total RNA was isolated using TRIzol reagent , according to the manufacturer’s protocol. Reverse-transcription (RT) of RNA was performed on a Veriti PCR system, using the High Capacity cDNA Reverse Transcription kit and random RT primers . Real-time quantitative PCR reactions were performed on a QuantStudio 12K Flex Real-Time PCR System , using custom primers . Data were expressed as means ± standard error of the mean and analyzed by independent two-tailed (Student’s) C. sativa L. denoted KC Dora, cultivated in north-eastern Romania ): 0.87 , 1.25–1.31 , 1.48 , 1.59–1.68 , 1.90–2.11 , 2.31 , 3.04 , 3.83 , 4.42–4.45 , 5.09 , 6.02  and 8.66  and 13C-NMR ): 13.9 (CH-5″), 19.2 (CH3-10), 21.9 (CH-4″), 23.2 (CH3-7), 29.4 (CH-5), 30.2 (CH-4 and CH-2″), 30.9 (CH-3″), 34.9 (CH-1″), 35.5 (CH-1), 43.6 (CH-6), 106.6 (CH-3′ and CH-5′), 109.6 (CH-9), 114.1 (C-1′), 126.8 (CH-2), 129.9 (C-3), 140.1 (C-4′), 149.1 (C-8) and 156.2 (C-2′ and C-6′).A sample of 60 mg crude hemp oil was processed by flash chromatography see  in order solvent . The NMRw/w) CBD and 13.54% (w/w) CBDA  CBDA .C. sativa L. plants, and is produced by cannabidiolic acid synthase from cannabigerolic acid. Through a slow decarboxylation process, which takes place spontaneously at room temperature in plants, CBDA is partially transformed into CBD [As it was mentioned before, CBDA is the most abundant cannabinoid in into CBD . Moreoveinto CBD .w/w) CBD and 4.52% (w/w) CBDA after 1 h and 17.82% (w/w) CBD and 3.8% (w/w) CBDA after 2 h, respectively, representing 8.6 and 8.91 mg CBD/g vegetal material, which is 35.4 and 36.5 times higher than reported for hemp seed oil  [In order to obtain a therapeutically valuable product, characterized by a higher concentration of CBD compared to CBDA, CBDA in hemp oil could be subjected to a decarboxylation reaction. For this purpose, Citti et al.  performeaterial) . Decarboaterial) .In the literature, CDB’s antioxidant role is based on two main mechanisms which are specific for phenol derivatives: one is based on electron transfer to reduce any compound and the second one is hydrogen donation to quench free radicals ,24. Thes2●− or HO● [In brief, to determine the antioxidant properties of natural extracts, in the literature there are many methods based on the two mentioned main mechanisms: one is the reduction of a metallic species  by the natural compound, and another is the reaction of the natural compound with a free radical, such as O− or HO● . In orde2+ chelating activity, FRAP assay, O2●− scavenging activity, HO● scavenging ability and lipid peroxidation inhibitory assay , followed by the formation of Perl’s Prussian complex (intense blue color) in the presence of Fe= 0.9463 , being i2●− (mildly reactive) and HO● (highly reactive), which are generated in different metabolic processes and may lead to lipid peroxidation and tissue damage [The antioxidant capacity of vegetal bioactive compounds can be estimated also by the compounds’ capacity to neutralize ROS with a short half-life, such as Oe damage .2●− and HO● super reactive radicals, agreeing with other published data [In our case, as it can be observed in hed data ,27,28, w2●− and HO● through free radical chain reactions [The last method applied for the determination of scavenging activity toward ROS was the lipid peroxidation assay, which is initiated by Oeactions . The amoTaken together, these results show that CBD-enriched hemp oil has a high antioxidant activity, being capable of reducing iron, scavenging free radicals and inhibiting lipid peroxidation.C. sativa L., cultivated in north-eastern Romania, with maximum yield of CBDA decarboxylation.Cytotoxicity on normal fibroblasts (NHDF) and malignant cell lines  of CBD-enriched hemp oil was determined using MTS assay after 48 h incubation. The CBD-enriched hemp oil samples of different CBD concentrations used for cell treatments were obtained by serial dilutions of oil extracted from The results show that CBD-enriched hemp oil promoted the proliferation of normal fibroblasts at concentrations up to 15 µg CBD/mL, followed by cytotoxic effects at higher concentrations , being iRegarding the cell viability of malignant cells, its decrease started at a concentration of 5 µg CBD/mL, in a more pronounced or lesser manner, depending on the malignant cell line. Thus, the viability of the HOS cell line decreased dramatically even at a concentration of 5 µg CBD/mL, while for HepG2, HeLa and MeWo cells at 10 µg CBD/mL .Taking into consideration these results, it can be concluded that the range of optimal concentrations is between 5 and 15 µg CBD/mL as fibroblast proliferation is not affected, while a decrease in viability of cancer cells can be observed.50), meaning in our case the amount of CBD necessary to decrease the cell viability by 50%. Another important parameter is the selectivity index (SI) of compounds toward cell lines. When SI values are higher than 2, a good selectivity of the compound toward a certain cancer line can be considered and, also, the higher the value, the better selectivity to the cell line is considered, while SI values < 2 suggest general toxicity, thus affecting normal cells as well [50 normal cells/IC50 studied cancer cells.Additionally, an important parameter in studying the anticancer effect of different compounds is the half-maximal inhibitory concentration on cell viability  being at least three times lower than the one obtained for NHDF cells (IC50 = 26.65), suggesting that CBD-enriched hemp oil is selective towards osteosarcoma and could be used as an alternative or adjuvant treatment. To the best of our knowledge, CBD’s or hemp oil’s cytotoxic effects have not been investigated in osteosarcoma. However, some research groups have determined that a synthetic cannabinoid  induced cell cycle arrest and endoplasmic reticulum stress [Data presented in m stress  and had m stress .50 and SI values in HeLa and HepG2 cells , and, by increasing the CBD concentrations, cell shrinkage and detachment, cytoplasmic condensation and vesicle formation were observed . MeWo ceAO/EB staining allows visualization of apoptotic body formation and nuclear changes characteristic of apoptosis . AcridinIn order to determine the capacity of CBD-enriched hemp oil to induce apoptosis in different malignant cell lines, MeWo, HeLa, HepG2, HOS and NHDF cells were treated with different doses of CBD-enriched hemp oil for 48 h and AO/EB staining was applied , accordiFrom MeWo cells  displayeC. sativa extracts induce apoptosis in HeLa cells.HeLa cells  exhibiteStarting at 5 µg/mL, chromatin condensation and nuclear fragmentation occurred in HepG2 cells , accompaHOS cells exhibited chromatin condensation, cell shrinkage and detachment starting from 10 µg CBD/mL, as well as apoptotic bodies’ formation (stained red) starting from 15 µg CBD/mL . Moreove−ΔΔCq method [50 calculated for each cell line) for 48h  by real-time quantitative PCR see , and calq method . For thi 48h see .50 of CBD-enriched hemp oil for 48 h  and normal fibroblasts (NHDF) treated with cell line-specific IC50 of CBD-enriched hemp oil  cells, and it was significantly higher in HeLa and HepG2 cells , after CBD-enriched hemp oil treatment for 48 h, GPX1 gene expression was significantly reduced c, but GS2O2 production was observed, an important conclusion can be drawn, namely that CBD-enriched hemp oil induces apoptosis in HOS cells by ROS-independent mechanisms.In treated HOS cells, SOD1, CAT, GPX1 and GSR gene expressions were largely unaffected by CBD-enriched hemp oil treatment . If we cC. sativa L. denoted KC Dora, cultivated in north-eastern Romania, and after vacuum evaporation of the solvent, a yield of 5.09% from vegetal material was obtained. The content and the purity of CBD in extracted hemp oil were determined by flash chromatography and 1H- and 13C-NMR, respectively. We found the most efficient decarboxylation conditions for CBDA from hemp oil , which yields hemp oil enriched in CBD without significant degradation of bioactive compounds.In conclusion, hemp oil was extracted with ethanol at room temperature from The obtained CBD-enriched hemp oil was capable of reducing iron, scavenging free radicals and inhibiting lipid peroxidation in oxidative conditions, suggesting that it could protect against oxidative damage in normal cells. Conversely, CBD-enriched hemp oil induced ROS production in cancer cells, but more extensive studies need to be undertaken in order to elucidate the exact mechanisms. Furthermore, CBD-enriched hemp oil promoted the proliferation of normal fibroblasts when the concentration of CBD was up to 15 µg/mL, while being cytotoxic for cancer cells. To the best of our knowledge, this is the first study to investigate the cytotoxic effects of CBD or hemp oil on osteosarcoma cells, and the results show that CBD-enriched hemp oil was selective for osteosarcoma cells and induced apoptosis in a dose-dependent manner, by p53- and ROS-independent mechanisms.CBD-enriched hemp oil promoted apoptosis in the studied malignant cell lines through p53-independent mechanisms, in contradiction with other published data . Due to To the best of our knowledge, the effect of CBD-enriched hemp oil on antioxidant enzymes’ gene expression in MeWo, HeLa, HepG2 and HOS cells has not been investigated. Our study revealed that CBD-enriched hemp oil modulated the gene expression of antioxidant enzymes in a cell line-specific manner. Our findings suggest normal fibroblasts treated with CBD-enriched hemp oil have greater cellular resilience to oxidative stress, mainly through an increase in glutathione levels, in contrast with malignant melanoma cells. Moreover, our results suggest that CBD-enriched hemp oil induces apoptosis in MeWo cells by ROS-related mechanisms. Additionally, our results suggest that CBD-enriched hemp oil could be used as a pretreatment or adjuvant in adenocarcinoma (HeLa) and hepatocellular carcinoma (HepG2) therapies, as well as in topical administration in melanoma treatment.Our study aimed to extend the knowledge in the field, and our results give new insights on the biological effects of CBD-enriched hemp oil, recommending it as adjuvant or treatment in various cancers, especially for osteosarcoma, with numerous health benefits and very few side effects. CBD-enriched hemp oil should be considered a versatile nutraceutical with applications in cancer management and treatment and, although CBD is the major pharmacologically active compound in CBD-enriched hemp oil, this product should be viewed as a complex mixture of bioactive molecules with synergic properties.


Myelodysplastic syndromes (MDS) is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells. It has been demonstrated that apolipoproteins A1(ApoA1) are associated with disease risk in many cancer types. However, there still lacks evidence regarding the link between ApoA1 and MDS. This study was designed to investigate the prognostic value of pretreatment ApoA1 levels in MDS patients.We retrospectively analyzed a cohort of 228 MDS patients to explore the prognostic value of the serum ApoA1 levels at diagnosis. Patients were divided into the high ApoA1 group and the low ApoA1 group. The prognostic significance was determined by univariate and multivariate Cox hazard models.P < 0.0001) along with a higher frequency of TP53 mutation (P = 0.002). Based on univariate analysis, age (≥ 60 years), gender , lower levels of hemoglobin (< 10 g/dl), HDL (≤0.91 mmol/L), higher bone marrow blast percentage (> 5%), higher IPSS-R scores and poorer karyotype were significantly associated with decreased OS. However, low ApoA1 level did not influence leukemia-free survival . Multivariate Cox proportional hazards regression analysis indicated that low ApoA1 level (≤ 1.02 g/L) was also an independent adverse prognostic factor for OS in MDS (P = 0.034).MDS patients with low ApoA1 levels had significantly shorter overall survival (OS, Decreased ApoA1 level predicts a poor prognosis of MDS patients and thus provides a novel evaluation factor for them that is independent of the IPSS-R system. TP53, SRSF2, IDH2 and ASXL1 were also demonstrated to be valuable in predicting the prognosis of MDS [Myelodysplastic syndromes (MDS), characterized by ineffective hematopoiesis that is manifested by morphologic dysplasia in hematopoietic cells and peripheral cytopenia(s), is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells with a high risk of transforming to secondary acute myeloid leukemia (AML) . The proThe tumor microenvironment interacts with tumor cells and plays a crucial role in tumorigenesis and development. By mediating complex signaling pathways, tumor microenvironment regulates the expression of various pro-inflammatory cytokines, chemokines, and angiogenic factors, all of which promote tumor growth, invasion and metastasis . MDS alsClinical and follow-up data of 228 patients who were newly diagnosed with MDS in Ningbo First Hospital from 2009 to 2019 were collected. Diagnosis and classification of MDS and leukemic transformation were determined according to the 2016 WHO classification [Peripheral blood was drawn after strict fasting of at least 6 h. Serum ApoA1 level was measured using turbidimetric immunoassay. The reagents were tested by using Beckman’s ApoA1 kit withtheir instructions, under an automatic biochemical analyzer (Beckman AU5800).Morphology of MDS myeloid cells was observed through Wright-Giemsa stained bone marrow smears. It was evaluated subjectively by light microscopy at low power (10 × objectives) for overall quality and distribution, before further analysis at high power (100 × oil objectives) for the differential count.BM cells were collected and cultured in RPMI-1640 medium supplemented with 20% newborn calf serum for 24 h. R-banded metaphases and the karyotypes were identified at least 20 metaphases for normal karyotype and at least 10 metaphases for abnormal karyotype according to the International System for Human Cytogenetic Nomenclature (2016) (ISCN2016) . The karNRAS, DNMT3A, SF3B1, IDH1, IDH2, TET2, EZH2, JAK2, CBL, ETV6, TP53, SRSF2, ASXL1 and RUNX1 was performed using the next-generation sequencing. Variants with a variant allele frequency of <1% were excluded from the analysis. Multiplex PCR was used to amplify and construct sample library, high-throughput sequencing was performed on the Ion Proton platform, and bioinformatics analysis was performed with reference to PolyPhen, HG19, 1000 genomes, COSMIC, ClinVar, dbSNP databases. Gene mutation detection was completed by Kindstar Global Medical Laboratory .Molecular analysis was performed as a part of the routine clinical work-up. Mutational analysis for 14 common genes of MDS including U and categorical variables by Chi-squared test. The cutoff point of ApoA1 was calculated using the X-Tile software [P-value under the log-rank test) was 1.02 g/L. The P-value of < 0.05 was considered statistically significant.Statistical analyses were performed by SPSS 26.0. OS was calculated from the date of initial diagnosis of MDS to the date of death, last follow-up or acquiring allo-HSCT. Leukemia-free survival (LFS) was determined from the date of diagnosis to the date of leukemia transformation, last follow-up or acquiring allo-HSCT. OS and LFS were analyzed using the Kaplan-Meier method and were compared using the log-rank test. Multivariable analyses were performed using the Cox proportional hazard regression model. Differences in the distribution of continuous variables between categories were analyzed by Mann-Whitney software . The optThe data of 228 MDS patients, including 95 females and 133 males were collected over 10 yearswith a median age of 62 years (range 16–90 years). The median OS of these patients was 27  months and 26 of them (11.4%) progressed to AML. Based on the 2016 WHO classification, all MDS patients were classified as follows: 23(10.1%) of MDS-SLD, 63(27.6%) of MDS-MLD, 15(6.6%) of MDS-RS, 59(25.9%) of MDS-EB1, 48(21.1%) of MDS-EB2, 6(2.6%) of MDS-del(5q) including del(5q) alone or with 1 additional abnormality except − 7 or del(7q), and 14 (6.1%) of MDS-U. Besides, 194 patients were stratified into IPSS-R risk groups as follows: 13 (6.7%) at very low risk, 36(18.6%) at low risk, 67(34.5%) at intermediate risk, 41(21.1%) at high risk and 37(19.1%) at very high risk. Of these, the median IPSS-R score was 4.5(1.0–10.0). Detailed information was provided in Table P < 0.0001; Fig. P = 0.007), higher levels of CRP (P = 0.013) and fewer counts of HB (P < 0.0001), PLT (P = 0.005), ALB (P < 0.0001), CHO (P < 0.0001), HDL (P < 0.0001), LDL (P < 0.0001), and ApoB (P < 0.0001) along with higher risk distribution in terms of IPSS-R (P = 0.035). Additionally, the WHO subtype between these two groups had a significant difference (P = 0.039). There were no significant differences in other factors between the two groups , TP53 (10.9%), RUNX1 (12.5%), SF3B1 (7.8%), TET2 (7.8%), DNMT3A(6.3%), IDH2 (4.7%), SRSF2 (4.7%), NRAS(3.1%), EZH2 (3.1%), CBL (3.1%), IDH1 (1.6%), JAK2 (1.6%) and ETV6 (0.0%) . Of note, the low ApoA1 group showed a higher mutation frequency of TP53 compared with the high ApoA1 group . There was no difference between the two groups in other 13 gene mutation (data not shown).The mutation profile of 14 dominant genes were detected in 64 patients, 41(64.1%) of whom harbored at least one mutated gene. The mutation rates of the 14-target genes as follows: , RUNX1 1.5%, SF3BP < 0.0001; Fig. P = 0.367; Fig. Compared with the high ApoA1 group, the median OS in the low ApoA1 group was significantly shorter , male gender (P = 0.012), higher-risk IPSS-R cytogenetic (P = 0.012), higher BM blast percentage , higher IPSS-R score (P < 0.0001), lower levels of HB(< 10 g/dl) (P = 0.005), lower levels of HDL (≤0.91 mmol/L) (P = 0.002),and ApoA1 .In univariate analysis, the OS was adversely associated with older age , higher BM blast percentage , higher-risk IPSS-R cytogenetic (P = 0.005), were adverse factors while a low ApoA1 level was a significant prognostic factor for worse OS (P = 0.034)  Table . TherefoTP53 mutation rate, higher CPR levels as well as lower levels of HB, PLT, ALB, CHO, HDL, LDL and ApoB. We observed that ApoA1 was closely correlated with HDL. We have added HDL to our cohort. Decreased serum ApoA1 levels correlated with a shorter survival period in MDS, indicating that lower serum ApoA1 level reflects a poor prognosis in MDS patients. The Cox regression analysis revealed that the ApoA1 level was an independent prognostic factor for MDS patients.Based on the obtained results, we showed that pretherapy serum ApoA1 at a low level was associated with higher BM blast percentage, higher IPSS-R scores, higher The metabolic patterns of tumor cells, including lipid metabolism are different from those of normal cells. It has been demonstrated that lipids play an important role in the occurrence and development of malignant tumors. ApoA1 is synthesized predominantly in the liver and the small intestine, which is the predominant protein of plasma HDL . ApoA1 nTP53 gene, located in the 17p13 chromosomal region is one of the major tumor suppressor genes and is often inactivated by deletion and/or mutation in many tumors, including hematologic malignancies [TP53 was 5–10% in MDS [TP53 mutation in MDS is strongly associated with poor treatment outcomes [TP53 is known to play a role in lipid metabolism [TP53 had the role in enhancing lipid catabolism while inhibiting its anabolism. In our cohort, we found that ahigh TP53 gene mutation rate was correlated with the decrease of serum ApoA1 in MDS. Due to applying a 14-mutations panel to do the MDS mutation screening, the detected gene mutation rate is low, which is in line with the study of Mengyi Du [gnancies . The mut% in MDS . TP53 muoutcomes . TP53 istabolism . Goldstetabolism  found thengyi Du .It is well known that IPSS-R was widely used in measuring the prognosis of MDS. Although ApoA1 was reported to be a prognostic factor in several malignancies, to the best of our knowledge, an association between ApoA1 and the prognosis of MDS patients has not been reported to date. In this study, we found that the ApoA1 level in MDS patients was lower than that in controls, and was proved to be an independent predictor of OS.Furthermore, ApoA1 could function as an independent prognostic factor of MDS, also it is a common and convenient indicator in pretreatment examination. In addition, this study provides a new idea for the prognostic evaluation of MDS, and provides a potential therapeutic target.TP53 mutation and was associated with a poor prognosis in MDS patients. ApoA1 as a prognostic factor could provide convenience for evaluating the prognosis of MDS patients and be a useful supplement to IPSS-R. Thus, lipid metabolism-oriented therapeutics might be promising strategies for MDS patients. As this study is a retrospective analysis, it is only valid for generating a hypothesis, and the value of ApoA1 should be validated in large prospective trials.We demonstrated that decreased ApoA1 level was accompanied by a higher frequency of


Statins are the first-line lipid-lowering therapy for reducing cardiovascular disease (CVD) risk. A plasma lipid ratio of two phospholipids, PI(36:2) and PC(18:0_20:4), was previously identified to explain 58% of the relative CVD risk reduction associated with pravastatin, independent of a change in low-density lipoprotein-cholesterol. This ratio may be a potential biomarker for the treatment effect of statins; however, the underlying mechanisms linking this ratio to CVD risk remain unclear. In this study, we investigated the effect of altered cholesterol conditions on the lipidome of cultured human liver cells (Hep3B). Hep3B cells were treated with simvastatin (5 μM), cyclodextrin (20 mg/mL) or cholesterol-loaded cyclodextrin (20 mg/mL) for 48 h and their lipidomes were examined. Induction of a low-cholesterol environment via simvastatin or cyclodextrin was associated with elevated levels of lipids containing arachidonic acid and decreases in phosphatidylinositol species and the PI(36:2)/PC(18:0_20:4) ratio. Conversely, increasing cholesterol levels via cholesterol-loaded cyclodextrin resulted in reciprocal regulation of these lipid parameters. Expression of genes involved in cholesterol and fatty acid synthesis supported the lipidomics data. These findings demonstrate that the PI(36:2)/PC(18:0_20:4) ratio responds to changes in intracellular cholesterol abundance per se, likely through a flux of the n-6 fatty acid pathway and altered phosphatidylinositol synthesis. These findings support this ratio as a potential marker for CVD risk reduction and may be useful in monitoring treatment response. StatinsDue to the strong relationship between elevated low-density lipoprotein cholesterol (LDL-C) levels and coronary events, the beneficial effects of statins have been largely attributed to a reduction of circulating LDL-C . By inhiMore recent preclinical investigations have suggested that statins mediate additional beneficial actions beyond their ability to lower LDL-C levels. Proposed mechanisms include a reduction in endothelial nitric oxide synthase, improvement and restoration of endothelial function, enhanced stability of atherosclerotic plaques and decreased oxidative stress and vascular inflammation ,10,11. TIn one of the largest lipidomic studies to date, Jayawardana and colleagues characterised changes in the plasma lipidome in response to pravastatin treatment to define the relationship between statin treatment, plasma lipids and risk reduction of future cardiovascular events in secondary prevention . The autThis study aimed to better understand the mechanisms by which statins modulate the PI(36:2)/PC(18:0_20:4) lipid ratio. To achieve this, we developed a cell-based model that reflected the statin-induced changes observed in the human plasma lipidome by Jayawardana et al. . Hep3B cSimvastatin treatment was associated with a significant decrease in cholesterol, total CE and the PI(36:2)/PC(18:0_20:4) ratio A. It wasp = 0.00001; p = 0.0001; p = 0.00001; To investigate whether the effects of simvastatin on the lipid ratio were a downstream consequence of cholesterol lowering or the result of an off-target effect of simvastatin, cellular cholesterol levels were modulated using an alternative approach. Methyl-ß-cyclodextrin was utilised to deplete cells of membrane cholesterol, which resulted in a comparable, yet more exaggerated lipidomic profile to that seen with simvastatin . A stronTo assess whether the addition of cholesterol would result in opposing effects to that observed when cells were depleted of cholesterol, cells were treated with the cholesterol donor, cholesterol-loaded cyclodextrin (COH-cyclodextrin). Interestingly, this did not appear to produce the same magnitude of effect on the lipidome as seen with simvastatin or cyclodextrin . Whilst HMGCR, the rate-limiting enzyme in cholesterol synthesis, LDLR, a key receptor involved in LDL-C uptake, and ABCA1, a marker of cholesterol efflux, were assessed as readouts of the sterol regulatory element binding protein 2 (SREBP2) and liver X receptor (LXR) pathways, respectively. Expression of INSIG1, which resides in the endoplasmic reticulum and regulates cholesterol synthesis, and ACAT2, an enzyme that esterifies cholesterol, was also assessed. Both HMGCR and LDLR mRNA expression increased in response to simvastatin  and cyclodextrin , whilst expression decreased in response to COH-cyclodextrin compared to simvastatin  (ABCA1 decreased when intracellular cholesterol levels were lowered via simvastatin (p < 0.01 vs. control) and significantly increased in response to COH-cyclodextrin (p < 0.0001 vs. statin). Interestingly, mRNA expression of INSIG1 was similar to that seen with HMGCR and LDLR (FASN), a SREBP-1c target gene, only slightly increased in response to elevated cholesterol (p < 0.05 vs. control). ELOVL5 is a protein involved in the elongation of very long-chain fatty acids. Specifically, it converts dihomo-γ-linolenic acid (20:3) into AA in the n-6 fatty acid pathway. Our data demonstrates an inverse relationship between ELOVL5 mRNA expression and total cholesterol levels  and cyclodextrin (p < 0.05 vs. control). Expression of MBOAT7, a gene involved in PI synthesis, showed an increase with simvastatin and cyclodextrin treatments compared to control (p < 0.01 vs. control); however there was no significant difference in expression with COH-cyclodextrin treatment. Finally, expression of ACAT2, the enzyme involved in cholesterol esterification, was consistent with the other SREBP targets, as it increased in response to lowered COH environments and returned to baseline (control) when COH levels increased  A,B. Convand LDLR D. Expresl levels F, similancreased H. These mic data E,F.ACAT2 further endorsed the lipidomics data, indicating that the reduced cholesterol and CE levels sparked an increase in ACAT2 expression, likely in an attempt to recover the low CE levels. Many species containing AA increased after simvastatin treatment, suggesting a flux through the n-6 fatty acid pathway was occurring. PC and TG ether lipids ((PC(O), PC(P) and TG(O)), appeared to be the primary lipid classes containing AA species that increased. This is in line with their known function as storage depots for polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid  and AA [Lipids are the major structural components of all organelle and cellular membranes. Whilst membranes are highly organised, they undergo constant remodeling in response to extra- and intra-cellular stimulation . Simvast) and AA ,16,17. D) and AA ,19. As m) and AA . Interes) and AA . Experimcid DHA; :6 and AA) and AA ,24. ThusHMGCR, LDLR) confirmed a similar effect on endogenous cholesterol levels between simvastatin and cyclodextrin treatments. Hep3B cells treated with cyclodextrin demonstrated a comparable lipid profile to simvastatin. The levels of PI(36:2) and PC(18:0_20:4) decreased and increased respectively with cyclodextrin treatment, resulting in an overall decrease in the PI(36:2)/PC(18:0_20:4) ratio. A greater number of species containing AA increased after cyclodextrin, aligning with the ability of cyclodextrin to rapidly remove cholesterol from cellular membranes, faster than physiological cholesterol acceptors [ABCA1, an LXR target gene that promotes cholesterol efflux, and a significant increase in cholesterol and CE species, confirmed the success of the COH-cyclodextrin treatment. Most PI species increased after treatment whilst the majority of species containing AA decreased. Changes in AA species containing palmitic acid showed an opposing effect as they increased in response to COH-cyclodextrin treatment. This suggests an upregulation of de novo lipogenesis in response to the increased cellular cholesterol.Altering cholesterol conditions via different treatments (cyclodextrin and COH-cyclodextrin) provided insight into whether changes in the PI(36:2)/PC(18:0_20:4) ratio were an off-target effect of statins, or the consequence of cholesterol lowering. Cyclodextrin induces a low-cholesterol phenotype by specifically extracting cholesterol from the outer leaflet of the plasma membrane . This mecceptors . HoweverELOVL5, the enzyme that catalyses the first and rate-limiting step involved in the long-chain fatty acid elongation cycle of the n-6 pathway, supports this. Further analysis suggests that this flux is likely caused by the SREBP pathway. SREBPs are a family of membrane-bound transcription factors and are considered the primary regulators of cholesterol and unsaturated fatty acid synthesis [−/−ELOVL5 mice, Moon and colleagues further demonstrated that endogenously synthesised PUFA are key regulators of SREBP-1c activation and fatty acid synthesis in the liver [FASN.The underlying mechanisms driving this change in the PI(36:2)/PC(18:0_20:4) lipid ratio appear to be diverse and complex. The increase in species containing AA, including PC(18:0_20:4), across multiple lipid classes, suggests that modulation of the n-6 fatty acid pathway is occurring. Expression of ynthesis . Statinsynthesis . Using Ehe liver . This ishe liver . This suhe liver . This isWhilst most PI species decreased in response to simvastatin and increased after COH-cyclodextrin, the underlying mechanisms remain unclear. Jayawardana et al. proposed that pravastatin reduced PI species via modulation of CDP-diacylglycerol synthesis, an important intermediate in phospholipid biosynthesis . Work byWhilst the majority of the findings in this study support the observations of Jayawardana et al., there are some differences to be noted. Jayawardana et al. observed a significant decrease in plasma TG species whilst our data demonstrated an increase in cellular TG species. As simvastatin drastically reduces cholesterol and CE levels, formation of VLDLs may be hindered. This would result in the observed accumulation of cellular TGs as their secretion is reduced. Differences between simvastatin and cyclodextrin treatments were also observed, however, these differences were consistent with their unique mechanisms of lowering cholesterol. For example, CE decreased after simvastatin treatment and increased with cyclodextrin. As discussed previously, cyclodextrin does not inhibit cholesterol synthesis. This means endogenous cholesterol is still available for esterification into CE, however, we would expect CE to gradually decrease due to hydrolytic activity of cholesterol esterase in an attempt to increase the cells’ depleted cholesterol levels. The magnitude of changes across various lipid classes in response to simvastatin and cyclodextrin treatments were also markedly different. This is consistent with the ability of cyclodextrin to rapidly reduce cholesterol levels by 80–90% . SurprisOur study has several limitations. As our cell model involved harvesting cells following a 48 h treatment, it is important to consider that many of the changes in lipids may not be in response to the simvastatin treatment, but rather in response to changes in feedback mechanisms to negate the effects of the cholesterol inhibition. Similarly, whilst Hep3B cells are human-derived liver cells, they are an immortalised cell line with known metabolic differences to animals and humans. Further validation studies in primary hepatocytes at multiple time points would be important in understanding the effect of statins on lipid abundance in cells with similar characteristics to an in vivo model. Additionally, this study focused on gene expression to validate flux through metabolic pathways, however, assessment of protein levels would offer further insight into the regulation of such pathways. Finally, the difference in potency between statins has been detailed extensively. In this study, we used simvastatin and whilst the results suggest this is a class effect of statins, alternate statins should also be tested to validate this.v/v) Foetal Bovine Serum  and passaged every 2 to 3 days. Cells were passaged a maximum of 20 times before being discarded. Cells were maintained at 37 °C and 5% CO2. At 90% confluency, cells were dissociated using trypsin  and seeded at a density of 2.3 × 105 cells per well in a 6-well plate for lipidomic analysis or 2.45 × 105 per well in a 6-well plate for RNA isolation studies. Treatments were added 24 h after plating to ensure cells had adhered to the wells. Cells were harvested for analysis 48 h following the addition of treatments. Cell culture experiments for RNA isolation were repeated 3 times with 2–4 technical replicates per treatment. For lipidomics, the experiment was performed once with 6 technical replicates per treatment.The immortalised human hepatoma cell line, Hep3B, was used for cell culture experiments . Hep3B cells were cultured in DMEM supplemented with 10% (2+/ and Mg2+ and treated with DMEM supplemented with 10% (v/v) lipoprotein-deficient foetal bovine serum  plus the relative treatment. Treatments were as follows: 5 µM simvastatin  dissolved in dimethyl sulfoxide (DMSO)  plus 10 µM mevalonate  dissolved in ethanol  (statin treatment), 20 mg/mL methyl-ß-cyclodextrin plus vehicle (0.1% DMSO and 0.02% ethanol) (cyclodextrin treatment) and 20 mg/mL cholesterol-loaded methyl-ß-cyclodextrin plus vehicle (0.1% DMSO and 0.02% ethanol) (COH-cyclodextrin treatment) or vehicle alone (DMSO and ethanol) (control). Methyl-ß-cyclodextrin and cholesterol-loaded methyl-ß-cyclodextrin  were prepared as follows: 5% methyl-ß-cyclodextrin (w/v) in H2O +/− 15 mg/mL cholesterol in 100% ethanol (methyl-ß-cyclodextrin: cholesterol at 10:1) was stirred for 30 min at 80 °C on a heat block before being dried down using a Savant SPD121P SpeedVac . Prior to experiments, treatments were freshly dissolved in molecular grade water.After plating for 24 h, Hep3B cells were washed in PBS without CaHep3B cells were harvested in 200 µL of cold PBS on ice. Once collected, cells were disrupted by sonication with a Misonix S-4000 Sonicator  for 10 s at amplitude 25. Protein concentrations were determined using a BCA assay, according to the manufacturer’s protocol (Pierce Protein Methods)  and as described by Brown et al. . Aliquot3/MeOH)  solvent extraction as described by Weir et al. [3:MeOH (2:1) and 10 µL of the internal standard mix (2O) and technical quality control (TQC) samples (pooled PQC extracts) were distributed throughout the randomised samples (1:10) to measure variation in the extraction process and mass spectrometry analysis. Extracts were mixed for 10 min on a rotary mixer, sonicated in a water bath at room temperature for 30 min, left to stand on a bench for 20 min and then centrifuged at 15,000 rcf for 10 min at 20 °C. The supernatant was transferred to a 96-well plate and dried using a Savant SPD121P SpeedVac. Samples were reconstituted in a 1:1 mixture of water saturated butanol and methanol containing 10 mM ammonium formate. Samples were centrifuged at 1000 rcf for 5 min at 20 °C before the supernatant was transferred into glass vials with 0.2 mL micro-inserts for further analysis.Lipids were isolated using a single phase chloroform:methanol  using an Agilent 1290 HPLC coupled to an Agilent 6490 triple quadrupole mass spectrometer. The settings were as follows: gas temperature 150 °C, gas flow 17 L/min, nozzle pressure 20 psi, sheath gas temperature 200 °C, sheath gas flow 10 L/min, capillary voltage 3500 V, nozzle voltage 1000 V. Liquid chromatography was performed on a Zorbax Eclipse Plus RRHD C18, 1.8 μM, 100 × 2.1 mm column (Agilent Technologies) using solvents A and B consisting of water:acetonitrile:isopropanol, 50:30:20 and 1:9:90 respectively, both containing 10 mM ammonium formate, with solvent A also containing 0.05 μM medronic acid. The column was heated to 45 °C and the auto-sampler regulated to 25 °C. Lipid extracts (1 µL) were injected and separated under gradient conditions with a flow rate of 400 µL/min. The gradient was as follows: 15% solvent B to 50% solvent B over 2.5 min, increase to 57% solvent B over 0.1 min, increase to 70% solvent B over 6.4 min, increase to 93% solvent B over 0.1 min, increase to 96% solvent B over 1.9 min, increase to 100% solvent B over 0.1 min, and hold at 100% solvent B for 0.9 min. Solvent B was then decreased to 15% over 0.2 min, held at 15% solvent B for 0.7 min, until next injection. Total time was 13.5 min with the first 1.2 min of each analytical run diverted to waste.A total of 662 lipid species across 39 lipid classes were measured using dynamic multiple reaction monitoring (dMRM) where data was collected for a retention time window specific to each lipid species . Resultsrcf for 15 min at 4 °C. The separated aqueous phase was transferred into a microfuge tube and 500 µL of room temperature isopropanol  was added. Samples were inverted 10 times and left to rest for 30 min at room temperature before being centrifuged at 14,000 rcf for 15 min at 4 °C. The supernatant was then discarded leaving an RNA pellet. The pellet was washed by adding 1 mL 75% (v/v) ethanol then centrifuged at 4 °C for 5 min at 12,000 rcf. The supernatant was removed before repeating these steps a further two times. Once the final wash had been completed, the pellet was centrifuged at 14,000 rcf for 1 min at 4 °C to remove any remaining ethanol. Pellets were then dried on a heat block at 55 °C for 10 min before resting on the bench for a further 5 min. The RNA pellet was resuspended in 12 µL of warmed molecular grade water and quantified using the NanoDrop  to assess RNA purity and quantity. Samples with 230/260 ratios above 1.8 and 260/280 ratios above 1.7 were used.Hep3B cells were harvested on ice in 500 µL of RNAzol (prepared in house) and transferred into 1.5 mL microfuge tubes. Samples were incubated at room temperature for 5 min before 100 µL chloroform  was added to phase separate the RNA. Samples were shaken rapidly, incubated for 3 min at room temperature, then centrifuged at 12,000 To generate cDNA, 2 µL (50 ng/µL) random hexamers  was added to 750–1000 ng RNA and samples were incubated at 70 °C for 5 min. After incubation, 5× first-strand buffer, 0.1 M DTT, 10 mM dNTP, 40 U/μL Ribosafe and 200 U/μL M-MLV reverse transcriptase  were added, and samples were placed at 25 °C for 10 min, 37 °C for 50 min, and 70 °C for 10 min on the Applied Biosystems thermocycler. cDNA samples were then diluted with molecular grade water to 5 ng/μL for qPCR analysis in a 384-well plate. A master mix of SYBR Green , and the appropriate primer sets for target genes were prepared; 10 ng of cDNA was added to each well and qPCR was performed using the Applied Biosystems Quant Studio 7 real-time PCR machine. Conditions were as follows: Hold stage: 95 °C for 20 s PCR stage: 60 °C for 20 s; Melt Curve stage: 95 °C for 15 s, decrease to 60 °C for 1 min and increase back to 95 °C for 15 sec for 40 cycles. Quantification of a given gene was calculated using the ∆∆CT method. Data were normalised to the reference gene, Ribosomal Protein Lateral Stalk Subunit P0 (RPLP0), and expressed as fold change compared to the control group. Primer sequences were validated using the Basic Local Alignment Search Tool (BLAST) and are available in Supplementary Data .t-tests were performed on 662 lipids. Mean differences and 95% confidence intervals were then converted to log2 fold difference for interpretation of results. Lipidomics data are presented as log2 fold difference ± SD. All p-values were corrected for multiple comparisons using the false discovery rate method of Benjamini and Hochberg [p < 0.05 was considered significant.Samples for all mass spectrometry-based analysis were randomised prior to data acquisition. Quantification of lipid species was determined using R (3.4.0) analytical software. Lipid concentrations were normalised to total cellular protein content and log2 transformed prior to statistical analysis. Student’s Hochberg . qPCR daELOVL5 and FASN gene expression suggest that it may involve modulation of the n-6 fatty acid pathway, via SREBP-1c, and inhibition of PI synthesis. Changes in the n-6 fatty acid pathway and subsequent increases in PUFAs may also explain some of the additional cardioprotective effects of statins. The PI(36:2)/PC(18:0_20:4) ratio may therefore serve as a putative surrogate marker for some of these pleiotropic effects on secondary outcomes, and could be useful in monitoring treatment response to statins. Despite the need for further investigations, it is clear that understanding these mechanisms may be important in uncovering the full treatment effect of statins.In summary, our findings have important implications for understanding the mechanisms underlying changes in the plasma PI(36:2)/PC(18:0_20:4) lipid ratio in response to statin treatment. We observed significant changes in multiple lipid species across 40 lipid classes with simvastatin treatment. Importantly, species containing AA increased, whilst free cholesterol, CE and the PI(36:2)/PC(38:4) ratio decreased. Replication of the low-cholesterol phenotype seen with simvastatin via the use of cyclodextrin resulted in a similar yet more exaggerated lipidomic profile. Conversely, the high cholesterol environment induced by cholesterol-loaded cyclodextrin exhibited a contrasting lipid profile. These data suggest that changes in intracellular cholesterol abundance and its downstream metabolites are the primary mediators of the lipid ratio, rather than a specific off-target effect of the statin itself. The mechanisms by which cholesterol mediates the constituents of the ratio PI(36:2) and PC(18:0_20:4) remain unclear, however, changes in


A retrospective observational study was conducted to assess the prevalence of maturity onset diabetes of the young (MODY) in a large paediatric population of Southern Italy newly diagnosed with diabetes. Clinical and genetic features of the identified MODY patients were also described.Genetic testing was performed in children and adolescents newly diagnosed with diabetes who presented autoantibody negativity and fasting C-peptide levels ≥ 0.8 ng/mL. Patients with a low insulin daily dose and optimal glycaemic control after two years from diabetes onset were also investigated for monogenic diabetes, regardless of their autoimmunity status and/or C-peptide levels.A prevalence of 6.5% of MODY was found. In particular, glucokinase-MODY was the most common type of MODY. The mean age at diagnosis was 9.1 years. Clinical presentation and biochemical data were heterogeneous also among patients belonging to the same MODY group.We found a relatively high prevalence of MODY among paediatric patients with a new diagnosis of diabetes in comparison to literature data. Our findings highlight that a more detailed clinical evaluation along with easier and less expensive approachability to genetic testing may allow diagnosing an increasing number of MODY cases. A correct, prompt diagnosis is crucial to choose the most appropriate treatment and offer adequate genetic counselling. Maturity onset diabetes of the young (MODY) is a rare condition characterized by autosomal dominant inheritance which is usually diagnosed before 25 years of age . AccordiHNF1A), glucokinase (GCK), hepatocyte nuclear factor 4-alpha (HNF4A), and hepatocyte nuclear factor 1-beta (HNF1B) genes are the most frequently identified aetiologies of MODY. Other MODY genes include: PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, ABCC8, and KCNJ11.MODY accounts for 1–5% of all cases of diabetes and 1–6% of paediatric diabetes cases –13. MutaGCK mutational spectrum includes over 700 mutations distributed throughout the gene [HNF4A gene, resulting in a progressive β-cellular dysfunction [HNF1A mutations have been described [GCK-MODY, known as MODY 2, is the commonest subtype of monogenic diabetes in paediatric age, especially among Caucasian populations . The GCKthe gene . Patientthe gene . HNF1A-Mthe gene , 17. Hypthe gene . Thus fathe gene , 20. HNFfunction . HNFA4-Mfunction . More thescribed . Patientescribed .Aim of this study was to assess the prevalence of MODY among a large paediatric population of Southern Italy newly diagnosed with diabetes. Secondary outcome of the study was to describe the main clinical and genetic features of the identified MODY patients.A retrospective analysis was conducted to select all patients who were admitted at our Paediatric Centre of Diabetes between January 2009 and February 2021 with a new diagnosis of diabetes. The study was conducted in accordance with the Helsinki Declaration, good clinical practice and all applicable laws and regulations. The study protocol was approved by the  Ethics Committee of the Medical University of Messina. The diagnosis of diabetes was made according to the current criteria of the ISPAD Clinical Practice Consensus Guidelines: classic symptoms of diabetes or hyperglycaemic crisis, with plasma glucose concentration ≥ 11.1 mmol/L (200 mg/dL) or fasting plasma glucose ≥ 7.0 mmol/L (≥ 126 mg/dL) or two hour postload glucose ≥ 11.1 mmol/L (≥ 200 mg/dL) during an oral glucose tolerance test (OGTT) or glycated haemoglobin (HbA1c) > 6.5% (48 mmol/mol) [Diabetes-specific islet cell autoantibodies (ICA), glutamic acid decarboxylase 65 autoantibodies (GADA), tyrosine phosphatase-like insulinoma antigen 2 (IA2), insulin (IAA), and β-cell-specific zinc transporter 8 autoantibodies (ZnT8) were measured. The presence of ICA was determined by immunofluorescence assay, while GADA, IA2, IAA, and ZnT8 were evaluated with enzyme linked immunosorbent assay. The identification of predisposing human leukocyte antigen (HLA) alleles  was also performed in every patient. In cases with the suspicion of monogenic diabetes, genetic tests were performed. In accordance with previous studies , selectiABCC8, APPL1, BLK, CEL, GATA4, GATA6, GCK, HNF1A, HNF1B, HNF4A, INS, PDX1, KCNJ11, KLF11, LMNA, NEUROD1, PAX4, PPARG, RFX6, TRMT10A, WFS1, ZFP57, EIF2AK3, FOXP3, GLIS3, IER3IP1, NEUROG3, PTF1A, SLC19A2, SLC2A2. Raw data obtained from the genetic investigations were evaluated according to American College of Medical Genetics and Genomics guidelines. Confirmation studies were performed for variants that were considered to be pathogenic, or likely pathogenic, using Sanger sequencing.Genetic testing for monogenic diabetes was performed in two different laboratories located in Reggio Calabria and San Giovanni Rotondo, Italy. Analysis of monogenic diabetes causing genes was performed using the “HaloPlex HS Target Enrichment System” for sample preparation, which was thereafter sequenced on NextSeq 500 through 2 × 150 bp paired-end sequencing. The generated sequencing data was mapped to the human genome reference hg19. Thirty genes were analysed: During the study period, a total of 565 children and adolescents were newly diagnosed with diabetes. Genetic tests for monogenic diabetes were performed in 68 patients. Of these, 59 (86.8%) were negative for diabetes-specific autoantibodies, 46 (67.6%) had fasting C-peptide level ≥ 0.8 ng/mL, 63 92.6%) were not on insulin therapy or required insulin daily dose < 0.5 IU/kg along with an optimal glycaemic control (HbA1c < 6.5% or 48 mmol/mol).Thirty-seven patients (6.5%) were diagnosed as MODY. In particular, 2 had a diagnosis of HNF4A-MODY (5.4%), 30 were diagnosed as GCK-MODY (81%) and the remaining 5 were HNF1A-MODY (13.5%). Mean age at diagnosis of MODY was 9.1 years (ranging from 0 to 16 years). Genotypic and phenotypic features of MODY patients are summarized in Tables .6% were HFN4A-MODY at the age of 16 and 12, respectively. Diagnostic investigations started after discovering an occasional hyperglycaemia in one of the two siblings. At that time, her HbA1c was 6.1% (43 mmol/mol) and an OGTT confirmed the diagnosis of diabetes (2-h postload blood glucose 12.1 mmol/l). Levels of the diabetes-specific autoantibodies were all negative. HLA was not predisposing to T1D. On the suspicion of monogenic diabetes, genetic testing was performed. Genetic analysis revealed a frameshift mutation (HNF4A c.364_365insG) in exon 3 of the HNF4A gene. Thus, genetic investigations were extended to her families, revealing the same mutation in her brother, who was asymptomatic. Both siblings had normal liver function without morpho-structural alterations. Treatment with gliclazide was started at low daily dose. Thus far, after a 5-year-follow-up, both patients have optimal glycaemic control—last year HbA1c value < 6% (42 mmol/mol)—thus, oral hypoglycaemic agent withdrawal has been tried.Two siblings  had a diagnosis of A total of 30 children were identified as GCK-MODY during the study period. Of these, 13 were male. Mean BMI Z-score at start of follow-up was − 0.2 ± 1.1. Genetic analysis leading to diagnosis of monogenic diabetes was performed at the age of 8.5 ± 4.5 years. Mean HbA1c at diagnosis was 6.5 ± 0.5% (48 ± 6 mmol/mol), while basal C-peptide level was 0.8 ± 0.5 ng/ml. Oral glucose tolerance tests of these patients showed a fasting blood glucose of 6.26 ± 0.93 mmol/l with a postload blood glucose after 120 min of 9.22 ± 1.89 mmol/l. All patients were investigated for diabetes-specific autoantibodies and the presence of T1D predisposing HLA: 2 patients were positive for GADA and one of these also had ICA and IA2; 10 patients showed a predisposing HLA. Mean weight at birth of the components of this group was 2.95 ± 0.6 kg. Of note, one patient diagnosed with GCK-MODY at the age of 6 years was also affected by cystic fibrosis . An infaHFN1A gene mutation were found in our study population. Their clinical features are summarized in Table HFN1A gene (HFN1A-c.709A > G) [HFN1A gene affecting exons 1, 3, 4 and 7 in every patient.Five patients with 09A > G) . Other pOver the past two decades, improved knowledge and awareness of monogenic diabetes, along with the development of increasingly more accurate and worldwide available molecular diagnostic techniques, have led to an increased estimate of the prevalence of MODY. The identification of genetic diagnosis is extremely important when choosing the most proper treatment plan. It is well established that patients affected by GCK-MODY do not require any pharmacological treatment , while pOur 12-year-long experience identified a prevalence of MODY of 6.5% among a population of children and adolescents newly diagnosed with diabetes. This finding is consistent with a previous Italian multicentre study, which found a prevalence of 5.5% . ConversIn our centre, genetic investigations for monogenic diabetes were performed in presence of a clinical suspicion of non-T1D, without any strict criteria. We can speculate that serum C-peptide value alone is too stringent as it is well-known to be correlated with patients’ age and BMI . Basal sInterestingly, we found a relatively high number of patients belonging to the MODY group who presented HLA haplotype predisposing to T1D. Data from other studies show that the presence of HLA alleles DR3 and DR4 is not rare among MODY patients, ranging from 20 to 35.1% , 36. TheGCK gene mutations, confirming previous evidence among Caucasian populations [Regarding the frequency among MODY forms, most of our MODY patients have ulations , 31, 38.ulations , 13, 29 ulations . The ItaIn our study, mean HbA1c at diagnosis in GCK-MODY patients was lower than in HNF1A-MODY, confirming the results of another study , in whicIt may be thought that dysglycaemia in obese children indicates a case of T2D. However, auxological parameters from the MODY group show a significant number of patients presenting with a condition of overweight or obesity. A study on a cohort of obese and overweight patients with a clinical diagnosis of T2D reported that 4.5% of them were actually affected by monogenic diabetes . Our datGCK gene cause a higher glycaemic set point of insulin secretion resulting in mild fasting hyperglycaemia, in contrast to other subtypes which are characterized by impaired insulin secretion and high glycaemic response after meals and during OGTT [To date, there are few data , 42 on OThe coexistence of GCK-MODY and cystic fibrosis is very rare. To the best of our knowledge, only two other cases of children suffering from both cystic fibrosis and GCK-MODY have been reported in literature , 44. It Limitations to this study involve the retrospective design and the restriction to the sole population of Southern Italy, mostly composed of Caucasian ethnicity. Unfortunately, some clinical data during the follow-up period, such as C-peptide levels, are not available. Finally, due to the technology used for genetic investigations, some gene variations characterized by large gene deletions may have been overlooked. This group of large genomic rearrangements, including 17q12 microdeletions, have been described in literature as responsible for the majority of cases of HNF1B-MODY, known as MODY 5 , 47.Our findings suggest that a detailed clinical evaluation  could be more useful than restricted criteria as a first approach to screen diabetic children who need genetic testing for MODY. Finally, we speculate that easier and less expensive approaches to genetic investigations will allow to diagnose an increasing number of cases in the near future. A correct, prompt diagnosis is essential to start the most appropriate treatment, to offer adequate genetic counselling, and also to facilitate the diagnosis of monogenic diabetes in asymptomatic family members.


Halomonas species, which are aerobic, alkaliphilic, and moderately halophilic bacteria, produce diverse biochemicals. To identify food-related Halomonas strains for bioremediation and the industrial production of biochemicals, 20 strains were isolated from edible seashells, shrimp, and umeboshi (pickled Japanese plum) factory effluents. All isolates were phylogenetically classified into a large clade of Halomonas species. Most isolates, which grew in wide pH (6–13) and salt concentration (0–14%) ranges, exhibited the intracellular accumulation of poly(3-hydroxybutyrate) granules. The characteristics of these isolates varied. A020 isolated from umeboshi factory effluents exhibited enhanced stress tolerance and proliferation and comprised two plasmids. IMZ03 and A020 grew to more than 200 OD600, while IMZ03 produced 3.5% 3-hydroxybutyrate in inorganic medium supplemented with 10% sucrose. The mucus of TK1-1 cultured on agar medium comprised approximately 64 mM of ectoine. Whole-genome sequencing of A020 was performed to elucidate its origin and genomic characteristics. The genome analysis revealed a region exhibiting synteny with a large virus genome isolated from the ocean, but did not identify any predictable pathogenic genes. Therefore, saline foods and related materials may be suitable resources for isolating Halomonas strains exhibiting unique, useful, and innocuous features. Halomonas are Gram-negative, aerobic, mesophilic, alkaliphilic, and moderately halophilic bacteria. The first species of this genus to be isolated was Halomonas elongata from a solar salt facility  conditions -3-hydroxybutyrate (3HB)  and Ruditapes philippinarum (Japanese clam) were harvested from fisheries at Kada  and Kawaguchi  in Wakayama and Kumamoto, Japan, which were approximately 60 and 450 km away from the umeboshi factories, respectively  and Iwamoto Foods (33°47'6.5''N 135˚20'37.3''E), Wakayama, Japan. The two factories are located approximately 2 km from each other and 4 km from the nearest coast. Halomonas sp. KM-1 (FERM BP-10995) was a kind gift from Dr. Kawata, the National Institute of Advanced Industrial Science and Technology.Halomonas species were cultured and isolated in SOT-base medium  supplemented with 3.0% (w/v) sucrose  unless otherwise specified. Agar concentrations in SOT and SOT2 agar media were 2.0 and 1.5%, respectively. The composition of umeboshi seasoning medium was 1.5% agar and 50% umeboshi effluent, which contained 7.6% NaCl, 2.5% citrate, and 37% sugar (including 8.0% honey), with a pH of 3.0. pH was adjusted to 7.0 with NaOH. The composition of Luria-Bertani (LB) medium was as follows (for 1 L): 10.0 g peptone, 5.0 g yeast extract, and 10.0 g NaCl. sucrose . Liquid g at 25°C for 10 min. Pellets were resuspended in 1/40 volume of the supernatant and resuspended samples were left undisturbed for 20 min. The supernatant was collected and used as a bacterial suspension. The soft parts of seashells (5 g) were soaked in 10 mL of phosphate-buffered saline  and soaked samples were left undisturbed for 10 min. The supernatant was collected and used as a bacterial suspension. Soil and sediments (10 g) were resuspended in 20 mL of H2O and resuspended samples were left undisturbed for 10 min. The supernatant was collected and used as a bacterial suspension.Umeboshi effluents were obtained from tanks and comprised a mixture of various umeboshi seasoning liquids used for the re-pickling of traditional umeboshi before processing in activated sludge facilities. Samples were centrifuged at 10,600×The bacterial suspension and seawater (100 μL) were spread on SOT and SOT2 agar media supplemented with 3.0% sucrose and incubated at 30°C for 3–8 days. Bacterial colonies were subjected to colony isolation twice using the same media.DNA fragments encoding the 16S rRNA gene were amplified by a polymerase chain reaction (PCR) with the following primers: 5′-aaactgaagagtttgatcatggc-3′ and 5′-aggtgatccagccgcaggtt-3′. The DNA sequences of the PCR products were elucidated using the same primers as those used for PCR. Genus identification was performed using the Basic Local Alignment Search Tool (BLAST) . The chemical composition of the granules was identified using gas chromatography  analysis using a HPLC system  equipped with a Aminex®HPX-87H column (Bio-Rad). HPLC conditions were as follows: column temperature, 35°C; mobile phase, 5 mM sulfuric acid; flow rate, 0.6 mL min–1; detector, UV detector; detection wavelength, 210 nm.PHA production was examined using Nile red staining  equipped with an Inertsil Amide column for amino acid separation (GL Science). HPLC conditions were as follows: column temperature, 45°C; mobile phase, 75% acetonitrile; flow rate, 0.6 mL min–1; detector, UV detector; detection wavelength, 210 nm. ESI-TOF MS analyses were performed using a Triple TOF 5600+ system (AB Sciex) equipped with ESI in the positive ion mode. HPLC fractions were applied to TOF MS and ions were monitored in the range of 50 to 500 m/z.Ectoine concentrations were measured using HPLC and electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). Mucus samples (1.0 g) of TK1-1 and other Halomonas isolates were elucidated using a whole-genome shotgun strategy according to a previously described method with minor modifications . Genomic DNA was fragmented and subjected to PCR-free library preparation using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs). The library was sequenced using the HiSeq system (Illumina) to obtain 10.5 M pair-end reads (150 bases per read). Short DNA reads were assembled using the CLC Genomics Workbench (Qiagen), which generated 51 contigs (>500 bases and >0.5 copy number) with 219,903 bp of N50 and a total of 3,905,596 bp (coverage 398×). The complete genomic DNA sequence of Halomonas sp. A020 was analyzed using a previously described method , 7 from R. philippinarum (Japanese clam), 1 from Pandalus latirostris (shrimp), and 8 from effluents of traditional umeboshi manufacturers  multiplied under a wide range of salt concentrations (2.5–11.5%), growth temperatures (15–40°C), and pH (6–13) . All umeHalomonas species for industrial applications. Most of the isolates exhibited Nile red-stained intracellular granule accumulation. However, the timing of the highest accumulation and the number of intracellular granules varied between different isolates . TK1-1 appeared to more efficiently produce ectoine without optimization than H. elongata DSM2581 , temperatures (up to 40°C), and salt concentrations (0–12%), and higher PHB and 3HB productivities. Moreover, there were two plasmids in the A020 genome  among Halomonas strains, 369 were conserved among certain Halomonas and other bacterial strains, and 127 did not exhibit similarities in databases.Compared with other isolates,  and ter   encoded a large membrane-bound protein , which comprised 20 repeats of a repetitive sequence with 227 amino acids in the N-terminal region and a type I secretion C-terminal target domain (COG2931) in the C-terminal region. The A020_36530 gene was conserved among various Gammaproteobacteria strains, particularly Pseudomonas and some Halomonas strains. However, the number of repetitive sequences varied between the strains. The functions of this protein are unknown.Analyses using the Kyoto Encyclopedia of Genes and Genomes metabolic pathway revealed all genes involved in PHB metabolism in the A020 genome and these genes were conserved among phylogenetically distant strains, such as Halomonas strains from edible seafood harvested from Japanese seashores and umeboshi factory effluents. Approximately 103 live Halomonas bacteria were detected in 1 g of seafood. Seafood is traditionally consumed without boiling or baking in Japan, which suggests that Halomonas is not a causative agent of food poisoning in humans. A genome analysis of A020 isolates did not reveal any predictable pathogenic genes. Umeboshi isolates are potential candidates for the development of a safe and low-cost bioremediation system for effluents containing high concentrations of salt and organic compounds, including umeboshi factory effluents.The present study is the first to isolate 20 Eight umeboshi isolates were phylogenetically classified into a large clade with other seafood isolates. The genome of the umeboshi isolate A020 comprised a region that exhibited synteny with that of a marine phage. Activated sludge processing facilities for umeboshi factory effluents were located a few kilometers away from the nearest coast. Therefore, the origin of umeboshi isolates may be a nearby ocean  Identification of New https://doi.org/10.1264/jsme2.ME21052Supplementary Material