Euterpe oleraceae) is a potential source of dietary phytochemicals beneficial to health. This study aimed to screen the nutraceutical potential of the acai berry, in the form of aqueous and ethanolic extracts, for the ability to inhibit acetyl- and butyryl-cholinesterase (ChE) enzymes and scavenge free radicals via 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) or 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assays. In addition, this study aimed to quantify the acai berry’s antioxidant potential via hydrogen peroxide or hydroxyl scavenging, nitric oxide scavenging, lipid peroxidation inhibition, and the ability to reduce ferric ions. Total polyphenol and flavonoid contents were also determined. Acai aqueous extract displayed a concentration-dependent inhibition of acetyl- and butyryl-cholinesterase enzymes. Both acai extracts displayed useful concentration-dependent free radical scavenging and antioxidant abilities, with the acai ethanolic extract being the most potent antioxidant and displaying the highest phenolic and flavonoid contents. In summary, extracts of the acai berry contain nutraceutical components with anti-cholinesterase and antioxidant capabilities and may therefore provide a beneficial dietary component that limits the pathological deficits evidenced in AD.Alzheimer’s disease (AD) is characterised by progressive neuronal atrophy and the loss of neuronal function as a consequence of multiple pathomechanisms. Current AD treatments primarily operate at a symptomatic level to treat a cholinergic deficiency and can cause side effects. Hence, there is an unmet need for healthier lifestyles to reduce the likelihood of AD as well as improved treatments with fewer adverse reactions. Diets rich in phytochemicals may reduce neurodegenerative risk and limit disease progression. The native South American palm acai berry ( Approximately one million people are affected by neurodegenerative diseases (NDDs) in the United Kingdom, and more than 50 million people suffer worldwide from dementia ,2. TheseThe pathogenicity of AD is complex, with pathomechanisms that include oxidative stress, excitotoxicity, impaired mitochondrial function, and disrupted cholinergic signalling, as well as toxic peptide and protein accumulations ,15,16,17post-mortem brain tissue from AD patients 2 provides a determination of the reducing capacity of a plant compound [The direct reduction of Fe[(CN)compound . The redThe total phenolic content (TPC) and total flavonoid content (TFC) of the acai aqueous and ethanolic extracts were quantified and are included in The present study evaluated the nutraceutical and, hence, the therapeutic potential of acai berry extracts via their ability to act as ChEIs; also assessed were the additional benefits of free radical scavenging and antioxidant activities. The current first-line treatment for mild-to-moderate AD is to treat the cholinergic deficit experienced by AD patients via a transient inhibition of AChE to increase the signal longevity of the neurotransmitter acetylcholine (ACh) ,25,56. H50 of 13.8 µg/mL) and, to a lesser extent, BuChE (estimated IC50 of 6.378 mg/mL). Interestingly, the agent(s) binding the cholinesterases presumably reached a point of saturation at an approximate concentration of 0.01 µg/mL, such that further enzymatic inhibition was limited. This may result from finite binding at the esteratic and/or peripheral binding sites of AChE or BuChE [The aqueous extract from the acai pulp contained an agent(s) capable of inhibiting both AChE , whereas rivastigmine, which was originally extracted from a medicinal plant, is a relatively potent AChE and BuChE inhibitor  [50 < 20 µg/mL as high potency, moderate potency as an IC50 > 20 µg/mL but < 200 µg/mL, and low potency as an IC50 > 200 µg/mL but < 1000 µg/mL [50 value for galantamine, reported in the literature as approximately 2 µM (or 0.575 µg/mL), multiplied by a factor of 10 [50 of 13.8 µg/mL, was an extract of high potency and therefore has potential use as an effective ChEI.Chemicals able to target and simultaneously inhibit both AChE and BuChE, rather than AChE alone, may offer improved clinical efficacy to combat the increased levels of BuChE in AD patients, with a reduction in side effects ,69,70,71ctively) ,73. It w00 µg/mL . The cutor of 10 . Accordi•OH, H2O2, and •NO free radicals, and they exhibited ferric ion reduction. Similarly, other independent studies have reported the high antioxidant capacity of the acai berry against superoxide (O2•−) and peroxyl radicals (RO2) [2−) in mouse brain BV-2 microglial cells in vitro [In addition to ChEI activities, the acai extracts displayed useful radical scavenging and antioxidant activities; they were able to scavenge DPPH, ABTS, ls (RO2) . Acai alls (RO2) . Acai puin vitro . Acai flin vitro . In addiin vitro . It is ain vitro .The current study also demonstrated that acai extracts have anti-lipid peroxidation effects, in support of studies in vitro  and in vResearch has considered the chemical composition of the acai berry and its antioxidant potential and has detected the presence of numerous polyphenols and flavonoids, such as anthocyanins ,78,80,81All chemicals were purchased from Sigma  unless otherwise specified.An ethanolic extract of acai berry was prepared by the maceration of 300 mg/mL of commercially available freeze-dried acai pulp and skin powder purchased from NaturaleBio  in 70% ethanol for 48 h. The macerated sample was shaken 3 times daily to assist solvation and then filtered using a bottle-top filter. Filtrates were dried at 45–50 °C for 24 h in a water bath to obtain the ethanolic dry extracts ,82. The −6 µg/mL to 1000 µg/mL) was mixed with 150 µL of 0.38 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), 3 µL of 0.5 U/mL AChE enzyme from Electrophorus electricus (electric eel)  or BuChE enzyme from equine serum , and 43 µL of phosphate buffer . Samples were incubated for 20 min at room temperature; then, the reaction was initiated by the addition of 4 µL of 35 mM of acetylthiocholine iodide (ATCI) substrate for AChE or butyrylthiocholine iodide (BTCI) substrate for BuChE, and the absorbance was measured at 412 nm every 30 s for 5 min using a Varioskan™ LUX multimode microplate reader . Reagent blanks were performed in the absence of AChE or BuChE. The positive (inhibitor) control for AChE assays was an organophosphate pesticide, azamethiphos  at 5 mM, capable of the irreversible inhibition of AChE [50) of AChE or BuChE activity were determined. The assays were performed in duplicate for at least three independent experiments, after which a mean was calculated.Based on the method of Ellman et al. (1961) , the abi of AChE . For BuC of AChE . The perThe antioxidant capacities of acai aqueous and ethanolic extracts over the concentration range of 0.01–4000 µg/mL were evaluated by monitoring the ability to reduce the stable free radical di(phenyl)-iminoazanium (DPPH) according to a previous publication . DPPH wa•+ was determined according to the procedure of Acharya (2017) [•+) [•+ was calculated using the following equation:The ability of the acai extracts to scavenge ABTSa (2017) , with so17) [•+) . The sol2O2 either directly through mitochondrial metabolism or indirectly from the environment. H2O2 is widely considered a cytotoxic agent, and the rapid breakdown of H2O2 can produce a •OH that can initiate lipid peroxidation and cause protein and DNA damage [2O2 scavenging ability of the acai extracts was measured using the method of Alam et al. (2013) [2O2 solution prepared in PBS was added, followed by 20 µL of standard or acai extracts at a concentration range of 1–4000 µg/mL. The mixture was incubated for 10 min, and the absorbance was read at 230 nm using a Varioskan™ LUX multimode microplate reader . The following equation was used to calculate the percentage of H2O2 scavenging:Humans are exposed to HA damage . The H2O. (2013) , with mo•OH scavenging activity was evaluated using the method described by Bajpai et al. (2015) [3+–ascorbate–ethylenediaminetetraacetic acid–H2O2 system to produce hydroxyl radicals. In a total volume of 200 µL, the reaction mixture contained 50 µL of 2-deoxy2-ribose sugar (12 mM), 20 µL of ferric chloride (FeCl3) (1 mM), 20 µL of ethylenediaminetetraacetic acid (EDTA) (1 mM), 20 µL of L-ascorbic acid (1 mM), 50 µL of H2O2 (8 mM), 30 µL of PBS, and 10 µL of standard or acai extracts at a concentration range of 1–4000 µg/mL. A volume of 40 µL of 2.8% trichloroacetic acid (TCA) and 2-thiobarbituric acid (TBA) (0.5% in 0.025 M sodium hydroxide solution) was added to the reaction mixture after 45 min at 37 °C, and the mixture was incubated at 85 °C for 15 min to generate a pink chromogen that resulted from the reaction of TBA with degraded sugar, i.e., a ‘malondialdehyde-like’ compound [The acai extracts’ . (2015) , with mocompound . After c•NO radicals. •NO radicals can be produced by sodium nitroprusside (SNP) decomposing in an aqueous solution at pH (7.2) [•NO quantities under aerobic conditions as NO reacts with oxygen to produce nitrates [The procedures described by Unuofin et al. (2018) and Jimoh et al. (2019) ,94 were pH (7.2) . The Grinitrates . Briefly2•− anion, •OH, and the H2O2 radical, trigger LPO, which damages cell membranes and produces numerous secondary products that are neurotoxic, resulting in neuronal death via necrosis or apoptosis [Reactive oxygen species (ROS) such as Opoptosis . Moreovepoptosis . The abipoptosis . Briefly3+) to ferrous ions (Fe2+) was used to estimate the reducing capacity of acai extracts. The acai extract concentrations were assessed over a concentration range of 0.001–8000 µg/mL. Each assay data point contained 4 µL of each acai extract, 400 µL of phosphate buffer , and 250 µL of 1% potassium ferricyanide. After the incubation of the mixture at 50 °C for 20 min, 250 µL of 10% trichloroacetic acid was added. The samples were centrifuged at 3000 rpm for 10 min. Then, 100 µL of the supernatant was transferred to a 96-well microtiter plate and mixed with 100 µL of double-distilled water and 20 µL of freshly prepared (0.1%) ferric chloride (FeCl3) solution. Then, the formation of Perl’s Prussian blue was read at 700 nm, according to Nwidu et al. (2018) [The ability to reduce ferric ions (Fe. (2018)  using a Based on the Folin–Ciocalteu reagent (FCR) method, the total phenolic content was determined spectrophotometrically at 760 nm according to Nwidu et al. (2018) . In this3) forms acid-stable complexes with flavone and flavonol keto groups and their hydroxyl groups [3 produces acidic compounds with orthodihydroxyl groups in flavonoid A- or B-rings [This colourimetric method is based on the principle that aluminium chloride (AlCll groups . Further B-rings . The tot B-rings . The pos50). The statistical analysis comparing different groups was performed using one-way ANOVA tests with Tukey’s multiple comparisons post-test via PRISM v7 . A p-value of below 0.05 was defined as the level of statistical significance for all analyses.Results were expressed as means ± standard error of the mean (SEM) in each treatment and control group. Non-linear regression analysis was used to calculate the concentration of acai extracts producing 50% inhibition (ICTo summarise, the acai berry contains a range of phytochemicals that likely contribute to its anti-cholinesterase and antioxidant activities. This study suggests that the acai aqueous extract could be further fractionated, and its compounds identified for their potential use as a medication alternative for AD therapy, due to the potent ChEI activity and powerful antioxidant capabilities. However, the limitation of this study is that the work was performed in vitro; future in vivo analyses are required, particularly those that mimic NDDs such as AD.


Our study demonstrates that the TNFα-stimulated urothelial cells recapitulate key observations found in the bladders of patients with IC/BPS, underpinning their utility as a suitable in vitro model for understanding IC/BPS mechanisms and confirming the role of TNFα signaling as an important component of the associated pathology. The present study also identifies novel upregulated gene targets of TNFα in urothelial cells, including genes encoding the acute phase protein SAA, complement component C3, and the cytokine receptor IFNGR1, which could be exploited as therapeutic targets of IC/BPS. Altogether, our study provides a reference database of the phenotype of urothelial cells in an inflammatory environment that will not only increase our knowledge of their role in IC/BPS, but also advance our understanding of how urothelial cells shape tissue immunity in the bladder.Urothelial cells of the urinary bladder play a critical role in the development and progression of interstitial cystitis/bladder pain syndrome (IC/BPS), a chronic and debilitating inflammatory disease. Given the lack of data on the exact phenotype and function of urothelial cells in an inflammatory setting (as in IC/BPS), we performed the first in-depth characterization of these cells using RNA sequencing, qPCR, ELISA, Western blot, and immunofluorescence. After TNFα stimulation, urothelial cells in the  Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic disorder, generally characterized by discomfort or pain in the bladder and surrounding pelvic region, often accompanied by increased micturition, urgency, and nocturia . IC/BPS The exact etiology and pathophysiology of IC/BPS still remain elusive, however, a substantial number of studies have suggested injury or dysfunction of the urinary bladder urothelium and continuous cycle of inflammation as key components , 7. The in vitro models of bladder urothelial cells frequently use TNFα to mimic an inflammatory milieu found in IC/BPS   and F12 (HAM) , supplemented with 5% fetal bovine serum (FBS), and 4 mM GlutaMAX . Cells repeatedly tested negative for mycoplasma infection using MycoAlert mycoplasma detection kit . For the experiments, RT4 cells were seeded in 6-well plates with (for IF) or without (for RNA and protein extraction and measurement of protein levels in supernatants) coverslips or 96-well plates (for viability assay) at a seeding density of 5×104 cells/cm2 and grown until reaching 80-90% confluency (approximately 3-4 days). To mimic a proinflammatory environment, cells were treated with 20 ng/ml human recombinant TNFα  for 24 h in serum-free basal media, as previously described  were grown in 75 cmescribed –21. Untr2 cell culture flasks in basal UroM medium, consisting of equal parts of MCDB153 medium  and A-DMEM , supplemented with 2.5% FBS , 0.1 mM phosphoethanolamine, 15 µg/mL adenine, 0.5 µg/mL hydrocortisone, 5 µg/mL insulin , and 4 mM glutamax . For experiments, NPU cells were seeded in 6-well plates with (for IF) or without (for RNA and protein extraction and measurement of protein levels in supernatants) coverslips or 96-well plates (for viability test) at a seeding density of 1×105 cells/cm2 and grown until reaching 80-90% confluency (approximately 4-5 days). To mimic a proinflammatory environment, cells were treated with 20 ng/ml porcine recombinant TNFα  for 48 h in serum-free basal media. Untreated cells, grown in serum-free basal media served as controls. All cell cultures were maintained at 37°C in a humidified atmosphere at 5% CO2.NPU cells were established from porcine urinary bladders (n=5), obtained independently from a local abattoir as described in –29. The ® Luminescent Cell Viability Assay  following manufacturer’s instructions. Luminescent signal proportional to the amount of ATP present was subsequently measured using a microplate reader . Viability assay was performed in triplicates in 3 independent experiments (RT4) or 3 biological replicates (NPU). The results were expressed as percentage of luminescence signal intensity of untreated controls (set to 100).For viability assay, RT4 or NPU cells were seeded in 96-well plates and grown until reaching 80-90% confluency. Subsequently, cells were treated with increasing concentrations of TNFα  in serum-free basal media for 24 h (RT4 cells) or 48 h (NPU cells). Cell viability was determined using CellTiter-GloFor RNA seq and qPCR, total RNA was isolated from RT4 (n=8 independent experiments) and NPU cells , grown on 6-well plates, using Quick-RNA Microprep Kit , according to manufacturer’s instructions with on column genomic DNA digestion. The concentration and purity of isolated RNA were assessed with Qubit RNA Broad Range Assay Kit on Qubit Flex Fluorimeter  and NanoDropTM 1000 (Thermo Fisher Scientific), respectively.® UltraTM RNA Library Prep Kit for Illumina  following manufacturer’s recommendations. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS  according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced to a read depth of 26 million reads/sample, on an Illumina Novaseq 6000 platform, generating paired-end 150 bp reads fastq files. Original raw data can be found in the NCBI Gene Expression Omnibus database with the accession number GSE202576.RNA seq was performed in RT4 cells (n=3 independent experiments) by Novogene Co., Ltd. . Prior to library preparation, the integrity of RNA, isolated from RT4 cells was assessed using the RNA Nano 6000 Assay Kit and the Bioanalyzer 2100 system . A total amount of 0.5 µg RNA per sample was used as an input material for the RNA sample preparations. RNA included in the analysis had a RNA integrity number (RIN) ≥ 7. Sequencing libraries were generated using NEBNextRaw data (raw reads) of fastq format were firstly processed through Novogene Co., Ltd. in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapters, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. High quality reads were aligned to human (Ensembl GRCh38.p13) reference genome using Hisat2 (v2.0.5).FeatureCounts (v1.5.0-p3) was used to count the reads numbers mapped to each gene. The fragments per kilobase of transcript sequence per millions base pairs sequenced (FPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene. RNA seq reads were of good quality with ~ 93% reads mapping uniquely to the reference genome.Differential expression analysis of two groups  was performed by Novogene Inc., using DESeq2 R package (1.20.0). The p value was calculated using the negative binomial distribution and the resulting p values were adjusted using the Benjamini-Hochberg’s approach for controlling the false discovery rate (FDR). The clusterProfiler (v. 3.8.1) software was used for enrichment analysis of differentially expressed genes, including Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Pathways with adjusted p values less than 0.05 were considered as significantly enriched by differentially expressed genes. The protein-protein interaction network was constructed for differentially expressed genes by searching STRING protein interaction database.® 480 PCR System in LightCycler® 480 Multiwell Plates 384 , using self-designed primers  and 5x HOT FIREPol EvaGreen qPCR Mix Plus . Sequences of primers used for qPCR are listed in The expression profile of selected genes was confirmed using qPCR performed on RNA samples isolated from RT4 and NPU cells. For RT4 cells, three independent experiments employed in RNA seq and five additional experiments  were used. For NPU cells, RNA was isolated from 5 biological replicates (TNFα-treated and controls). Reverse transcription of 1 ug of total RNA/sample was performed with Promega Reverse Transcription System Kit  following manufacturer’s instructions. qPCR analysis was performed in triplicates on LightCyclerFor F-actin and IFNGR1 labeling, both RT4 and NPU cells were seeded in 6–well plates with coverslips. After 24 h (RT4) or 48 h (NPU) of TNFα-treatment, cells were fixed in 4% paraformaldehyde in PBS. After fixation, the samples were washed in PBS and blocked with 1% BSA/PBS, at room temperature for 1 h. The samples were then incubated with phalloidin conjugated with FITC  for 1 h, at RT, and washed in PBS. For immunolabeling with anti-IFNGR1, the samples were permeabilized for 10 min with 0.1% TritonX-100  prior to blocking in 1% BSA/PBS, at RT, 1 h. Subsequently, samples were incubated with anti-IFNGR1 rabbit monoclonal antibody , diluted 1:100 in 1% BSA/PBS overnight at 4°C. For negative control, the primary antibodies were omitted and samples were incubated in 1% BSA in PBS at 4°C overnight. After washing in PBS, secondary goat anti-rabbit antibodies , diluted 1:400 in 1% BSA/BPS were added for 2 h, at RT. After washing in PBS, the samples were mounted in Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI)  for DNA labeling. The samples were analyzed with a fluorescence microscope AxioImager.Z1 equipped with ApoTome . Immunofluorescence images shown here are representative of 3 independent experiments (RT4) and 3 biological replicates (NPU).The supernatants of RT4 and NPU cells, seeded in 6-well plates and treated with/without TNFα for 24 h or 48 h, were collected, centrifuged  and stored at -80°C until analysis. Enzyme-linked immunoassays (ELISA) were performed using commercial ELISA kits in duplicates, according to manufacturer’s instructions. Absorbance at 450 nm with a reference wavelength set at 570 nm was measured on a microplate reader . The following ELISA kits were used in the present study: human IL8 ELISA MAX™ Deluxe Set, human IL1A MAX™ Deluxe Set, human IL32 MAX™ Deluxe Set, human CXCL10 ELISA MAX™ Deluxe Set , human CXCL1 Duo Set Kit , human complement C3 ELISA Kit , and porcine IL8/CXCL8 Quantikine ELISA Kit .For western blots, RT4 and NPU cells were seeded in 6-well plates and treated with/without TNFα for 24 or 48 h, respectively. After the treatment, the RT4 and NPU cells were collected and lysed in ice-cold RIPA lysis buffer , containing a cocktail of protease and phosphatase inhibitors . Total protein levels were quantified using the Pierce BCA Protein Assay Kit . Equivalent concentrations of protein (20 µg/lane) were separated using 4–20% Novex WedgeWell Tris-Glycine Gels  and then transferred onto a nitrocellulose membrane . The membranes were blocked in blocking buffer consisting of 5% skim milk in 0.1% Tris Buffered saline/Tween 20 (TBS-T) for 2 h at RT and incubated overnight at 4°C with primary antibodies against IFNGR1  and anti-α-tubulin . The next day, the membranes were washed with TBS-T and immediately incubated for 1 h at RT with secondary antibodies conjugated with horseradish peroxidase . Visualization of the protein bands was performed using the SuperSignal West Pico Chemiluminescent Substrate , and the iBright FL1500 imaging system . iBright Firmware 1.7.  was used to perform the densitometric analysis, normalized to the expression of α-tubulin, used as a loading control. Western blot analyses shown here are representative of 5 independent experiments (RT4) and 4 biological replicates (NPU).Statistical analysis was performed using Graph Pad Prism software 8.01 . The normality of data distribution was investigated by the Shapiro-Wilk test. Due to the normal distribution of the data, summary statistics are expressed as means and standard deviations (SD) unless otherwise stated. One-sample t-test  was used to compare the x-fold change of treated versus control (set to 1) groups. Statistical differences between two groups were calculated using unpaired t-test with  or without  Welch’s correction for variables with normal data distribution. All tests were two-tailed and p values of <0.05 were regarded as statistically significant.in vitro model of RT4 urothelial cells (in vitro model included measurement of gene expression of two inflammatory markers (IL1β and IL8) known to be upregulated in urothelial cells after TNFα stimulation . Differential expression analysis identified 199 differentially expressed genes , of which 127 were upregulated and 72 were downregulated , regulation of inflammatory response, and acute inflammatory response. The top five enriched GO molecular functions included receptor ligand activity, cytokine receptor binding/activity, chemokine receptor binding/activity, endopeptidase regulatory activity, and G protein-coupled receptor binding   receptors  in RT4 cells , which iin vitro model of IC/BPS, they may not accurately reflect IC/BPS pathology due to phenotype changes caused by the tumor microenvironment , and the cytokine receptor IFNGR1 , have previously been shown to provide early recognition of pathogens and elicit a rapid and robust proinflammatory immune response to combat urinary tract infections  also in and TNFα , 43, whiand TNFα –47, possand TNFα , who perand TNFα . Their oin vitro model recapitulates key observations found in the urinary bladders of individuals with IC/BPS, as shown in the present study. Several enriched pathways and processes, observed in the bladder of patients with IC/BPS, especially those with Hunner’s lesions, including complement and coagulation cascades, cytokine-cytokine receptor interaction, chemokine signaling, granulocyte chemotaxis and migration, inflammatory response, TLR-, NOD-like receptor-, NFkB-and TNF-signaling pathways , which are available in sufficient amounts and have many structural, molecular and physiological properties that correspond to normal human urothelial cells  compared to the concentration of TNFα measured in urine (approximately 2-4 pg/ml) of patients with IC/BPS  and reached statistical significance vs untreated cells. When lower concentrations of TNFα were used (for example 2 ng/ml), only a slight increase in secreted IL8 levels was detected compared to untreated cells, so higher TNFα concentrations were necessary in order to be able to describe this particular in vitro model and compare our findings with the findings determined in the bladders of patients with IC/BPS. Although the in vitro model described here recapitulated the key findings from the transcriptomes of bladders from patients with IC/BPS, especially those with Hunner’s lesions in whom the inflammatory component is more prominent, caution is still required when interpreting these results due to the high TNFα concentrations used. A lower concentration of TNFα, used repeatedly over a longer period of time might better mimic the low-grade inflammatory environment observed in IC/BPS. Future studies should examine whether urothelial cells can elicit a prominent inflammatory response when stimulated with lower, clinically relevant concentrations of TNFα, using more sensitive methods for detection, such as single cell RNA seq, and 3D in vitro models that better mimic the structure of the bladder and account also for interactions between different cell types. Urothelial cells in such models might be much more sensitive also to lower concentrations of TNFα, such as the ones measured in urine samples of IC/BPS patients. Second, RT4 cell line, isolated from non-invasive grade I transitional cell carcinoma and NPU cells used were not terminally differentiated and thus may not fully reflect the characteristics and functionality of normal bladder urothelial cells  and inflammation was induced over a short time period of 24 h. It should be noted here that the concentration of TNFα, used in the current (20 ng/ml) and previously published h IC/BPS . The conal cells , 52, 73.al cells . Third, e sample . Our stuhttps://www.ncbi.nlm.nih.gov/) with accession number GSE202576.The datasets presented in this study can be found in online NCBI repository   and MRIC UL IP-0510 Infrastructure program.The authors would like to acknowledge the support of Nada Pavlica Dubarič in Western blot experiments, Sanja Čabraja in cell culture experiments and Nina Visočnik in performing ELISA assays.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Diabetic ketoacidosis (DKA) is a triad of uncontrolled hyperglycemia, metabolic acidosis, and increased total body ketone concentration. It is a well-known manifestation of type 1 diabetes mellitus (T1DM). However, it can also be the first presentation of type 2 diabetes mellitus (T2DM). This subtype of diabetes shares the characteristics of both T1DM and T2DM and is called 'Flatbush diabetes,' also known as 'ketosis-prone T2DM.' This article highlights the importance of early identification of ketosis-prone T2DM (KPD). We describe two cases of African American men with no past medical histories who presented with unproved DKA as the first presentation of T2DM requiring initial intensive insulin therapy, which was gradually withdrawn with the addition of metformin. Both patients are currently insulin independent for more than 15 months. It is an essential clinical entity to recognize as insulin independence positively impacts the quality of life and decreases the economic burden on the health care system.  It has been recognized since the 1980s that for some patients hospitalized with diabetic ketoacidosis (DKA), their clinical courses were more like those with type 2 diabetes mellitus (T2DM) . We, theThis article was previously presented as a meeting abstract at the Endocrine Society's annual meeting (ENDO) 2022 on 06/12/2022.Case 1A 65-year-old African American man presented to the emergency room experiencing polydipsia, polyphagia, and polyuria for two weeks. These were the results of his examination on presentation: initial fingerstick glucose (FS) >500mg/dL, beta-hydroxybutyrate (BHB) of 4.32mmol/L, urine ketone positive, PH of 7.305, bicarbonate at 18mEq/L, anion gap (AG) of 29mEq/L, serum osmolality of 368mOsm/Kg, glucose of 1262mg/dL, hemoglobin A1c (HbA1C) of 12.9%, and body mass index (BMI) of 26.36kg/m2. The DKA mixed with hyperosmolar hyperglycemic syndrome (HHS), and new-onset T2DM was diagnosed. Precipitating factors such as infections, Covid-19, and myocardial infarction were ruled out. The patient was admitted to the medical intensive care unit (MICU) and received an insulin drip and fluid. He recovered uneventfully and was discharged on aspart 8u three times a day (TID) and glargine 20u every night at bedtime (QHS), and followed closely in our endocrinology clinic.At one month of follow-up, fasting glucose reading from the log book was around 100mg/dL, pre-meal glucose ranged between 90-120mg/dL, glargine was decreased to 17u QHS, aspart was decreased to 7u TID, and metformin 1000 mg twice a day (BID) was started. At the three-month follow-up, HbA1C improved significantly to 6.7% without significant hypoglycemic episodes. The insulin dose was further reduced, and metformin 1000mg BID was continued. The B-cell autoantibodies were negative, and the C-peptide was 2.6nmol/L. The HbA1C was improved to 5.8% on a subsequent visit in the same year. Therefore, insulin was gradually withdrawn, and the metformin dose was decreased with close monitoring. The patient's hemoglobin level and renal function were within normal limits in all clinical follow-ups. Current medications include metformin 500mg BID and atorvastatin 40mg, with the most recent HbA1C at 6.0%. The patient’s current BMI is 27.41 kg/m2. He has been in remission for 18 months  presented to the emergency room with fatigue, polyuria, and polydipsia for the past three days. On arrival, these were his results: glucose of 1299mg/dL, BHB of 6.45mmol/L, urine ketone>80, PH at 7.248, bicarbonate of 15mEq/L, AG of 33mEq/L, HbA1C at 11.1%, and BMI of 37.7kg/m2. He was diagnosed with unprovoked DKA and new-onset DM. The patient recovered uneventfully and was discharged with aspart 6 units TID and glargine 25 units QHS.He was seen two months later, and his fasting glucose was around 70mg/dL, so aspart was discontinued, glargine was decreased to 18 units QHS, and metformin 500mg BID was started. At the three-month follow-up, HbA1C dropped to 6.2% with medications and lifestyle changes. The patient reported only a few hypoglycemic episodes. Glargine was gradually decreased and stopped within a year of his diagnosis. Islet cell antibodies and GAD antibodies were negative. The C-peptide level was 6nmol/L. The patient’s current BMI is 37.57kg/m2. His current medications are metformin 1000mg BID and atorvastatin 10mg daily. His most recent HbA1C was 5.5%. He has been in remission for 15 months  autoantibodies (A+ or A-), and β-cell functional reserve (β+ or β-). The course of disease progression mainly depends on their β-cell functional reserve. Although A+β− and A−β− patients are genetically different, they both lack β-cell function. Their clinical course is similar to T1DM, and they require lifelong insulin therapy. For A-β+ and A+β+ patients, because they have a normal β-cell function, their clinical course is like those with T2DM, insulin dose can be slowly reduced, even discontinued, and an oral agent can be started if patients’ glucose remains at the goal ,11.Among KPD, the A−β+ group is the largest; nearly half of these patients present to the hospital with new-onset diabetes and develop DKA without precipitating factors (unprovoked A−β+ KPD), whereas the remainder has a history of diabetes and develop ketoacidosis due to stress, acute illness, or medication noncompliance (provoked A−β+ KPD) . PatientSince many patients with KPD present with ketoacidosis, there is a high probability of it being misdiagnosed as T1DM. If that happens, these patients will be unnecessarily treated with long-term insulin as physicians will be under the impression that they need lifelong insulin for T1DM. Therefore, they are at an increased chance of developing side effects of insulin treatment i.e., hypoglycemia and weight gain. Long-term insulin injection and frequent self-monitoring can also decrease their quality of life, causing career limitations and economic burdens to patients and the healthcare system . So it iIt is vital to recognize ketosis-prone diabetes clinically as many patients present with diabetic ketoacidosis, mainly if they are non-Caucasians. However, KPD is challenging to diagnose in the acute hospital setting. For patients with suspected ketosis-prone diabetes, β-cell autoantibodies and C-peptide level should be obtained from one to three weeks at the first outpatient visit to help determine KPD subgroups and predict patients’ prognoses. These patients should also follow up with an endocrinologist closely. This acute dysfunction is temporary in significant amounts of people and is followed by robust recovery of β-cell function. However, the mechanism of ketosis-prone diabetes remains unknown and merits further study.


Lelliottia amnigena.Here, we report the genome sequence of PTJIIT1005, isolated from a polluted site on the Yamuna River, Delhi. The genome is complete and consists of ~4.5 Mbp with a GC content of 52.62%, 4,259 protein-coding genes, 76 tRNAs, and 4 rRNAs. Strain PTJIIT1005 shows 98.89% average nucleotide identity (ANI) with  Lelliottia is composed of Gram-negative, rod-shaped gammaproteobacteria (Lelliottia amnigena (formerly referred to as Enterobacter amnigenus) members are facultatively anaerobic bacilli in nature that have been isolated from water and different food products and are known to cause bacterial infection in humans , Delhi, India , in May 2018. This strain was grown and maintained on nitrate agar medium at 30°C to 32°C under anaerobic conditions . A single colony was inoculated into 5 mL nitrate broth and incubated at 32°C and 200 rpm in a shaker incubator. DNA was extracted using the NucleoSpin microbial DNA kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Then, genomic libraries were prepared for sequencing using the QIAseq FX DNA library kit (Qiagen). Genome sequencing was performed using the Illumina NovaSeq 6000 platform. A paired-end (2 × 150-bp) sequencing library was prepared and then sequenced on the NovaSeq 6000 platform (Illumina). A total of 9,365,132 raw reads were obtained, providing 280× genome coverage. The fastp v0.20.1 tool was used to filter the reads. Of these, 8,596,940 Illumina reads were This genome sequence was assembled into 85 contigs comprising 4,552,797 bp, with a GC content of 52.62%. It contains 4,259 coding sequences, 76 tRNAs, 4 rRNAs, 1 transfer-messenger RNA (tmRNA), and 2,857 functional proteins. Details of the genome assembly can be found in The RAST tool  was usedThis strain has nitrate remediation traits that can contribute to the removal of nitrate from water.Lelliottia amnigena (GenBank accession number NZ_CP077236.1).The average nucleotide identity (ANI)  analysisJAKRZN000000000 . The Illumina raw reads have also been deposited at the Sequence Read Archive (SRA) under BioProject accession number PRJNA830830 and BioSample accession number SAMN27738499.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under accession number


OsAmy1A, OsAmy1C, and OsAmy3C in the salt-sensitive IR28 cultivar. Salt stress differentially regulated the expression of GA biosynthetic genes. Salt stress increased antioxidant activity in all rice genotypes tested, except in IR28. GA3 mitigates the effect of salt stress, rescuing seed germination and growth attributes. GA3 significantly increased bioactive GA content in Nagdong and pokkali (50 µM) and Cheongcheong and IR28 (100 µM) cultivars. The α-amylase genes were also significantly upregulated by GA3. Similarly, GA3 upregulated OsGA2ox1 and OsGA2ox9 expression in the Cheongcheong and salt-sensitive IR28 cultivars. The present study demonstrated that salt stress inactivates bioactive GA – inhibiting germination and seedlings growth – and decreases bioactive GA content and GSH activity in IR28 and Pokkali cultivars. Further, GA3 significantly reversed the effects of 120 mM NaCl salt stress in different rice genotypes. The current study suggested that the known coastal area salinity concentration can be significantly recovered with the application of exogenous GA3. Thus, it could be possible to grow eco-friendly rice close to the coastal zone in order to reduce the damage caused by salinity.The overall effects of gibberellic acid (GA3) with NaCl on different rice genotypes are inadequately understood. The present study determines the effect of different GA3 (50 and 100 µM) concentrations on the morphophysiological, molecular and biochemical effects of 120 mM NaCl salt stress in rice seedlings. Salt stress reduced germination percentages and seedling growth and decreased bioactive GA content. It also downregulated the relative expression of α-amylase-related genes –  Oryza sativa L.) worldwide. One estimate is that relatively 6 and 20% of barren and irrigated land, are usually affected by salinity  decreases various physiological processes in maize such as reducing the length of root and shoot, fresh and dry weights, chlorophyll and carotenoid contents, K+ ion accumulation, and affects the activities of antioxidant enzymes while enhancing oxidative damage and high Na+ accumulation, however, the exogenously applied GA3 improved their growth attributes, decrease oxidative stress, increased K+ ion content, induces antioxidant genes expression and activities  ent-kaurene synthase (KS), ent-kuarene 19-oxidase (EKO), GA12-aldehyde 7-oxidase (GA7ox), GA20-oxidase (GA20ox), GA3ox-hydroxylase (GA3ox), and GA 2-oxidase (GA2ox)  .There are some enzymatic and non-enzymatic antioxidants that play a vital role in minimising or alleviating high ROS accumulation in a well-organized plant system . EnzymatRice is one of the most important staple foods for over half of the world’s population (IRR) and has an impact on the livelihoods and economies of several billion people. In recent years, salinity stress has become a major driver of decreasing rice growth and yield worldwide, particularly in South Korea. Therefore, in the current study, we used two famous rice cultivars, Cheongcheong and Nagdong, from a plant molecular breeding lab (South Korea), and two additional cultivars, the salt-sensitive IR28, and salt-tolerant Pokkali. This study determined the effect of salt stress on these rice genotypes, with or without exogenous GA3 application. Furthermore, the GA metabolism, effects of salt on GA metabolism, expression of α-amylase and GA biosynthesis-related genes, and antioxidant activity were investigated in the different rice cultivars in this study.Oryza sativa L.) cultivars, Cheongcheong, Nagdong, IR28, and Pokkali (Gyehwa-20) were used in this study. Cheongcheong and Nagdong are two well-known rice cultivars generally used in South Korea. Pokkali is a one of unique salt-resistant cultivar that is cultivated worldwide in waterlogged and seaside areas, and IR28 is a salt-sensitive cultivar. The rice genotypes Cheongcheong and Nagdong were used for evaluation in this study. Seeds of all rice genotypes surface sterilised with 70% ethanol, 5% sodium hypochlorite, for 1 and 20 min. Sterilised clean seeds were germinated in Petri dishes (9 cm in diameter) according to the method described by  was used as the internal control gene for normalization.Seeds of each rice cultivar were grown in double-distilled water in Petri-dishes containing an autoclaved filter paper. After 14 days, seedlings were treated with 15 ml of distilled water (control group), 120 mM NaCl, 120 mM NaCl + 50 µM GA3, or 120 mM NaCl + 100 µM GA3; three replicates were performed for each cultivar per condition. After 6 h, the roots and shoots samples were collected and RNA extracted from the root and shoot regions using RNeasy Plant Mini Kit  the manufacturer’s guidelines. A qPCRBIO cDNA Synthesis Kit was used for first-stand cDNA synthesis, using 500 ng total RNA. Quantitative real-time PCR (qRT-PCR) was performed using the Steponeplus Real-time PCR System , Biofact 2x Real-Time PCR Master Mix (including SYBR Green I) , and primers specific to the selected genes Table 1.Seeds from the four different rice genotypes were grown for 14 days in a growth room, in either distilled water (control group), 120 mM NaCl, and treated with 50 or 100 µM GA3. Subsequently, rice seedlings were collected and immediately ground in liquid nitrogen using a pestle and mortar, Endogenous GAs were quantified described by .2O2 (50 µM) was mixed with 100 µl sample crude extract. After incubating this reaction mixture at 25°C for 5 min, 5% H2SO4 (v/v) was added to stop the enzymatic reaction. The resulting absorbance was measured spectrophotometrically at 420 nm.Rice cultivars were grown for two weeks at 28°C in Petri dishes containing distilled water (control), 120 mM NaCl, and with 50 or 100 µM GA3 treatment. Seedlings samples were collected after 14 days, and 200 mg  frozen tissue samples were ground immediately in a liquid nitrogen; samples were then homogenised in 2 ml 10% trichloroacetic acid. After centrifugation at 10,000 × g for 15 min, 0.1 ml supernatant was moved into 3 ml 150 mM monosodium phosphate buffer and 0.5 ml Ellman’s reagent and incubated at 30°C for 5 min. Relative absorbance was then measured spectrophotometrically at 420 nm. For POD activity briefly, 500 mg seedlings samples were ground in liquid nitrogen. Following this, 0.1 M potassium phosphate buffer (pH 6.8) was added to the samples, which were then centrifuged at 4°C for 15 min, at 5000 rpm. A reaction mixture containing 0.1 M potassium phosphate buffer (pH 6.8), 50 µl pyrogallol (50 µM), and 50 µl HGroup differences were analysed using an independent sample one-way or two-way ANOVA, followed by Turkey’s multiple comparisons test, with at least three replicates. p values < 0.05 were considered statistically significant. All data are expressed as the mean ± SE.). In contrast, seedling growth in the salt-stressed Cheongcheong and salt-sensitive IR28 cultivars significantly increased with both 50 and 100 µM GA3 treatment. However, 100 µM GA3 more effectively enhanced seedling growth in the Cheongcheong and salt-sensitive IR28 cultivars .Seed germination in rice genotypes under salt stress and treated with exogenous GA3 was analysed at 7 and 14 d. Salt stress drastically reduced the seed germination percentage in both Cheongcheong and IR28 cultivars compared to the distilled water controls, whereas Nagdong and Pokkali cultivars responded well to salinity . When treated with 120 mM NaCl + 50 µM GA3, the Nagdong cultivar contained high levels of GA1, GA3, GA4, and GA7. The salt-tolerant Pokkali cultivar contained high levels of GA1, GA3 and GA7 when treated with 120 mM NaCl + 50 µM GA3 than treated with 120 mM NaCl only . Conversely, there was no significant difference between the levels of bioactive GAs observed in the control and 50 µM GA3-treated seedlings in the Nagdong and Pokkali cultivars .GA1 and GA4 are considered the essential bioactive forms of GA in rice. Generally, salinity stress decreases the bioactive GA content in germinating seeds. As we observed an effect of both 120 mM NaCl, and 50 or 100 µM exogenous GA3 on different rice seeds, we measured the levels of bioactive GAs, namely GA1, GA3, GA4, and GA7, under these conditions. To first examine whether the bioactive GA content was decreased by salinity, the levels of GA1, GA3, GA4, and GA7 in rice seedlings were determined after 14 days. NaCl treatment decreased the level of bioactive GAs in different rice seedlings compared to those in the distilled water controls –D. We oOsAmy1A, OsAmy1C, and OsAmy3C, help in starch degradation during seed germination. To investigate how salinity may increase or decrease the expression of α-amylase-related genes that are closely related to α-amylase-related production, the effects of NaCl and NaCl + 50 or 100 µM GA3 treatment on α-amylase gene expression were analysed. The results showed that after 6 h NaCl treatment, the expression levels of all α-amylase genes were significantly decreased in the roots and shoots of the salt-sensitive IR28 cultivar, whereas in the shoots of the Cheongcheong and Pokkali cultivars, no significant difference were observed compared to the control, Under NaCl stress, the α-amylase gene was significantly upregulated in the roots and shoots of the Nagdong cultivar compared to that in other cultivars. The exogenous application of 50 and 100 µM GA3 rescued the NaCl stress-induced decreases in α-amylase gene expression . However, 120 mM NaCl stress significantly decreased GSH content in the salt-sensitive IR28, and the salt-tolerant Pokkali cultivars . In contrast, compared to the control group, other rice cultivars except the salt-sensitive IR28 cultivar showed significantly increased POD activity in response to NaCl stress . Exogenous GA3, at both 50 and 100 µM, could attenuate the effects of NaCl stress, significantly increasing GSH content in the rice seedlings of all cultivars, except the Nagdong cultivar . Similarly, 50 and 100 µM exogenous GA3 increased POD activity in Nagdong and IR28 seedlings compared to the activity in NaCl-only treated seedlings. However, in the Cheongcheong and Pokkali cultivars, there was no difference observed in POD activity with the additional application of exogenous GA3, compared to the 120 mM NaCl-only condition .The activity of two vital antioxidants, GSH and POD, was analysed in different rice cultivar seedlings either under control conditions or treated with 120 mM NaCl ± exogenous GA3. We observed that compared to controls, 120 mM NaCl stress significantly increased GSH activity in the Cheongcheong and Nagdong cultivars . In seeds from the GA signalling repressor mutant slr1(a null mutant for Della), α-amylase gene expression levels were similar to those of GA-treated wild-type seeds, regardless of GA treatment . However, we suggest that exogenous GA3 does not significantly upregulate all antioxidant activity. In the present study, we noticed that under, salt stress with exogenous GA3 application, different rice genotypes differentially regulated antioxidant activity .A recent study suggests that a balanced redox status in rice would be the consequence of a synergetic activity of both enzymatic and non-enzymatic antioxidant mechanisms, resulting in tolerance to Pb-induced oxidative damage . Under oactivity Figure 6Global agriculture is at greater risk from crop loss due to soil salinization, particularly for rice. The current Petri dish-based investigation showed the impact of 120 mM NaCl treatment along with exogenous GA3 in various rice cultivars. We found that rice cultivars germination rates, seedling, and GA content were all considerably reduced by 120 mM NaCl stress. The expression of genes linked to GA production and inactivation, antioxidants, and α-amylase was also observed to change amongst rice genotypes during salt stress. In the present study, we found that 50 and 100 µM GA3 significantly alleviated the effects of 120 mM NaCl stress among rice genotypes. According to findings from the current study, salt-tolerant Pokkali and Nagdong cultivars should be treated with 50 µM GA3 and salt-sensitive IR28 and Cheongcheong cultivars should be treated with 100 μM GA3. The current study provided evidence that the exogenous GA3 application can significantly rescue the salinity effect, and the rice can be grown environmentally friendly near coastal areas.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MF planned, designed, and performed the study, analysed the data, and wrote the findings. MK helped with GAs analysis, and D-DZ helped with data collection. SA helped with the antioxidant analysis. E-GK, Y-HJ, J-RP contributed to the experimental resources, I-JL and K-MK drafted and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a National Research Foundation of Korea grant funded by the Korean Government (NRF-2021M3E5E6022715).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Thereafter, measures of oxidative damages, such as ROS production, antioxidant expression levels, and apoptosis, were evaluated. Butein effectively increased cell viability and rescued the cells from oxidative damage through the inhibition of ROS production, apoptosis, and increased antioxidant expression. Furthermore, butein dramatically inhibited mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which are the main ROS inducers. Nrf2 protein translocated from the cytosol to the nucleus and consequently activated its target genes as oxidative stress suppressors. These findings demonstrate that butein has potential antioxidant effects in H9c2 cardiomyoblasts, suggesting that it could be used as a therapeutic substance for the treatment of cardiac diseases.Oxidative stress, defined as an imbalance between reactive oxygen species (ROS) production and the antioxidant defense system, contributes to the pathogenesis of many heart diseases. Therefore, oxidative stress has been highlighted as a therapeutic target for heart disease treatment. Butein, a tetrahydroxychalcone, has potential biological activities, especially antioxidant properties. However, the effect of butein on oxidative-stressed heart cells has been poorly studied. Thus, we sought to identify the antioxidant effects of butein in H9c2 cardiomyoblasts. To elucidate these antioxidant effects, various concentrations of butein were used to pretreat H9c2 cells prior to H Briefly2O2 were fixed with 4% paraformaldehyde  for 30 min at room temperature (RT). Then, 10 µg/mL Hoechst dye (ThermoFisher) was added for 30 min at 37 °C. The nuclei were imaged by a fluorescence microscope .The detection of apoptotic cells was determined by nuclear staining using Hoechst 33342 dye . Briefly5 cells per well and were then treated with butein and/or H2O2. After that, 1 µM DCFH-DA dye was treated to the cells for 30 min at 37 °C after washing thrice with PBS. Finally, they were imaged with a fluorescence microscope (Olympus).To detect intracellular ROS production, DCFH-DA (ThermoFisher) dye was used . Briefly2O2. After that, the cells were added to 5 µM MitoSOX reagent for 10 min at 37 °C. After washing thrice with PBS, they were then imaged by a live-cell imaging system .The ROS in mitochondria was observed by using MitoSOX Red mitochondrial superoxide indicator (ThermoFisher) . Briefly2O2, the cells were fixated with 4% PFA for 10 min at RT. They were then permeabilized with 1% Triton X-100 in PBS for 10 min. After blocking by 5% BSA for 1 h, the cells were incubated with Nrf2 primary antibody  overnight at 4 °C followed by incubation with a tetramethylrhodamine (TRITC)-conjugated secondary antibody  for 1 h at RT. After that, the nuclei were stained with 4,6-diamidino-2-phenyindole  and were further imaged by a fluorescence microscope (Olympus).After treatment with butein and/or H2O2 were lysed in RIPA buffer  supplemented with the protease inhibitors (Roche) and phosphatase inhibitors (Roche Diagnostics). The proteins were separated by SDS-PAGE and transferred onto PVDF membranes . After blocking with 5% BSA in TBST buffer for 1 h at RT, they were incubated with the appropriate primary antibodies overnight at 4 °C. The membranes were further incubated with HRP-conjugated secondary antibodies . The ECL reagents and a UVITEC system  were used to develop and visualize the protein bands. The antibodies are listed in The cells that treated with butein and/or H2, 10 mM KCl, 0.5 mM DTT, 0.05% NP40, and pH 7.9) supplemented with protease inhibitors followed by incubating for 10 min on ice. The nuclear pellet was harvested by centrifugation at 3000× g for 10 min at 4 °C. After the pellet was washed thrice, it was suspended with the lysis buffer , and pH 7.9). After incubating for 30 min on ice, it was collected after centrifugation at 24,000× g for 20 min at 4 °C.Nuclear fractionation was performed using a nuclear-extraction reagents kit according to the manufacturer’s instructions (abcam). In brief, the cells were lysed with hypotonic buffer  after extracting total RNA by Monarch total RNA extraction kit (Promega). The qPCR kit from Kapa Biosystems  with a qRT-PCR machine  was used. GAPDH gene was used for normalization. The primer sequences are shown in p values less than 0.05 were considered statistically significant.Values are shown as the SEM. Statistical analysis was performed using one-way ANOVA with a Bonferroni post hoc test using Prism 8.02 . To determine whether butein is toxic to cells, they were treated with various concentrations of butein ranging from 0.5 to 10 µM for 24, 48, and 72 h. An MTT assay was performed to determine the viability of cells treated with butein. The results showed that treatment with all concentrations and times of butein did not significantly change the cell viability a–c. Ther2O2, which is commonly used to evoke cellular oxidative damage [2O2 to elucidate the protective effects of butein and were applied to the MTT assay. The cell viability of cells treated with 500 µM H2O2 alone was significantly reduced, with a 55.2% survival rate, compared with that of control cells   . These r2O2-treated cells, nuclear staining and Western blotting against apoptosis-related proteins were performed using cells treated with 500 µM H2O2 or cells pretreated with 1, 3, and 5 µM butein followed by exposure to 500 µM H2O2. Nuclear staining was performed using Hoechst 33342 dye to detect nuclear condensation as a main feature of apoptosis. The apoptotic cells were significantly increased in H2O2-only-treated cells  (2O2-treated cells (0.2-fold change in Bcl-2/Bax in H2O2-only-treated cells vs. controls). Notably, these changes in expression levels were significantly attenuated by pretreatment with butein   a,b. Convl cells) a,b. The h butein c,d. Withontrols) c,e,f.2O2. The results showed that intracellular ROS production detected by green fluorescence was markedly increased in 500 µM H2O2-only-treated cells. However, pretreatment with butein significantly inhibited ROS production. In particular, 5 µM butein pretreatment reduced ROS production to similar levels in control cells     a,b. In cd cells) a,b. Simid cells) c,d. Coll2O2, PERK and eIF2α proteins were activated because the phosphorylation of these proteins was upregulated   d. Howeveed cells d. CollecHydnophytum formicarum Jack in 2008, butein has been reported to possess antioxidant properties [2O2.In recent years, antioxidant therapy has been attracting attention to improve heart function to protect against a variety of cardiovascular diseases, such as heart failure, myocardial infarction, and ischemic heart diseases . As partoperties . Many properties ,28, nephoperties , obesityoperties , diabeteoperties , and hypoperties . In partoperties . However2O2, which is an oxidative stress inducer. Since previous findings indicated that oxidative stress causes cell apoptosis [First, we demonstrated that butein markedly increased the viability of cardiac cells after treatment with Hpoptosis , we then2O2. Although H2O2 has a critical role in the normal function of cells through acting on various cell signaling pathways [2O2. Consistent with the suppression of ROS production, treatment with butein also attenuated the decreased levels of the typical antioxidants, including catalase, SOD1, and SOD2 by exposure to H2O2. Thus, these results suggest that butein effectively blocks oxidative stress caused by H2O2 in H9c2 cardiomyoblasts.We next elucidated the anti-oxidative effects of butein in cardiac cells with oxidative stress induced by Hpathways , it alsopathways . Herein,2O2, contributing to oxidative stress. Under normal conditions, mitochondria utilize an exquisite network of ROS scavenging systems to eliminate mitochondrial ROS including SODs, catalase, the GSH-PX system, and the PRX/Trx system [2O2 treatment caused mitochondrial dysfunction and increased mitochondrial ROS production. Notably, pretreatment with butein significantly attenuated mitochondrial dysfunction and ROS production.Increasing evidence has suggested that mitochondrial dysfunction is closely associated with the incidence of oxidative stress . Indeed,x system . Howeverx system . In acco2O2-treated H9c2 cardiomyoblasts. Herein, we detected that H2O2-treated cardiac cells showed the activation of or increased expression levels of several ER stress-related proteins. Notably, these changes were dramatically blocked by pretreatment with butein. Collectively, our results demonstrated that butein effectively protected H9c2 cardiomyoblasts from oxidative stress through the inhibition of myocardial dysfunction and ER stress.The ER is a specialized subcellular organelle that is essential for protein folding, secretion, and post-translational modifications . This prTo elucidate the mechanism of the effects of butein against oxidative-stress-inducible H9c2 cells, we focused on the Nrf2-related signaling pathway. Nrf2, which is a transcriptional factor, has beneficial actions, such as antioxidant and anti-inflammatory responses, against numerous pathological conditions by upregulating the expression of a variety of cytoprotective genes . In part2O2. This antioxidant effects of butein are mediated by the preservation of mitochondrial function and inhibition of the ER stress through the activation of the Nrf2 signaling pathway. Therefore, we propose that butein might contribute to the development of a therapeutic strategy for the treatment of oxidative stress in heart diseases.The present study demonstrated that butein rescues cardiac cells from the oxidative damage induced by H


G6PC1) gene and is associated with hepatocellular adenoma (HCA) formation. Data on risk factors for HCA occurrence in GSDIa are scarce. We investigated HCA development in relation to sex, G6PC1 genotype, and serum triglyceride concentration (TG).Glycogen storage disease type Ia (GSDIa) is an inborn error of carbohydrate metabolism caused by pathogenic variants in the glucose-6-phosphatase catalytic subunit 1 (G6PC1 variant (PSV); and median TG during childhood .An observational study of patients with genetically confirmed GSDIa ≥12 years was performed. Patients were categorised for sex; presence of 2, 1, or 0 predicted severe p = 0.045). Two-thirds of patients with GSDIa carried 2 PSVs, 20% carried 1, and 13% carried none. Neither the number of PSVs nor any specific G6PC1 variants were associated with HCA occurrence. Childhood TG was 3.4 (3.0–4.2) mmol/L in males vs. 5.6 (4.0–7.9) mmol/L in females (p = 0.026). Childhood TG >5.65 mmol/L was associated with HCA development at younger age, compared with patients with childhood TG <5.65 mmol/L . Cox regression analysis including TG, sex, and TG–sex interaction correction revealed childhood TG >5.65 mmol/L as an independent risk factor for HCA development .Fifty-three patients  were included, of which 26 patients developed HCA at a median (IQR) age of 21 (17–25) years. At the age of 25 years, 48% of females and 30% of males had developed HCA  is a rare, inherited metabolic disease that can be complicated by liver tumours (hepatocellular adenomas), which in turn may cause bleeding or progress to liver cancer. Risk factors associated with hepatocellular adenoma formation in patients with GSDIa are largely unknown. In our study, we found that high serum triglyceride concentrations during childhood, but not specific genetic variants, were associated with increased risk of hepatocellular adenoma diagnosis later in life.  •G6PC1).Glycogen storage disease Ia (GSDIa) is a metabolic disease caused by mutations in glucose-6-phosphatase catalytic subunit 1 (•Patients with GSDIa often develop hepatocellular adenoma (HCA), with unclear risk factors.•Metabolic control in GSDIa is commonly evaluated through serum triglyceride concentration (TG).•Patients with GSDIa with high childhood TG had increased risk and earlier onset of HCA.•G6PC1 genotype were not associated with HCA.Sex-associated hypertriglyceridaemia and  G6PC1) gene.,[5]Glycogen storage disease type Ia  is a rare, inborn error of carbohydrate metabolism caused by pathogenic variants in the glucose-6-phosphatase catalytic subunit 1 .,[15]HCAs are rare, benign liver tumours, with size (>5 cm)-dependent associated complications consisting of hepatic haemorrhage, or transformation to hepatocellular carcinoma.G6PC1 is a single-copy gene, with 5 exons coding for 357 amino acids.G6PC1 expression is restricted to the liver, kidney, and intestine.G6PC1 catalytic domain  have shown to completely abolish glucose-6-phosphatase (G6Pase) function, whereas truncating (nonsense) variants either abolish or greatly impair G6Pase function.,G6PC1 genotype has been linked to the severity of the metabolic phenotype of GSDIa, no specific G6PC1 variants have definitively been associated with HCA formation.Improved dietary management in GSDIa has resulted in improved metabolic control, which is commonly evaluated through serum triglyceride concentration (TG). Prolonged suboptimal metabolic control (hypertriglyceridaemia >5.65 mmol/L or 500 mg/dl) has been associated with HCA development.G6PC1 genotype, and metabolic control on HCA development is as yet unknown. The aim of this study was to assess the association between sex, type of G6PC1 variants, TG in childhood, and HCA formation in a nationwide cohort of patients with genetically confirmed GSDIa.Because longitudinal data on HCA incidence in GSDIa patients are scarce, the association and potential interaction of sex, G6PC1 genetic analysis by traditional Sanger sequencing or next-generation sequencing. Strengthening the Reporting of Observational Studies in Epidemiology guidelines were adhered to for study design and manuscript preparation.a priori approval by the Medical Ethical Committee of the University Medical Center Groningen (UMCG-MEC 2019-119). The study was registered before initiation in the UMCG research registry (UMCG-RR #202000465). All patient data were collected and processed in accordance with Dutch privacy laws.A nationwide, retrospective, observational, multicentre cohort study of patients with GSDIa was performed between 1969 and September 2021. The metabolic expert centres of 7 Dutch university medical centres provided information on patients followed-up. Inclusion criteria were as follows: current age ≥12 years, availability of diagnostic imaging, and GSDIa diagnosis based on HCA was diagnosed by either magnetic resonance imaging (MRI), histopathology, or both. The date of the first HCA diagnosis was retrospectively adjusted to the first tumour observation on ultrasound, in case of later diagnosis on MRI or histopathology. The largest HCA diameter was measured by ultrasound and/or MRI according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECISTv1.1) criteria.G6PC1 variants were categorised according to both the molecular characteristics of the genetic variants and the G6Pase location. All G6PC1 missense variants in the active site  and all G6PC1 nonsense variants (regardless of location) were categorised as predicted severe variants (PSVs). Patients’ G6PC1 genotypes were categorised as 0, 1, or 2 PSVs. G6PC1 variants accounting for 50% or more of observed variants in the cohort  were grouped and compared with all other variants.Birth cohorts were defined as the older or current treatment era as previously reported.,TGs were measured at the local laboratories according to standard practice. TG data were expressed in mmol/L. Longitudinal childhood TGs were calculated as the median of measurements per 6 months per patient. Of included patients, a single childhood TG was calculated per patient as the median of all measurements obtained and available before the age of 12 years. Childhood TGs were categorised into low and high childhood TGs, defined as those patients with median childhood TG above or below 5.65 mmol/L (500 mg/dl), according to the previous definition.,www.graphpad.com). Dichotomous data were presented as proportions. Continuous variables were reported as median with IQR. Categorical variables were expressed as number (n) and percentage (%). Statistical analysis was performed using R version 4.1.0, including the ‘survival’ and ‘survminer’ packages. Univariate survival analyses were performed using Kaplan–Meier analyses and the log-rank test. Multivariate survival analyses were performed using Cox proportional hazards models. Parameters with 2-tailed p <0.05 were considered statistically significant.Patients were categorised in groups according to sex , number of PSVs , childhood TG (low/high), and birth cohort (older/current). Study data were collected from individual patient records and managed using REDCap electronic data capture tools  hosted at the UMCG.G6PC1 variant analysis available (n = 10). Fifty-three patients from 46 families were included for data analysis, with a median follow-up time of 32 (22–43) years (Seventy-seven patients with GSDIa from 66 families were diagnosed at the outpatient clinic of the 7 participating centres. Twenty-four patients were excluded because of current age <12 years (n = 8), no imaging performed (n = 6), or no 3) years . Most pa3) years .Fig. 1Flp = 0.98; HCA was diagnosed in 26 of 53 GSDIa patients (49%), at a median age of 21 (17–25) years. The lowest age of HCA diagnosis was 13 years A. No difp = 0.045; p = 0.69). Among patients without adenomatosis, male patients had a median of 6 (2–7) HCAs, compared with 3 (1–5) for females (p = 0.22). The largest median HCA size was 41 (15–104) mm in males and 28 (19–47) mm in females (p = 0.64).HCA formation was more common in female than in male patients with GSDIa , p.Gln347X (17%), and p.Gln27ArgfsX9  mmol/L, respectively; p = 0.026; vs. 1 PSV p = 0.79; 1 vs. 2 PSVs p = 0.65; 0 vs. 2 PSVs p = 0.38). Patients with GSDIa who developed HCA had a median childhood TG of 4.6 (4.0–7.8) mmol/L, compared with 3.2 (2.3–3.4) mmol/L for patients with GSDIa and without HCA diagnosis  mmol/L for patients with or those without history of lipid-lowering drug use, respectively (p = 0.56). Kaplan–Meier survival analysis did not reveal a significant difference in time to HCA diagnosis between patients with and those without historical use of lipid-lowering drugs (log-rank p = 0.18).Childhood TG data were available in 23 patients with GSDIa , with a median childhood TG of 3.9 (3.2–5.8) mmol/L. In total, 14 of the 23 patients developed HCA during follow-up. Male patients with GSDIa had a significantly lower median childhood TG than female patients with GSDIa . In this model, patients with GSDIa with a median childhood TG of >5.65 mmol/L had an HR of 4.6 (95% CI 1.3–16.3) for lifetime HCA development (p = 0.018). Because females had a higher median childhood TG than males (p = 0.028).Fifty percent cumulative HCA incidence was 18 years for patients with GSDIa with a median childhood TG of >5.65 mmol/L, compared with 33 years for patients with GSDIa with a median childhood TG of <5.65 mmol/L , whereas HCA regression has been observed after strict dietary management.,,G6PC1 genotype in relation to HCA development, however, has not been reported thus far. In our study, G6PC1 variants and G6Pase impairment, indirectly quantified through the number of PSVs, were not significantly associated with HCA development. We also did not identify a ‘hotspot’ for pathogenic G6PC1 variants that was associated with HCA development. No novel genetic variations were identified in this cohort, and more than half of patients were diagnosed with 2 PSVs.vs. 1 vs. 2 PSVs) or grouped (0/1 vs. 2 PSVs) did not reveal as a particular risk factor for HCA development.There is only limited research on genotype–phenotype correlations for GSDIa.[3]G6PC1 variant combinations including 23 subjects with homozygous G6PC1 variants. Although many patients in our study display unique combinations of G6PC1 variants and despite a relatively low number of inclusions, lessons can be learned from patients with homozygosity for specific G6PC1 variants. For example, the median age of HCA diagnosis was 16 (15–17) years for p.Arg83Cys homozygotes (n = 10), as compared with 27 (26–27) years for p.Gln347X homozygotes  treatment strategies, including dietary therapy, have evolved over time. Increased treatment efficacy influenced both metabolic control and overall survival of patients, including those with more severe metabolic phenotypes. However, stratification by birth cohort did not reveal any significant differences in HCA occurrence. Second, patient-specific heterogeneity in environmental and/or genetic factors may have resulted in residual confounding that could not be accounted for owing to the retrospective nature of this study. Third, TG analysis is likely more often performed in patients with metabolic dysregulation, as this parameter is measured frequently during hospital admissions or outpatient department evaluations. We have mitigated this aspect by calculating medians for all childhood TG data, and a median per 6 months for longitudinal measurements, yielding similar results.G6PC1 genotype, parameters of biomedical control, and sex to long-term complications. We have analysed a subset of those traits, and more investigations are needed on the alternate complications such as nephropathy and biomedical outcome markers such as lactate, uric acid, and continuous glucose monitoring parameters. These are urgently warranted to compose a set of person-centred outcomes for patients with GSDIa, to standardise future data collections, to identify important endpoints for clinical trials, and to evaluate novel treatments in the future.[43]The data in this study may assist in patient-centred (dietary) management and follow-up, as we have identified subgroups of patients especially vulnerable for HCA development. This study illustrates the importance of correlating the multifactorial processes that define the complex human GSDIa phenotype, including the G6PC1 genotype. Recognition of these risk factors may assist in further development of individual monitoring strategies for GSDIa.In conclusion, in patients with GSDIa, high childhood TG was associated with an increased risk of HCA, and earlier onset of HCA development, independent of sex-associated hypertriglyceridaemia, and 10.13039/501100001721University of Groningen as a MD/PhD (MD-PhD 16-24). MHO was supported by a VIDI grant (917.15.350) from the 10.13039/501100003246Dutch Research Council and holds a Rosalind Franklin Fellowship from the 10.13039/501100001721University of Groningen.FP was supported by a Junior Scientific Masterclass grant from the Study design: MPDH, FP, MHO, HJV, TGJD, VEM. Data collection: MPDH, FP. Analysis: MPDH, FP. Drafting of manuscript: MPDH, FP, MHO, HJV, TGJD, VEM. Drafting of figures: MPDH, FP. Feedback on data analyses: MHO, HJV, TGJD, VEM. Feedback on the manuscript: MCGJB, CEMH, MCHJ, JGL, ML, AJMR, MAEMW. Assistance with data collection: MCGJB, CEMH, MCHJ, JGL, ML, AJMR, MAEMW.The data that support the findings of this study are available from the corresponding author, VEM, upon reasonable request..There are no conflicts of interest to be reportedPlease refer to the accompanying ICMJE disclosure forms for further details.


These novel polyesters have been characterized in-depth using ATR-FTIR, 1H, and 13C NMR and XRD spectroscopies and thermal analysis (TGA and DSC). They showed enhanced thermal stability (up to 330 °C), and the glass transition temperature increased with the content of FDCA from ca. 9 to 60 °C. Regarding their (bio)degradation, the enzymatic conditions lead to the highest weight loss compared to simulated seawater conditions, with values matching 27% vs. 3% weight loss after 63 days of incubation, respectively. Copolymerization of biobased FDCA, Is, and DDA represents an optimal approach for shaping the thermal/(bio)degradation behaviors of these novel polyesters.Polys incorporating aliphatic moieties represent a promising family of polyesters, typically entirely based on renewable resources and with tailored properties, notably degradability. This study aims to go beyond by developing poly(isosorbide 2,5-furandicarboxylate- All (co)polyesters were dissolved in that solvent mixture (0.1 g per 20 mL) and kept at 140 °C for 20 min to achieve complete dissolution. The intrinsic viscosity was determined by the ratio of specific viscosity and samples solution concentration: t0 and t1 are the solvent mixture elution time of the solvent mixture and (co)polyesters solutions, respectively.Intrinsic viscosity [−1) and aluminum pans to encapsulate the samples (ca. 5 mg). The calorimeter was calibrated for heating temperature with approximately 10 mg of each of the following metals: 99.999% pure indium, mT =156.60 °C, and 99.999% pure lead mT = 327.47 °C. Scans were conducted with a heating rate of 10 °C·min−1 in the temperature range of −40 to 250 °C.Differential Scanning Calorimetry analysis (DSC) thermograms were obtained with a Pyris Diamond DSC calorimeter from Perkin Elmer , using nitrogen as purging gas (20 mL·min−1) and heated at a constant rate of 10 °C·min−1 from room temperature up to 800 °C.Thermogravimetric analyses (TGA) were carried out with a Shimadzu TGA50 analyzer  equipped with a platinum cell, using platinum pans to encapsulate the samples. Thermograms were recorded under a nitrogen flow (20 mL·min3), placed in a foldable stainless-steel materials pocket of Triton technology, was performed with a Tritec 2000 DMTA Triton equipment  operating in the single cantilever bending geometry. Tests were carried out in multifrequency mode (1 and 10 Hz), from −80 to 250 °C, at 2 °C·min−1. The gT of the PIsFDD (co)polyesters were determined from the maximum of Tan δ vs. T curve, at 1 Hz.Dynamic Mechanical Thermal Analysis (DMTA) of thick samples  analyses were carried out using the Philips X’pert MPD instrument operating with CuKα radiation (λ = 1.5405980 Scanning electron microscopy (SEM) images of the surface of the films were acquired using a field emission gun-SEM Hitachi SU70 microscope  operating at 4 kV. Samples were deposited onto a sample holder and coated with evaporated carbon twice.2 of ca. 16–31 mg) of (co)polyesters  incubated on 10 mL of NutriSelectTM Basic sea salts’ solution  at ca. 18 °C. Pure hydrolytic and enzymatic experiments were carried out with film specimens (0.5 × 0.5 cm2 of ca. 16–31 mg), using 10 mL of a phosphate buffer solution , additionally having porcine pancreas lipase (1 mg·mL−1) in the case of enzymatic degradation tests, and incubated at 37 °C for 63 days. The specimens were taken out from the solutions at regular intervals (each 3 or 7 days), rinsed thoroughly with distilled water, dried under vacuum for 3 days, and weighed. In order to prevent saturation, the buffer solution was renewed every 7 days. The weight-loss percentage was calculated as follows: In vitro simulated seawater experiments were carried out using square-shape specimens -co- (PIsFDD) (co)polyesters were herein prepared for the first time using biobased monomers, namely isosorbide, dimethyl 2,5-furandicarboxylate (FDCA derivate), and 1,12-dodecanedioic acid and the conditions described in −1, assigned to the C=O stretching vibration, characteristic of ester groups, and another typical band at 1271 cm−1, assigned to the C-O-C stretching mode also from ester groups. The presence of these two bands, as well as the absence of a noticeable OH bond stretching near 3400 cm−1, confirms the success of these polymerizations. It is also observed that the relative intensity of the two bands near 2976 and 2820 cm−1  increased with the content of 1,12-dodecanedioate (DD) in the (co)polyester backbone, as evidenced in −1 (=C-H) and 1575 cm−1 (C=C), decreased instead with the DD amount increment (or furan units decrease). The ATR FTIR spectra of all (co)polyesters are available as Electronic The elucidation of the expected chemical structure of the newly prepared (co)polyesters was confirmed by ATR FTIR spectroscopy. Accordingly, the spectra of 1H NMR spectra (δ ~ 7.25 ppm). In the case of those polymers incorporating 1,12-dodecanedioate units (PIsDD and PIsFDDs), their spectra show several typical proton resonances at C2H, C(O)CH2C2H and mid-chain C2H, respectively). All spectra show the isosorbide methylene proton resonances, split into four multiplets in the neighborhood of furan moiety, at δ ~ 5.40, 5.07, 4.69, 4.14, and 3.99 ppm ((bH + eH), (gH), (dH), (cH), and (fH), respectively). These resonances chemically deviate to higher fields when the isosorbide unit is linked to the DD moiety  and 1,12-dodecanedioate (%DD) moieties effectively incorporated in the copolyesters’ backbone is of fundamental importance for the correct interpretation of structure-properties relationships. Therefore, the molar percentage of each diacid monomer effectively incorporated in the copolyester was assessed using Results summarized in θ = 5.1°, 17.9°, 19.5°, 21.2°, 24.8°, similarly to previously reported data [θ ≈ 19°. Nevertheless, a broad peak near 2θ ≈ 18.4° is detected for the 70/30 copolymer, evidencing some order. The very rigid PIsF and PIsFDD 90/10 (co)polymers are also essentially amorphous [θ ≈ 18°) is detected, they also show a broad peak evidencing some order.The XRD patterns of the novel PIsFDD 10/90 and 20/80 copolyesters, as well as the PIsDD homopolymer counterpart , i.e., tted data . Oppositmorphous , most prIn regard to the thermal behavior, the DSC traces displayed in Oppositely, the copolymers with higher amounts of stiff furan moieties (PIsFDD 70/30), showed no melting feature in their DSC traces, displaying only a glass transition slightly below 60 °C, in agreement with its amorphous character as highlighted before on XRD patterns.gT at approximately 157 °C [gT by restricting the mobility of the chains. DMTA traces  and the maximum degradation temperature  are summarized in d,5%T). They exhibited a single weight loss step with d,maxT values ranging between 404 and 424 °C. Td,max of the PIsDD homopolymer is quite consistent with what has been reported before in the literature  [d,5%T = 300 °C; d,maxT = 398 °C) [d,maxT. A similar trend was also reported before for similar polyesters [In order to assess the thermal stability of PIsFDD (co)polyesters, a TGA analysis of all polymers was carried out . The dec 423 °C) . In gene 398 °C) . Furtherlyesters .To further investigate the potential of these biobased PIsFDD (co)polyesters and assess their (bio)degradation behavior under an aqueous environment, their degradability in three different conditions, namely: (a) in PBS at pH 7.4, (b) enzymatic degradation in the presence of porcine pancreatic lipase, at pH 7.4, and (c) simulated seawater conditions  were tested. These latter experimental conditions are quite relevant considering that today a high percentage of polymer debris ends up in aquatic environments, particularly in the ocean. Gravimetric  and SEM The enzymatic degradation prompted the highest weight loss for all polymers studied, with values matching 27% weight loss after 63 days of incubation of PIsDD films, followed by PIsFDD 10/90 with ~16%. These results are in line with those previously reported for poly degradation studies under similar conditions  and enhaSimulated seawater conditions were conducted to the overall lowest weight losses (around 3%), despite the slightly higher pH value. In this case, salinity must play a role. Therefore, one can anticipate the need for the correct disposal of these polymers after their use.gT polymer resistance to degradation under similar aqueous conditions. However, it is important to note that PIsF is a very brittle material that hampered enzymatic degradation gravimetric studies after 35 days of incubation due to the impossibility of handling the material.Overall, PIsF homopolymer showed the lowest weight loss ; regardlFollowing a similar trend, it was found that, in general, increasing the F moiety content in the copolyesters backbone led to a decrease in weight loss in all degradation conditions, with the exception of PIsFDD 10/90 degradation under hydrolytic degradation in PBS, which loses more weight than PIsDD, despite the absolute value under enzymatic conditions is higher. This finding can be related to a lower crystallinity of PIsFDD 10/90 compared to PIsDD, which may facilitate water diffusion into the polymeric matrix, promoting increased hydrolytic ester cleavage. The same was not observed, however, with the enzymatic studies, probably because, in this case, the specificity of the enzymes also played a role. Lipases are well-known for their higher affinity for degrading polymers with aliphatic moieties rather than (hetero)aromatic ones .The surface morphologies of (co)polyesters films before and after degradation in PBS, under porcine pancreas enzymatic degradation, and in simulated sea conditions were analyzed at the microscopic scale using SEM .Based on these findings, the degradation under enzymatic conditions is obviously more severe than under hydrolytic conditions (PBS and simulated sea water), demonstrating that the porcine pancreas lipase has effectively contributed to the degradation of these polyesters.1H NMR, were clearly controlled by the feed molar ratio. Depending on the F/DD moiety ratio, they acquired amorphous or semi-crystalline structures, and thermal properties varied accordingly. DMTA analysis showed that the glass transition values increased with the increasing amount of stiff furan moiety incorporated in the polymer backbone chain (from ≈ 0.2 °C to 73.8 °C) as expected for stiff furan ring-bearing polymers. Furthermore, all PIsFDDs had good thermal stability, as high as d,5%T ~ 330 °C, in line with PEF. Regarding their (bio)degradation, the enzymatic conditions lead to the highest weight loss compared to hydrolytic conditions (PBS and simulated seawater conditions), demonstrating that porcine pancreas lipase has effectively contributed to the degradation of these polyesters. Some degradation is, however, detected both under PBS incubation and simulated seawater conditions. Based on their properties, PIsFDDs stand out as promising biobased polymers with potential utility in environmentally friendly plastic applications. Further characterization, such as mechanical properties and degradation in soil, is planned for the future.In this work, aiming at obtaining fully biobased polyesters featuring a combination of good thermal properties with improved (bio)degradability, poly (co)polyesters series were successfully synthesized using the two-step bulk polymerization approach and typical reaction temperature for such systems (or even slightly lower than previously reported in the literature ). Their