Natrinema altunense 4.1R is a halophilic archaeon isolated from the endorheic saline lake systems, Sebkhas, located in arid and semi-arid regions of Tunisia. It is an ecosystem characterized by periodic flooding from subsurface groundwater and fluctuating salinities. Here, we assess the physiological responses and genomic characterization of N. altunense 4.1R to UV-C radiation, as well as osmotic and oxidative stresses. Results showed that the 4.1R strain is able to survive up to 36% of salinity, up to 180 J/m2 to UV-C radiation, and at 50 mM of H2O2, a resistance profile similar to Halobacterium salinarum, a strain often used as UV-C resistant model. In order to understand the genetic determinants of N. altunense 4.1R survival strategy, we sequenced and analyzed its genome. Results showed multiple gene copies of osmotic stress, oxidative stress, and DNA repair response mechanisms supporting its survivability at extreme salinities and radiations. Indeed, the 3D molecular structures of seven proteins related to responses to UV-C radiation , saline stress , and oxidative stress (superoxide dismutase SOD) were constructed by homology modeling. This study extends the abiotic stress range for the species N. altunense and adds to the repertoire of UV and oxidative stress resistance genes generally known from haloarchaeon.Halophilic archaea are polyextremophiles with the ability to withstand fluctuations in salinity, high levels of ultraviolet radiation, and oxidative stress, allowing them to survive in a wide range of environments and making them an excellent model for astrobiological research. The online version contains supplementary material available at 10.1007/s10709-023-00182-0. Halobacteria) thrive in arid and semi-arid saline and hypersaline environments, such as solar salterns, hypersaline marshes and saline ponds , mismatch repair (MMR), and homologous recombination repair (HRR), in addition to the light repair mechanism such as photoreactivation , singlet oxygen (O2), peroxide H2O2 and hydroxyl radicals (HO·) , including superoxide radicals  and osmotic stress. Its genome was sequenced, and the genetic determinants of abiotic stress tolerance were identified. In addition, the 3D molecular structures of putative proteins implicated in UV-C radiation, osmotic and oxidative stresses were constructed by homology modeling. To our knowledge, this work provides the first report on molecular basis of N. altunense species responses to abiotic stress.While mechanisms and pathways implicated in abiotic stress defense were described in detail for some Halobacteria members, very few to no studies were reported on tolerance mechanisms in the halophilic archaeon genus 4·7H2O, 20.0; FeCl2·4H2O, 0.036; NaCl, 250; agar, 15 pH = 7.4) based on the serial dilution technique , 0.25 mM of each dNTP, 0.5 µM of each primer, 0.1 µg of chromosomal DNA, and 1 U of Taq DNA polymerase (Fermentas), was used to in 50 µl to perform PCR reactions programmed as follows: 95 °C for 5 min; 35 cycles of , and a final extension step at 72 °C for 10 min. PCR products were purified using QIAquick PCR Purification (Qiagen) kit and the clean product was Sanger sequenced with an automated capillary ABI Biosystem 3130 . The 16S rRNA gene sequence obtained was compared to sequences deposited EzBioCloud server , in December 2020. At the time of sampling, the salt pan water had a salinity of 29.5 g/L, pH of 7.48, and was 25 °C. Physicochemical characteristics of the saline water are shown in Table S1. Isolation was performed on DSM-97 medium  on DSM-97 agar plates for 10–20 days. The optimal temperature was determined by incubating the cells at 25, 30, 37, 40, 55, and 60 °C, keeping the pH 7.4. The optimal pH was determined at 5.0, 6.0, 8.0, 9.0, 10.0, and 11.0, incubating the cells at 37 °C. The salinity- effect was assessed at 0, 5, 7, 8, 10, 15, 20, 25, 30, and 36% w/v NaCl, growing the cells at pH 7.4 and 37 °C.The growth conditions were determined by cultivating N. altunense 4.1R strain was in optimal growth conditions in DSM-97 medium (25% NaCl) at 42 °C until early exponential phase cultures (OD 0.8), then 1 ml of cell suspension was irradiated in the dark  on ice at 36, 72, 108 and 216 J/m2. Cells were then left to recover at 42 °C both in light and dark conditions. A non-irradiated control was processed identically. Cell viability was determined by counting colonies on DSM-97 agar plates after incubation at 42 °C for 15−30 days. Escherichia coli DH5 α was used as sensitive control and was grown on Bertani (LB) medium (at 37 °C). At least three independent measurements were conducted for determining radiation resistance profiles.UV-C radiation tolerance was measured according to the method of Baliga  for a final concentration of 10–1 to 10–6 cells/ml. Dilutions were spread on DSM-97 agar plates containing different concentrations of H2O2 . Colonies were counted after incubation for 1–3 weeks at 42 °C, and numbers were compared to control plates without H2O2. Three independent biological replicates were performed for each treatment.We used the method described by Kaur and his colleagues . Whole genome sequencing was performed on Illumina HiSeq 2000 platform using MiSeq V3 kit (600 cycle) using the services of Inqaba Biotechnical Industries. Adaptor sequence removal, trimming, error correction, and assembly were performed using SPAdes  under GenBank accession numbers NZ_JNCS00000000.1 , JXAN00000000.1  and NZ_AOIK00000000.1 . In silico DNA-DNA hybridization (DDH) values were calculated by the Genome-to-Genome Distance  using the BLAST + method  to the String database at high confidence score (≥ 0.7) , UvrB (WP_1301696694.1), and UvrC (WP_130169688.1); photolyase (WP_130169120.1), trehalose-6-phosphate synthase OtsA (WP_130171241.1), trehalose-phosphatase OtsB (WP_130171242.1), and superoxide dismutase SOD (WP_007109425.1) were modelled. The 3D structures were built using YASARA software  with 99.68% of similarity . Results of 16S rDNA gene sequencing and phylogenetic assignment to the closest related species showed that 4.1R strain is most close to Natrinema altunense 4.1R grew in salinities ranging from 8 to 36% (w/v) and in temperatures from 25 to 50 °C but grew optimally at 45 °C. The optimal pH was 7.4, although the strain was able to grow in a range of 6–10 . The results showed no difference between recovery under light or dark conditions Fig. B, indica2O2 , N. altunense JCM 12,890 (AOIK00000000) and N. altunense 1A4-DGR (JXAN00000000.1), this functional profile is similar .An overview of the genome features of strain 4.1R is shown in Table N. altunense AJ2 (JNCS01000019) as its closest relative with 98.03% ANI similarity and 82.2% isDDH. These values are within the cut-offs to define species (95% ANI)  and 3135 single-copy gene clusters indicating few duplication events before speciation  were of unknown functions. The rest (23%) were categorized on the basis of orthologous group annotation (COG) into 3 classes belonging to metabolic processes with uneven distribution (i) class P (67. 3%) responsible for inorganic ion transport metabolism (ii) class F (23.07%) involved in nucleotide transport metabolism (iii) class C (9.6%) involved in energy production and conversion. Globally these clusters lost in strain 4.1R probably evolved additional functions in relation with ecological niche adaptation.OrthoVenn was used to construct a N. altunense 4.1R genome encodes several genes potentially involved in osmotic stress response  (n = 8), mismatch repair (MMR) (n = 11), and homologous recombinational repair (HRR) (n = 17) were identified (Table S5). These include: (i) UvrABCD excinucleases, UvrD helicase, and DNA ligase (ligA) involved in the NER pathway; (ii) DNA mismatch repair proteins  and RecJ-like exonuclease involved in MMR pathway; (iii) DNA double-strand break repair protein, ATPase and nuclease , Bipolar DNA helicase (HerA), DNA repair and recombination protein , and recombinase (RecA), all involved in HHR pathway. These results highlight the mosaic nature of the DNA repair pathways in N. altunense 4.1R.Putative genes involved in protective mechanisms, such as membrane pigments, including carotenoid C50 and bacteriorubine, have also been identified. In fact, genes encoding carotenoid biosynthesis enzymes, including 15-cis-phytoene synthase (crtB) (locus tag ELS17_RS03615), 1-hydroxy-2-isopentenylcarotenoid 3,4-desaturase (crtD), lycopene beta-cyclase (crtY) (locus tag ELS17_RS02895), phytoene desaturase (crtI) (ELS17_RS15240) and beta-carotene 15,15’-dioxygenase, Brp/Blh family  were found in the genome. Several genes needed for the biosynthesis of bacterioruberin have been identified, including lycopene elongase (locus tag ELS17_RS02895) and bisanhydrobacterioruberin hydratase .2O2 into a neutral element (H2O). As well, non-enzymatic antioxidants that are capable of trapping free radicals, e.g., glutathione, thioredoxin and glutaredoxin , gas vesicle structural protein (GvpA) (locus tag ELS17_RS10385) and gas vesicle protein GvpFL (locus tag ELS17_RS10420).To cope with oxidative stress, microorganisms use defense mechanisms including antioxidant enzymes as well as nonenzymatic elements. Strain 4.1R genome encodes the enzymatic antioxidants superoxide dismutase (SOD), catalase (Cat), catalase/ Peroxidase HPI (Cat/Prx HPI), glutaredoxins (GRx), peroxiredoxins (PRxs), glutathione-S-transferases (GSTs), alkyl hydroperoxide reductase (AhpC), all of them transform the generated HAnalysis of protein–protein interactions indicated possible interactions between the different proteins involved in UVr-C, osmotic and oxidative stress tolerance simultaneously Fig. . In factThe 3D molecular structures of seven proteins were constructed by homology modeling. These proteins are related to responses to UV-C radiation , saline stress  and trehalose-phosphatase (OtsB)), and oxidative stress (superoxide dismutase (SOD)) Table . AccordiGeobacillus stearothermophilus  Fig. D. The maPyricularia oryzae  model was obtained as a tetramer and as a complex with uridine diphosphate (UDP) and trehaolose-6-phosphate (T6P) Fig. A. The teMycobacterium tuberculosis  model was obtained as a monomer binding a magnesium metal ion Fig. B. The maPyrobaculum aerophilum (Lee The superoxide dismutase (SOD) model obtained is a tetrameric structure with a magnesium binding site in each subunit Fig. C. The mailum Lee . Both prilum Lee C.N. altunense 4.1R strain isolated from Sabkhat Ennaouel that enable tolerance to abiotic stresses, including osmotic, UV-C radiation, and oxidative (H2O2).Haloarchaea constitutes one of the most predominant groups of the microbial community present in hypersaline habitats  with 99.68% of similarity. AJ2 was isolated from a high-altitude salt lake in Xinjiang, China  and isDDH (82.2%) similarities values with the genome sequence of N. altunense AJ2 (NZ_JNCS00000000.1), these values are within the cut-offs used to define species (95% ANI) , with optimal growth at concentrations ranging from 20 to 30% (w/v). Haloarchaea are obligate halophiles that need a minimum of 8% w/v NaCl to grow +/H+-antiporter, the uptake of K+ via trk systems  and the potassium efflux using kef system  is known to be responsible for the release of cytoplasmic ions (Booth & Louis + transport system (KdpB) was absent in Natrinema sp. J7-2 strain. Indeed, a large number of genes homologous to Na+/H+ antiporters, and monovalent cation/H+ antiporters were identified N. altinense 4.1R synthesizes trehalose using the OtsAB pathway like reported in other haloarchaea such as Haladaptatus paucihalophilus , proton/glutamate, sodium/glutamate, and Na+/solute , mismatch repair (MMR), and homologous recombination repair (HRR) , Uvr-C (acting as exinucleases), UvrD (a DNA helicase II), and DNA ligase (ligA). These genes were previously identified in certain halophiles, such as Halobacterium cutirubrum, Halobacterium halobium, H. volcanii, and H. salinarum , and hydroxyl radicals (·OH) which affect the different cellular components including proteins, nucleic acid, cell wall components and membrane lipids  compared to H. mediterranei and H. salinarum NRC1, which tolerated up to 25 mM and 30 mM H2O2, respectively , gas vesicle structural protein (GvpA), and gas vesicle protein (GvpFL), as well as genes responsible for the construction of the gas vesicle wall and as regulatory proteins, were found. Carotenoids are also widely known for their remarkable antioxidant properties because they can quench free radicals  such as superoxide (·O2 −), hydrogen peroxide , saline stress (OtsA and OtsB), and oxidative stress (SOD) have shown highly conserved regions, mainly in the catalytic sites and cofactor binding sites, which strongly support their predicted functions. It is also remarkable the computational prediction of oligomeric forms such as UvrA, OtsA, and SOD. This study gives insights to gain, in the future, an in-depth understanding of the regulation of metabolic pathways under stressful conditions.In this study, we assessed the physiological response of Supplementary file1 (PDF 1043 KB)Below is the link to the electronic supplementary material.


Their structures were elucidated by extensive spectroscopic analysis, including HR-MS, 1D and 2D NMR, CD spectrum, as well as comparison with literature data. Compound 1 exhibited significant antiviral activity against PRV (Pseudorabies virus) with an IC50 value of 2.056 μM and therapeutic ratio at 14.98, suggesting that it might have potential for development of an antiviral agent. Moreover, compound 1 displayed the strongest inhibition against PRV protein among the tested napyradiomycins in the indirect immunofuorescence assay. Compounds 3 and 4 showed higher activities against swine pathogenic Streptococcus suis than the positive control penicillin G sodium salt, with MIC values of 3.125 and 6.25 μg/mL, respectively. Compounds 1 and 3–6 exhibited moderate antibacterial activity against the swine pathogenic Erysipelothrix rhusiopathiae, with MIC values ranging from 25 to 50 μg/mL. Two new napyradiomycins derivatives, napyradiomycin A4 ( In recent decades, infection with pathogens has caused huge economic losses to the swine industry. Pseudorabies virus (PRV), a herpes virus, is one of the most important pathogens in swine ,2. The sStreptococcus suis and Erysipelothrix rhusiopathiae, can also cause economic losses and zoonotic diseases. S. suis is an important zoonotic pathogen that causes several pathological conditions such as septicemia, meningitis, arthritis and pneumonia , a methine multiple proton at δH 2.53 (m), four sets of methylene protons , and five methyl protons . The 13C NMR spectrum , two oxygenated aromatic carbons (δC 164.8 and 164.4), three unsaturated methine carbons , six quaternary carbons , two aliphatic methine carbons (δC 58.9 and 41.1), four methylene carbons , and five methyl carbons . Combined with the 2D-NMR spectrum of 1 (3)  in 2, and a methyl group in A90915G (5) is replaced by the hydroxymethyl group  in 2. The 1H-1H COSY correlations  25 D −4.2 (c 0.1 MeOH); IR (KBr) υmax 3360, 2926, 2855, 2359, 1698, 1636, 1614, 1456, 1385, 1371, 1258, 1186, 1134, 1074, 1015, 862, 725 cm−1; UV (MeOH) λmax (log ε): 249 (4.14), 270 (2.99), 359 (1.59) μM; ESIMS m/z 497.4, 499.2, 501.4 [M + H]+  (1H and 13C NMR data see c 0.3 MeOH): λmax (Δε): 220 nm (−0.69), 250 nm (+0.31), 273 nm (−0.27), 300 nm (+0.23), 330 nm (−0.04), 360 nm (+0.15).Napyradiomycin A4  ; 1H and 2). Yellow oil; [α]25 D +59.35 (c 0.2 MeOH); UV (MeOH) λmax (log ε): 251 (2.15), 270 (1.57), 363 (0.86) nm; ESIMS m/z 441.6 [M − H]−  (1H and 13C NMR data see c 0.2 MeOH): λmax (Δε): 220 nm (−0.29), 252 nm (+0.21), 270 nm (−0.03), 295 nm (+0.09), 335 nm (−0.07), 362 nm (+0.24).A80915H  ; 1H and 2 atmosphere. The PRV was obtained from Professor Tan Chen of Huazhong Agricultural University. The virus was propagated in Marc-145 cells and stored at −80 ℃ until used. DMEM containing high glucose levels and Sodium Pyruvate was purchased from Hyclone. FBS was purchased from Gemini. Ribavirin, 0.25% tyrisin and MTT were purchased from Solarbio.Marc-145 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 IU/mL penicillin, at 37 °C in 5% COThe anti-Pseudorabies Virus antibody (ab3534), goat anti-rabbit IgG H&L (Alexa Fluor 488) (ab150077), and DAPI staining solution (ab228549) were purchased from Abcam.The supernatant was discarded upon Marc proliferation; cells were washed with PBS, and trypsin was added until the cells were digested into singles at 37 ℃. Cells were dispersed in DMEM with 10% FBS and cultured at 37 ℃ for 2–3 days.450μM value/cell control average OD450μM value) × 100%. Compounds were added to monolayer in different concentration. After 48h incubation, the cell viability (%) was calculated by MTT assay, and the cytotoxic effect (CC50) was analyzed by SPSS 16.0 software. Compounds in different concentrations  and PRV (100 TCID50) were added to monolayer at 37℃. After 72h incubation, the cell viability (%) was calculated by MTT assay, and the half maximal inhibitory concentration (IC50) was analyzed by SPSS 16.0 software. Ribavirin was used as a positive control.Cell viability was assessed using the MTT according to the protocol. Briefly, cells in 96-well plates were treated with compounds in different concentration for 48 h. Assays were performed in triplicate. Then, MTT was added to the cells and they were incubated in the dark for 4 h at 37 ℃. The supernatant was discarded and DMSO was added. After 10 min incubation, the OD 450 of each well was read using a spectrophotometer (Thermo). The cell viability (%) was calculated as (compound average OD50/IC50.Therapeutic index (TI) was calculated as CCThe monolayers of Marc-145 cells cultured in 24-well plates were treated with PRV and drug mixture. After 24 h incubation, the cells were washed with PBS twice and fixed with cold absolute ethanol at 4 °C for 15 min, then permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 1% BSA in PBS for 1 h. The cells were then incubated with 1:200 anti-Pseudorabies Virus antibody in PBS containing 1% BSA at 4 °C overnight. After washing three times with PBST, the cells were stained with 1:2000 goat anti-rabbit IgG H&L. To show the nucleus of the cells, DAPI  was used to stain the cellular DNA. The fluorescent images were observed using an inverted fluorescence microscope ).1–7 were determined against the four bacterial strains  following the Clinical and Laboratory Standards Institute (CLSI) guidelines [® 3599 Corning, Corning, NY, USA) using MHB . The final concentration of the culture was 5 × 105 colony-forming units (CFU)/mL, and measurements were repeated at least in triplicate. Penicillin G sodium salt and cefpirome sulfate were purchased from Solarbio.The minimum inhibitory concentrations of idelines . The mic


AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function.Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10 Aquaporin-9 (AQP9) is a member of the major intrinsic protein family of small transmembrane channels and displays broad substrate specificity. It is permeable to water and several neutral solutes, including glycerol ,2. We ha−/−Aqp9 knockout mice are healthy and show few phenotypes. In a C57BL/6 genetic background the only known effect of Aqp9 gene deletion is an elevation of plasma glycerol  describes AQP9-expressing cells incubated in 1% DMSO , the inhibitor vehicle [AQP9 inhibitor] describes cells incubated at respective inhibitor concentration, and [CHO] describes recordings from CHO control cells that do not express ectopic AQP9 [Whereby pic AQP9 .10 × His-hAQP9) as well as a codon-optimized version of the construct were identified in Pichia pastoris. Prior to purification by Ni2+-NTA affinity chromatography, n-decyl-β-D-maltopiranoside (DM) was used to solubilize the protein from isolated membranes. For details see High-expression clones of a recombinant human AQP9 bearing a 10-histidine tag fused at its N-terminus  under a nitrogen stream while rotating and drying under vacuum for 4 h at room temperature (RT). The resulting thin lipid film was hydrated in 1 mL of reconstitution buffer  containing 2 mM ß-mercaptoethanol and incubated for 2 h, at RT. The suspension was then vortex-mixed and incubated on ice for 10 min. Aliquots of 200 µL (5 mg/mL lipids) were shock-frozen in liquid nitrogen and thawed on ice for 2 h before sonication in a bath sonicator for 120 min to obtain multilamellar liposomes. A reconstitution mixture was prepared at RT by sequentially adding the purified 10×His-hAQP9 protein , DM (1.8 mM) and liposome buffer . The 10×His-hAQP9 protein was omitted for empty liposomes. The mixture was vortex mixed, incubated on ice for 30 min and slowly injected through a 23-gauge needle into 22 mL of liposome buffer to dilute the detergent and force the protein to reconstitute into proteoliposomes. Multilamellar proteoliposomes/empty liposomes were collected by centrifugation for 1 h at 100,736× g at 4 °C. The supernatant was discarded and the resulting pellet resuspended in 1.5 mL of liposome buffer. The diameter of the liposome specimens was determined by electron microscopy. Briefly, pellets of proteoliposomes or empty liposomes were fixed in a mixture of 3% paraformaldehyde and 1% glutaraldehyde in 100 mM PBS at pH 7.4 for 4 h, at 4 °C. Specimens were post-fixed in 1% OsO4 in PBS for 60 min, at 4 °C. Fixed specimens were dehydrated in ethanol and then embedded in Epon . Ultrathin sections were mounted on Cu/Rh mesh grids. The sections were stained with uranyl acetate and lead citrate and observed with a Zeiss EM 109 electron microscope . Ten to fifteen micrographs were made from each gravitational pellet of liposome specimen and mean diameter was measured over an overall micrograph surface of about 2000 µm2. The diameter of the prepared liposome specimens was also measured using an N5 Submicron Particle Size Analyzer . Before the stopped-flow light-scattering analysis (see below) samples were passed through a 27-gauge needle.Purified protocol . BrieflyPgly, cm/s), was calculated as:For measuring glycerol permeability of 10×His-hAQP9 proteoliposomes and empty liposomes, respectively, scattered light intensity time courses reflecting liposome volume change were recorded at 20 °C, 530 nm using an SFM-20 stopped-flow reaction analyzer  with a dead time of 1.6 ms and 99% mixing efficiency in <1 ms. Glycerol permeability was measured, as described previously , by mixiS and V denote liposome surface and volume, and τ is the exponential time constant fitted to the liposome swelling phase, corresponding to glycerol entry, of the light- scattering time course. Inhibitors and respective vehicles  were incubated with liposomes/proteoliposomes for 10 min before stopped-flow light-scattering recordings.g, 4 °C) was prepared within 45 min from blood collected in Li-heparin tubes and stored on ice until processing. Plasma samples were stored frozen at −20 °C before analysis, using the Free Glycerol Colorimetric/Fluorometric Assay Kit , as described previously [RG100204 was formulated in tetraethylene glycol  at 5, 10, and 20 mg/mL, respectively. At t = 0, four groups of 10 mice were removed from food, followed by oral gavage at 2.5 mL/kg with the respective RG100204 formulation, or TEG (0 mg/mL group). Mice were kept fasting throughout the experimental period. Blood samples were collected from the tail vein of restrained animals at 90, 180, 270, and 360 min post-dosing. Blood glucose was measured using a handheld glucometer , and documented manually. Plasma . Mass spectrometry was performed on a Q Exactive Plus (Orbitrap) accurate mass spectrometer equipped with a heated electrospray (H-ESI) interface (ThermoFisher Scientific) connected to a PC running the standard software Xcalibur 4.0.27.19. Analytes were separated on a Kinetex Phenyl-Hexyl, 2.6 µm, 50 × 2.1 mm analytical column . The HPLC was performed in the gradient mode using acetonitrile as organic phase (A) and 10 mM ammonium acetate in water as aqueous phase (B). The pump flow rate was set to 600 µL/min. The HPLC gradient A:B was 0–0.1 min 5:95, 0.4–1.7 min 97:3, 1.8–2.5 min 5:95. A generic tune file was used as MS tune file. Diisooctyl phthalate (m/z 391.28429) was used as lock mass, the MS was operated in the positive full-scan mode. Accurate mass of the monitoring ions ±5 mDa was used for the test item and internal standard peak integration. Full MS-SIM analysis was applied with the m/z ranges. Analyzer settings were as follows: max. trap injection time, 80 ms, sheath gas 40, aux gas 10, sweep gas 2, spray voltage 3.8 kV, capillary temperature 350 °C, heater 350 °C.Calibration standards and QCs were prepared by spiking 20 µL of drug-free blank blood with 2.4 µL of RG100204 dissolved in DMSO at 200 µg/mL. Unknown samples, zero samples, and blanks were spiked with 2.4 µL DMSO. A volume of 40 µL acetonitrile containing griseofulvin (600 ng/mL) was added to each calibration standard, QC, zero sample and unknown sample, while 40 µL acetonitrile was added to blanks. Samples were mixed and centrifuged for 10 min at 6000× In vivo dosing of RG100204, as well as plasma sampling, blood glucose, and MS bioanalysis was conducted by Pharmacelsus Gmbh, Saarbrücken, Germany.Dose responses were calculated after log transformation of compound concentrations and fitting to a sigmoidal log-inhibitor vs. response function with a standard hillslope of −1.t test, with p < 0.01 denoting presence of a statistically significant difference.Stopped-flow recordings were performed at least in triplicate. All data resulted from at least four independent preparations and were expressed as mean ± SEM. Differences between experimental groups were examined for statistical significance using the Student’s In vivo data were analyzed by 2-way analysis of variance (ANOVA), considering time points as repeated measures, followed by Bonferroni post-tests. RG100204 plasma concentration was analyzed by linear regression. Calculations and plots were prepared in GraphPad Prism 5.0 . Data were expressed as means ± SD.50 water permeability ~2 × 10−7 M vs. ~5 × 10−8 M, IC50 glycerol permeability ~4 × 10−7 M vs. ~5 × 10−8 M). Thus, inhibition of permeability by RG100204 was comparable for the two channel substrates, water and glycerol. Moreover, inhibition of glycerol permeability by RG100204 was more efficacious, compared to phloretin (remaining permeability −1.3% ± 0.4 vs. 16.2% ± 2.6).In order to characterize the potency and efficacy of RG100204 for inhibition of AQP9 permeability, water permeability and glycerol permeability were measured in calcein-loaded CHO cells ectopically expressing human AQP9, as well as in control CHO cells, without AQP9 expression . CompareAQP9 in the yeast P. pastoris, and to reconstitute the purified recombinant protein 10×His-hAQP9 into unilamellar liposomes prepared from E.coli polar lipids  of the proteoliposomes and control liposomes by the stopped-flow light-scattering method, after applying a 150 mM inwardly directed glycerol gradient. Pgly) between empty liposomes treated with phloretin and vehicle-treated empty liposomes (1.38 ± 0.17 × 10−6 cm/s and 1.98 ± 0.39 × 10−6 cm/s, respectively; −6 cm/s; p < 0.01; Pgly value of the proteoliposomes was significantly reduced by pre-treatment with the positive control inhibitor phloretin, applied at 0.7 mM (2.10 ± 0.21 × 10−6 cm/s, −52.5%; p < 0.01; Pgly compared with vehicle treated proteoliposomes (1.26 ± 0.33 × 10−6 cm/s and 4.43 ± 0.34 × 10−6 cm/s, respectively; −71.5%; p < 0.01; Functionality of the AQP9 channel in the proteoliposomes was established by measuring the glycerol permeability coefficient (Aqp9 gene deletion on blood glucose and plasma glycerol. Diabetic C57BKS db/db mice were exposed to three different oral doses of RG100204, while removing mice from food, to mimic conditions that were previously established in -/- db/dbAqp9 mice [In order to investigate if RG100204 is suitable as a tool compound for studying AQP9 function in vivo, we tested previously observed effects of P. pastoris before incorporation into artificial proteoliposomes. RG100204 strongly reduced glycerol permeability in this cell-free system, thereby corroborating RG100204 as an AQP9 inhibitor.In the current study, we describe the characterization of RG100204 as a novel, specific inhibitor of human aquaporin-9 water and glycerol permeability in a calcein-quenching- based cell assay and by stopped-flow light-scattering using hAQP9 proteoliposomes. We have previously described a short list of compounds with similar characteristics ,17. In g2+-NTA affinity chromatography and verified by Western blotting (see Furthermore, as the tested proteoliposomes essentially do not contain any other protein except for AQP9, which was purified by Niting see , these a−/−Aqp9 mice [Aqp9 deletion, e.g., the blood glycerol elevating effect in −/−Aqp9 mice [−/−Aqp9 mice have been demonstrated in a mixed C57Bl6 × BKS genetic background [Aqp9 and a blood glucose modifier gene could have caused these observations. In the current study, db/db mice of the BKS background were used, which develops a severe diabetic phenotype, in contrast to db/db mice of the Bl6 background [Moreover, previously identified AQP9 inhibitors were not suitable as tool compounds for in vivo experimental work in mouse models . Much of−/− mice ,22. Whil−/− mice  result f−/− mice . Moreoveckground , thus thckground . An obsedb/db mouse plasma glycerol was seen after oral administration of the compound. A blood glucose-lowering effect was also observed, although it was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest this heterocyclic compound as a novel tool for investigating the physiological and pathophysiological role of AQP9 in health and disease. These findings complement our recent demonstration of RG100204 blocking the role of AQP9 in sepsis [Altogether, our results demonstrate functional inhibition of AQP9 by RG10204 both in vitro and in vivo. A dose-dependent elevation of n sepsis ,24.


Solanecio gigas (S. gigas) stem bark essential oil (EO), as well as the effectiveness of EO and extracts  against human pathogenic bacteria and their antioxidant activity. The GC-MS analysis identified 23 components, accounting for 98.7% of the total oil containing Methylene chloride (49.2%), sabinene (10.5%), 1-nonene (11.3%), Terpinen-4-ol (6.9%), Camphene (4.3%), γ-terpinene (3.6%), α-phellandrene (2.9%) β-myrcene (2.6%), 1,2,5-Oxadiazol-3-carboxamide, 4,4′-azobis-2,2′-dioxide (2.4%), α-terpinene (1.9%), 1-Octanamine, N-methyl- (1.9%), ρ-cymene (1.6%) as major components. The antibacterial efficacy of the EO and extracts  was demonstrated by the inhibitory zones (8.5 ± 0.47–23.3 ± 0.36 and 7.2 ± 0.25–22.0 ± 0.45 mm), respectively. The MIC values of the extracts and the EO were 120–150 and 240 to <1100 μg/ml, respectively. The EO also demonstrated a significant antibacterial impact. The EO and methanolic extract had free radical scavenging activities with IC50 value, 13.8 ± 0.48 and 4.2 ± 0.04 μg/ml, respectively. In comparison to the other extracts, the methanolic extract had the greatest phenolics (100.2 ± 0.13 μg GAE/mg of dry extract) and flavonoid contents (112.1 ± 0.18 μg CE/mg of dry extract).Herbal medication developed from natural resources has to have antibacterial and antioxidant effects. The aim of this research is to look at the chemical makeup of  The search for natural-source compounds with pharmacological properties has resulted in the discovery of one of the many new molecules with critical applications , 2. EthiAlso, natural polyphenol compounds are in high demand because of their potential to treat a number of disorders, including diabetes, cardiovascular problems, anti-cancer, anti-inflammatory, and antibacterial properties. In addition, they have potential applications in the food and pharmaceutical industries , 6.Antioxidants have long been used as food additives to prevent food degradation. There are also compounds that react with free radicals, neutralizing them and avoiding or reducing their harmful effects in the human body . Lipid oAlternative antibiotics, such as plant-based drugs, are being investigated as a possible replacement for traditional antibiotics. As a result, substantial research has been conducted to assess the antimicrobial impact of EOs and extracts, which have demonstrated the potential to suppress the growth of a variety of harmful microorganisms .S. gigas (Vatke) C. Jeffrey (Asteraceae) is a large rosette plant or shrub with soft woody stems that grows up to 4 meters tall /(Abs control)] × 100.  The % antioxidant activity calculated as  stable radicals . 3 mL of50) was derived. As a positive control, an ascorbic acid methanol solution was employed.All of the tests and analyses were performed three times. From the graph plotting inhibition percentage against extract concentrations, the extract concentration providing 50% inhibition  or standard error of mean (SEM), excel, and chemdraw . The results were thought to be statistically significant.S. gigas stem bark yielded different percent based on room temperature dry weight by different solvents. The percentage quantities of the extracts with methanol, ethyl acetate and chloroform solvents were presented in The extracts of S. gigas exhibited positive results from ten phytochemical tests. Nine phytochemical tests were positive in the extract of the plant. The stem bark methanolic extract of the plant showed the presence of alkaloids, flavonoids, steroids, tannins, saponins, anthraquinones, terpenoids, glycosids, and phenolic compounds, and absence of reducing sugar , γ-terpinene (3.6%), β-myrcene (2.6%), 1,2,5-Oxadiazol-3-carboxamide, 4,4′-azobis-, 2,2′-dioxide (2.4%), α-terpinene (1.9%), 1-Octanamine, N-methyl-(1.9%), ρ-cymene (1.6%) (Yellowish oil was obtained by hydro-distilling the stem bark of e (1.6%) , and aloAntioxidant, antibacterial, and other biological properties have been found for phenolic compounds. Folin-Ciocalteu technique was used to evaluate total phenolic components in extracts, which were expressed as Gallic acid equivalents (GAEs).μg GAE/mg), followed by ethyl acetate extract (57 ± 0.02 μg GAE/mg). The chloroform extract contains the smallest phenolic chemicals (27.4 ± 0.53 μg CE/mg). Total flavonoids in catechin equivalent were calculated (CE). The maximum concentration of flavonoids was found in S. gigas methanol extract (112.1 ± 0.18 μg CE/mg), followed by Ethyl acetate extract (67.2 ± 0.24 μg CE/mg) and chloroform extract (40.3 ± 0.23 μg CE/mg).S. gigas stem bark EO and extracts against the microorganisms used. As shown in E. coli (ATCC 25922) was found to be highest inhibited bacterial pathogen by the EO with their respective diameter zones of inhibition of 23.3 ± 0.36 mm, and the rest of the bacterial strains, K. pneumonia (ATCC 27853), S. aureus (ATCC 31488) and S. pyogenes (ATCC 27853) were inhibited in a good way, with diameter of zones of inhibition ranging from 17.2 ± 0.40–19.3 ± 0.52 mm by this EO of the plant.The presence of inhibitory zones was used to qualitatively and quantitatively analyze the antibacterial activity of In addition, the methanolic and ethyl acetate extract shown antibacterial action against the bacteria tested. Also, at 200 mg/ml, the diameters of methanolic and ethyl acetate extract zones of inhibition against the studied microorganisms were 17.5 ± 0.50–22.0 ± 0.45 mm and 17.9 ± 0.17–21.9 ± 0.51 mm , respectμg/ml, the EO had a great antibacterial action against two Gram-negative bacteria and two Gram-positive bacteria, respectively. In contrast to chloroform extract, ethyl acetate and methanol extracts had a significant antibacterial impact. Methanol, ethyl acetate, and chloroform extract all had better antibacterial effects against all of the tested bacterial strains with MICs ranging from 240 to 1100 μg/ml, but chloroform extract had a mild to moderate antibacterial effect against all Gram-negative and Gram-positive bacteria with MIC values ranging from 1000 to 1100 μg/ml, respectively.As shown in S. gigas concentrations result in a larger inhibition ratio (percentage). The ascorbic acid standard (AA = 4.7 ± 0.02 μg/ml) was used to compare the IC50 values of EO and methanolic extract. The best antioxidant activity is shown by a reduced IC50 value. Methanolic extract and EO both had IC50 values of 4.2 ± 0.04 and 13.8 ± 0.48 g/ml, respectively. Methanolic extract was found to be superior to both EO and the standard (ascorbic acid) in terms of free radical scavenging.The DPPH test was performed to evaluate the free radical scavenging activities of the methanolic extract and EO, and the reaction followed a concentration-dependent pattern Figures  and 4.  Phytochemicals such as alkaloids, flavonoids, steroids, tannins, saponins, anthraquinones, terpenoids, glycosids, and phenolic compounds were discovered in plant methanol extract. These are recognized to have both therapeutic and bioactive characteristics. Several researchers have reported the analgesic , antispaPlant based secondary metabolites such as EO and extracts are widely used in the food industry and considered Generally Recognized as Safe (GRAS). Various publications have documented the antimicrobial activity of the EOs and plant extracts . Also, sS. aureus (ATCC 31488) and S. pyogenes (ATCC 27853), as well as Gram-negative bacteria such as E. coli (ATCC 25922) and K. pneumonia (ATCC 27853). This might be the result of the major components such as Methylene chloride (49.2%), sabinene (10.5%), 1-nonene (11.3%), Terpinen-4-ol (6.9%), Camphene (4.3%), α-phellandrene (2.9%), γ-terpinene (3.6%), β-myrcene (2.6%), 1,2,5-Oxadiazol-3-carboxamide, 4,4′-azobis-, 2,2′-dioxide (2.4%), α-terpinene (1.9%), 1-Octanamine, N-methyl- (1.9%), ρ-cymene (1.6%) present in the stem bark EO of S. gigas  and S. pyogenes (ATCC 27853), E. coli (ATCC 25922) and K. pneumonia (ATCC 27853) were found sensitive to the essential oil and the extracts. The investigated plant EO showed maximum antibacterial activity against Gram-negative, E. coli, and K. pneumonia [S. aureus and S. pyogenes. The present results agree with that of [S. gigas flower can inhibit different bacterial and fungal species.EO, which are odorous and volatile products of plant secondary metabolism, have wide applications in the food flavoring and preservation industries . In addineumonia  than Gra that of  who repoIt's possible that the difference in sensitivity between Gram-negative and Gram-positive bacteria is due to differences in cell wall structure. The cell wall of Gram-positive bacteria is made up of 70–100 layers of peptidoglycans . PeptidoS. gigas stem bark. This is because methanol extract has a higher concentration of bioactive chemicals than other organic extracts. Several studies have stressed the importance of phenolic compounds in scavenging free radicals [Furthermore, the DPPH radical is a free radical that has been widely used as a tool to estimate the free radical scavenging activity of antioxidants. When antioxidants interact with DPPH, they either transfer electrons or hydrogen atoms to it, neutralizing its free-radical nature , 46. In radicals .Organic extracts may be more beneficial than isolated constituents because other compounds present in the extracts can change the chemical or biological properties of bioactive individual components , 49. In S. gigas stem bark. According to the researchers, these molecules could be utilized as preservatives to prevent food-borne diseases as well as natural antioxidants to reduce oxidative stress in humans. The results of this study suggest that stem bark EO and extracts could be used in the food industry. The natural origins of the EO and extracts, which consumers find comforting and are good for the environment, as well as the extremely low risk of pathogens developing resistance to the mixture of components that make up the oil and extracts, which appear to have a wide range of antibacterial mechanisms, are the main reasons for their suitability. As a result of these beneficial properties, food safety and shelf life may be increased. This applies to medications as well as the food and cosmetics industries. As a result of the aforesaid findings, it may be inferred that natural antibacterial and antioxidant agents generated from S. gigas stem barks will be suitable for food industry applications, as plant-derived natural antimicrobials have been used by mankind for ages with little or no toxicity. The antioxidant and antibacterial activities of the extracts were impacted by the relative amounts of TPC and TFC, as well as the EO, due to the presence of major components. As a result, further rigorous study is needed to isolate the beneficial component, conduct toxicity testing, and conduct clinical trials.Alkaloids, flavonoids, steroids, tannins, saponins, anthraquinones, terpenoids, glycosids, and phenolic compounds were found in a methanolic extract of


Eucalyptus nitens wood samples were subjected to hydrothermal processing to obtain soluble saccharides from the hemicellulosic fraction. The hemicellulose-derived saccharides were employed as substrates for furfural production in biphasic media made up of water, methyl isobutyl ketone, and one acidic ionic liquid (1-butyl-3-methylimidazolium hydrogen sulfate or 1-(3-sulfopropyl)-3-methylimidazolium hydrogen sulfate). The reactions were carried out in a microwave-heated reactor to assess the effects of the most influential variables. Under selected operational conditions, the molar conversions of the precursors into furfural were within the range of 77–86%. The catalysts conserved their activity after reutilization in five consecutive reaction cycles. Sustainability and environmental concerns derived from the intensive use of fossil resources foster the use of renewable resources as feedstocks for the industry. Among these problems, climate change is one of the main ones to combat. Other problems linked to the intensive use of fossil resources are their limited availability, supply insecurity, and the volatility of prices. In this context, the European Commission has adopted the European Green Deal , a stratVegetal biomass, and particularly lignocellulosic materials (LCMs), are renewable and potential feedstock for the industry that could help to meet the objectives of a carbon-neutral economy by 2050, as proposed by the European Commission. These materials can be treated following the biorefinery approach. According to the International Energy Agency HSO4; or 1-(3-sulfopropyl)-3-methylimidazolium hydrogen sulfate, denoted [C3SO3Hmim]HSO4. Reactions were performed in a stirred, microwave-heated reactor, and the effects of the selected variables  were assessed for optimal furfural production.This study deals with the production of furfural from hemicellulose-derived saccharides obtained via the hydrothermal treatment of E. nitens wood was performed as per Penín et al. HSO4 or [C3SO3Hmim]HSO4) as a catalyst.The aqueous phase from 4/L aqueous phase. Based on preliminary experiments (data not shown), the effects of the relative amount of organic solvent (OAR in the range of 1–4 g/g) were assessed by performing treatments at 180 °C for 30 min in media containing 1.16 mol [C4mim]HSOThe experimental data showed a slight FC increase when OAR increased from 1 to 2, almost no variation in FC when OAR increased from 2 to 3, and a small FC decrease when OAR increased from 3 to 4. Based on these results, OAR = 2 was considered optimal, owing to the good balance between furfural conversion, limitation of solvent usage, and increased volumetric concentration of the target product. Owing to this, the rest of the experiments were performed at OAR = 2 g/g.4/L (4/L aqueous phase).To assess the effects of the catalyst concentration, five additional experiments were performed at 180 °C for 30 min with OAR = 2, in media containing 0.106–1.16 mol [C4mim]HSO4/L . The max4/L aqueous phase, the concentrations of furfural in the aqueous and organic phases were 0.612 ± 0.015 and 3.48 ± 0.004 g/L, respectively .When the catalyst loading decreased to 0.847 mol [C4mim]HSO4/L, FC reached 69.5 ± 0.46% . The experiments performed with CC of 0.212 or 0.106 mol [C4mim]HSO4/L aqueous phase led to overall furfural molar conversions of 61.3 ± 0.07 and 55.9 ± 0.65%, respectively. Since no significant conversions were found when using catalyst concentrations of 1.16 or 0.847 mol/L aqueous phase , further experiments were carried out while keeping CC constant at 0.847 mol/L aqueous phase.Lower catalyst loadings resulted in decreased furfural concentrations, as can be observed in a|) were as follows: %FC in organic phase, |δ|range 0.02–0.71, |δa|= 0.35; %FC in aqueous phase, |δ|range 0.00–0.14, |δa|= 0.07; xylose concentration in g/L, |δ|range 0.01–0.51, |δa|= 0.19.As a result of biphasic treatments, most xylooligomers were converted into xylose, which appeared concentrated in the aqueous phase . Concern4/L aqueous phase. a|= 0.73; xylose concentration in g/L, |δ|range 0.00–0.38, |δa|= 0.20.Based on the above information, a new set of 8 assays were performed at 180 °C for reaction times up to 45 min in media formulated with 0.847 mol [C4mim]HSO4 media, whereas Wang et al. HSO4 instead [C4mim]HSO4. For the sake of simplicity, the first experiment of the set was performed under the conditions  identified as optimal in the previous section for operation with [C4mim]HSO4, and the effects of the catalyst charge were assessed. The experimental results are shown in 3Hmim]HSO4 boosted the furfural-consuming reactions and suggesting that milder operational conditions would result in improved furfural production. Following this idea, a new experiment was performed cutting the catalyst concentration by half (CC = 0.329 mol/L liquid phase). Although the furfural concentrations in both phases increased by factors of 1.31–1.38, the resulting FC (67.6 ± 1.56%) was still below the target range, suggesting that lower catalyst charges could improve the results. To confirm this idea, a new set of experiments were performed with lower catalyst concentrations , keeping the rest of operational variables constant. When CC decreased from 0.164 to 0.049 mol [C3SO3Hmim]HSO4/L, FC increased from 70.9 ± 0.30 up to 82.5 ± 0.91%, whereas lower CC (0.033 or 0.016 mol [C3SO3Hmim]HSO4/L) led to decreased yields (below 77%). The deviations observed for the data in a|= 0.88; %FC in aqueous phase, |δ|range 0.00–0.19, |δa|= 0.07; xylose concentration in g/L, |δ|range 0.00–0.04, |δa|= 0.01.In order to assess the possible benefits derived from using a catalyst of higher acidity, a new set of experiments were performed using [C3SO3Hmim]HSO4/L was considered optimal and, therefore, was employed in further experiments.Based on these results, CC = 0.049 mol [C3SO3Hmim]HSO4 concentration leading to the highest FC was lower than in the previous case, as a consequence of its higher activity. The deviations observed for the data in a|= 0.88; xylose concentration in g/L, |δ|range 0.01–0.54, |δa|= 0.09.In order to assess the effects of the reaction time, experiments lasting up to 30 min were performed in media containing the optimal catalyst concentration, keeping the rest of the operational variables unchanged. The results  led to a3Hmim]HSO4 has been employed in the literature as a catalyst for furfural manufacture from commercial substrates. Matsagar et al. HSO4) as catalysts for furfural manufacture from xylose in water–GVL. The best result (FC = 78.12%) was obtained using 1-propylsulfonic-3-methylimidazolium chloride at 140 °C for 180 min. Recently, López et al. [3Hmim]HSO4 and MIBK to yield furfural at near 78% molar conversion.[C3SOr et al.  employedn et al.  assessedz et al.  obtained4/L or 0.049 mol [C3SO3Hmim]HSO4/L aqueous phase, 180 °C, 5 min). The organic and aqueous phases obtained in the experiment were separated via decantation, and the aqueous phase  was filtered. The filtrate was extracted with ethyl acetate, and subjected to vacuum evaporation to recover the catalyst as the non-volatile fraction. The recovered catalyst was employed in further experiments (after supplementation with fresh AIL when necessary to compensate for losses).The possibility of reusing the catalyst in consecutive reaction cycles was assessed from reaction media obtained under the conditions considered optimal , confirming that the catalyst kept most of its activity after five runs. The assays performed with recovered [C3SO3Hmim]HSO4 resulted in FC in the range 68.5–85.6% . The deviations observed for the data in 4, |δ|range 0.17–3.32, |δa|= 1.45; %FC for experiments with [C3SO3Hmim]HSO4, |δ|range 0.19–0.55, |δa|= 0.34; xylose concentration in g/L, |δ|range 0.01–0.54, |δa|= 0.09. The increased experimental error observed in these experiments was ascribed to the utilization of very small amounts of catalyst.E. nitens wood operating in biphasic media catalyzed using [C4mim]HSO4 or [C3SO3Hmim]HSO4. The conversion of potential substrates into furfural was in the range of 77–86%. These results showed that the comparatively high acidity of [C3SO3Hmim]HSO4 enabled operation under mild conditions . Both AIL considered in this study kept most of their catalytic activity after reutilization in five consecutive reaction cycles.In conclusion, high furfural conversions were obtained from the hemicellulose fraction of E. nitens wood samples were provided by ENCE . Hydrothermal treatments were performed under the conditions reported as optimal [4 or [C3SO3Hmim]HSO4), and processed in a stirred MARS 6 microwave reactor under the desired reactions conditions. The reactions and the analyses were performed in duplicate. The experimental tasks are summarized in  optimal . The liqThe liquid phases from hydrothermal treatments , as well as the aqueous and organic phases from furfural production runs, were analyzed via HPLC operating as per Peleteiro et al. .The aqueous phase from the first reaction medium obtained in the set of experiments was filtered through a 0.2 µm membrane, extracted with ethyl acetate, and subjected to evaporation in a vacuum oven at 50 °C for 48 h. The AIL recovered as the non-volatile fraction was supplemented with fresh catalyst when necessary to compensate for losses and employed in the next run.


Recently, numerous studies have synthesized Fe3O4/GO nanomaterials for biological diagnosis and treatments, including photothermal therapy and magnetic thermal therapy. However, the biosafety of the synthesized Fe3O4/GO nanomaterials still needs to be further identified. Therefore, this research intended to ascertain the cytotoxicity of Fe3O4/GO after treatment with different conditions in HBE cells. The results indicated the time-dependent and concentration-dependent cytotoxicity of Fe3O4/GO. Meanwhile, exposure to Fe3O4/GO nanomaterials increased reactive oxygen species (ROS) levels, calcium ions levels, and oxidative stress in mitochondria produced by these nanomaterials activated Caspase-9 and Caspase-3, ultimately leading to cell apoptosis.Iron oxide (Fe Cell count kit-8 (cck-8) and Fluo-4AM were purchased from Beyotime Biotechnology. 2'7'-dichlorofluorescein diacetate (DCFH-DA), Mitosox red, and 4', 6-diamidine-2-phenylindole dihydrochloride (DAPI) were provided by Sigma–Aldrich. Antibodies of Caspase-9/Caspase-3 were obtained from a protein technology company. The secondary antibody used Alexa Fluor 488 conjugated goat anti-mouse and Cy3 conjugated goat anti-rabbit, which were purchased from Servicebio. Calcein-AM/PI and Annexin-V/PI double staining kits were purchased from Dojindo laboratories.3O4/GO, glycine is used as a linker. First, 20 mg Fe3O4 nanospheres were dispersed in 0.5 mg/ml water, ultrasonic treatment until uniform dispersion, and then functionalized with glycine so that the -NH2 group was attached to its surface. 20 mg GO sample was ultrasonically stripped in 60 ml H2O to generate a homogeneous GO aqueous suspension. The carboxyl groups on the surface of GO were then activated by 8 mg N-hydroxysuccinimide (NHS) and 10 mg 1-(3-dimethylaminopropyl-1)-3-ethylcarbondiimide (EDC). The mixture of modified Fe3O4 and GO was stirred for 2 h, and the resulting product was centrifuged, washed with water and ethanol several times, and dried at 100°C.In order to produce Fe3O4/GO were evaluated by dynamic light scattering (DLS) via Malvern (Zetasizer Nano S90) in water and DMEM, respectively. The Zeta potential of Fe3O4/GO was analyzed by Malvern, Zetasizer Nano S90.The images of NP morphology were obtained using a transmission electron microscope . Fourier infrared spectroscopy (FTIR) was analyzed by IRTracer-100, Japan. Hydrodynamic sizes of Fe2.HBE cells and Ad12-SV40 2B (BEAS-2B) cells were obtained from American Type Culture Collection . Cells were cultured in DMEM containing 10% FBS and 1% penicillin and streptomycin at 37°C with 5% CO3O4/GO in DMEM was added to each well at a dose of 0, 10, 20, 50, 100, and 200 μg/ml for 3 replicates per group. HBE cells were tested after 6, 12, 24, and 48 h of co-incubation and BEAS-2B cells were tested after 12, 24 h of co-incubation later according to the manufacturer’s instructions.Cell viability was measured by cck-8. HBE cells and BEAS-2B cells were seeded in 96-well plates overnight and Fe3O4/GO  in DMEM was added and then coincubated after 24 h. Then, it was cleaned with PBS three times, then prepared DCFH-DA staining solution was added and incubated for 15 min. It was cleaned with PBS three times, observed, and analyzed by using a confocal microscope  (Ex: 505 nm Em: 525 nm).Oxidative stress changes are represented by reactive oxygen species (ROS). The fluorescence intensity of DCFH-DA is the most commonly used method to detect intracellular ROS levels. HBE cells were seeded in confocal dishes overnight. Fe3O4/GO  in DMEM was added and then coincubated after 24 h. Then, it was cleaned with PBS three times, prepared Mitosox red and DAPI staining solution was added and incubated for 15 min. It was cleaned with PBS three times, observed, and analyzed via a confocal microscope  .Mitosox red is a mitochondrial superoxide indicator. HBE cells were seeded in confocal dishes overnight. Fe2+ levels were detected by Fluo-4AM. HBE cells were seeded in confocal dishes overnight. Fe3O4/GO  was added and then co-incubated for 24 h. Then, it was cleaned with PBS three times and then prepared Fluo-4AM staining solution was added, and incubated for 15 min. It was cleaned with PBS three times, observed, and analyzed under a confocal microscope  (Ex: 494 nm Em: 516 nm).Ca3O4/GO  in DMEM was added and then coincubated for 24 h. Then, it was cleaned with PBS three times and prepared calcein-AM/PI staining solution was added and incubated for 15 min. It was cleaned with PBS three times, observed, and analyzed under a confocal microscope  .Dead/live cells were analyzed by using a calcein-AM/PI double staining kit. HBE cells were seeded in confocal dishes overnight. Fe3O4/GO  in DMEM was added and then coincubated for 24 h. Then, cells were stained with Annexin-V FITC/PI and analyzed by confocal microscope.  .HBE cells were seeded in confocal dishes overnight. Fe3O4/GO (200 μg/ml) in DMEM was added and then co-incubated for 24 h. Then, cells were immunofluorescence stained by Caspase-3 antibody and Caspase-9 antibody and observed under a confocal microscope .HBE cells were seeded in confocal dishes overnight. Fe3O4 particles prepared by a coprecipitation method have a diameter of approximately 10 nm. However, due to the interaction between coulomb force and van der Waals force among the nanoparticles, a few of the nanoparticles exhibit the agglomeration phenomenon  water and DMEM, respectively by DLS. As shown in 3O4/GO was 1,325 nm in dH2O. There is a certain aggregation in DI water, so the measured particle size is larger. Due to the presence of serum in the medium, the serum protein such as albumin could form a corona which stabilization of the materials in the suspension  of Fe3O4/GO, and cell viability was detected after 6, 12, 24, and 48 h by cck-8 assay. As shown in 3O4/GO exposure at 200 μg/ml. In short periods, even high concentrations of Fe3O4/GO can have a rational biosafety profile. After 12 h, we can significantly infer that cell viability was decreased to 47.55% after the highest concentration of Fe3O4/GO exposure shown in 3O4/GO was time-dependent. In 3O4/GO stimulation at the concentration of 200 μg/ml. It is worth noting that cell viability was less than 60% after 48 h, even at low concentrations (20 μg/ml). However, other reports indicated that these nanoparticles had the potential to produce toxic effects in cells, and their toxic effects were related to their size, concentration, time, shape, and the cell type , hydroxyl radicals (HO•), and hydrogen peroxide] levels could enhance in human cells after exposure to Fe3O4/GO nanosheets. Literature also unravels that iron oxide nanoparticles can induce cytotoxicity by activating oxidative stress responses  and PI (red fluorescent) represented dead cells . Figure 3O4/GO, which was characterized via Annexin-V/PI in 3O4/GO exposure, fluorescence of Annexin-V and PI were obviously increased response to Fe3O4/GO nanoparticle concentration, which were concordant with the results of the cck-8 assay. It indicated high concentration Fe3O4/GO can lead to HBE cell apoptotic (Apoptosis detection was performed to verify the death mode of cells after incubation with Fepoptotic .3O4/GO can stimulate HBE cells to produce oxidative stress, calcium influx, and mitochondrial superoxide, ultimately leading to apoptosis, and these phenomena are concentration-dependent. In order to explore the specific pathway through which Fe3O4/GO stimulates HBE cell genesis, we characterized the expression levels of Caspase-9 and Caspase-3.So far, we have confirmed that Fe3O4/GO, the fluorescence of Caspase-9 and Caspase-3 was significantly enhanced in 2+-ER stress led to mitochondrial dysfunction, which promoted the activation of Caspase-9 and activation of Caspase-3, resulted in cell apoptosis (After coincubation with Fepoptosis .2+ levels, which can trigger a series of mitochondrial related events, including endoplasmic reticulum stress, and mitochondrial dysfunction, then activated Caspase-9/Caspase-3 relate apoptosis, these proteins are important in apoptotic pathway (3O4/GO, ROS levels increased and Ca2+ levels enhanced, lead to mitochondrial dysfunction, and then, resulting in Caspase-9/Caspase-3 related apoptotic of HBE cells.ROS are produced in cells via a variety of mechanisms. High intracellular ROS increased Ca pathway  (Hailan  pathway . Our stu3O4/GO. Specifically, after exposure to high concentration of Fe3O4/GO nanomaterials, ROS levels and Ca2+ influx enhanced, and then, mitochondrial dysfunction, thereby leading to cell apoptosis via the Caspase-9/Caspase-3 pathway ultimately. The results also demonstrated that the cytotoxicity of Fe3O4/GO was in time-dependent and concentration-dependent manners. Therefore, it is still a challenging task in the future to transform Fe3O4/GO nanocomposites with cytotoxicity into biocompatible Fe3O4/GO nanocomposites.In this study, the obtained results elaborated the cytotoxicity effects of Fe


SC-enantiomer of dimethyl α-hydroxy-benzylphosphonate, and the optical purity was retained. A part of the acyloxyphosphonates was tested against eight tumor cell lines of different tissue origin at c = 50 μM concentration. The compounds elicited moderate cytostatic effect against breast, skin, prostate, colon, and lung carcinomas; a melanoma cell line; and against Kaposi’s sarcoma cell lines. Then, dose-dependent cytotoxicity was assayed, and benzoylation of the α-hydroxy group was identified as a moiety that increases anticancer cytotoxicity across all cell lines. Surprisingly, a few analogues were more toxic to multidrug resistant cancer cell lines, thus evading P-glycoprotein mediated drug extrusion.An efficient method applying acyl chlorides as reagents was developed for the acylation of the hindered hydroxy group of dialkyl α-hydroxy-benzylphosphonates. The procedure did not require any catalyst. A few acylations were also performed with the  A simil4c*: 31P NMR (CDCl3) δ 20.15; δPracemic (CDCl3) 18.0 +found = 281.0553, calculated: 281.0555, C11H15O5PNa; [α]D25 = −43.3 .l3) 18.0 ; [M + H]4b*: 31P NMR (CDCl3) δ 20.18; [M + H]+ = 273, [M + Na]+found = 295.0712, calculated: 295.0711, C12H17O5PNa; [α]D25 = −44.0 .4d*: 31P NMR (CDCl3) δ 20.23; δPracemic (CDCl3) 20.1 +found = 343.0711, calculated: 343.0711, C16H17O5PNa; [α]D25 = +26.2 .l3) 20.1 ; [M + H]2. The cells were grown to confluency and then divided into 96-well tissue culture plates  with the initial cell number of 5.0 × 103 cells/well. After 24 h incubation at 37 °C, the cells were treated with the compounds in 200 μL final volume containing 1.0 v/v% DMSO  at 50 μM concentration overnight, whereas control cells were treated with serum-free medium only, or with DMSO (c = 1.0 v/v%) at the same conditions. After incubation, the cells were washed twice with serum-free medium. Subsequently, the cells were cultured for additional 72 h in 10% serum containing medium at 37 °C; then, the MTT  solution  was added to each well. The respiratory chain [620 values were subtracted from OD540 values. The percent of cytostasis was calculated with the following equation:treated and ODcontrol correspond to the optical densities of the treated and the control wells, respectively. In each case, two independent experiments were carried out with four parallel measurements. Statistical analysis of data was performed using Student’s t test at the 95% confidence level.In vitro cytostatic effect of the compounds was studied on MDA-MB-231 human breast adenocarcinoma , A431 hury chain  and othery chain  reduce Mry chain . The amory chain . After 3The 143/B osteosarcoma cell line was a kind gift of Dr. József Balla . Mes-Sa and Mes-Sa/Dx5 uterine sarcoma cell lines were obtained from ATCC  in 2012, where they were characterized by DNA fingerprinting. A431 was obtained from ATCC as well. We purchased CAKI-1, HCT116, MDA-MB-231, and OVCAR-8 from NIH NCI from the NCI-60 cell line panel. 143/B cells were maintained in MEM; A431, Mes-Sa, and Mes-Sa/Dx5 cell lines were maintained in DMEM. NCI-60 panel cell lines were cultivated in RPMI. Media were supplemented with 10% FBS, 5 mmol/L glutamine, and 50 units/mL penicillin and streptomycin. Mes-Sa cells were engineered by lentiviral transduction to stably express the fluorescent protein mCherry, while Mes-Sa/Dx5 cells express eGFP (see more in reference ). After A total 2500 cells/well were seeded in 20 µL of the 143/B, A431, and NCI-60 panel cell lines on 384-well plates. Cells settled for 24 h, when serial dilution of the drugs were added in an additional 40 µL. After 72 h incubation, we added PrestoBlue cell viability reagent  for an hour in a final volume of 5%. Fluorescent intensity of the reagent was read by a PerkinElmer EnSpire microplate reader  at 555 nm excitation and 585 nm emission wavelengths. pIC50 values were calculated by sigmoidal curve fitting by our custom program, written by Judit Sessler in C#.The fluorescent Mes-Sa mCherry and Mes-Sa/Dx5 eGFP cell lines were seeded and cocultured on 384-well plates with a density of 2 × 1250 cells/well. Cells settled for 24 h when serial dilutions of the drugs were added in an additional 40 µL (with or without tariquidar). After 144 h incubation, the fluorescent intensity of mCherry  and eGFP  was measured. pIC50 values were calculated by our custom program. Liquid handling was performed by a Hamilton StarLet robot.Sc-enantiomer, the optical purity was preserved. To evaluate structure–activity relations, the hydroxyphosphonate derivatives were first tested for cytostatic activity at a low concentration range utilizing eight different cell lines. Their activity was cell- and concentration-dependent. Based on the SAR observed, substitution of hydrogens in the phenyl ring, preferably with trifluoromethyl-group, and more preferably in the meta- and ortho-positions [In summary, a series of hydroxyphosphonates was converted to the corresponding acyloxyphosphonates by the catalyst-free reaction with different acyl chlorides. During the acylation of the ositions , could i


Background: Erythritol is a zero-calorie sweetener that is widely used in the food, pharmaceutical, and medical industries. Crude glycerol is the main by-product of biodiesel, and the effective utilization of crude glycerol will help to improve biodiesel viability. Previous studies on the production of erythritol from Y. lipolytica using crude glycerol as a carbon source have focused on optimizing the fermentation process of the mutant Y. lipolytica Wratislavia K1, while metabolic engineering has not been successfully applied.Results: To this end, we engineered the yeast Y. lipolytica to increase the productivity of this strain. Wild strains tolerant to high concentrations of crude glycerol were screened and identified. A series of rational metabolic approaches were employed to improve erythritol production. Among them, the engineered strain Y-04, obtained by tandem overexpression of GUT1 and GUT2, significantly increased glycerol assimilation by 33.3%, which was consistent with the results of RT-qPCR analysis. The effects of tandem overexpression of GUT1, GUT2, TKL1, and TAL1 on erythritol synthesis were also evaluated. The best results were obtained using a mutant that overexpressed GUT1, GUT2, and TKL1 and knocked out EYD1. The final Y-11 strain produced 150 g/l erythritol in a 5-L bioreactor with a yield and productivity of 0.62 g/g and 1.25 g/l/h, respectively. To the best of our knowledge, this is the highest erythritol yield and productivity from crude glycerol ever reported in Y. lipolytica.Conclusion: This work demonstrated that overexpression of GUT1, GUT2, and TKL1 and knockdown of EYD1 could be used to improve crude glycerol utilization and erythritol synthesis in Y. lipolytica. The process parameters such as erythritol yield and productivity were significantly elevated, which is valuable for industrial applications. Crude glycerol, as a carbon source, could efficiently restrict the synthesis of by-products while enhancing the generation of erythritol, compared to glucose. This indicates considerable potential for synthesizing value-added products from crude glycerol by Y. lipolytica. Y. lipolytica has a clear genetic background and proven tools for molecular modification : yeast nitrogen base (YNB) 1.7, (NH4)2SO4 5, glucose 20. Note: add appropriate antibiotics or nutrients (400 μg/ml hygromycin B or 700 μg/ml bleomycin or 0.1 g/l leucine) to the medium before inoculation or plate coating. High glycerol medium (g/L): crude glycerol 500, yeast extract 5. Medium glycerol medium (g/L): crude glycerol 300, yeast extract 5. Erythritol fermentation medium (g/L): crude glycerol 250, yeast extract 1, NH4Cl 5, KH2PO4 0.25, MgSO4·7H2O 0.5. Erythritol fermentation medium : pure glycerol 250, yeast extract 1, NH4Cl 5, KH2PO4 0.25, MgSO4·7H2O 0.5. Erythritol fermentation medium : glucose 250, yeast extract 1, NH4Cl 5, KH2PO4 0.25, MgSO4·7H2O 0.5. The crude glycerol in this work was obtained from biodiesel waste. The composition of crude glycerol is (v/v): glycerol 80–85%, sodium salt 2.0%, methanol 10–15%, other organic matter 2.5%, and water 2%. 0.1 mol/l lithium acetate: accurately weigh 1.02 g of lithium acetate, dissolve in 90 ml of distilled water, adjust pH 6.0 with acetic acid, then fix the volume to 100 ml, sterilize at 115°C for 20 min and store at −20°C. 40% PEG4000: accurately weigh 20 g of PEG4000, dissolve in 30 ml of 0.1 mol/l lithium acetate (pH 6.0), dissolve fully, fix the volume to 50 ml, sterilize at 115°C for 20 min and store at −20°C.All strains used in this study are listed in Additional file 1: Ku70 and LEU2, two primer pairs (Ku70-sg-1 and LEU2-sg-1) were designed to construct plasmids pCAS1yl-∆Ku70 and pCAS1yl-∆LEU2, respectively. The Ku70-UP-F/R, Ku70-DOWN-F/R, LEU2-UP-F/R, and LEU2-DOWN-F/R primer pairs were used to amplify the upstream and downstream homologous arms of 1,000 bp each from Y. lipolytica 2,021,417 genomic DNA. The hygB gene from Coccidioides posadasii was synthesized in pUC-GW to construct pUC-GW-hygB by Genewiz  with codon optimization. The hygB gene cloned into KpnI linearized pINA1269 using the hygB-F/R primer pair to generate plasmid pINA1269-hygB. The fragment of hp4d-hygB-XPR2t from pINA1269-hygB was then amplified using the hp4d-hygB-XPR2t-F/R primer pair. A fusion fragment Ku70 UP-hp4d-hygB-XPR2t-Ku70 DOWN and LEU2 UP-hp4d-hygB-XPR2t- LEU2 DOWN was subsequently generated. The fusion fragments were cloned into PmeI linearized pCAS1yl-∆Ku70 and pCAS1yl-∆LEU2 to generate pCAS2yl-∆Ku70 and pCAS2yl-∆LEU2, respectively. The LEU2 sgRNA expression cassette from pCAS2yl-∆LEU2 was then amplified with LEU2 F/R primer pair and subsequently cascaded with the Ku70 sgRNA expression cassette in pCAS2yl-∆Ku70 to obtain pCAS2yl-∆Ku70∆LEU2 for Y. lipolytica Y01 transformation  until the cell survival was less than 0.05%, and the cells were inoculated onto YPD plates at 30°C. After the mutagenic strains were cultured, the strains with larger and thicker colonies were selected, numbered, and inserted into a test tube for storage. In contrast, the corresponding strains were inoculated into 250 ml shake flasks (50 ml Erythritol fermentation medium) at 30°C, 220 r/min for 120 h, and the erythritol titers were determined.The wild-strain Y. lipolytica as described previously . Cells were harvested by centrifugation at 6000 × g for 5 min. The cells were then washed with sterile water, resuspended the cells with 1 ml 0.1 mol/l lithium acetate and incubated for 10 min at room temperature for transformation. The transformation mix was added to the cells in the following order: 10 μl 10 mg/ml ssDNA, 1 μg linearized plasmid DNA, 700 μl 40% PEG4000. The centrifuge tube was vortexed until the cell pellet was completely mixed. Cells were incubated at 30°C for 30 min and then heat shocked in a water bath at 42°C for 30 min. Cells were centrifuged to remove the transformation mix and resuspended in 100 μl of sterile water. Cells were then plated on the appropriate selection agar plates. Colonies were verified by PCR and then selected for erythritol production.The lithium acetate method was used to transform eviously . In brieSeed culture was carried out in a 500 ml flask containing 50 ml of YPD medium on a rotary shaker at 30°C and 220 r/min for 20 h. An inoculum of 20% was introduced into a shake flask containing 30 ml of the Erythritol fermentation medium. Shake flasks were performed on a rotary shaker at 30°C and 220 r/min for 120 h. An inoculum of 20% was introduced into a bioreactor containing 1.6 l of the Erythritol fermentation medium. Batch cultivations were carried out in a 5-L fermenter  at 30°C with a working volume of 2 l. The aeration rate was fixed at 1.0 l/min. The stirrer speed was adjusted to 800 rpm and the dissolved oxygen concentration was maintained at 20–30%. pH was maintained automatically at 3.0 by the addition of 20% (w/v) of NaOH solution. All cultures were carried out in three replications.2  eluted with 80% acetonitrile as the mobile phase at a flow rate of 1.0 ml/min. The detector was a RID detector coupled to a detector temperature of 35°C and a column temperature of 40°C. The mass yield (YERY) and volumetric productivity (QERY) of erythritol and glycerol consumption were calculated based on previous studies . The sample volume was 20 μl using an amino column 70 Å NH studies .Y. lipolytica according to the manufacturer’s instructions using the Bead-Beat Total RNA Mini kit . The TranScriba kit  was used to synthesize the cDNA strands.Total RNA was extracted from 2O. Reaction conditions were as follows:95°C for 3 min, 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, 2–4 steps of 40 cycles, and melting curve phases: 94°C for 15 s, 60°C for 60 s, 95°C for 30 s, 60°C for 15 s. Each qPCR reaction was performed in technical replicates.Quantitative PCR (qPCR) was performed in a 7,500 real-time PCR thermal cycler  using an SYBR®Green B PCR MasterMix . Each reaction contained 0.5 μl of cDNA template, 5 μl of SYBR®Green B PCR MasterMix, and 0.5 μl of forwarding and reversed primers, which were made up to 10 μl using ddHY. lipolytica, producing 10 ± 0.07 g/l erythritol in shake flask fermentation  in the NHEJ pathway was absent  for Y. lipolytica Y01-ΔKu70ΔLEU2  and GUT2 (YALI0B13970g), respectively. These two enzymes are mainly involved in glycerol assimilation in the erythritol synthesis pathway . However, the expression levels of GUT1 and GUT2 were only slightly up-regulated in the co-expression strain, which may be due to the mutual coordination between genes to maintain the dynamic balance of the cells. The results were similar to those obtained in previous studies , crude glycerol consumption was increased by 33% compared to the control strain  and Y-03(pINA1269-GUT2), the erythritol titers were 35 g/l and 31 g/l, which increased by 29.6 and 14.8% compared to the control strain, achieving YERY 0.39 g/g and 0.35 g/g, QERY 0.292 g/l/h and 0.258 g/l/h, respectively . For strain Y-04, the erythritol titer, YERY, and QERY were 51.9, 56.7, and 52% higher than the control strain  and TAL1 (YALI0F15587g), respectively , the erythritol titer, YERY and QERY increased to 40 g/l, 0.45 g/g and 0.333 g/l/h, which were increased by 48.1, 50 and 48% compared to the control strain, respectively (TKL1) is more important in promoting erythritol synthesis. TKL1 obtained the best results in terms of reduced fermentation time and improved erythritol production compared to overexpression of other genes. In addition, Y. lipolytica, and overexpression of TKL1 resulted in a 2-fold improvement in erythritol synthesis. In light of these findings, we further investigated the effect of tandem overexpression of multiple genes on erythritol synthesis.Based on these results, we evaluated the effect of ectively , D. ThisGUT1 and GUT2 enhanced crude glycerol assimilation and improved erythritol synthesis in the engineered strain. Overexpression of TKL1 significantly increased erythritol titer, YERY, and QERY. Co-expression of multiple key genes has been shown to improve cellular metabolic performance. Therefore, to further enhance erythritol production, GUT1, GUT2, and TKL1 or GUT1, GUT2, and TAL1 were overexpressed in tandem in strains Y-08 and Y-09. For strain Y-08, the erythritol titer, YERY, and QERY reached 65 g/l, 0.56 g/g, and 0.541 g/l/h, which were increased by 58.5, 19.2, and 58.2% compared to the control strain Y-04, respectively , erythritol titer, YERY, and QERY were reduced by 15.3, 9.1, and 15.3% compared to the strain Y-08  gene encoding erythritol dehydrogenase is involved in the catabolic pathway of erythritol  to produce erythritol was evaluated. Cell growth, crude glycerol consumption, erythritol titer, YERY, and QERY of the fermentation process were monitored , the National Natural Science Foundation of China (Nos. 32071470 and 32100055), the Natural Science Foundation of Jiangsu Province (No. BK20210464), the National first-class discipline program of Light Industry Technology and Engineering (No. LITE2018-06), the Program of Introducing Talents of Discipline to Universities (No. 111-2-06), and Postgraduate Research & Practice Innovation Program of Jiangsu Provence (KYCX22_2370).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. 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The failure of current chemotherapeutic agents for pancreatic cancer (PCa) makes it the most aggressive soft tissue tumor with a 5-year survival of slightly above 10% and is estimated to be the second leading cause of cancer death by 2030.Objective: The main aim was to synthesize, characterize and evaluate the anticancer activity of 1,3-bistetrahydrofuran-2yl-5FU (MFU).Methods: MFU was synthesized by using 5-fluorouracil (5-FU) and tetrahydrofuran acetate, and characterized by nuclear magnetic resonance (NMR), micro-elemental analysis, high-performance liquid chromatography (HPLC), and liquid chromatography with mass spectrophotometry (LC-MS). MFU and Gemcitabine hydrochloride (GemHCl) were tested for antiproliferative activity against MiaPaca-2 and Panc-1 cell lines.Results: The half-minimum inhibitory concentration (IC50) of MFU was twice lower than that of GemHCl when used in both cell lines. MiaPaca-2 cells (MFU-IC50 = 4.5 ± 1.2 μM vs. GemHCl-IC50 = 10.3 ± 1.1 μM); meanwhile similar trend was observed in Panc-1 cells (MFU-IC50 = 3.0 ± 1 μM vs. GemHCl-IC50 = 6.1 ± 1.03 μM). The MFU and GemHCl effects on 3D spheroids showed a similar trend (IC50-GemHCl = 14.3 ± 1.1 μM vs. IC50-MFU = 7.2 ± 1.1 μM) for MiaPaca-2 cells, and (IC50-GemHCl = 16.3 ± 1.1 μM vs. IC50-MFU = 9.2 ± 1.1 μM) for Panc-1 cells. MFU significantly inhibited clonogenic cell growth, and induced cell death via apoptosis. Cell cycle data showed mean PI for GemHCl (48.5–55.7) twice higher than MFU (24.7 to 27.9) for MiaPaca-2 cells, and similarly to Panc-1 cells. The in-vivo model showed intensely stained EGFR (stained brown) in all control, GemHCl and MFU-treated mice bearing subcutaneous PDX tumors, however, HER2 expression was less stained in MFU-treated tumors compared to GemHCl-treated tumors and controls. Mean tumor volume of MFU-treated mice (361 ± 33.5 mm3) was three-fold lower than GemHCl-treated mice (1074 ± 181.2 mm3) bearing pancreatic PDX tumors.Conclusion: MFU was synthesized with high purity and may have potential anticancer activity against PCa.The online version contains supplementary material available at 10.1186/s12885-022-10449-y. PCa is the most aggressive and scourging soft tissue tumor worldwide, with a mean age of onset between the 5th and 6th decades of life in the US . AccordiFluoropyrimidines (FP) are increasingly making up the chemotherapeutic backbone of neoadjuvant, adjuvant, and palliative treatment for PCa . 5-FluorAlthough GemHCl is the preferred first-line treatment of PCa singly or in combination with other anticancer drugs , 5-FU reThis study successfully synthesized, characterized, and evaluated the invitro anticancer activity of FP derivative, 1,3 bistetrahydrofuran-2-yl-5-FU (MFU) in PCa cell lines. Viability studies conducted in 2D and 3D MiaPaca-2 and Panc-1 models demonstrated significant sensitivity to MFU compared with GemHCl. Further, clonogenic studies revealed a remarkable damage to MFU treated MiaPaCa-2 cells than GemHCl treated ones. Equally, we demonstrated pathway of cell death (apoptosis vs necrosis) and the expression of different cellular proteins using western blot analysis following treatment with MFU. Finally, we analyzed the expression of EGFR, HER2 and VEGFR using immunohistochemistry techniques following intravenous treatment of PDX mouse models bearing human pancreatic cancer tumors.5-FluoroUracil (5-FU) was purchased from AK Scientific . Pancreatic cancer MiaPaCa2 (ATCC® CRL1420™) and Panc-1 (ATCC® CRL1469™) were bought from American Type Culture Collection (ATCC) . All other solvents and reagents for analysis were purchased from Sigma Aldrich .The synthesis of MFU followed a method described by Zasada et al., with some modifications . To a soThe MFU synthesized with the method mentioned earlier was analyzed by proton and carbon-13 Nuclear Magnetic Resonance (NMR) spectroscopy, Micro-elemental analysis, High Performant Liquid Chromatography (HPLC), and High-Resolution Mass Spectrometry (HRMS). The melting point for the solid MFU compound was determined. The purity of MFU was calculated to be 99.6%, as determined by NMR and elemental analysis. Elemental analysis was performed by an independent lab . HPLC and HRMS were formed by an independent laboratory to confirm NMR findings.1H NMR : 7.36 , 6.60–6.67 , 5.97–5.99 , 4.29–4.36 , 4.20–4.26 , 3.93–4.04 , 2.44–2.54 , 2.33–2.42 , 2.20–2.28 , 2.03–2.13 , 1.86–1.96 .MFU: 13C NMR  δ (a mixture of two rotamers): 157.28 , 157.17 , 148.2, 148.66, 139.94 , 139.87 , 121.75 , 121.58 , 87.97, 87.87, 85.20, 84.99, 70.75, 70.74, 70.27, 70.21, 33.04, 32.92, 28.78, 28.73, 26.51, 26.47, 23.76, 23.70. MP = (114-115 °C), Rf value = 0.36 (100% ethyl-acetate). HPLC (H20/ACN/TEAA (80:15:5), % purity = 100%, RT = 1.85 min) MS (M + H) + = 270.08. Molecular formula: C12H15FN2O4, Mol wt: 270.02. Elemental analysis: Calculated for C12H15FN2O4: C 53.33, H 5.59, N 10.37; Found: C 53.20, H 5.61, N 10.40.l-glutamine, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PenStrep) [3 cells per well in quintuplicate for each drug concentration level and incubated at 5% CO2 and a temperature of 37 °C. At 80–85% confluence, both MiaPaca-2, and Panc-1 cells were treated with MFU. A stock solution of MFU was prepared with distilled water and serially diluted with the growth medium to prepare varied concentrations: thus 80, 40, 20, 10, and 5 μM. Both cells were treated with 200 μL of each drug concentration in quintuplicate and incubated for 48 h. At termination, 20 μL of 0.05% resazurin sodium salt (Alamar blue®) was added and incubated at optimum conditions  for four h [Both cell lines were incubated in Dulbecco’s modified Eagle medium (DMEM) with high glucose and enStrep)  and 2.5%4 and 2.0 × 104 in 100 μL/well of the growth medium, respectively, the volume of cell suspension in each well plate was then made up to 200 μL with growth medium and centrifuged at 1500rpms and incubated for 24 h to allow for spheroid formation. At treatment, 100 μL of supernatant was replaced with the drug in the growth medium prepared as described under 2D viability studies of Mia Paca-2 and Panc-1 cells and incubated for 48 h. At termination, 50 μL of 0.05% resazurin sodium salt (Alamar blue®) was added to each well and gently dispersed by pipetting and incubated for 4 h. Fluorometric analysis was measured as described above.MiaPaca-2 and Panc-1 cells were plated in 3D formation 96-well plates, Nunclon Sphera® at seeding density of 5.0 × 104 and 2.0 × 104 in 100 μL/well) in treatment media were seeded into the wells of a 96 U Nunclon Sphera plate . After 24 h, equivalent amounts of varying concentrations of MFU (0–10 μM) were supplemented to each well of the 96 U plate. The ability of the cells to form viable spheroids was examined at 48 h post-exposure to MFU. The viabilities of the spheroids were determined by staining them with Acridine Orange (AO)/Ethidium Bromide (EB) (5 μg/mL) solution and then capturing fluorescent and bright-field images. The ratios of the fluorescent intensities of AO over EB for the respective MFU concentrations used were computed, and the data was graphed using GraphPad Prism 5.0.MiaPaCa-2 and Panc-1 cell suspensions (5.0 × 10The incubation of MFU (concentration 50 μm) with HLMs was performed in phosphate buffer (pH 7.4) at 37 °C. Then, the incubated samples were shaken for a specified time at 150 rpm. The optimization of conditions for carrying out the process included the incubation time (0–120 min), the concentration of HLM (20 mg/mL), and the reaction buffer consisting of an enzymatic reaction cofactor NADP/NADPH. The blank samples were of the same composition as the test samples; however, they did not contain any of the studied biologically active compounds. The process was terminated with 200 μL of ice-cold methanol. Thereafter, samples were immediately prepared and analyzed with the use of HPLC-UV/MS. All samples were prepared in triplicate. All samples were incubated and treated as described above and as also described in detail as per the manufacturer’s protocol (ThermoFisher Scientific).l-glutamine, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PenStrep) and 2.5% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Additionally, MFU was added to the culture media at the concentrations 12.5 μm, 25 μm, and 50 μm for 24 h and 48 h. Cells were cultured at 37 °C in humidified atmosphere of 5% CO2. Cells were plated in T25 culture flask at the density of 1.5 × 105 cells per cm2 and grown for 48 h. Then the medium was aspirated at 24 or 48 h. Finally, MFU and potential metabolites were analyzed after filtration from the culture medium.Cells (Mia Paca-2 and Panc-1) were cultured in Dulbecco’s modified Eagle medium (DMEM) with high glucose and 4 cells/mL and left at 37 °C for 24 h to attach. Cells were exposed to MFU (0–10 μM) for 48 h, followed by washing in PBS and labeling with FITC-conjugated Annexin V for 20 min in the dark. Cells were then washed and analyzed using a Becton Dickinson FACSort flow cytometer with CellQuest software .In the morphologic analysis, Annexin V/propidium iodide staining was used as per the manufacturer’s instructions to study the mode of cancer cell death upon exposure to MFU. Mia Paca-2 and Panc-1 cells were seeded into a 25 mL culture flask at 2.0 × 105 cells in a 25 mL flask and allowed to grow until 75–80% confluence. Cells were incubated with different concentrations of GemHCl and MFU (0–10 μM) for 48 h at 37 °C and 5% CO2. After treatment, cells were collected by trypsinization, fixed gently in 70% ethanol drop-wisely, and then stored at -20 °C overnight. Then cells were washed in PBS and stained with 0.5 mL PI/RNase for 30 min at room temperature in the dark. Cells were then collected and analyzed using a Becton Dickinson FACSort flow cytometer with CellQuest software .The percentage of cells in the G1 (apoptotic cells), S, and G2/M phases was determined by DNA flow cytometry. In brief, Mia Paca-2 and Panc-1 cells were plated at 1 × 10Panc-1 cells were plated in a T25 flask and cultured for 2 days, respectively, and different concentrations of MFU were added for 48 h. The corresponding cells were collected, washed with PBS, and lysed with RIPA cell lysis buffer to extract the total proteins. The extracted proteins were loaded and subjected to electrophoresis, transferred to a PDVF membrane, the blots were then cut at appropriate molecular sizes prior to hybridization and incubated in the primary antibody overnight. The following day, the primary antibody was recovered, and the corresponding secondary antibody incubated. The immune complexes were detected using enhanced chemiluminescence solution (Biorad) and visualized using the ChemiDoc™ XRS+ imaging system (Bio-Rad).Eight-week-old mice were obtained from the Jackson Laboratory . The mice were housed in a virus-free environment with indoor light and temperature controlled and provided access to food and water ad libitum for 1 week before treatment started. All procedures with mice were in strict accordance with the National Institutes of Health Guide for the care and Use of Laboratory Animals and the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines. This was approved by the Florida A&M University Animal Care and Use Committee.n = 12). Xenografts were allowed to grow to a maximum of 1.5 cm before implantation to the flank of the new host.Tumor tissue was surgically implanted in the left flank of immune-compromised mice as previously described . A viabln = 5/group) once tumor volumes became palpable and reached a range of 70–100 mm [2)/2, where V is volume (mm3), W (width) is the smaller of two perpendicular tumor axes and the value L (length) is the larger of two perpendicular axes. Tumor growth volumes were calculated for each treatment group.In this study, mice bearing surgically implanted tumors were randomized into groups as control, GemHCl and MFU (0–100 mm . Baselin2) flow to the chamber was adjusted to 3 l per minute for 2 to 3 minutes and observed each mouse for lack of respiration and faded eye color. The CO2 flow was maintained for a minimum of 1 minute after respiration was.Carbon dioxide (COceased and followed by decapitation with scissors. The tumors were then incised and prepared for immunohistochemistry studies.histowiz.com) using a Standard Operating Procedure and fully automated workflow. Samples were processed, embedded in paraffin, and sectioned at 4μm [Tumor samples excised from the mice, washed with PBS, placed in 10% buffered formalin for 24 h, and transferred to 70% ethanol for histopathological analysis. Immunohistochemistry was performed by Histowiz Inc. . When necessary, results were presented in simple tables, graphs, and bar charts.All results were presented in the form of means ± SEM. The difference between GemHCl and MFU treatment groups was analyzed using ANOVA, and where necessary, significance was considered for 1H and 13C NMR  and 1.04 ppm (−C-N on 2″), representing the amide linkage contributed by the tetrahydrofuran acetate on the 5-FU cyclic structure. The introduced tertiary amine carbonyl carbon in MFU displays a characteristic C-13 NMR peak at 87.87–87.97 ppm and 84.98–85.19 ppm for 2′ and 2″ carbonyl carbon on MFU, respectively. These peaks confirmed the successful conjugation of tetrahydrofuran acetate on 5-FU.We successfully synthesized MFU Fig.  in good The elemental analysis of MFU is presented in Table The HPLC purity for MFU was greater than 99.6%  treated groups, respectively. While IC50 values were expectedly higher in the treated 3D MiaPaCa-2 culture compared with that of 2D MiaPaCa-2 culture  showed lower IC50 values compared with GemHCl treated 3D MiaPaCa-2 culture (IC50 = 14.3 ± 1.1 μM). A similar trend was observed for studies performed with Panc-1 cells with similar findings in 2D  and 3D  cultures  in MiaPaCa-2 and Panc-1 cells via 2D culture and 3D spheroids using the Alamar blue assay. As shown in Fig. 50 values also demonstrate better cell death in 3D by MFU when compared to GemHCl  over 24 h, we realized that more than half the initial concentration of MFU in all cases was intact after 24 h treatment in both cell lines  exhibited significantly lower tumor growth compared with the mean tumor volume of GemHCl-treated group (1074 ± 181.2 mm3) especially on week 7 and 8. Figure Mice bearing pancreatic cancer PDX tumor were treated over a period of 6 weeks. In the MFU treated group, a significant tumor growth suppression was observed compared with the control and GemHCl treated groups  are highly expressed in many solid tumors, including PCa. These receptors are reported to be associated with cancer cell proliferation, survival, migration, and differentiation and a factor of poor prognosis . In thisGemHCl and fluoropyrimidines have been widely documented and demonstrated to suppress tumor growth effectively and significantly in PCa cell lines –34. Thei50 values of MFU were significantly lower than corresponding values for GemHCl, implying that MFU has higher antiproliferative activity against 2D and 3D MiaPaca-2 and Panc-1 cultures.MFU was successfully synthesized and characterized using NMR, elemental analysis, HPLC, and HRMS  for its delivery and accumulation in PCa cells and thus under-expression of these receptors contributes to the resistance of GemHCl to PCa . The higIn addition, results of cell-induced death or apoptotic studies further support the assertion that MFU exhibits strong cytotoxic effects in 2D and 3D spheroids compared to GemHCl. To further evaluate the stability of MFU, we treated Miapaca-2 and Panc-1 cells at different concentrations of MFU over 24 h and used HPLC to determine the concentration of unchanged MFU. Human liver microsomes were used to determine the systemic stability of MFU. We found that MFU was very stable both in vitro and in human liver microsomal studies with more than 80% remaining intact after 2 h, suggestive that it is more stable compared to GemHCl as reported in literature , 24, 25.50) that were twice lower than GemHCl in both monolayer and spheroid treated PCa cell lines used. The ability of MFU to inhibit PCa Cell lines is not surprising as MFU was designed to mimic activities of other existing fluoropyrimidines like Fluorouracil which is a component of one of the currently used regimens in clinical practice (FOLFIRINOX) [To further assess the anti-survival effects of MFU, we examined the viability panel of PCa cells grown as monolayers and spheroids. In these assays, we observed a dose-dependent inhibition of the viability of PCa cell lines. To gauge the clinical potential of MFU, we compared their efficacy to GemHCl currently in clinical use for the treatment of PCa: MFU induced cell death at concentrations (ICFIRINOX) . The tet50 in spheroids compared to GemHCl and clonogenic studies performed.The previous observation that MFU inhibits the viability of PCa cells led us to investigate the mechanism by which the MFU causes the death of these cells. Programmed cell death (apoptosis) is orchestrated by a series of committed signaling events that ultimately lead to the activation of proteases known as Caspases. After conducting apoptotic and necrosis studies, it was evident that MFU causes cell death through apoptosis. We further investigated the expression of pro-apoptotic and anti-apoptotic protein expression in the cells after exposing them to MFU over 48 h. We investigated the expression of BCL-XL, BAX, and Caspase-9; we obtained a decreasing trend in BCL-XL expression, while an increasing trend was observed in the expression of BAX and Caspase-9, indicating apoptosis was the mechanism of cell death through the activation of mitochondria (intrinsic) pathway of apoptosis. Furthermore, the expression of p53 tumor suppressor protein was assessed, showing an increase in the expression of p53 compared to control. We observed dose-independent regulation of uncontrolled cell growth by the activation of p53 in treated cells compared to control. We also observed a dose dependent increase in the expression of FADD in MFU treated cells, this corresponds with literature as 5-FU increases the expression of FADD in the extrinsic pathway of apoptosis . FinallyIn addition to inducing apoptosis, anti-inflammatory, suppressing survival, and resistance to treatment, effective cancer agents, must have chemo-preventive properties to avoid tumor relapse. This was equally investigated in assays where we pre-treated PCa cells with MFU and GemHCl and then examined their survival and growth to form colonies. In these assays, MFU markedly suppressed cell survival and growth compared to GemHCl, indicating that MFU is a potential chemo-preventive agent.3) compared to GemHCl-treated mice (1074 ± 181.2 mm3) at the end of the studies . This extraordinary tumor efficacy of MFU could be explained by two major factors: i) absence of free NH2-group on MFU which rendered the DPD enzyme ineffective to metabolize MFU. This event most likely allowed for prolong circulation, increased bioavailability and improved therapeutic efficacy of MFU and ii) conjugation of THF to 5-FU may have imparted some degree of lipophilicity to MFU which may have facilitated its delivery to cancer cells. EGFR, VEGF, and HER2 are tyrosine kinase receptors highly expressed in several solid tumors, including PCa [Anti-tumor activity of MFU after the 40th day exhibited a significant inhibition of tumor growth compared with GemHCl. There was a significant decrease in tumor volume for MFU-treated mice : 7.36 , 6.60–6.67 , 5.97–5.99 , 4.32 , 4.23 , 3.93–4.04 , 2.44–2.54 , 2.33–2.42 , 2.20–2.28 , 2.03–2.13 , 1.86–1.96 . Figure 2.13C NMR analysis for MFU: 13C NMR  δ (a mixture of two rotamers): 157.28 , 157.17 , 148.2, 148.66, 139.94 , 139.87 , 121.75 , 121.58 , 87.97, 87.87, 85.20, 84.99, 70.75, 70.74, 70.27, 70.21, 33.04, 32.92, 28.78, 28.73, 26.51, 26.47, 23.76, 23.70. Figure 3. Micro-elemental analysis of MFU showing the percent by mass of the elements carbon, hydrogen and nitrogen in the structure of MFU compared to theoretical estimates. Figure 4. HPLC analysis of MFU showing a sharp peak at retention time of 1.85, performed by an independent laboratory using acetonitrile/water at a gradient of 20–90% with triethylammonium acetate (50 mM) additive. Figure 5. HRMS analysis of MFU. Supplement Table 1. Pyrimidine derivatives currently used as anticancer in clinical settings. Figure 6. NFKB taking at 1 sec (a), 2 secs (b), 3 secs (c) and 4 secs (d). Figure 7. FADD images taken at 1 sec (a), 6 secs (b), 12 secs (c) and 18 secs (d). Figure 8. B-actin images taken at 1 sec (a), 2 secs (b), 3 secs (c) and 4 secs (d). Figure 9. Images of BAX taken at 1 sec (a), 6 secs (b), 12 secs (c) and 18 secs (d). Figure 10. Caspase-9 images taken at 1 sec (a), 6 secs (b), 12 secs (c) and 18 secs (d). Figure 11. p53 images taken at 1 sec (a), 3 secs (b), 6 secs (c) and 9 secs (d). Figure 12. BCL-XL images taken at 1 sec (a), 3 secs (b), 6 secs (c) and 9 secs (d). Figure 13. In vitro studies comparing the efficiency of 5-FU and MFU. a) 2D culture of MiaPaca-2 cells, b) 2D culture of Panc-1 cells, c) 3D culture of Miapaca-2 cells and d) 3D culture of Panc-1 cells.